rowid,run.accession,experiment.accession,sample.accession,study.accession,bioproject,study.title,study.alias,study.type,study.abstract,study.attributes,study.PMIDs,sample.description,sample.title,sample.alias,sample.centername,sample.attributes,GEOsample.title,GEOsample.dataprocessing,GEOsample.source,GEOsample.treatmentprotocol,GEOsample.extractprotocol,GEOsample.growthprotocol,GEOsample.characteristics,GEOsample.accession,experiment.title,experiment.alias,experiment.library_name,experiment.design_description,experiment.library_construction_protocol,experiment.attributes,experiment.library_strategy,experiment.library_source,experiment.library_selection,experiment.library_layout,experiment.platform,experiment.instrument_model,experiment.spot_descriptor,experiment.study_ref,run.title,run.attributes,run.filename,run.semantic_name,run.total_bases,run.total_spots,run.alias,run.read_lengths,run.base_counts,run.r1_length,run.r2_length,run.r3_length,run.r4_length,run.Acount,run.Ccount,run.Gcount,run.Tcount,run.Ncount,run.experiment,run.pool_member,submission.accession,submission.srasource,submission.bioprojectsource,seqdetective.n_mates,seqdetective.mapping_rate.mate1,seqdetective.mapping_rate.mate2,seqdetective.nofeature_rate.mate1,seqdetective.nofeature_rate.mate2,seqdetective.sparsity.mate1,seqdetective.sparsity.mate2,seqdetective.pos_strand_rate.mate1,seqdetective.pos_strand_rate.mate2,seqdetective.readlen.mate1,seqdetective.readlen.mate2,seqdetective.judgement.mate1,seqdetective.judgement.mate2,seqdetective.judgement.reason,platform_family,instrument_generation,read_bias,selection_class,prep_kit,sc_or_bulk,tech_class,technology,tech_variant,submission.bioprojectsource.country,earliest_date,devstage_curation,devstage_curation_coarse,tissue_curation,tissue_curation_coarse
19476,ERR14208827,ERX13611047,ERS22979745,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,Scrambled 1 sample,SAMEA117628607,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 004|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 004|scientific name:Danio rerio,,,,,,,,,Raw reads: Scrambled 1 sample,webin reads Scrambled 1 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: Scrambled 1 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,F04.bam,bam,8497741.0,104283.0,webin reads Scrambled 1 sample,0:81.49,A:2628864;C:1112827;G:2146146;T:2609768;N:136,81,,,,2628864,1112827,2146146,2609768,136,ERX13611047,ERS22979745,ERA31123309,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19477,ERR14208813,ERX13611033,ERS22979748,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,ythdf2KO 1 sample,SAMEA117628610,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 007|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 007|scientific name:Danio rerio,,,,,,,,,Raw reads: ythdf2KO 1 sample,webin reads ythdf2KO 1 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: ythdf2KO 1 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,D09.bam,bam,673213184.0,8122011.0,webin reads ythdf2KO 1 sample,0:82.89,A:180836688;C:107076381;G:193711319;T:191586188;N:2608,82,,,,180836688,107076381,193711319,191586188,2608,ERX13611033,ERS22979748,ERA31123295,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19478,ERR14208811,ERX13611031,ERS22979755,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,GFP 6 sample,SAMEA117628617,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 014|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 014|scientific name:Danio rerio,,,,,,,,,Raw reads: GFP 6 sample,webin reads GFP 6 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: GFP 6 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,H01.bam,bam,337553787.0,3153658.0,webin reads GFP 6 sample,0:107.04,A:81816649;C:55524538;G:113686171;T:86525390;N:1039,107,,,,81816649,55524538,113686171,86525390,1039,ERX13611031,ERS22979755,ERA31123293,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19479,ERR14208821,ERX13611041,ERS22979752,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,ythdf2KO 5 sample,SAMEA117628614,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 011|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 011|scientific name:Danio rerio,,,,,,,,,Raw reads: ythdf2KO 5 sample,webin reads ythdf2KO 5 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: ythdf2KO 5 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,G10.bam,bam,226771232.0,2050630.0,webin reads ythdf2KO 5 sample,0:110.59,A:56944731;C:30766256;G:83820146;T:55239221;N:878,110,,,,56944731,30766256,83820146,55239221,878,ERX13611041,ERS22979752,ERA31123303,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19480,ERR14208818,ERX13611038,ERS22979750,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,ythdf2KO 3 sample,SAMEA117628612,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 009|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 009|scientific name:Danio rerio,,,,,,,,,Raw reads: ythdf2KO 3 sample,webin reads ythdf2KO 3 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: ythdf2KO 3 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,G08.bam,bam,351425328.0,3771763.0,webin reads ythdf2KO 3 sample,0:93.17,A:83933175;C:50138103;G:128300798;T:89051913;N:1339,93,,,,83933175,50138103,128300798,89051913,1339,ERX13611038,ERS22979750,ERA31123300,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19481,ERR14208826,ERX13611046,ERS22979758,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,YTHDF2 3 sample,SAMEA117628620,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 017|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 017|scientific name:Danio rerio,,,,,,,,,Raw reads: YTHDF2 3 sample,webin reads YTHDF2 3 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: YTHDF2 3 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,E01.bam,bam,181017083.0,1670383.0,webin reads YTHDF2 3 sample,0:108.37,A:38910474;C:31607688;G:67095095;T:43403227;N:599,108,,,,38910474,31607688,67095095,43403227,599,ERX13611046,ERS22979758,ERA31123308,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19482,ERR14208815,ERX13611035,ERS22979749,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,ythdf2KO 2 sample,SAMEA117628611,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 008|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 008|scientific name:Danio rerio,,,,,,,,,Raw reads: ythdf2KO 2 sample,webin reads ythdf2KO 2 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: ythdf2KO 2 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,D10.bam,bam,282414832.0,2675188.0,webin reads ythdf2KO 2 sample,0:105.57,A:73651155;C:41694223;G:96485766;T:70582706;N:982,105,,,,73651155,41694223,96485766,70582706,982,ERX13611035,ERS22979749,ERA31123297,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19483,ERR14208804,ERX13611024,ERS22979742,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,GFP 1 sample,SAMEA117628604,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 001|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 001|scientific name:Danio rerio,,,,,,,,,Raw reads: GFP 1 sample,webin reads GFP 1 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: GFP 1 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,F01.bam,bam,8036009.0,97716.0,webin reads GFP 1 sample,0:82.24,A:2445867;C:1074319;G:2086526;T:2429153;N:144,82,,,,2445867,1074319,2086526,2429153,144,ERX13611024,ERS22979742,ERA31123286,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19484,ERR14208829,ERX13611049,ERS22979747,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,Scrambled 3 sample,SAMEA117628609,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 006|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 006|scientific name:Danio rerio,,,,,,,,,Raw reads: Scrambled 3 sample,webin reads Scrambled 3 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: Scrambled 3 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,F06.bam,bam,90051514.0,910768.0,webin reads Scrambled 3 sample,0:98.87,A:30132090;C:13700682;G:16323331;T:29894332;N:1079,98,,,,30132090,13700682,16323331,29894332,1079,ERX13611049,ERS22979747,ERA31123311,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19485,ERR14208809,ERX13611029,ERS22979753,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,GFP 4 sample,SAMEA117628615,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 012|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 012|scientific name:Danio rerio,,,,,,,,,Raw reads: GFP 4 sample,webin reads GFP 4 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: GFP 4 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,G11.bam,bam,72278736.0,751207.0,webin reads GFP 4 sample,0:96.22,A:17589630;C:11153310;G:25212154;T:18323416;N:226,96,,,,17589630,11153310,25212154,18323416,226,ERX13611029,ERS22979753,ERA31123291,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19486,ERR14208806,ERX13611026,ERS22979743,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,GFP 2 sample,SAMEA117628605,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 002|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 002|scientific name:Danio rerio,,,,,,,,,Raw reads: GFP 2 sample,webin reads GFP 2 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: GFP 2 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,F02.bam,bam,19196592.0,230075.0,webin reads GFP 2 sample,0:83.44,A:5957309;C:2709511;G:4434875;T:6094552;N:345,83,,,,5957309,2709511,4434875,6094552,345,ERX13611026,ERS22979743,ERA31123288,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19487,ERR14208810,ERX13611030,ERS22979754,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,GFP 5 sample,SAMEA117628616,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 013|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 013|scientific name:Danio rerio,,,,,,,,,Raw reads: GFP 5 sample,webin reads GFP 5 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: GFP 5 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,G12.bam,bam,431708790.0,5173015.0,webin reads GFP 5 sample,0:83.45,A:119077902;C:68329173;G:126879243;T:117420803;N:1669,83,,,,119077902,68329173,126879243,117420803,1669,ERX13611030,ERS22979754,ERA31123292,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19488,ERR14208828,ERX13611048,ERS22979746,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,Scrambled 