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 32806,SRR29478752,SRX24989905,SRS21691708,SRP515053,PRJNA1126173,Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood brain barrier integrity,GSE270309,Transcriptome Analysis,The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. Overall design: 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension . Sorting and RNA extraction was performed on TgBACscar2ba:KalTA4; UAS mKate2,,pubmed:39289367,,head scarb2a mut rep 3,GSM8339369,,source name:head|tissue:head|cell type:scarb2a+ cells|genotype:scarb2a mut|geo loc name:missing|collection date:missing,head scarb2a mut rep 3,done using the User friendly Transcriptomic Analysis Pipeline Assembly: danRer11 Supplementary files format and content: countsMatrix normalized.txt: expression dataset txt file contaning normalized UMI counts table for all samples.,head,,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,tissue:head|cell type:scarb2a+ cells|genotype:scarb2a mut,GSM8339369,GSM8339369: head scarb2a mut rep 3; Danio rerio; RNA Seq,GSM8339369 r1,GSM8339369,1,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP515053,,,scarb2_mut3_R2.fastq.gz scarb2_mut3_R1.fastq.gz,fastq fastq,590803877.0,7118119.0,GSM8339369 r1,0:75 1:8,A:171152826;C:118382558;G:138973475;T:162287717;N:7301,75,8,,,171152826,118382558,138973475,162287717,7301,SRX24989905,SRS21691708,SRA1904886,"Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science","Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science",2,0.86187,0.0,0.06751,0.0,0.80081,1.0,0.47421,,75,8,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2024-06-20,Hatching,Embryo,Head,Nervous System 32807,SRR29478753,SRX24989904,SRS21691707,SRP515053,PRJNA1126173,Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood brain barrier integrity,GSE270309,Transcriptome Analysis,The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. Overall design: 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension . Sorting and RNA extraction was performed on TgBACscar2ba:KalTA4; UAS mKate2,,pubmed:39289367,,head scarb2a mut rep 2,GSM8339368,,source name:head|tissue:head|cell type:scarb2a+ cells|genotype:scarb2a mut|geo loc name:missing|collection date:missing,head scarb2a mut rep 2,done using the User friendly Transcriptomic Analysis Pipeline Assembly: danRer11 Supplementary files format and content: countsMatrix normalized.txt: expression dataset txt file contaning normalized UMI counts table for all samples.,head,,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,tissue:head|cell type:scarb2a+ cells|genotype:scarb2a mut,GSM8339368,GSM8339368: head scarb2a mut rep 2; Danio rerio; RNA Seq,GSM8339368 r1,GSM8339368,1,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP515053,,,scarb2_mut2_R2.fastq.gz scarb2_mut2_R1.fastq.gz,fastq fastq,560228337.0,6749739.0,GSM8339368 r1,0:75 1:8,A:161395198;C:113034644;G:131199008;T:154592664;N:6823,75,8,,,161395198,113034644,131199008,154592664,6823,SRX24989904,SRS21691707,SRA1904886,"Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science","Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science",2,0.85633,0.0,0.06116,0.0,0.80438,1.0,0.47226,,75,8,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2024-06-20,Hatching,Embryo,Head,Nervous System 32808,SRR29478754,SRX24989903,SRS21691706,SRP515053,PRJNA1126173,Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood brain barrier integrity,GSE270309,Transcriptome Analysis,The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. Overall design: 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension . Sorting and RNA extraction was performed on TgBACscar2ba:KalTA4; UAS mKate2,,pubmed:39289367,,head scarb2a mut rep 1,GSM8339367,,source name:head|tissue:head|cell type:scarb2a+ cells|genotype:scarb2a mut|geo loc name:missing|collection date:missing,head scarb2a mut rep 1,done using the User friendly Transcriptomic Analysis Pipeline Assembly: danRer11 Supplementary files format and content: countsMatrix normalized.txt: expression dataset txt file contaning normalized UMI counts table for all