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Single cell expression data from ICM cells of mouse blastocysts

ABSTRACT: Transcriptional reactivation of the paternal X chromosome occurs in specific cells of the mouse blastocyst between E(mbryonic day)3.5 and E4.5 during pre- to peri-implantation development. While the trophectoderm (TE) and the primitive endoderm (PE) maintain Xist RNA expression and thereby imprinted silencing of genes on the Xp, the epiblast (EPI) cells within the inner cell mass (ICM) gradually downregulate Xist and undergo XCR. To identify differentially expressed genes with potential roles in the XCR process, we performed single-cell expression profiling of ICM cells of blastocysts prior (E3.5, EPI Xist+), during (E4.25, EPI Xist+/Xist-) and after (E4.5, EPI Xist-) XCR. Single cell cDNAs were then assigned to cell-type of origin using qPCR for lineage-specific marker genes (epiblast: Nanog+, PE: Gata6+, TE: Cdx2+). Cells were considered of male sex if they expressed the male marker Eif2s3y or female sex in absence of Eif2s3y and presence of Xist expression in PE cells of the same embryo. Furthermore, female E4.5 epiblast cDNAs were classified according to Xist expression as before (Xist+) or after (Xist-) downregulation during XCR.

ORGANISM(S): Mus musculus

PROVIDER: GSE208430 | GEO | 2022/11/13

REPOSITORIES: GEO

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Project description:Differentiation of totipotent embryonic cells into the first cell lineages (trophectoderm (TE) and inner cell mass (ICM), and further into epiblast (EPI) and primitive endoderm (PE)), although well studied, is only beginning to be understood at the molecular level. An in depth understanding of the regulation of these early differentiation events would enable improvements in the quality of in vitro produced embryos, as well as the study and application of bovine embryonic stem cells. The objective of this work was to perform single cell RNASeq on bovine blastocysts derived in vivo (IVV), in vitro in a conventional in vitro production system (IVC), and in vitro in reduced nutrient media (IVR). All in vitro culture was serum free. In vivo embryonic cells could be assigned based on Leiden clustering analysis, although there was some overlap in expression of marker genes. The two TE groups identified clearly separated in a uniform manifold approximation and projection (UMAP) plot from EPI groups. Putative epiblast cells could be identified. In in vitro embryos produced in a conventional system, considerable overlap in expression of marker genes made Leiden clustering difficult, but a selected count could be used for UMAP to tentatively identify one TE, 2 EPI and 2 PE groups. In assigned cell types we observed higher expression of the identifying marker genes, but some marker genes were expressed in all cell types. In IVC embryos, TE markers clustered while PE and EPI marker genes overlapped. In IVR embryos, again there was considerable overlap between marker gene expression. Like IVC embryos, TE was easily identified by clustering in IVR, but EPI and PE were too similar to distinguish. In summary, the methods used here could partially distinguish putative cell types in bovine blastocysts, although marker gene expression was not always discreet. This may not be surprising given differentiation events are in progress in the blastocyst stage embryo. TE cells were more easily distinguished than EPI and PE cells, which would support this hypothesis.

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Single cell expression data from ICM cells of mouse blastocysts

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