2 sample,SAMEA117628608,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 005|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 005|scientific name:Danio rerio,,,,,,,,,Raw reads: Scrambled 2 sample,webin reads Scrambled 2 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: Scrambled 2 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,F05.bam,bam,32695808.0,357320.0,webin reads Scrambled 2 sample,0:91.50,A:10989624;C:4719485;G:5954777;T:11031603;N:319,91,,,,10989624,4719485,5954777,11031603,319,ERX13611048,ERS22979746,ERA31123310,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19489,ERR14208819,ERX13611039,ERS22979751,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,ythdf2KO 4 sample,SAMEA117628613,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 010|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 010|scientific name:Danio rerio,,,,,,,,,Raw reads: ythdf2KO 4 sample,webin reads ythdf2KO 4 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: ythdf2KO 4 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,G09.bam,bam,254231539.0,2766205.0,webin reads ythdf2KO 4 sample,0:91.91,A:63984512;C:40328422;G:86431821;T:63485754;N:1030,91,,,,63984512,40328422,86431821,63485754,1030,ERX13611039,ERS22979751,ERA31123301,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19490,ERR14208808,ERX13611028,ERS22979744,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,GFP 3 sample,SAMEA117628606,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 003|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 003|scientific name:Danio rerio,,,,,,,,,Raw reads: GFP 3 sample,webin reads GFP 3 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: GFP 3 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,F03.bam,bam,35932988.0,419944.0,webin reads GFP 3 sample,0:85.57,A:11252929;C:5321445;G:8032297;T:11325814;N:503,85,,,,11252929,5321445,8032297,11325814,503,ERX13611028,ERS22979744,ERA31123290,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19491,ERR14208825,ERX13611045,ERS22979757,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,YTHDF2 2 sample,SAMEA117628619,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 016|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 016|scientific name:Danio rerio,,,,,,,,,Raw reads: YTHDF2 2 sample,webin reads YTHDF2 2 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: YTHDF2 2 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,D12.bam,bam,626993184.0,7599502.0,webin reads YTHDF2 2 sample,0:82.50,A:182701108;C:91008174;G:172822143;T:180459253;N:2506,82,,,,182701108,91008174,172822143,180459253,2506,ERX13611045,ERS22979757,ERA31123307,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
19492,ERR14208822,ERX13611042,ERS22979756,ERP167299,PRJEB83709,YTHDF2 and ASD DM,3296f78e-e1c0-43ea-968c-53fe04e67615,Other,Among autistic individuals  a subphenotype of disproportionate megalencephaly ASD DM seen at three years of age is associated with co occurring intellectual disability and poorer prognoses later in life. However  many of the genes contributing to ASD DM have yet to be delineated. In this study  we identified additional ASD DM candidate genes with the aim to better define the genetic etiology of this subphenotype of autism. We expanded the previously studied sample size of ASD DM individuals ten fold by including probands from the Autism Phenome Project and Simons Simplex Collection  totaling 766 autistic individuals meeting the criteria for megalencephaly or macrocephaly and revealing 154 candidate ASD DM genes harboring de novo protein impacting variants. Our findings include fourteen high confidence autism genes and seven genes previously associated with DM. Five impacted genes have previously been associated with both autism and DM  including CHD8 and PTEN. By performing functional network analysis  we expanded to additional candidate genes  including one previously implicated in ASD DM PIK3CA as well as 184 additional genes previously implicated in ASD or DM alone. Using zebrafish  we modeled a de novo tandem duplication impacting YTHDF2  encoding an N6 methyladenosine m6A mRNA reader  in an ASD DM proband. Testing zebrafish CRISPR knockdown led to reduced head/brain size  while overexpressing YTHDF2 resulted in increased head and brain size matching that of the proband. Single cell transcriptomes of YTHDF2 gain of function larvae point to reduced expression of Fragile X syndrome associated FMRP target genes globally and in the developing brain  providing insight into the mechanism underlying autistic phenotypes. We additionally discovered a variant impacting a different gene encoding an m6A reader  YTHDC1  in our ASD DM cohort. Though we highlight only two cases to date  our study provides support for the m6A RNA modification pathway as potentially contributing to this severe form of autism.,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,,Single cell RNA sequencing of zebrafish heads,YTHDF2 1 sample,SAMEA117628618,UNIVERSITY OF CALIFORNIA - DAVIS,ENA first public:2025 02 01|INSDC center name:UNIVERSITY OF CALIFORNIA   DAVIS|INSDC status:public|Submitter Id:Sample 015|collection date:2022 08 07|common name:zebrafish|dev stage:72 hpf|geographic location country and/or sea:USA|sample name:Sample 015|scientific name:Danio rerio,,,,,,,,,Raw reads: YTHDF2 1 sample,webin reads YTHDF2 1 sample,,unspecified,,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina NovaSeq 6000,,ERP167299,Raw reads: YTHDF2 1 sample,ENA FIRST PUBLIC:2025 02 01|ENA LAST UPDATE:2025 02 01,D11.bam,bam,120748751.0,1368887.0,webin reads YTHDF2 1 sample,0:88.21,A:34957526;C:19985010;G:32927086;T:32878659;N:470,88,,,,34957526,19985010,32927086,32878659,470,ERX13611042,ERS22979756,ERA31123304,UNIVERSITY OF CALIFORNIA - DAVIS|European Nucleotide Archive,UNIVERSITY OF CALIFORNIA - DAVIS,,,,,,,,,,,,B,,usable mapping rate,illumina,novaseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_generic,generic-scrnaseq-only,,United States,2025-02-01,Larval,Larval,Head,Nervous System
25107,SRR25605432,SRX21332628,SRS18578260,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep3,GSM7702835,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702835,GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq,GSM7702835 r1,GSM7702835,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_GFP_pos_S4_L001_R2_001.fastq.gz 2707_GFP_pos_S4_L001_R1_001.fastq.gz 2707_GFP_pos_S4_L001_I2_001.fastq.gz 2707_GFP_pos_S4_L001_I1_001.fastq.gz,fastq fastq fastq fastq,21603309774.0,156545723.0,GSM7702835 r1,0:10 1:10 2:28 3:90,A:3960451458;C:3105338977;G:3480072824;T:3541218595;N:2033216,10,10,28,90,3960451458,3105338977,3480072824,3541218595,2033216,SRX21332628,SRS18578260,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.94286,,0.14175,,0.78395,,0.51541,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25108,SRR25605433,SRX21332628,SRS18578260,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep3,GSM7702835,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702835,GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq,GSM7702835 r1,GSM7702835,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_GFP_pos_S4_L002_I1_001.fastq.gz 2707_GFP_pos_S4_L002_I2_001.fastq.gz 2707_GFP_pos_S4_L002_R1_001.fastq.gz 2707_GFP_pos_S4_L002_R2_001.fastq.gz,fastq fastq fastq fastq,22413465270.0,162416415.0,GSM7702835 r2,0:10 1:10 2:28 3:90,A:4104405257;C:3220235633;G:3625448968;T:3667329471;N:58021,10,10,28,90,4104405257,3220235633,3625448968,3667329471,58021,SRX21332628,SRS18578260,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.94239,,0.14234,,0.78338,,0.50616,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25109,SRR25822232,SRX21332628,SRS18578260,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep3,GSM7702835,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702835,GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq,GSM7702835 r1,GSM7702835,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_GFP_pos_S4_L001_R2_002.fastq.gz 2707_GFP_pos_S4_L001_R1_002.fastq.gz 2707_GFP_pos_S4_L001_I2_002.fastq.gz 2707_GFP_pos_S4_L001_I1_002.fastq.gz,fastq fastq fastq fastq,21424338264.0,155248828.0,GSM7702835 r3,0:10 1:10 2:28 3:90,A:3928077020;C:3077672525;G:3451667186;T:3513734857;N:1242932,10,10,28,90,3928077020,3077672525,3451667186,3513734857,1242932,SRX21332628,SRS18578260,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.9433,,0.14242,,0.78328,,0.52384,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25110,SRR25822233,SRX21332628,SRS18578260,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep3,GSM7702835,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702835,GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq,GSM7702835 r1,GSM7702835,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_GFP_pos_S4_L002_I1_002.fastq.gz 2707_GFP_pos_S4_L002_I2_002.fastq.gz 2707_GFP_pos_S4_L002_R1_002.fastq.gz 2707_GFP_pos_S4_L002_R2_002.fastq.gz,fastq fastq fastq fastq,22542479952.0,163351304.0,GSM7702835 r4,0:10 1:10 2:28 3:90,A:4130174287;C:3235362228;G:3643457599;T:3691295761;N:1327485,10,10,28,90,4130174287,3235362228,3643457599,3691295761,1327485,SRX21332628,SRS18578260,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.94183,,0.14124,,0.78301,,0.50517,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25111,SRR25605434,SRX21332627,SRS18578259,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep3,GSM7702834,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702834,GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq,GSM7702834 r1,GSM7702834,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_pos_Mcherry_S3_L001_R2_001.fastq.gz 2707_pos_Mcherry_S3_L001_R1_001.fastq.gz 2707_pos_Mcherry_S3_L001_I2_001.fastq.gz 2707_pos_Mcherry_S3_L001_I1_001.fastq.gz,fastq fastq fastq fastq,20161522758.0,146097991.0,GSM7702834 r1,0:10 1:10 2:28 