samples.,head,,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,tissue:head|cell type:scarb2a+ cells|genotype:scarb2a mut,GSM8339367,GSM8339367: head scarb2a mut rep 1; Danio rerio; RNA Seq,GSM8339367 r1,GSM8339367,1,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP515053,,,scarb2_mut1_R1.fastq.gz scarb2_mut1_R2.fastq.gz,fastq fastq,536045042.0,6458374.0,GSM8339367 r1,0:75 1:8,A:152863781;C:109210501;G:126922310;T:147041910;N:6540,75,8,,,152863781,109210501,126922310,147041910,6540,SRX24989903,SRS21691706,SRA1904886,"Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science","Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science",2,0.86464,0.0,0.06577,0.0,0.80359,1.0,0.48395,,75,8,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2024-06-20,Hatching,Embryo,Head,Nervous System 32809,SRR29478755,SRX24989902,SRS21691705,SRP515053,PRJNA1126173,Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood brain barrier integrity,GSE270309,Transcriptome Analysis,The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. Overall design: 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension . Sorting and RNA extraction was performed on TgBACscar2ba:KalTA4; UAS mKate2,,pubmed:39289367,,head wt rep 4,GSM8339366,,source name:head|tissue:head|cell type:scarb2a+ cells|genotype:wt|geo loc name:missing|collection date:missing,head wt rep 4,done using the User friendly Transcriptomic Analysis Pipeline Assembly: danRer11 Supplementary files format and content: countsMatrix normalized.txt: expression dataset txt file contaning normalized UMI counts table for all samples.,head,,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,tissue:head|cell type:scarb2a+ cells|genotype:wt,GSM8339366,GSM8339366: head wt rep 4; Danio rerio; RNA Seq,GSM8339366 r1,GSM8339366,1,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP515053,,,scarb2_wt4_R1.fastq.gz scarb2_wt4_R2.fastq.gz,fastq fastq,723943430.0,8722210.0,GSM8339366 r1,0:75 1:8,A:206155698;C:145344454;G:172171717;T:200262536;N:9025,75,8,,,206155698,145344454,172171717,200262536,9025,SRX24989902,SRS21691705,SRA1904886,"Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science","Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science",2,0.86958,0.0,0.0737,0.0,0.79928,1.0,0.47906,,75,8,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2024-06-20,Hatching,Embryo,Head,Nervous System 32810,SRR29478756,SRX24989901,SRS21691704,SRP515053,PRJNA1126173,Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood brain barrier integrity,GSE270309,Transcriptome Analysis,The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. Overall design: 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension . Sorting and RNA extraction was performed on TgBACscar2ba:KalTA4; UAS mKate2,,pubmed:39289367,,head wt rep 3,GSM8339365,,source name:head|tissue:head|cell type:scarb2a+ cells|genotype:wt|geo loc name:missing|collection date:missing,head wt rep 3,done using the User friendly Transcriptomic Analysis Pipeline Assembly: danRer11 Supplementary files format and content: countsMatrix normalized.txt: expression dataset txt file contaning normalized UMI counts table for all samples.,head,,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,tissue:head|cell type:scarb2a+ cells|genotype:wt,GSM8339365,GSM8339365: head wt rep 3; Danio rerio; RNA Seq,GSM8339365 r1,GSM8339365,1,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP515053,,,scarb2_wt3_R1.fastq.gz scarb2_wt3_R2.fastq.gz,fastq fastq,560881132.0,6757604.0,GSM8339365 r1,0:75 1:8,A:158992539;C:112696361;G:133852695;T:155332822;N:6715,75,8,,,158992539,112696361,133852695,155332822,6715,SRX24989901,SRS21691704,SRA1904886,"Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science","Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science",2,0.8706,0.0,0.07425,0.0,0.80038,1.0,0.47161,,75,8,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2024-06-20,Hatching,Embryo,Head,Nervous System 32811,SRR29478757,SRX24989900,SRS21691703,SRP515053,PRJNA1126173,Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised blood brain barrier