Spatiotemporal sequence of mesoderm and endoderm lineage segregation during mouse gastrulation

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).

前中胚层（anterior mesoderm, AM）与定型内胚层（definitive endoderm, DE）祖细胞是小鼠胚胎原条（primitive streak, PS）处胚层形成过程中最早特化的胚胎细胞类型。遗传实验表明，两种细胞谱系均源自表达Eomes的祖细胞，其分离过程依赖于不同水平的NODAL信号通路活性。然而，此类细胞的确切时空出现模式以及谱系分离的分子细节仍有待阐明。本研究结合遗传命运标记、成像技术与单细胞RNA测序（single cell RNA sequencing, scRNA-seq），追踪受Eomes调控的细胞类型的转录特征，并明确其谱系分化轨迹。研究结果显示，原肠运动首日穿过原条的所有细胞均表达Eomes；AM与DE的特化发生于细胞从Eomes阳性祖细胞脱离原条之前，且呈现独特的时空分布模式。单细胞RNA测序分析进一步证实，AM与DE谱系从作为最终共同双潜能祖细胞的Eomes阳性细胞中实现了即时且完全的分离。整体实验设计：本研究对胚胎发育第6.75天的576枚手动挑取的小鼠胚胎细胞，以及胚胎发育第7.5天的1172个经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）得到的小鼠胚胎细胞开展了单细胞RNA测序。其中，手动挑取细胞的测序采用CEL-Seq2实验流程，而荧光分选细胞的测序则采用mCEL-Seq2实验流程——该流程是CEL-Seq2的自动化微型化版本（Hashimshony等，2016；Herman等，2018）。