Fig. 2. Mesoderm and endoderm form by distinct molecular programs.
a, Schematic of FACS of early-, mid- and late-streak-stage FVF embryos for scRNA-seq analysis (n = 79 for early- to mid-streak-stage embryos and n = 24 for mid- to late-streak-stage embryos). b, UMAP plot with RNA velocity arrows, coloured by CellRank’s metastable state assignment. Each shown tissue is either in the initial (epiblast (Epi)), intermediate (posterior epiblast (pEpi)) or final state (AME, definitive endoderm, lateral plate mesoderm (LPM) and nascent endothelium (NE)). c, UMAP showing CellRank’s fate probabilities for different tissues as pie charts (n = 9,794 cells). The partitions of each pie chart show the previously identified initial, intermediate or final state. Dashed lines indicate significant connections between clusters (PAGA graph model). Arrows indicate consistent RNA velocity between two clusters. The thickness of each line shows the confidence of the model. The solid line without an arrowhead suggests a transition along the velocity between clusters but not unique flow. IM, intermediate mesoderm; PGC, primordial germ cell; PM, paraxial mesoderm. d, Scatter plot of lineage drivers, showing the correlation of gene expression for the lineages definitive endoderm and LPM, computed using CellRank. The top 50 correlated genes are indicated by dashed horizontal and vertical lines. e, Stacked violin plots showing the gene expression distribution (columns) with definitive endoderm, EMT, EMT inhibitors and cell adhesion genes of all tissues (rows) (n = 2,215 (posterior epiblast); n = 2,198 (paraxial mesoderm); n = 1,183 (LPM); n = 701 (definitive endoderm); n = 389 (intermediate mesoderm); n = 350 (transitory progenitors); n = 278 (AME); n = 91 (nascent endothelium)). The colours correspond to normalized median gene expression for each group. f, UMAP plots coloured by the log[counts per million + 1] normalized gene expression. g, Schematic of endoderm and mesoderm differentiation of TGFP/+; Foxa2tagRFP/+ mESCs. h, Heatmap of FACS-sorted endodermal and mesodermal subpopulations expressing different levels of CD24 at days 2 and 4, showing RNA expression levels of pluripotency, endoderm, mesoderm and EMT genes in mESCs (ES), early (definitive endoderm progenitor (DEP)/mesoderm progenitor (MP)) and late (definitive endoderm (DE)/mesoderm (MES)) endoderm and mesoderm cells. The coloured boxes indicate differentially expressed genes (DEGs) in DEP versus mesoderm progenitor (green and orange, respectively) or definitive endoderm versus mesoderm (blue and red, respectively) and whether Foxa2 binds (pink) or binds and regulates them (purple).