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Parallel scRNA-seq and genetic recording reveals lineage decisions in early mouse embryogenesis

ABSTRACT: How cell fate is specified at early stages of development is a fundamental question in biology. We apply deep transcriptomic profiling to map cell lineages at single cell resolution in embryoid bodies (EBs); a spontaneous, manipulatable, in vitro system for embryonic development. We predict the overall cell fate trajectory and define the transcriptomic landscape of cells at each critical branch. We present a dual barcoding system, comprising a static cellular barcode and inducible genetic recorder, and leverage long-read sequencing to obtain both barcodes and transcriptome from each single cell. Using this system, we confirm overall trajectory architecture, and validate key branch points, including the early isolation of the primordial germ cell (PGC) like lineage from preimplantation epiblast-like cells. Our data suggest DNA methylation is a critical determinant of PGC cell fate choice before the postimplantation epiblast cell state is determined. Together, our data provide a high resolution lineage decision map in an organoid model of early embryogenesis, with implications for the developing embryo.

ORGANISM(S): Mus musculus

PROVIDER: GSE140890 | GEO | 2020/08/07

REPOSITORIES: GEO

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Project description:The generation of properly functioning gametes in vitro, a key goal in developmental/reproductive biology, requires multi-step reconstitutions of complex germ cell development. Based on the logic of primordial germ cell (PGC)-specification, we demonstrate here the generation of PGC-like cells (PGCLCs) in mice with robust capacity for spermatogenesis from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pre-gastrulating epiblasts, but distinct from epiblast stem cells (EpiSCs). The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs are a meticulous capture of those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-beta 3 and SSEA1 as markers that purify PGCLCs with spermatogenic capacity free from tumorigenic undifferentiated cells. With the reconstitution of PGC specification pathway from the naive inner cell mass state, our study defines a paradigm for the essential step of in vitro gametogenesis. We performed this analysis to reveal the characters of the cells that we created in this study, epiblast-like cells (EpiLCs) and primordial germ cells-like cells (PGCLCs). Because EpiLCs were induced from embryonic stem cells (ESCs), and equivalent to pre-gastrulating epiblast (embryonic day [E] 5.5-6.0) in vivo (embryo), ESCs and epiblast were included in this analysis. Epiblast stem cells (EpiSCs) are a culture cell type derived from epiblast, and were also included. PGCLCs were supposed to be equivalent to E9.5 PGCs based on reporter fluorescent transgene expressions and epigenetic properties, and therefore E9.5 PGCs were also inckuded in this analysis. Because epiblast and E9.5 PGCs are of a small number of cells in embryos (a few hundred to thousand cells), cDNAs were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research) for microarray analyses. We took two biological replicate for each cell type.

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Parallel scRNA-seq and genetic recording reveals lineage decisions in early mouse embryogenesis

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