Eomesodermin dependence delineates the formation of spatially and functionally diverse extra-embryonic hematovascular tissues [scRNA-seq: chimera]

During mouse gastrulation, extraembryonic mesoderm (ExEM) contributes to the extraembryonic yolk sac (YS) and allantois, both of which are essential for successful gestation. Although the genetic networks coordinating intra-embryonic mesodermal subtype specification are well-studied, the mechanisms driving ExEM diversification are poorly understood. Here, we reveal that embryoid body (EB) in vitro differentiation generates two distinct lineages of mesodermal cells matching YS and allantois respectively. Combining in vitro models with in vivo chimeric embryo analysis, we discover that Eomesodermin (Eomes) regulates the formation of a subset of YS-fated ExEM but is dispensable for allantois formation. Furthermore, simultaneous disruption of Eomes and T impedes the specification of any YS or allantois mesoderm, indicating compensatory roles for T during allantois formation when Eomes is disrupted. Our study highlights previously unrecognized functional and mechanistic diversity in ExEM diversification and endothelial development and introduces a tractable EB model to dissect the signaling pathways and transcriptional networks driving the formation of key extraembryonic tissues. Overall design: Single-cell RNA sequencing was performed on chimeric embryos containing Eomes KO and wild-type (WT) cells. Chimeras were produced by injecting WT blastocysts with tdTomato+ Eomes KO embryonic stem cells (ESCs). Embryos were collected in several pools (replicates) at around stage E8.5 and then dissociated into single-cell suspensions. The cells were sorted using FACS based on tdTomato expression to distinguish between WT (tdTom-) and Eomes KO (tdTom+) cells in each pool. The sorted cells were then prepared for scRNA-seq using the 10x Genomics 3' v3 kit.