3:90,A:3649819934;C:2958286158;G:3267504300;T:3271384686;N:1824112,10,10,28,90,3649819934,2958286158,3267504300,3271384686,1824112,SRX21332627,SRS18578259,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.94851,,0.1483,,0.80012,,0.52549,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25112,SRR25605435,SRX21332627,SRS18578259,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep3,GSM7702834,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702834,GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq,GSM7702834 r1,GSM7702834,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_pos_Mcherry_S3_L002_I1_001.fastq.gz 2707_pos_Mcherry_S3_L002_I2_001.fastq.gz 2707_pos_Mcherry_S3_L002_R1_001.fastq.gz 2707_pos_Mcherry_S3_L002_R2_001.fastq.gz,fastq fastq fastq fastq,20708273928.0,150059956.0,GSM7702834 r2,0:10 1:10 2:28 3:90,A:3746088654;C:3037032642;G:3368938151;T:3353289242;N:47351,10,10,28,90,3746088654,3037032642,3368938151,3353289242,47351,SRX21332627,SRS18578259,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.94778,,0.14581,,0.80028,,0.52885,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25113,SRR25822230,SRX21332627,SRS18578259,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep3,GSM7702834,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702834,GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq,GSM7702834 r1,GSM7702834,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_pos_Mcherry_S3_L001_I1_002.fastq.gz 2707_pos_Mcherry_S3_L001_I2_002.fastq.gz 2707_pos_Mcherry_S3_L001_R1_002.fastq.gz 2707_pos_Mcherry_S3_L001_R2_002.fastq.gz,fastq fastq fastq fastq,20078121078.0,145493631.0,GSM7702834 r3,0:10 1:10 2:28 3:90,A:3634984131;C:2943891505;G:3255149272;T:3259243920;N:1157962,10,10,28,90,3634984131,2943891505,3255149272,3259243920,1157962,SRX21332627,SRS18578259,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.94884,,0.14622,,0.80099,,0.52572,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25114,SRR25822231,SRX21332627,SRS18578259,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep3,GSM7702834,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep3,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702834,GSM7702834: mcherry enriched rep3; Danio rerio; RNA Seq,GSM7702834 r1,GSM7702834,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,2707_pos_Mcherry_S3_L002_I1_002.fastq.gz 2707_pos_Mcherry_S3_L002_I2_002.fastq.gz 2707_pos_Mcherry_S3_L002_R1_002.fastq.gz 2707_pos_Mcherry_S3_L002_R2_002.fastq.gz,fastq fastq fastq fastq,20884392978.0,151336181.0,GSM7702834 r4,0:10 1:10 2:28 3:90,A:3779370721;C:3059502508;G:3396295214;T:3383865877;N:1221970,10,10,28,90,3779370721,3059502508,3396295214,3383865877,1221970,SRX21332627,SRS18578259,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.94896,,0.14559,,0.8002,,0.51418,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25115,SRR25605436,SRX21332626,SRS18578258,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep2,GSM7702833,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702833,GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq,GSM7702833 r1,GSM7702833,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py|options:  allowEarlyFileEnd,1907_GFP_pos_S2_L001_I1_001.fastq.gz 1907_GFP_pos_S2_L001_I2_001.fastq.gz 1907_GFP_pos_S2_L001_R1_001.fastq.gz 1907_GFP_pos_S2_L001_R2_001.fastq.gz,fastq fastq fastq fastq,22174775364.0,160686778.0,GSM7702833 r1,0:10 1:10 2:28 3:90,A:4207510387;C:3050740898;G:3362227609;T:3839333241;N:1997885,10,10,28,90,4207510387,3050740898,3362227609,3839333241,1997885,SRX21332626,SRS18578258,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.93287,,0.11844,,0.81087,,0.55264,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25116,SRR25605437,SRX21332626,SRS18578258,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep2,GSM7702833,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702833,GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq,GSM7702833 r1,GSM7702833,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py|options:  allowEarlyFileEnd,1907_GFP_pos_S2_L002_I1_001.fastq.gz 1907_GFP_pos_S2_L002_I2_001.fastq.gz 1907_GFP_pos_S2_L002_R1_001.fastq.gz 1907_GFP_pos_S2_L002_R2_001.fastq.gz,fastq fastq fastq fastq,22047847518.0,159767011.0,GSM7702833 r2,0:10 1:10 2:28 3:90,A:4174013532;C:3033959926;G:3357962867;T:3813042664;N:52001,10,10,28,90,4174013532,3033959926,3357962867,3813042664,52001,SRX21332626,SRS18578258,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.93359,,0.11585,,0.81087,,0.55705,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25117,SRR25822228,SRX21332626,SRS18578258,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep2,GSM7702833,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702833,GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq,GSM7702833 r1,GSM7702833,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,1907_GFP_pos_S2_L001_I1_002.fastq.gz 1907_GFP_pos_S2_L001_I2_002.fastq.gz 1907_GFP_pos_S2_L001_R1_002.fastq.gz 1907_GFP_pos_S2_L001_R2_002.fastq.gz,fastq fastq fastq fastq,22010894568.0,159499236.0,GSM7702833 r3,0:10 1:10 2:28 3:90,A:4176090595;C:3026940106;G:3336506731;T:3814128890;N:1264918,10,10,28,90,4176090595,3026940106,3336506731,3814128890,1264918,SRX21332626,SRS18578258,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.93457,,0.11733,,0.81032,,0.55203,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25118,SRR25822229,SRX21332626,SRS18578258,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,gfp enriched rep2,GSM7702833,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,gfp enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702833,GSM7702833: gfp enriched rep2; Danio rerio; RNA Seq,GSM7702833 r1,GSM7702833,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,1907_GFP_pos_S2_L002_I1_002.fastq.gz 1907_GFP_pos_S2_L002_I2_002.fastq.gz 1907_GFP_pos_S2_L002_R1_002.fastq.gz 1907_GFP_pos_S2_L002_R2_002.fastq.gz,fastq fastq fastq fastq,22227220746.0,161066817.0,GSM7702833 r4,0:10 1:10 2:28 3:90,A:4209198596;C:3055507768;G:3382717459;T:3847294849;N:1294858,10,10,28,90,4209198596,3055507768,3382717459,3847294849,1294858,SRX21332626,SRS18578258,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.93428,,0.11629,,0.81014,,0.56014,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25119,SRR25605438,SRX21332625,SRS18578257,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep2,GSM7702832,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702832,GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq,GSM7702832 r1,GSM7702832,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,1907_pos_pos_S1_L001_I1_001.fastq.gz 1907_pos_pos_S1_L001_I2_001.fastq.gz 1907_pos_pos_S1_L001_R1_001.fastq.gz 1907_pos_pos_S1_L001_R2_001.fastq.gz,fastq fastq fastq fastq,25141955454.0,182188083.0,GSM7702832 r1,0:10 1:10 2:28 3:90,A:4942067104;C:3446689328;G:3778138998;T:4227670796;N:2361244,10,10,28,90,4942067104,3446689328,3778138998,4227670796,2361244,SRX21332625,SRS18578257,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.90803,,0.11273,,0.81872,,0.54345,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25120,SRR25605439,SRX21332625,SRS18578257,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep2,GSM7702832,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702832,GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq,GSM7702832 r1,GSM7702832,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,1907_pos_pos_S1_L002_R2_001.fastq.gz 1907_pos_pos_S1_L002_R1_001.fastq.gz 1907_pos_pos_S1_L002_I2_001.fastq.gz 1907_pos_pos_S1_L002_I1_001.fastq.gz,fastq fastq fastq fastq,25433730786.0,184302397.0,GSM7702832 r2,0:10 1:10 2:28 3:90,A:4980326106;C:3488986366;G:3842594871;T:4275245265;N:63122,10,10,28,90,4980326106,3488986366,3842594871,4275245265,63122,SRX21332625,SRS18578257,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.91035,,0.11175,,0.82016,,0.52814,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25121,SRR25822226,SRX21332625,SRS18578257,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep2,GSM7702832,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702832,GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq,GSM7702832 r1,GSM7702832,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,1907_pos_pos_S1_L001_R2_002.fastq.gz 1907_pos_pos_S1_L001_R1_002.fastq.gz 1907_pos_pos_S1_L001_I2_002.fastq.gz 1907_pos_pos_S1_L001_I1_002.fastq.gz,fastq fastq fastq fastq,24834656088.0,179961276.0,GSM7702832 r3,0:10 1:10 2:28 3:90,A:4878672093;C:3403443000;G:3732878174;T:4180051888;N:1469685,10,10,28,90,4878672093,3403443000,3732878174,4180051888,1469685,SRX21332625,SRS18578257,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.90776,,0.11325,,0.81852,,0.54011,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
25122,SRR25822227,SRX21332625,SRS18578257,SRP454539,PRJNA1004255,Unique activities of two overlapping PAX6 retinal enhancers,GSE240575,Transcriptome Analysis,Enhancers play a critical role in development by precisely modulating spatial  temporal  and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease  however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development  with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive  redundant  or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers  HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos  we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping  these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active  we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique  we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.,,pubmed:37643867,,mcherry enriched rep2,GSM7702832,,source name:Eye|tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf|geo loc name:missing|collection date:missing,mcherry enriched rep2,Cell Ranger v6.1.2 was used to perform alignment  filtering  barcode counting  and UMI counting. A custom reference genome was created for alignment using cellranger mkref  combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al.  2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size  detected genes  and mitochondrial reads McCarthy et al.  2017. Cells with detected genes ≥ 500  library size ≥ 800  and mitochondrial reads ≤ 10% were retained. Within the processed dataset  mean reads per cell = 11239  and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al.  2019; Farnsworth et al.  2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types.  Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al.  2018. SCTransform was used to perform log normalisation  scaling  and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used  with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs  using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples  we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al.  2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes  and other known genes for each cell type  using information from the literature and ZFIN Sprague et al.  2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function  using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files  matrix files  Seurat object RDS file,Eye,,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.,,tissue:Eye|genotype:NRE eGFP/HS5 mCherry|developmental stage:48 hpf,GSM7702832,GSM7702832: mcherry enriched rep2; Danio rerio; RNA Seq,GSM7702832 r1,GSM7702832,1,NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf  embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF  and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C  then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution  and once with FACSmax Amsbio. In a final 500 µl FACSmax  the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris  and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,NextSeq 2000,,SRP454539,,loader:fastq load.py,1907_pos_pos_S1_L002_I1_002.fastq.gz 1907_pos_pos_S1_L002_I2_002.fastq.gz 1907_pos_pos_S1_L002_R1_002.fastq.gz 1907_pos_pos_S1_L002_R2_002.fastq.gz,fastq fastq fastq fastq,25609437216.0,185575632.0,GSM7702832 r4,0:10 1:10 2:28 3:90,A:5015380345;C:3509305882;G:3867598814;T:4308011415;N:1510424,10,10,28,90,5015380345,3509305882,3867598814,4308011415,1510424,SRX21332625,SRS18578257,SRA1702612,"Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh","Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh",1,0.90907,,0.11192,,0.81994,,0.50902,,90,,B,,usable mapping rate,illumina,nextseq_v2,3prime,cdna_unspecified,unknown,sc,single_cell_droplet,10x,,United Kingdom,2023-08-10,Hatching,Embryo,Eye,Sensory System
26558,SRR26173859,SRX21885960,SRS18977085,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F17 R1,GSM7804200,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F17 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804200,GSM7804200: V2a sample2 354 F17 R1; Danio rerio; RNA Seq,GSM7804200 r1,GSM7804200,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F17_R1.fastq.gz,fastq,26979490.0,627430.0,GSM7804200 r1,0:43,A:7493119;C:5872644;G:6013311;T:7600416;N:0,43,,,,7493119,5872644,6013311,7600416,0,SRX21885960,SRS18977085,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.82979,,0.33034,,0.94253,,0.53995,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26559,SRR26173860,SRX21885959,SRS18977083,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F16 R1,GSM7804199,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F16 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804199,GSM7804199: V2a sample2 354 F16 R1; Danio rerio; RNA Seq,GSM7804199 r1,GSM7804199,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F16_R1.fastq.gz,fastq,26312130.0,611910.0,GSM7804199 r1,0:43,A:7117249;C:5957278;G:6103577;T:7134026;N:0,43,,,,7117249,5957278,6103577,7134026,0,SRX21885959,SRS18977083,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87071,,0.23579,,0.90678,,0.52226,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26560,SRR26173861,SRX21885958,SRS18977084,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F15 R1,GSM7804198,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F15 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804198,GSM7804198: V2a sample2 354 F15 R1; Danio rerio; RNA Seq,GSM7804198 r1,GSM7804198,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F15_R1.fastq.gz,fastq,32073872.0,745904.0,GSM7804198 r1,0:43,A:8695962;C:7255833;G:7422516;T:8699561;N:0,43,,,,8695962,7255833,7422516,8699561,0,SRX21885958,SRS18977084,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.86262,,0.24391,,0.90881,,0.52511,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26561,SRR26173862,SRX21885957,SRS18977081,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F14 R1,GSM7804197,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F14 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804197,GSM7804197: V2a sample2 354 F14 R1; Danio rerio; RNA Seq,GSM7804197 r1,GSM7804197,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F14_R1.fastq.gz,fastq,31158875.0,724625.0,GSM7804197 r1,0:43,A:8769852;C:6707813;G:6866255;T:8814955;N:0,43,,,,8769852,6707813,6866255,8814955,0,SRX21885957,SRS18977081,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8739,,0.27254,,0.9246,,0.54185,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26562,SRR26173863,SRX21885956,SRS18977082,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F13 R1,GSM7804196,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F13 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804196,GSM7804196: V2a sample2 354 F13 R1; Danio rerio; RNA Seq,GSM7804196 r1,GSM7804196,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F13_R1.fastq.gz,fastq,41289804.0,960228.0,GSM7804196 r1,0:43,A:11114918;C:9358398;G:9568761;T:11247727;N:0,43,,,,11114918,9358398,9568761,11247727,0,SRX21885956,SRS18977082,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.82861,,0.29409,,0.93235,,0.49485,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26563,SRR26173864,SRX21885955,SRS18977079,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F12 R1,GSM7804195,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F12 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804195,GSM7804195: V2a sample2 354 F12 R1; Danio rerio; RNA Seq,GSM7804195 r1,GSM7804195,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F12_R1.fastq.gz,fastq,30569259.0,710913.0,GSM7804195 r1,0:43,A:8286806;C:6854804;G:7028453;T:8399196;N:0,43,,,,8286806,6854804,7028453,8399196,0,SRX21885955,SRS18977079,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80426,,0.29136,,0.94123,,0.54305,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26564,SRR26173865,SRX21885954,SRS18977080,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F11 R1,GSM7804194,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F11 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804194,GSM7804194: V2a sample2 354 F11 R1; Danio rerio; RNA Seq,GSM7804194 r1,GSM7804194,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F11_R1.fastq.gz,fastq,29626097.0,688979.0,GSM7804194 r1,0:43,A:8063765;C:6645347;G:6803778;T:8113207;N:0,43,,,,8063765,6645347,6803778,8113207,0,SRX21885954,SRS18977080,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.84972,,0.26423,,0.9234,,0.54087,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26565,SRR26173866,SRX21885953,SRS18977078,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 F10 R1,GSM7804193,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 F10 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804193,GSM7804193: V2a sample2 354 F10 R1; Danio rerio; RNA Seq,GSM7804193 r1,GSM7804193,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_F10_R1.fastq.gz,fastq,24594409.0,571963.0,GSM7804193 r1,0:43,A:6605642;C:5535165;G:5682586;T:6771016;N:0,43,,,,6605642,5535165,5682586,6771016,0,SRX21885953,SRS18977078,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.77683,,0.28872,,0.94633,,0.52513,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26566,SRR26173867,SRX21885952,SRS18977077,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E9 R1,GSM7804168,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E9 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804168,GSM7804168: V2a sample2 354 E9 R1; Danio rerio; RNA Seq,GSM7804168 r1,GSM7804168,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E9_R1.fastq.gz,fastq,33259941.0,773487.0,GSM7804168 r1,0:43,A:9004615;C:7506729;G:7674909;T:9073688;N:0,43,,,,9004615,7506729,7674909,9073688,0,SRX21885952,SRS18977077,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.85528,,0.32094,,0.89217,,0.50224,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26567,SRR26173868,SRX21885951,SRS18977076,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E8 R1,GSM7804167,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E8 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804167,GSM7804167: V2a sample2 354 E8 R1; Danio rerio; RNA Seq,GSM7804167 r1,GSM7804167,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E8_R1.fastq.gz,fastq,45348316.0,1054612.0,GSM7804167 r1,0:43,A:12143243;C:10459395;G:10612458;T:12133220;N:0,43,,,,12143243,10459395,10612458,12133220,0,SRX21885951,SRS18977076,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8783,,0.19318,,0.88572,,0.49101,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26568,SRR26173869,SRX21885950,SRS18977075,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E7 R1,GSM7804166,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E7 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804166,GSM7804166: V2a sample2 354 E7 R1; Danio rerio; RNA Seq,GSM7804166 r1,GSM7804166,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E7_R1.fastq.gz,fastq,30522002.0,709814.0,GSM7804166 r1,0:43,A:8167984;C:7033955;G:7127866;T:8192197;N:0,43,,,,8167984,7033955,7127866,8192197,0,SRX21885950,SRS18977075,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87804,,0.20762,,0.88418,,0.51381,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26569,SRR26173870,SRX21885949,SRS18977074,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E6 R1,GSM7804165,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E6 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804165,GSM7804165: V2a sample2 354 E6 R1; Danio rerio; RNA Seq,GSM7804165 r1,GSM7804165,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E6_R1.fastq.gz,fastq,39095858.0,909206.0,GSM7804165 r1,0:43,A:10597068;C:8804712;G:8974908;T:10719170;N:0,43,,,,10597068,8804712,8974908,10719170,0,SRX21885949,SRS18977074,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8093,,0.38297,,0.91545,,0.56195,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26570,SRR26173871,SRX21885948,SRS18977073,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E5 