integrity,GSE270309,Transcriptome Analysis,The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. The neurovascular unit NVU is a complex multicellular structure that helps maintain cerebral homeostasis and blood brain barrier BBB integrity. While extensive evidence links NVU alterations to cerebrovascular diseases and neurodegeneration the underlying molecular mechanisms remain unclear. Here we use zebrafish embryos carrying a mutation in Scavenger Receptor B2 a highly conserved endolysosomal protein expressed predominantly in Radial Glia Cells RGCs to investigate the interplay among different NVU components. Through live imaging and genetic manipulations we demonstrate that compromised acidification of the endolysosomal compartment in mutant RGCs leads to impaired Notch3 signaling thereby inducing excessive neurogenesis and reduced glial differentiation. We further demonstrate that alterations to the neuron/glia balance result in impaired VEGF and Wnt signaling leading to severe vascular defects hemorrhages and a leaky BBB. Altogether our findings provide novel insights into NVU formation and function and offer new avenues for investigating diseases involving white matter defects and vascular abnormalities. Overall design: 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension . Sorting and RNA extraction was performed on TgBACscar2ba:KalTA4; UAS mKate2,,pubmed:39289367,,head wt rep 1,GSM8339364,,source name:head|tissue:head|cell type:scarb2a+ cells|genotype:wt|geo loc name:missing|collection date:missing,head wt rep 1,done using the User friendly Transcriptomic Analysis Pipeline Assembly: danRer11 Supplementary files format and content: countsMatrix normalized.txt: expression dataset txt file contaning normalized UMI counts table for all samples.,head,,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,tissue:head|cell type:scarb2a+ cells|genotype:wt,GSM8339364,GSM8339364: head wt rep 1; Danio rerio; RNA Seq,GSM8339364 r1,GSM8339364,1,For each experimental condition a pool of 50 heads 3 biological replicates was dissected from euthanized 48 hpf scarb2a mutants and wt siblings and dissociated into a single cell suspension. post a brief enzymatic treatment with a cocktail of Liberase Blendzyme 3 Roche trypsin B BI and DNAseI Roche the cell suspension was strained through a 70µm filter and stained with SYTOXTM blue ThermoFisher for live/dead discrimination. 5000 live scarb2a+ single cells were sorted into 40 µl of lysis/binding buffer solution ThermoFisher containing RNase inhibitor RNasinTM Promega. FACS analysis and sorting were performed on a BD FACS Aria III using a 70µm nozzle. RNA was captured using Dynabeads™ mRNA DIRECT™ Purification Kit ThermoFisher prior to library preparation. A bulk adaptation of the MARS seq protocol was used to generate RNA libraries for the expression profile of scarb2+ mutant and wt cells. The RNA was further fragmented and transformed into a sequencing ready library by tagging the samples with Illumina sequences during ligation RT and PCR. The final library concentration was measured by Qubit TapeStation and qPCR for zebrafish actin as previously described. Sequencing was performed on a Nextseq500/550 High Output Kit v2.5 75 cycles Illumina; paired end sequencing and each sample was sequenced for 6M reads.,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP515053,,,scarb2_wt1_R1.fastq.gz scarb2_wt1_R2.fastq.gz,fastq fastq,539653799.0,6501853.0,GSM8339364 r1,0:75 1:8,A:153571518;C:110489477;G:128477853;T:147108152;N:6799,75,8,,,153571518,110489477,128477853,147108152,6799,SRX24989900,SRS21691703,SRA1904886,"Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science","Karina Yaniv lab, Immunology and Regenerative Biology, Weizmann Institute of Science",2,0.87359,0.0,0.07353,0.0,0.80184,1.0,0.47111,,75,8,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2024-06-20,Hatching,Embryo,Head,Nervous System 68656,SRR18097957,SRX14248907,SRS12068875,SRP360907,PRJNA809307,Generation of specialized blood vessels via lymphatic transdifferentiation,GSE197161,Transcriptome Analysis,The lineage and developmental