{"database": "metadata", "table": "run_metadata", "rows": [[10060, "ERR4795364", "ERX4665135", "ERS5281176", "ERP124847", "PRJEB41113", "Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma", "E-MTAB-9727", "Transcriptome Analysis", "The mesothelium forms epithelial membranes that line the bodies cavities and surround the internal organs. Mesothelia widely contribute to organ homeostasis and regeneration  and their dysregulation can result in congenital anomalies of the viscera  ventral wall defects  and mesothelioma tumors. Nonetheless  the embryonic ontogeny and developmental regulation of mesothelium formation has remained uncharted. Here  we combine genetic lineage tracing  in toto live imaging  and single cell transcriptomics in zebrafish to track mesothelial progenitor origins from the lateral plate mesoderm LPM. Our single cell analysis uncovers a post gastrulation gene expression signature centered on hand2 that delineates distinct progenitor populations within the forming LPM. Combining gene expression analysis and imaging of transgenic reporter zebrafish embryos  we chart the origin of mesothelial progenitors to the lateral most  hand2 expressing LPM and confirm evolutionary conservation in mouse. Our time lapse imaging of transgenic hand2 reporter embryos captures zebrafish mesothelium formation  documenting the coordinated cell movements that form pericardium and visceral and parietal peritoneum. We establish that the primordial germ cells migrate associated with the forming mesothelium as ventral migration boundary. Functionally  hand2 mutants fail to close the ventral mesothelium due to perturbed migration of mesothelium progenitors. Analyzing mouse and human mesothelioma tumors hypothesized to emerge from transformed mesothelium  we find de novo expression of LPM associated transcription factors  and in particular of Hand2  indicating the re initiation of a developmental transcriptional program in mesothelioma. Taken together  our work outlines a genetic and developmental signature of mesothelial origins centered around Hand2  contributing to our understanding of mesothelial pathologies and mesothelioma.", "ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14", null, "Protocols: Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 \u03bcm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 \u03bcl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5\u2032Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at \u221280\u00b0C.  In brief  ERCC Spike in RNA 0.02 \u03bcL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.", "Sample 4", "E MTAB 9727:Sample 4", null, "isolate:not applicable|organism:Danio rerio|sex:mixed|age:10|developmental stage:tailbud stage|organism part:lateral plate mesoderm plate|genotype:drl:mCherry|ENA FIRST PUBLIC:2021 04 01|ENA LAST UPDATE:2021 09 01", null, null, null, null, null, null, null, null, "NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma", "E MTAB 9727:Sample 4 s", "Sample 4 s", "Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma", "Wildtype stain and drl:mCherry positive zebrafish embryos were grown until tailbud stage and chorions were removed by incubating in 1 mg/mL Pronase Roche  followed by washing in E3 medium. Around 300 embryos were used to generate the sample. The E3 medium was replaced by 1X PBS and the embryos were stored on ice until further processing. The embryos were dissociated using 2 mg/mL collagenase IV Worthington in DMEM high glucose 4.5g/l and NaHCO3  without xxx glutamine and sodium pyruvate  Sigma Aldrich and incubated for 5 min in a water bath at 37oC. The embryonic tissues were triturated into a single cell suspension by pipetting carefully up and down. When the embryos were not yet dissociated sufficiently  they were incubated for another 5 min. Cells were filtered through a 35 \u03bcm cell strainer Falcon        round bottom tubes with cell strainer cap and centrifuged at 6000 rpm for 30 sec. Cell pellets were resuspended in 1X HBSS Gibco containing 2% FBS and subjected to an additional round of centrifugation and resuspension. post washing  the cells were resuspended in 1X PBS.  drl:mCherry positive cells were sorted using a FACS Aria III cell sorter BD Bioscience. Cells were gated based on size and forward scattering  to exclude debris and doublets. The gating for the negative population was determined based on wildtype tailbud staged embryos. See also Figure S2 for gate setting. The SORTseq single cell RNA sequencing protocol was carried out as described previously Muraro et al.  2016. Live mCherry positive single cells were sorted in four 384 well plates BioRad containing 5 \u03bcl of CEL Seq2 primer solution in mineral oil 24 bp polyT stretch  a 4 bp random molecular barcode UMI  a cell specific barcode  the 5\u2032Illumina TruSeq small RNA kit adaptor and a T7 promoter  provided by Single Cell Discoveries. post sorting  the plates were immediately placed on ice and stored at \u221280\u00b0C.  In brief  ERCC Spike in RNA 0.02 \u03bcL of 1:50000 dilution was added to each well before cell lysis with heat shocking. Reverse transcription and second strand synthesis reagents were dispensed using the Nanodrop II GC biotech. post generation of cDNA from the original mRNA  all cells from one plate were pooled and the pooled sample was amplified linearly with in vitro transcription. The amplified RNA was then reverse transcribed to cDNA using a random hexamer primers Hashimshony et al.  2016. To generate sequencing libraries  RPI series index primers were used for library PCR.", "Experimental Factor: replicate:lateral plate mesoderm plate 4", "RNA-Seq", "TRANSCRIPTOMIC SINGLE CELL", "RANDOM", "SINGLE", "ILLUMINA", "NextSeq 500", null, "ERP124847", "NextSeq 500 sequencing; Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma", "ENA FIRST PUBLIC:2022 01 14|ENA LAST UPDATE:2022 01 14", "SN4_AH2TW3BGX5_S1_R1_cat.fastq.gz", "fastq", 5410723202.0, 71707309.0, "E MTAB 9727:Sample 4", "0:75.46 1:0", "A:1330416803;C:531269504;G:734031980;T:2814959249;N:45666", 75, 0, null, null, 1330416803, 531269504, 734031980, 2814959249, 45666, "ERX4665135", "ERS5281176", "ERA3048543", "University of Zurich|European Nucleotide Archive", "University of Zurich|European Nucleotide Archive", 1, 0.33614, null, 0.21532, null, 0.99019, null, 0.41002, null, 76, null, "B", null, "usable mapping rate", "illumina", "nextseq", "unknown", "random_priming", "trueseq", "sc", "single_cell_plate", "celseq", null, "Switzerland", "2021-04-01", "Gastrula", "Embryo", "Embryo Imprecise", "All anatomical structures"]], "columns": ["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"], "primary_keys": ["rowid"], "primary_key_values": ["10060"], "units": {}, "query_ms": 10.31882799725281}