Gene Expression
Nebulas
Gene Expression NebulasSummary: In the early fetal stage, the gonads are bipotent and only later become the ovary or testis, depending on the genetic sex. Despite many studies examining how sex determination occurs from biopotential gonads, the spatial and temporal organization of bipotential gonads and their progenitors is poorly understood. Here, using lineage tracing in mice, we find that the gonads originate from a T+ primitive streak through WT1+ posterior intermediate mesoderm and appear to share origins anteriorly with the adrenal glands and posteriorly with the metanephric mesenchyme. Comparative single cell transcriptomic analyses in mouse and cynomolgus monkey embryos reveals the convergence of the lineage trajectory and genetic programs accompanying the specification of biopotential gonadal progenitor cells. This process involves sustained expression of epithelial genes and upregulation of mesenchymal genes, thereby conferring a unique epithelial/mesenchymal hybrid state. Our study provides key resources for understanding early gonadogenesis in mice and primates.
Overall Design: Single cell transcriptome analysis of cynomolgus monkey embryo using 10x chromium Single Cell Gene expression system.
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| Treatment Protocol: | - |
| Extract Protocol: | For preparation of E24 (CS11) embryos, the yolk sac and amnion were first carefully trimmed with forceps and ophthalmic scissors, and then the heart (and anterior tissues above the heart) and the posterior growth zone including the PS were removed. For E28 (CS13) and E31 (CS14) embryos, the heart (and anterior tissues above the heart), and hindlimb (and tissues beneath the hindlimb) were removed, and then the urogenital ridge containing the mesonephros, CE and a portion of the proximal mesentery were isolated. For E37 embryos (CS18), similarly to E28/31 embryos, after the anterior and posterior tissues were removed, the urogenital ridges were isolated en bloc. Subsequently, most mesonephros and mesentery were trimmed, and tissues consisting of predominantly GR with an attached portion of mesonephros, putative adrenal glands and proximal mesentery were used for downstream sample processing. Isolated embryonic fragments were washed twice with PBS and then minced with scissors in 500 μl of 0.1% trypsin/EDTA solution, then incubated for 9 min at 37°C with gentle pipetting every 3 min. After quenching of the reaction by addition of 500 μl of STO medium, cell suspensions were strained through a 70 µm nylon cell strainer and centrifuged for 220 g for 5 min. Fragments of cynomolgus monkey embryos at E24 (CS11), E28 (CS13), E31 (CS14) and E37 (CS18) were used for scRNA-seq with a Chromium Single Cell 3ʼ Reagent Kit (v2 chemistry). |
| Library Construction Protocol: | Cell pellets were resuspended in 0.1% BSA in PBS, counted, loaded into Chromium microfluidic chips and used to generate single cell gelbead emulsions with a Chromium controller (10x Genomics) according to the manufacturer’s protocol (Chromium Single Cell 3' Reagent Kits v3) |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | - |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Condition Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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