trajectory of a cell are key determinant s of cellular identity. In the vascular system endothelial cells ECs of blood and lymphatic vessels LVs differentiate and diversify to cater the different physiological demands of each organ . While LVs are known to originate from multiple origins lymphatic ECs LECs themselves are not known to generate other cell types . Here we u s e recurrent imaging and lineage tracing of ECs in zebrafish anal fins AF from early development through maturity to uncover an unexpected mechanism of specialized blood vessel formation through transdifferentiation of LECs . Moreover we demonstrate distinct functional implications for deriving AF vessels from either LECs or blood ECs uncovering a link between cell ontogeny and functionality. We further use scRNA seq to characterize the different cellular populations and transition states involved in the transdifferentiation process . Finally we show that akin to its normal development the vasculature is re derived from lymphatics during AF regeneration demonstr ating that LECs in adult fish retain both potency and plasticity for generating blood ECs . Overall our work highlights a new innate mechanism of blood vess el formation through LEC trans differentiation and provides in vivo evidence for a link between cell ontogeny and functionality in ECs Overall design: 80 anal fins from immature zebrafish were digested. Sorting and RNA extraction was performed on fli1a:dsRed positive cells,,pubmed:35614218,,Immature fin ECs plate3,GSM5910460,,source name:fli1a:dsRed positive cells|tissue:Anal fin|cell type:endothelial cells|Stage:II III,Immature fin ECs plate3,GRCz10 genome mapping was done using Bowtie2 with default parameters Demultiplexing and UMI count matrixes were based on 4bp pool barcodes in Read1 for each sample. 8bp of UMI with 7bp of cell barcodes in Read2 and performed as described in Jaitin et al. 2014 Genome build: GRCz10 Supplementary files format and content: AB006.txt AB007.txt AB008.txt: expression dataset txt files containing UMI counts table per plate. Single cell metadata p1.xslx: xslx file sheet1 pool barcode of plate1 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate1 analyzed cell names. Single cell metadata p2.xslx: xslx file sheet1 pool barcode of plate2 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate2 analyzed cell names. Single cell metadata p3.xslx: xslx file sheet1 pool barcode of plate3 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate3 analyzed cell names,fli1a:dsRed positive cells,,80 anal fins were dissected manually chopped with a sterile razor and enzymatically digested using Liberase I Trypsin B and DNaseI. Single cells were sorted into 384 well plates containing lysis solution UPW 8nM poly dT barcodes 0.1%triton and RNAse inhibitor MARS seq libraries were prepared as described Jaitin et al Science 2014,,tissue:Anal fin|cell type:endothelial cells|Stage:II III,GSM5910460,GSM5910460: Immature fin ECs plate3; Danio rerio; RNA Seq,GSM5910460 r1,GSM5910460,1,80 anal fins were dissected manually chopped with a sterile razor and enzymatically digested using Liberase I Trypsin B and DNaseI. Single cells were sorted into 384 well plates containing lysis solution UPW 8nM poly dT barcodes 0.1%triton and RNAse inhibitor MARS seq libraries were prepared as described Jaitin et al Science 2014,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP360907,,,BC3_S0_R1_001.fastq.gz BC3_S0_R2_001.fastq.gz,fastq fastq,5000226570.0,55558073.0,GSM5910460 r1,0:75 1:15,A:1398279779;C:1105834990;G:1278168650;T:1217071842;N:871309,75,15,,,1398279779,1105834990,1278168650,1217071842,871309,SRX14248907,SRS12068875,SRA1376737,Weizmann Institute of Science,"Department of Biological Regulation, Weizmann Institute of Science",2,0.75587,0.0,0.22384,0.0,0.87665,1.0,0.59533,,75,15,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2022-02-22,Undetermined,Adult,Fin,Surface Structure 68657,SRR18097958,SRX14248906,SRS12068874,SRP360907,PRJNA809307,Generation of specialized blood vessels via lymphatic transdifferentiation,GSE197161,Transcriptome