Fig. 5. Foxa2 activates Wnt inhibitors in endoderm.

a, Schematic of endoderm and mesoderm differentiation of T^GFP/+; Foxa2^tagRFP/+ mESCs. b, Heatmap of FACS-sorted endodermal and mesodermal subpopulations expressing different levels of CD24 at days 2 and 4, showing RNA expression levels of canonical Wnt signalling genes. c, Heatmap of endoderm and mesoderm from differentiations of T^GFP/+; Foxa2^tagRFP/+ cells, showing upregulation of EMT suppressors in endoderm. In b and c, the coloured boxes to the left indicate genes that were differentially expressed 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). d, Clustered heatmap showing the smoothed (sliding window of n = 100 cells) and scaled gene expression of Wnt signalling genes in mesoderm (paraxial mesoderm, intermediate mesoderm, LPM and nascent endothelium), posterior epiblast, transitory progenitors and definitive endoderm. e, Quadratic spline plots showing genes involved in activation and inhibition of Wnt signalling along diffusion pseudotime from epiblasts to posterior epiblasts to transitory progenitors to definitive endoderm. f,g, Maximum projections of control (representative of six embryos) (f) and Foxa2^Venus/Venus knockout (representative of eight embryos) (g) late-streak-stage aggregation embryos immunostained for GFP or Foxa2, Snail1 and Cer1. All samples were derived from biologically independent experiments. All shown confocal images are single planes of a z stack unless otherwise stated. Scale bars, 50 µm.