Abstract
The mammalian post-implantation embryo has been extensively investigated at the tissue level. However, to unravel the molecular basis for the cell-fate plasticity and determination, it is essential to study the characteristics of individual cells. In particular, the individual definitive endoderm (DE) cells have not been characterized in vivo Here, we report gene expression patterns in single cells freshly isolated from mouse embryos on days 5.5 and 6.5. Initial transcriptome data from 124 single cells yielded signature genes for the epiblast, visceral endoderm, and extra-embryonic ectoderm and revealed a unique distribution pattern of fibroblast growth factor (FGF) ligands and receptors. Further analysis indicated that early-stage epiblast cells do not segregate into lineages of the major germ layers. Instead, some cells began to diverge from epiblast cells, displaying molecular features of the premesendoderm by expressing higher levels of mesendoderm markers and lower levels of Sox3 transcripts. Analysis of single-cell high-throughput quantitative RT-PCR data from 441 cells identified a late stage of the day 6.5 embryo in which mesoderm and DE cells emerge, with many of them coexpressing Oct4 and Gata6 Analysis of single-cell RNA-sequence data from 112 cells of the late-stage day 6.5 embryos revealed differentially expressed signaling genes and networks of transcription factors that might underlie the segregation of the mesoderm and DE lineages. Moreover, we discovered a subpopulation of mesoderm cells that possess molecular features of the extraembryonic mesoderm. This study provides fundamental insight into the molecular basis for lineage segregation in post-implantation mouse embryos.
Highlights
The mammalian post-implantation embryo has been extensively investigated at the tissue level
Embryos containing only the EPI, visceral endoderm (VE), and EXE were dissociated into single-cell suspension after the parietal endoderm (PE) and ectoplacental cone (EPC) were removed with digestion. mRNA of each cell was reversetranscribed and amplified to obtain cDNA, and the expression of the EPI marker Oct4 (30 –34), the VE marker Gata6 [35,36,37], and the EXE marker Hand1 [38, 39] was examined by qRTPCR
We apply scRNA-Seq and high-throughput quantitative RT-PCR (qRT-PCR) approaches to investigate gene expression patterns in nearly 600 cells harvested from E5.5, E6.5_Early, and E6.5_Late mouse embryos
Summary
Unique transcriptional signatures of the EPI, VE, and EXE at the early post-implantation stage. Compared with Late EPI, 124 and 76 genes (one-sided Mann-Whitney U test, FDR Ͻ0.25) were enriched in ME and DE groups, respectively Both categories contained genes related to the GO term “tissue development” (Rank 2, p Ͻ 10Ϫ30, and Rank 2, p Ͻ 10Ϫ17, respectively), including genes such as Bmp, Cdh, Cxcr, Fn1, Gata, Gata, Lhx, and Tdgf (supplemental Table S6). We consistently noticed a positive correlation among genes Hand, Tbx, and Bmp when cells from E6.5_Late embryos were analyzed by PCA using the germ layer markers (supplemental Fig. S3, D and E). To precisely illustrate such a pattern, we analyzed the cells on the two branches of diffusion map (DE, ME, and Other clusters, together called MEN cells, all from E6.5_Late embryos) by PCA using RNA-Seq data of the 822 markers as an input. These identified genes could play important roles in the generation of EXEM and embryonic MEN cells during early embryonic development
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