Analysis,The lineage and developmental trajectory of a cell are key determinant s of cellular identity. In the vascular system endothelial cells ECs of blood and lymphatic vessels LVs differentiate and diversify to cater the different physiological demands of each organ . While LVs are known to originate from multiple origins lymphatic ECs LECs themselves are not known to generate other cell types . Here we u s e recurrent imaging and lineage tracing of ECs in zebrafish anal fins AF from early development through maturity to uncover an unexpected mechanism of specialized blood vessel formation through transdifferentiation of LECs . Moreover we demonstrate distinct functional implications for deriving AF vessels from either LECs or blood ECs uncovering a link between cell ontogeny and functionality. We further use scRNA seq to characterize the different cellular populations and transition states involved in the transdifferentiation process . Finally we show that akin to its normal development the vasculature is re derived from lymphatics during AF regeneration demonstr ating that LECs in adult fish retain both potency and plasticity for generating blood ECs . Overall our work highlights a new innate mechanism of blood vess el formation through LEC trans differentiation and provides in vivo evidence for a link between cell ontogeny and functionality in ECs Overall design: 80 anal fins from immature zebrafish were digested. Sorting and RNA extraction was performed on fli1a:dsRed positive cells,,pubmed:35614218,,Immature fin ECs plate2,GSM5910459,,source name:fli1a:dsRed positive cells|tissue:Anal fin|cell type:endothelial cells|Stage:II III,Immature fin ECs plate2,GRCz10 genome mapping was done using Bowtie2 with default parameters Demultiplexing and UMI count matrixes were based on 4bp pool barcodes in Read1 for each sample. 8bp of UMI with 7bp of cell barcodes in Read2 and performed as described in Jaitin et al. 2014 Genome build: GRCz10 Supplementary files format and content: AB006.txt AB007.txt AB008.txt: expression dataset txt files containing UMI counts table per plate. Single cell metadata p1.xslx: xslx file sheet1 pool barcode of plate1 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate1 analyzed cell names. Single cell metadata p2.xslx: xslx file sheet1 pool barcode of plate2 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate2 analyzed cell names. Single cell metadata p3.xslx: xslx file sheet1 pool barcode of plate3 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate3 analyzed cell names,fli1a:dsRed positive cells,,80 anal fins were dissected manually chopped with a sterile razor and enzymatically digested using Liberase I Trypsin B and DNaseI. Single cells were sorted into 384 well plates containing lysis solution UPW 8nM poly dT barcodes 0.1%triton and RNAse inhibitor MARS seq libraries were prepared as described Jaitin et al Science 2014,,tissue:Anal fin|cell type:endothelial cells|Stage:II III,GSM5910459,GSM5910459: Immature fin ECs plate2; Danio rerio; RNA Seq,GSM5910459 r1,GSM5910459,1,80 anal fins were dissected manually chopped with a sterile razor and enzymatically digested using Liberase I Trypsin B and DNaseI. Single cells were sorted into 384 well plates containing lysis solution UPW 8nM poly dT barcodes 0.1%triton and RNAse inhibitor MARS seq libraries were prepared as described Jaitin et al Science 2014,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP360907,,,BC2_S0_R1_001.fastq.gz BC2_S0_R2_001.fastq.gz,fastq fastq,4158365400.0,46204060.0,GSM5910459 r1,0:75 1:15,A:1056133752;C:954371027;G:1117373354;T:1029764959;N:722308,75,15,,,1056133752,954371027,1117373354,1029764959,722308,SRX14248906,SRS12068874,SRA1376737,Weizmann Institute of Science,"Department of Biological Regulation, Weizmann Institute of Science",2,0.76282,0.0,0.2382,0.0,0.87158,1.0,0.53888,,75,15,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2022-02-22,Undetermined,Adult,Fin,Surface Structure 68658,SRR18097959,SRX14248905,SRS12068873,SRP360907,PRJNA809307,Generation of specialized blood vessels via lymphatic