R1,GSM7804164,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E5 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804164,GSM7804164: V2a sample2 354 E5 R1; Danio rerio; RNA Seq,GSM7804164 r1,GSM7804164,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E5_R1.fastq.gz,fastq,33632880.0,782160.0,GSM7804164 r1,0:43,A:9099447;C:7602241;G:7747634;T:9183558;N:0,43,,,,9099447,7602241,7747634,9183558,0,SRX21885948,SRS18977073,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.83226,,0.28405,,0.90715,,0.52148,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26571,SRR26173872,SRX21885947,SRS18977072,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E4 R1,GSM7804163,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E4 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804163,GSM7804163: V2a sample2 354 E4 R1; Danio rerio; RNA Seq,GSM7804163 r1,GSM7804163,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E4_R1.fastq.gz,fastq,10823057.0,251699.0,GSM7804163 r1,0:43,A:3014035;C:2419189;G:2487845;T:2901988;N:0,43,,,,3014035,2419189,2487845,2901988,0,SRX21885947,SRS18977072,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.85783,,0.26946,,0.91033,,0.52459,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26572,SRR26173873,SRX21885946,SRS18977068,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E3 R1,GSM7804162,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E3 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804162,GSM7804162: V2a sample2 354 E3 R1; Danio rerio; RNA Seq,GSM7804162 r1,GSM7804162,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E3_R1.fastq.gz,fastq,49775381.0,1157567.0,GSM7804162 r1,0:43,A:13325978;C:11313973;G:11568082;T:13567348;N:0,43,,,,13325978,11313973,11568082,13567348,0,SRX21885946,SRS18977068,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80873,,0.29504,,0.93791,,0.52789,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26573,SRR26173874,SRX21885945,SRS18977070,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 E2 R1,GSM7804161,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 E2 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804161,GSM7804161: V2a sample2 354 E2 R1; Danio rerio; RNA Seq,GSM7804161 r1,GSM7804161,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_E2_R1.fastq.gz,fastq,44072334.0,1024938.0,GSM7804161 r1,0:43,A:11780930;C:10068667;G:10185567;T:12037170;N:0,43,,,,11780930,10068667,10185567,12037170,0,SRX21885945,SRS18977070,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.78009,,0.2621,,0.943,,0.54719,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26574,SRR26173875,SRX21885944,SRS18977071,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 D1 R1,GSM7804136,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 D1 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804136,GSM7804136: V2a sample2 354 D1 R1; Danio rerio; RNA Seq,GSM7804136 r1,GSM7804136,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_D1_R1.fastq.gz,fastq,49923.0,1161.0,GSM7804136 r1,0:43,A:12799;C:11604;G:11134;T:14386;N:0,43,,,,12799,11604,11134,14386,0,SRX21885944,SRS18977071,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.5773,,0.18478,,0.99472,,0.51097,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26575,SRR26173876,SRX21885943,SRS18977069,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 C24 R1,GSM7804135,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 C24 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804135,GSM7804135: V2a sample2 354 C24 R1; Danio rerio; RNA Seq,GSM7804135 r1,GSM7804135,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_C24_R1.fastq.gz,fastq,30735540.0,714780.0,GSM7804135 r1,0:43,A:7999390;C:7036767;G:7190113;T:8509270;N:0,43,,,,7999390,7036767,7190113,8509270,0,SRX21885943,SRS18977069,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.48457,,0.16974,,0.99328,,0.82979,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26576,SRR26173877,SRX21885942,SRS18977067,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 C23 R1,GSM7804134,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 C23 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804134,GSM7804134: V2a sample2 354 C23 R1; Danio rerio; RNA Seq,GSM7804134 r1,GSM7804134,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_C23_R1.fastq.gz,fastq,24386031.0,567117.0,GSM7804134 r1,0:43,A:6620599;C:5230020;G:5371347;T:7164065;N:0,43,,,,6620599,5230020,5371347,7164065,0,SRX21885942,SRS18977067,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.55183,,0.13531,,0.99105,,0.54904,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26577,SRR26173878,SRX21885941,SRS18977064,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 C22 R1,GSM7804133,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 C22 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804133,GSM7804133: V2a sample2 354 C22 R1; Danio rerio; RNA Seq,GSM7804133 r1,GSM7804133,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_C22_R1.fastq.gz,fastq,35984550.0,836850.0,GSM7804133 r1,0:43,A:9917644;C:7959027;G:8126203;T:9981676;N:0,43,,,,9917644,7959027,8126203,9981676,0,SRX21885941,SRS18977064,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.85473,,0.2804,,0.91165,,0.52909,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26578,SRR26173879,SRX21885940,SRS18977065,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 C21 R1,GSM7804132,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 C21 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804132,GSM7804132: V2a sample2 354 C21 R1; Danio rerio; RNA Seq,GSM7804132 r1,GSM7804132,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_C21_R1.fastq.gz,fastq,32265738.0,750366.0,GSM7804132 r1,0:43,A:8963112;C:7046873;G:7192067;T:9063686;N:0,43,,,,8963112,7046873,7192067,9063686,0,SRX21885940,SRS18977065,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8426,,0.36876,,0.91179,,0.58759,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26579,SRR26173880,SRX21885939,SRS18977066,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 C20 R1,GSM7804131,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 C20 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804131,GSM7804131: V2a sample2 354 C20 R1; Danio rerio; RNA Seq,GSM7804131 r1,GSM7804131,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_C20_R1.fastq.gz,fastq,33471157.0,778399.0,GSM7804131 r1,0:43,A:9073629;C:7510863;G:7675381;T:9211284;N:0,43,,,,9073629,7510863,7675381,9211284,0,SRX21885939,SRS18977066,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.82141,,0.28926,,0.93655,,0.49873,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26580,SRR26173881,SRX21885938,SRS18977062,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 C19 R1,GSM7804130,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 C19 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804130,GSM7804130: V2a sample2 354 C19 R1; Danio rerio; RNA Seq,GSM7804130 r1,GSM7804130,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_C19_R1.fastq.gz,fastq,30131304.0,700728.0,GSM7804130 r1,0:43,A:8036932;C:6893678;G:7034855;T:8165839;N:0,43,,,,8036932,6893678,7034855,8165839,0,SRX21885938,SRS18977062,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80896,,0.25245,,0.94067,,0.54072,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26581,SRR26173882,SRX21885937,SRS18977061,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 C18 R1,GSM7804129,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 C18 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804129,GSM7804129: V2a sample2 354 C18 R1; Danio rerio; RNA Seq,GSM7804129 r1,GSM7804129,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_C18_R1.fastq.gz,fastq,31050429.0,722103.0,GSM7804129 r1,0:43,A:8363024;C:7088111;G:7227301;T:8371993;N:0,43,,,,8363024,7088111,7227301,8371993,0,SRX21885937,SRS18977061,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87399,,0.20843,,0.89881,,0.49682,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26582,SRR26173883,SRX21885936,SRS18977063,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B17 R1,GSM7804104,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B17 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804104,GSM7804104: V2a sample2 354 B17 R1; Danio rerio; RNA Seq,GSM7804104 r1,GSM7804104,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B17_R1.fastq.gz,fastq,47093729.0,1095203.0,GSM7804104 r1,0:43,A:13057598;C:10185883;G:10457076;T:13393172;N:0,43,,,,13057598,10185883,10457076,13393172,0,SRX21885936,SRS18977063,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80343,,0.36098,,0.95059,,0.52562,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26583,SRR26173884,SRX21885935,SRS18977060,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B16 R1,GSM7804103,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B16 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804103,GSM7804103: V2a sample2 354 B16 R1; Danio rerio; RNA Seq,GSM7804103 r1,GSM7804103,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B16_R1.fastq.gz,fastq,34160791.0,794437.0,GSM7804103 r1,0:43,A:9178469;C:7729053;G:7901331;T:9351938;N:0,43,,,,9178469,7729053,7901331,9351938,0,SRX21885935,SRS18977060,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.79028,,0.25695,,0.94627,,0.51668,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26584,SRR26173885,SRX21885934,SRS18977059,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B15 R1,GSM7804102,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B15 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804102,GSM7804102: V2a sample2 354 B15 R1; Danio rerio; RNA Seq,GSM7804102 r1,GSM7804102,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B15_R1.fastq.gz,fastq,29152495.0,677965.0,GSM7804102 r1,0:43,A:7966546;C:6516464;G:6675123;T:7994362;N:0,43,,,,7966546,6516464,6675123,7994362,0,SRX21885934,SRS18977059,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.83832,,0.25969,,0.92673,,0.51661,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26585,SRR26173886,SRX21885933,SRS18977057,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B14 R1,GSM7804101,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B14 