SRA

Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the earliest embryonic cell types that are specified during germ layer formation at the primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both lineages segregate from Eomes expressing progenitors in response to different NODAL signaling levels. However, the precise spatiotemporal pattern of the emergence of these cell types and molecular details of lineage segregation remain unexplored. We combined genetic fate labeling and imaging approaches with single cell RNA sequencing (scRNA-seq) to follow the transcriptional identities and define lineage trajectories of Eomes dependent cell types. Accordingly, all cells moving through the PS during the first day of gastrulation express Eomes AM and DE specification occurs before cells leave the PS from Eomes positive progenitors in a distinct spatiotemporal pattern. ScRNA-seq analysis further suggest the immediate and complete separation of AM and DE lineages from Eomes expressing cells as last common bipotential progenitor. Overall design: Single cell RNA sequencing was performed on 576 hand-picked mouse embryonic cells at embryonic day 6.75 and 1172 FACS sorted cells from mouse embryos at embryonic day 7.5. Single-cell RNA sequencing was performed using the CEL-Seq2 protocol for hand-picked cells and mCEL-Seq2 protocol, an automated and miniaturized version of CEL-Seq2 for FACS-sorted cells (Hashimshony et al., 2016; Herman et al., 2018).