Embryonic patterning of the mouse during gastrulation and early organogenesis engenders the specification of anterior versus posterior structures and body laterality by the interaction of signalling and modulating activities. A group of cells in the mouse gastrula, characterised by the expression of a repertoire of "organiser" genes, acts as a source and the conduit for allocation of the axial mesoderm, floor plate and definitive endoderm. The organiser and its derivatives provide the antagonistic activity that modulates WNT and TGFbeta signalling. Recent findings show that the organiser activity is augmented by morphogenetic activity of the extraembryonic and embryonic endoderm, suggesting embryonic patterning is not solely the function of the organiser.

Gastrulation of the mouse embryo entails progressive restriction of lineage potency and the organization of the lineage progenitors into a body plan. Here we performed a high-resolution RNA sequencing analysis on single mid-gastrulation mouse embryos to collate a spatial transcriptome that correlated with the regionalization of cell fates in the embryo. 3D rendition of the quantitative data enabled the visualization of the spatial pattern of all expressing genes in the epiblast in a digital whole-mount in situ format. The dataset also identified genes that (1) are co-expressed in a specific cell population, (2) display similar global pattern of expression, (3) have lineage markers, (4) mark domains of transcriptional and signaling activity associated with cell fates, and (5) can be used as zip codes for mapping the position of single cells isolated from the mid-gastrula stage embryo and the embryo-derived stem cells to the equivalent epiblast cells for delineating their prospective cell fates.

Mouse epiblast stem cells (EpiSCs) can be derived from a wide range of developmental stages. To characterize and compare EpiSCs with different origins, we derived a series of EpiSC lines from pregastrula stage to late-bud-stage mouse embryos. We found that the transcriptomes of these cells are hierarchically distinct from those of the embryonic stem cells, induced pluripotent stem cells (iPSCs), and epiblast/ectoderm. The EpiSCs display globally similar gene expression profiles irrespective of the original developmental stage of the source tissue. They are developmentally similar to the ectoderm of the late-gastrula-stage embryo and behave like anterior primitive streak cells when differentiated in vitro and in vivo. The EpiSC lines that we derived can also be categorized based on a correlation between gene expression signature and predisposition to differentiate into particular germ-layer derivatives. Our findings therefore highlight distinct identifying characteristics of EpiSCs and provide a foundation for further examination of EpiSC properties and potential.

Understanding of the molecular drivers of lineage diversification and tissue patterning during primary germ layer development requires in-depth knowledge of the dynamic molecular trajectories of cell lineages across a series of developmental stages of gastrulation. Through computational modeling, we constructed at single-cell resolution, a spatio-temporal transcriptome of cell populations in the germ-layers of gastrula-stage mouse embryos. This molecular atlas enables the inference of molecular network activity underpinning the specification and differentiation of the germ-layer tissue lineages. Heterogeneity analysis of cellular composition at defined positions in the epiblast revealed progressive diversification of cell types. The single-cell transcriptome revealed an enhanced BMP signaling activity in the right-side mesoderm of late-gastrulation embryo. Perturbation of asymmetric BMP signaling activity at late gastrulation led to randomization of left-right molecular asymmetry in the lateral mesoderm of early-somite-stage embryo. These findings indicate the asymmetric BMP activity during gastrulation may be critical for the symmetry breaking process.