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804101,GSM7804101: V2a sample2 354 B14 R1; Danio rerio; RNA Seq,GSM7804101 r1,GSM7804101,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B14_R1.fastq.gz,fastq,23811379.0,553753.0,GSM7804101 r1,0:43,A:6532691;C:5215122;G:5353003;T:6710563;N:0,43,,,,6532691,5215122,5353003,6710563,0,SRX21885933,SRS18977057,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.74067,,0.3282,,0.95272,,0.49582,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26586,SRR26173887,SRX21885932,SRS18977058,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B13 R1,GSM7804100,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B13 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804100,GSM7804100: V2a sample2 354 B13 R1; Danio rerio; RNA Seq,GSM7804100 r1,GSM7804100,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B13_R1.fastq.gz,fastq,18141356.0,421892.0,GSM7804100 r1,0:43,A:4926550;C:4092334;G:4186281;T:4936191;N:0,43,,,,4926550,4092334,4186281,4936191,0,SRX21885932,SRS18977058,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87463,,0.22642,,0.89826,,0.50541,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26587,SRR26173888,SRX21885931,SRS18977056,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B12 R1,GSM7804099,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B12 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804099,GSM7804099: V2a sample2 354 B12 R1; Danio rerio; RNA Seq,GSM7804099 r1,GSM7804099,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B12_R1.fastq.gz,fastq,34932039.0,812373.0,GSM7804099 r1,0:43,A:9555191;C:7704451;G:7897587;T:9774810;N:0,43,,,,9555191,7704451,7897587,9774810,0,SRX21885931,SRS18977056,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80402,,0.30224,,0.93592,,0.53659,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26588,SRR26173889,SRX21885930,SRS18977055,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B11 R1,GSM7804098,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B11 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804098,GSM7804098: V2a sample2 354 B11 R1; Danio rerio; RNA Seq,GSM7804098 r1,GSM7804098,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B11_R1.fastq.gz,fastq,35987087.0,836909.0,GSM7804098 r1,0:43,A:9801738;C:8047161;G:8248020;T:9890168;N:0,43,,,,9801738,8047161,8248020,9890168,0,SRX21885930,SRS18977055,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.81805,,0.24526,,0.92092,,0.54523,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26589,SRR26173890,SRX21885929,SRS18977054,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 B10 R1,GSM7804097,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 B10 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804097,GSM7804097: V2a sample2 354 B10 R1; Danio rerio; RNA Seq,GSM7804097 r1,GSM7804097,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_B10_R1.fastq.gz,fastq,33252846.0,773322.0,GSM7804097 r1,0:43,A:8996915;C:7433025;G:7633859;T:9189047;N:0,43,,,,8996915,7433025,7633859,9189047,0,SRX21885929,SRS18977054,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.78892,,0.28622,,0.94237,,0.53023,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26590,SRR26173891,SRX21885928,SRS18977053,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A9 R1,GSM7804072,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A9 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804072,GSM7804072: V2a sample2 354 A9 R1; Danio rerio; RNA Seq,GSM7804072 r1,GSM7804072,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A9_R1.fastq.gz,fastq,23658600.0,550200.0,GSM7804072 r1,0:43,A:6447300;C:5342358;G:5472128;T:6396814;N:0,43,,,,6447300,5342358,5472128,6396814,0,SRX21885928,SRS18977053,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.86727,,0.23136,,0.8996,,0.52692,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26591,SRR26173892,SRX21885927,SRS18977051,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A8 R1,GSM7804071,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A8 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804071,GSM7804071: V2a sample2 354 A8 R1; Danio rerio; RNA Seq,GSM7804071 r1,GSM7804071,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A8_R1.fastq.gz,fastq,18645187.0,433609.0,GSM7804071 r1,0:43,A:5151340;C:4145072;G:4243308;T:5105467;N:0,43,,,,5151340,4145072,4243308,5105467,0,SRX21885927,SRS18977051,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.86561,,0.28633,,0.89438,,0.53463,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26592,SRR26173893,SRX21885926,SRS18977052,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A7 R1,GSM7804070,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A7 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804070,GSM7804070: V2a sample2 354 A7 R1; Danio rerio; RNA Seq,GSM7804070 r1,GSM7804070,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A7_R1.fastq.gz,fastq,42340466.0,984662.0,GSM7804070 r1,0:43,A:11373539;C:9660020;G:9840242;T:11466665;N:0,43,,,,11373539,9660020,9840242,11466665,0,SRX21885926,SRS18977052,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.81646,,0.24599,,0.93833,,0.57817,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26593,SRR26173894,SRX21885925,SRS18977050,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A6 R1,GSM7804069,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A6 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804069,GSM7804069: V2a sample2 354 A6 R1; Danio rerio; RNA Seq,GSM7804069 r1,GSM7804069,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A6_R1.fastq.gz,fastq,23486858.0,546206.0,GSM7804069 r1,0:43,A:6654369;C:4998242;G:5115579;T:6718668;N:0,43,,,,6654369,4998242,5115579,6718668,0,SRX21885925,SRS18977050,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.84122,,0.57245,,0.77794,,0.53266,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26594,SRR26173895,SRX21885924,SRS18977048,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A5 R1,GSM7804068,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A5 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804068,GSM7804068: V2a sample2 354 A5 R1; Danio rerio; RNA Seq,GSM7804068 r1,GSM7804068,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A5_R1.fastq.gz,fastq,46876450.0,1090150.0,GSM7804068 r1,0:43,A:12599291;C:10693086;G:10940020;T:12644053;N:0,43,,,,12599291,10693086,10940020,12644053,0,SRX21885924,SRS18977048,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.86707,,0.22085,,0.89412,,0.51257,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26595,SRR26173896,SRX21885923,SRS18977049,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A4 R1,GSM7804067,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A4 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804067,GSM7804067: V2a sample2 354 A4 R1; Danio rerio; RNA Seq,GSM7804067 r1,GSM7804067,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A4_R1.fastq.gz,fastq,43487233.0,1011331.0,GSM7804067 r1,0:43,A:11900913;C:9677048;G:9921622;T:11987650;N:0,43,,,,11900913,9677048,9921622,11987650,0,SRX21885923,SRS18977049,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8409,,0.27122,,0.91165,,0.55221,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26596,SRR26173897,SRX21885922,SRS18977047,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A3 R1,GSM7804066,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A3 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804066,GSM7804066: V2a sample2 354 A3 R1; Danio rerio; RNA Seq,GSM7804066 r1,GSM7804066,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A3_R1.fastq.gz,fastq,44287248.0,1029936.0,GSM7804066 r1,0:43,A:12065729;C:9907516;G:10143806;T:12170197;N:0,43,,,,12065729,9907516,10143806,12170197,0,SRX21885922,SRS18977047,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80982,,0.29232,,0.93675,,0.5661,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26597,SRR26173898,SRX21885921,SRS18977046,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample2 354 A2 R1,GSM7804065,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample2 354 A2 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804065,GSM7804065: V2a sample2 354 A2 R1; Danio rerio; RNA Seq,GSM7804065 r1,GSM7804065,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_354_A2_R1.fastq.gz,fastq,39822515.0,926105.0,GSM7804065 r1,0:43,A:10662390;C:9155016;G:9361757;T:10643352;N:0,43,,,,10662390,9155016,9361757,10643352,0,SRX21885921,SRS18977046,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.88377,,0.19244,,0.88627,,0.52412,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26598,SRR26173899,SRX21885920,SRS18977044,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 P1 R1,GSM7804040,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 P1 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804040,GSM7804040: V2a sample1 353 P1 R1; Danio rerio; RNA Seq,GSM7804040 r1,GSM7804040,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_P1_R1.fastq.gz,fastq,35622877.0,828439.0,GSM7804040 r1,0:43,A:10094838;C:7580955;G:7760837;T:10186247;N:0,43,,,,10094838,7580955,7760837,10186247,0,SRX21885920,SRS18977044,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.82753,,0.38743,,0.94085,,0.70753,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26599,SRR26173900,SRX21885919,SRS18977045,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 O24 R1,GSM7804039,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 O24 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804039,GSM7804039: V2a sample1 353 O24 R1; Danio rerio; RNA Seq,GSM7804039 r1,GSM7804039,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_O24_R1.fastq.gz,fastq,25162482.0,585174.0,GSM7804039 r1,0:43,A:6589034;C:5708817;G:5862315;T:7002316;N:0,43,,,,6589034,5708817,5862315,7002316,0,SRX21885919,SRS18977045,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.50646,,0.15797,,0.99086,,0.48626,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26600,SRR26173901,SRX21885918,SRS18977043,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 O23 R1,GSM7804038,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 O23 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804038,GSM7804038: