GSE136220
Single-cell transcriptomics reveals temporal dynamics of critical regulators of germ cell fate during mouse sex determination
Despite the importance of germ cell differentiation for sexual reproduction, gene networks underlying their fate remain unclear. Here, we describe a comprehensive characterization of gene expression dynamics during sex determination based on single-cell RNA sequencing on 14,750 XX and XY mouse germ cells between embryonic days 10.5 and 16.5. By computational gene regulation inference analysis, we identified sex-specific, sequential waves of master regulator genes during germ cells differentiation and unveiled that the meiotic initiator Stra8 is regulated by positive and negative master regulators acting in an antagonistic fashion. Consistent with the importance of the somatic environment, we found that rare adrenal germ cells exhibit delayed meiosis and display altered expression of master genes controlling the female and male genetic programs. Our study provides a molecular roadmap of germ cell sex determination at single-cell resolution will serve as a valuable resource for future studies of gonad development, function and disease. Overall design: This dataset contains primordial germ cells data from 28 10x single cell RNAseq captures on whole organs.  Urogenital ridges and adrenal glands were enzymatically dissociated at 37ºC for 20 and 40 minutes, respectively, using the Papain dissociation system (Worthington #LK003150). Cells were resuspended in DMEM 2%FBS, filtered through a 70 µm cell strainer and stained with the dead cell marker Draq7™ (Beckman Coulter, #B25595). Viable single cells were collected on a BD FACS Aria II by excluding debris (side scatter vs. forward scatter), dead cells (side scatter vs. Draq7 staining), and doublets (height vs. width). Testes and ovaries (from E12.5 to E16.5) were enzymatically dissociated at 37ºC during 15 minutes in Trypsin-EDTA 0.05% (Gibco #25300054), resuspended in DMEM 2%FBS and filtered through a 70 µm cell strainer. After counting, 3000 to 7000 single cells were loaded on a 10x Chromium instrument (10x Genomics). Single-cell RNA-Seq libraries were prepared using the Chromium Single Cell 3' v2 Reagent Kit (10x Genomics) according to manufacturer's protocol. Each condition (organ, sex and developmental stage) was performed in two biological independent replicates. Library quantification was performed using the Qubit fluorometric assay with dsDNA HS Assay Kit (Invitrogen). Library quality assessment was performed using a Bioanalyzer Agilent 2100 with a High Sensitivity DNA chip (Agilent Genomics). Libraries were diluted, pooled and sequenced on an Illumina HiSeq4000 using paired-end 26?+?98?bp as the sequencing mode. Libraries were sequenced at a targeted depth of 100 000 to 150 000 total reads per cell. Sequencing was performed at the Health 2030 Genome Center, Geneva.