transdifferentiation,GSE197161,Transcriptome Analysis,The lineage and developmental trajectory of a cell are key determinant s of cellular identity. In the vascular system endothelial cells ECs of blood and lymphatic vessels LVs differentiate and diversify to cater the different physiological demands of each organ . While LVs are known to originate from multiple origins lymphatic ECs LECs themselves are not known to generate other cell types . Here we u s e recurrent imaging and lineage tracing of ECs in zebrafish anal fins AF from early development through maturity to uncover an unexpected mechanism of specialized blood vessel formation through transdifferentiation of LECs . Moreover we demonstrate distinct functional implications for deriving AF vessels from either LECs or blood ECs uncovering a link between cell ontogeny and functionality. We further use scRNA seq to characterize the different cellular populations and transition states involved in the transdifferentiation process . Finally we show that akin to its normal development the vasculature is re derived from lymphatics during AF regeneration demonstr ating that LECs in adult fish retain both potency and plasticity for generating blood ECs . Overall our work highlights a new innate mechanism of blood vess el formation through LEC trans differentiation and provides in vivo evidence for a link between cell ontogeny and functionality in ECs Overall design: 80 anal fins from immature zebrafish were digested. Sorting and RNA extraction was performed on fli1a:dsRed positive cells,,pubmed:35614218,,Immature fin ECs plate1,GSM5910458,,source name:fli1a:dsRed positive cells|tissue:Anal fin|cell type:endothelial cells|Stage:II III,Immature fin ECs plate1,GRCz10 genome mapping was done using Bowtie2 with default parameters Demultiplexing and UMI count matrixes were based on 4bp pool barcodes in Read1 for each sample. 8bp of UMI with 7bp of cell barcodes in Read2 and performed as described in Jaitin et al. 2014 Genome build: GRCz10 Supplementary files format and content: AB006.txt AB007.txt AB008.txt: expression dataset txt files containing UMI counts table per plate. Single cell metadata p1.xslx: xslx file sheet1 pool barcode of plate1 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate1 analyzed cell names. Single cell metadata p2.xslx: xslx file sheet1 pool barcode of plate2 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate2 analyzed cell names. Single cell metadata p3.xslx: xslx file sheet1 pool barcode of plate3 sheet2 cell barcodes and coordinates required for MARS seq pipeline Jaitin et al. 2014 sheet3 plate3 analyzed cell names,fli1a:dsRed positive cells,,80 anal fins were dissected manually chopped with a sterile razor and enzymatically digested using Liberase I Trypsin B and DNaseI. Single cells were sorted into 384 well plates containing lysis solution UPW 8nM poly dT barcodes 0.1%triton and RNAse inhibitor MARS seq libraries were prepared as described Jaitin et al Science 2014,,tissue:Anal fin|cell type:endothelial cells|Stage:II III,GSM5910458,GSM5910458: Immature fin ECs plate1; Danio rerio; RNA Seq,GSM5910458 r1,GSM5910458,1,80 anal fins were dissected manually chopped with a sterile razor and enzymatically digested using Liberase I Trypsin B and DNaseI. Single cells were sorted into 384 well plates containing lysis solution UPW 8nM poly dT barcodes 0.1%triton and RNAse inhibitor MARS seq libraries were prepared as described Jaitin et al Science 2014,,RNA-Seq,TRANSCRIPTOMIC,cDNA,PAIRED,ILLUMINA,NextSeq 500,,SRP360907,,,BC1_S0_R1_001.fastq.gz BC1_S0_R2_001.fastq.gz,fastq fastq,4284501750.0,47605575.0,GSM5910458 r1,0:75 1:15,A:1172578653;C:976724863;G:1071723716;T:1062724053;N:750465,75,15,,,1172578653,976724863,1071723716,1062724053,750465,SRX14248905,SRS12068873,SRA1376737,Weizmann Institute of Science,"Department of Biological Regulation, Weizmann Institute of Science",2,0.73652,0.0,0.20176,0.0,0.86622,1.0,0.66697,,75,15,B,T,sc-like readlen,illumina,nextseq,unknown,cdna_unspecified,unknown,sc,single_cell_plate,marsseq,,Israel,2022-02-22,Undetermined,Adult,Fin,Surface Structure