V2a sample1 353 O23 R1; Danio rerio; RNA Seq,GSM7804038 r1,GSM7804038,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_O23_R1.fastq.gz,fastq,21378396.0,497172.0,GSM7804038 r1,0:43,A:5419690;C:4870276;G:5019946;T:6068484;N:0,43,,,,5419690,4870276,5019946,6068484,0,SRX21885918,SRS18977043,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.4177,,0.18826,,0.99344,,0.78066,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26601,SRR26173902,SRX21885917,SRS18977042,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 O22 R1,GSM7804037,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 O22 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804037,GSM7804037: V2a sample1 353 O22 R1; Danio rerio; RNA Seq,GSM7804037 r1,GSM7804037,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_O22_R1.fastq.gz,fastq,30321665.0,705155.0,GSM7804037 r1,0:43,A:8123534;C:6839326;G:6921711;T:8437094;N:0,43,,,,8123534,6839326,6921711,8437094,0,SRX21885917,SRS18977042,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.76574,,0.24445,,0.94014,,0.51811,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26602,SRR26173903,SRX21885916,SRS18977041,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 O21 R1,GSM7804036,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 O21 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804036,GSM7804036: V2a sample1 353 O21 R1; Danio rerio; RNA Seq,GSM7804036 r1,GSM7804036,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_O21_R1.fastq.gz,fastq,24544916.0,570812.0,GSM7804036 r1,0:43,A:6528543;C:5656479;G:5771413;T:6588481;N:0,43,,,,6528543,5656479,5771413,6588481,0,SRX21885916,SRS18977041,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87756,,0.17329,,0.88124,,0.51427,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26603,SRR26173904,SRX21885915,SRS18977040,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 O20 R1,GSM7804035,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 O20 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804035,GSM7804035: V2a sample1 353 O20 R1; Danio rerio; RNA Seq,GSM7804035 r1,GSM7804035,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_O20_R1.fastq.gz,fastq,27471324.0,638868.0,GSM7804035 r1,0:43,A:7349212;C:6154048;G:6323796;T:7644268;N:0,43,,,,7349212,6154048,6323796,7644268,0,SRX21885915,SRS18977040,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.7529,,0.26778,,0.94635,,0.5148,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26604,SRR26173905,SRX21885914,SRS18977039,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 O19 R1,GSM7804034,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 O19 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804034,GSM7804034: V2a sample1 353 O19 R1; Danio rerio; RNA Seq,GSM7804034 r1,GSM7804034,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_O19_R1.fastq.gz,fastq,24908137.0,579259.0,GSM7804034 r1,0:43,A:6706594;C:5634956;G:5764632;T:6801955;N:0,43,,,,6706594,5634956,5764632,6801955,0,SRX21885914,SRS18977039,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8525,,0.23739,,0.90694,,0.5194,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26605,SRR26173906,SRX21885913,SRS18977038,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 O18 R1,GSM7804033,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 O18 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804033,GSM7804033: V2a sample1 353 O18 R1; Danio rerio; RNA Seq,GSM7804033 r1,GSM7804033,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_O18_R1.fastq.gz,fastq,23328790.0,542530.0,GSM7804033 r1,0:43,A:6296932;C:5290824;G:5423100;T:6317934;N:0,43,,,,6296932,5290824,5423100,6317934,0,SRX21885913,SRS18977038,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87485,,0.19256,,0.88422,,0.46891,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26606,SRR26173907,SRX21885912,SRS18977037,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N17 R1,GSM7804008,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N17 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804008,GSM7804008: V2a sample1 353 N17 R1; Danio rerio; RNA Seq,GSM7804008 r1,GSM7804008,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N17_R1.fastq.gz,fastq,20379592.0,473944.0,GSM7804008 r1,0:43,A:5592768;C:4503044;G:4602811;T:5680969;N:0,43,,,,5592768,4503044,4602811,5680969,0,SRX21885912,SRS18977037,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.84619,,0.24373,,0.94095,,0.4423,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26607,SRR26173908,SRX21885911,SRS18977035,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N16 R1,GSM7804007,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N16 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804007,GSM7804007: V2a sample1 353 N16 R1; Danio rerio; RNA Seq,GSM7804007 r1,GSM7804007,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N16_R1.fastq.gz,fastq,14151343.0,329101.0,GSM7804007 r1,0:43,A:3796916;C:3223155;G:3290919;T:3840353;N:0,43,,,,3796916,3223155,3290919,3840353,0,SRX21885911,SRS18977035,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.85097,,0.21705,,0.93097,,0.52334,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26608,SRR26173909,SRX21885910,SRS18977036,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N15 R1,GSM7804006,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N15 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804006,GSM7804006: V2a sample1 353 N15 R1; Danio rerio; RNA Seq,GSM7804006 r1,GSM7804006,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N15_R1.fastq.gz,fastq,16536209.0,384563.0,GSM7804006 r1,0:43,A:4473687;C:3744698;G:3838620;T:4479204;N:0,43,,,,4473687,3744698,3838620,4479204,0,SRX21885910,SRS18977036,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.88744,,0.19834,,0.89966,,0.5281,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26609,SRR26173910,SRX21885909,SRS18977034,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N14 R1,GSM7804005,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N14 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804005,GSM7804005: V2a sample1 353 N14 R1; Danio rerio; RNA Seq,GSM7804005 r1,GSM7804005,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N14_R1.fastq.gz,fastq,16039215.0,373005.0,GSM7804005 r1,0:43,A:4355837;C:3605999;G:3695364;T:4382015;N:0,43,,,,4355837,3605999,3695364,4382015,0,SRX21885909,SRS18977034,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.86632,,0.22226,,0.90268,,0.5092,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26610,SRR26173911,SRX21885908,SRS18977033,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N13 R1,GSM7804004,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N13 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804004,GSM7804004: V2a sample1 353 N13 R1; Danio rerio; RNA Seq,GSM7804004 r1,GSM7804004,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N13_R1.fastq.gz,fastq,15280910.0,355370.0,GSM7804004 r1,0:43,A:4072742;C:3469231;G:3555056;T:4183881;N:0,43,,,,4072742,3469231,3555056,4183881,0,SRX21885908,SRS18977033,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.73874,,0.31529,,0.95988,,0.5442,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26611,SRR26173912,SRX21885907,SRS18977032,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N12 R1,GSM7804003,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N12 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804003,GSM7804003: V2a sample1 353 N12 R1; Danio rerio; RNA Seq,GSM7804003 r1,GSM7804003,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N12_R1.fastq.gz,fastq,10529926.0,244882.0,GSM7804003 r1,0:43,A:2835885;C:2354590;G:2390746;T:2948705;N:0,43,,,,2835885,2354590,2390746,2948705,0,SRX21885907,SRS18977032,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.74571,,0.36729,,0.9443,,0.77069,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26612,SRR26173913,SRX21885906,SRS18977030,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N11 R1,GSM7804002,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N11 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804002,GSM7804002: V2a sample1 353 N11 R1; Danio rerio; RNA Seq,GSM7804002 r1,GSM7804002,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N11_R1.fastq.gz,fastq,21706271.0,504797.0,GSM7804002 r1,0:43,A:5853146;C:4922477;G:5009970;T:5920678;N:0,43,,,,5853146,4922477,5009970,5920678,0,SRX21885906,SRS18977030,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8394,,0.23984,,0.93468,,0.51881,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26613,SRR26173914,SRX21885905,SRS18977031,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 N10 R1,GSM7804001,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 N10 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7804001,GSM7804001: V2a sample1 353 N10 R1; Danio rerio; RNA Seq,GSM7804001 r1,GSM7804001,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_N10_R1.fastq.gz,fastq,22772585.0,529595.0,GSM7804001 r1,0:43,A:6225918;C:5071489;G:5184079;T:6291099;N:0,43,,,,6225918,5071489,5184079,6291099,0,SRX21885905,SRS18977031,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.83486,,0.27703,,0.92433,,0.54887,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26614,SRR26173915,SRX21885904,SRS18977029,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M9 R1,GSM7803976,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M9 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803976,GSM7803976: V2a sample1 353 M9 R1; Danio rerio; RNA Seq,GSM7803976 r1,GSM7803976,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M9_R1.fastq.gz,fastq,19698730.0,458110.0,GSM7803976 r1,0:43,A:5282525;C:4494162;G:4591316;T:5330727;N:0,43,,,,5282525,4494162,4591316,5330727,0,SRX21885904,SRS18977029,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.8598,,0.23744,,0.90871,,0.52081,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26615,SRR26173916,SRX21885903,SRS18977028,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M8 R1,GSM7803975,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M8 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803975,GSM7803975: V2a sample1 353 M8 R1; Danio rerio; RNA Seq,GSM7803975 r1,GSM7803975,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M8_R1.fastq.gz,fastq,19646485.0,456895.0,GSM7803975 r1,0:43,A:5214519;C:4539724;G:4616920;T:5275322;N:0,43,,,,5214519,4539724,4616920,5275322,0,SRX21885903,SRS18977028,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.82407,,0.22367,,0.94298,,0.50209,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26616,SRR26173917,SRX21885902,SRS18977025,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M7 R1,GSM7803974,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M7 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803974,GSM7803974: V2a sample1 353 M7 R1; Danio rerio; RNA Seq,GSM7803974 r1,GSM7803974,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M7_R1.fastq.gz,fastq,7333994.0,170558.0,GSM7803974 r1,0:43,A:1958042;C:1671444;G:1690497;T:2014011;N:0,43,,,,1958042,1671444,1690497,2014011,0,SRX21885902,SRS18977025,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.77392,,0.29548,,0.95114,,0.51367,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26617,SRR26173918,SRX21885901,SRS18977027,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M6 R1,GSM7803973,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M6 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803973,GSM7803973: V2a sample1 353 M6 R1; Danio rerio; RNA Seq,GSM7803973 r1,GSM7803973,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M6_R1.fastq.gz,fastq,23546413.0,547591.0,GSM7803973 r1,0:43,A:6369803;C:5317401;G:5430197;T:6429012;N:0,43,,,,6369803,5317401,5430197,6429012,0,SRX21885901,SRS18977027,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.85602,,0.25223,,0.92654,,0.5306,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26618,SRR26173919,SRX21885900,SRS18977026,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M5 R1,GSM7803972,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M5 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803972,GSM7803972: V2a sample1 353 M5 R1; Danio rerio; RNA Seq,GSM7803972 r1,GSM7803972,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M5_R1.fastq.gz,fastq,32919424.0,765568.0,GSM7803972 r1,0:43,A:8685316;C:7623131;G:7771679;T:8839298;N:0,43,,,,8685316,7623131,7771679,8839298,0,SRX21885900,SRS18977026,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.80691,,0.23128,,0.95404,,0.45556,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26619,SRR26173920,SRX21885899,SRS18977024,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M4 R1,GSM7803971,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M4 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803971,GSM7803971: V2a sample1 353 M4 R1; Danio rerio; RNA Seq,GSM7803971 r1,GSM7803971,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M4_R1.fastq.gz,fastq,27743858.0,645206.0,GSM7803971 r1,0:43,A:7502879;C:6275733;G:6399007;T:7566239;N:0,43,,,,7502879,6275733,6399007,7566239,0,SRX21885899,SRS18977024,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.84534,,0.24169,,0.92131,,0.52033,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26620,SRR26173921,SRX21885898,SRS18977023,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M3 R1,GSM7803970,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M3 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803970,GSM7803970: V2a sample1 353 M3 R1; Danio rerio; RNA Seq,GSM7803970 r1,GSM7803970,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M3_R1.fastq.gz,fastq,31646753.0,735971.0,GSM7803970 r1,0:43,A:8540660;C:7169606;G:7344972;T:8591515;N:0,43,,,,8540660,7169606,7344972,8591515,0,SRX21885898,SRS18977023,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.86607,,0.22984,,0.90905,,0.52816,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26621,SRR26173922,SRX21885897,SRS18977019,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 M2 R1,GSM7803969,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 M2 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803969,GSM7803969: V2a sample1 353 M2 R1; Danio rerio; RNA Seq,GSM7803969 r1,GSM7803969,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_M2_R1.fastq.gz,fastq,18262487.0,424709.0,GSM7803969 r1,0:43,A:4905794;C:4167223;G:4257561;T:4931909;N:0,43,,,,4905794,4167223,4257561,4931909,0,SRX21885897,SRS18977019,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87727,,0.19789,,0.88327,,0.51404,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26622,SRR26173923,SRX21885896,SRS18977022,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 L1 R1,GSM7803944,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 L1 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803944,GSM7803944: V2a sample1 353 L1 R1; Danio rerio; RNA Seq,GSM7803944 r1,GSM7803944,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_L1_R1.fastq.gz,fastq,39261064.0,913048.0,GSM7803944 r1,0:43,A:10675714;C:8831605;G:9068828;T:10684917;N:0,43,,,,10675714,8831605,9068828,10684917,0,SRX21885896,SRS18977022,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.87519,,0.20246,,0.89696,,0.53416,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26623,SRR26173924,SRX21885895,SRS18977020,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 K24 R1,GSM7803943,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 K24 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803943,GSM7803943: V2a sample1 353 K24 R1; Danio rerio; RNA Seq,GSM7803943 r1,GSM7803943,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_K24_R1.fastq.gz,fastq,10877753.0,252971.0,GSM7803943 r1,0:43,A:2828581;C:2427642;G:2504205;T:3117325;N:0,43,,,,2828581,2427642,2504205,3117325,0,SRX21885895,SRS18977020,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.43358,,0.14805,,0.96483,,0.89489,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System
26624,SRR26173925,SRX21885894,SRS18977021,SRP463130,PRJNA1020854,Molecular blueprints for spinal circuit modules controlling locomotor speed,GSE243993,Transcriptome Analysis,The flexibility of motor actions is ingrained in the diversity of neurons and how they are organized into functional circuit modules  yet our knowledge of the molecular underpinning of motor circuit modularity remains limited. Locomotion is a motor behavior characterized by sudden changes in speed and strength enabled by the coordinated recruitment of different motoneuron subtypes. Here we use adult zebrafish to link the molecular diversity of motoneurons and the rhythm generating V2a interneurons with their modular circuit organization that is responsible for changes in locomotor speed. We show that the molecular diversity of motoneurons and V2a interneurons reflects their functional segregation into slow  intermediate or fast subtypes. Furthermore  we reveal shared molecular signatures between V2a interneurons and motoneurons of the three speed circuit modules. Overall  by characterizing how the molecular diversity of motoneurons and V2a interneurons relates to their function  connectivity and behavior  our study provides important insights not only into the molecular mechanisms for neuronal and circuit diversity for locomotor flexibility but also for charting circuits for motor actions in general. Overall design: To determine whether the functional subtypes of motoneurons and V2a Chx10+ interneurons are molecularly distinct  we performed single cell RNA sequencing respectively on adult islet1a:GFP and chx10:GFP transgenic zebrafish using SmartSeq2.,,pubmed:37919423,,V2a sample1 353 K23 R1,GSM7803942,,source name:Spinal cord|tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons|geo loc name:missing|collection date:missing,V2a sample1 353 K23 R1,The reads from each sequenced cell were mapped to the zebrafish reference genome “Danio rerio  Ensembl  GRCz11” using STAR version 2.5.3a. The resulting bam files were filtered to keep only uniquely mapped reads. Most of the following analysis was performed in R version 4.0.5  R core team  2022 using the Seurat package version 4.0.2. Assembly: GRCz11 Supplementary files format and content: .csv files with gene count matrixes; .txt and .csv metadata files and .rds files containing R objects from Seurat analysis,Spinal cord,,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,tissue:Spinal cord|cell line:Chx10:GFP|cell type:V2a interneurons,GSM7803942,GSM7803942: V2a sample1 353 K23 R1; Danio rerio; RNA Seq,GSM7803942 r1,GSM7803942,1,Adult animals 7 wpf of either sex were deeply anesthetized in a slush of frozen extracellular solution containing in mM: 134 NaCl  2.9 KCl  2.1 CaCl2  1.2 MgCl2  10 HEPES and 10 glucose  with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tgislet1a:GFP line  and two samples were prepared from the Tgchx10:GFP line. For each sample  6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM F12 medium Thermo Fisher  #11039021  osmolarity adjusted to 280 280 mOsm containing papain 10 U/ml Worthington biochem  #LK003178 on a heated shaker at 37°C for 15 min. DMEM/F 12 1 ml  280 290 mOsm was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min  and then re suspended in 0.5 ml of DMEM/F 12 280 290 mOsm post removal of the supernatant. Following mechanical trituration using fire polished Pasteur pipettes  the cell suspension was filtered through a cell 16 strainer 40 μm. The sample was kept at room temperature for 20 min post the addition of 0 1 ml of the nuclear DNA stain DRAQ5 Thermo Fisher  #65 0880 92. Using fluorescence activated cell sorting FACs  cells positive for GFP and DRAQ5 in each sample were sorted into a 384 wells plate containing a mild hypotonic lysis buffer 0.2% Triton X 100  2 U/ml RNase inhibitor and immediately snap frozen on ice  then stored at  80°C. Smart Seq2,,RNA-Seq,TRANSCRIPTOMIC SINGLE CELL,cDNA,SINGLE,ILLUMINA,Illumina HiSeq 2000,,SRP463130,,,SS2_18_353_K23_R1.fastq.gz,fastq,11967287.0,278309.0,GSM7803942 r1,0:43,A:3236216;C:2487815;G:2583125;T:3660131;N:0,43,,,,3236216,2487815,2583125,3660131,0,SRX21885894,SRS18977021,SRA1719948,"Neuroscience, Karolinaska Institutet","Neuroscience, Karolinaska Institutet",1,0.45352,,0.10712,,0.99249,,0.66514,,43,,B,,usable mapping rate,illumina,hiseq_era,unknown,cdna_unspecified,unknown,sc,single_cell_plate,smartseq,,Sweden,2023-09-25,Juvenile,Juvenile,Spinal Cord,Nervous System