Extended Data Fig. 6: Force-directed layouts of single E5.5 cells reveal relationships between EPI, visceral endoderm and ExE lineages.

a, Force-directed layouts of E5.5 data generated after pooling replicates, showing the relationship between EPI, visceral endoderm and ExE lineages. Cells are coloured by cell type. Black arrowheads mark cells that transdifferentiate from EPI to visceral endoderm. b, Plot showing the projection of EPI, visceral endoderm and ExE cells along the first two diffusion components. Distances between lineages were computed using multiscale distances. c, Plots highlighting the extremes of the diffusion components, serving as the boundaries of the phenotypic space for each lineage identity. d, Plots showing the shortest path-step sizes for paths from EPI to visceral endoderm (left) and EPI to ExE (right). e, Gene expression plots of AVE (Cer1 and Dkk1), visceral endoderm (Eomes, Foxa1 and Ttr), and visceral endoderm and EPI (Nodal) markers along EPI, and PrE and visceral endoderm lineages from E3.5–E5.5. Cells coloured on the basis of marker expression of indicated gene after MAGIC⁵⁰. f, Laser-scanning confocal images of E5.5 and E6.0 Sox2-cre^TG/+;Rosa26^mT/mG and Ttr-cre^TG/+;Rosa26^mT/mG embryos immunostained for GFP, RFP (red fluorescent protein, membrane-localized tdTomato) and GATA6 (a marker of endoderm identity). Cell nuclei stained with Hoechst, and membranes labelled with RFP. Yellow arrowheads point to cells of EPI origin that are present within the visceral endoderm epithelial layer (n = 10/20 GFP-positive cells in visceral endoderm of Sox2-cre^TG/+;Rosa26^mT/mG embryos; n = 0/27 GFP-positive cells in the EPI of Ttr-cre^TG/+;Rosa26^mT/mG embryos). Results validated in at least three independent experiments. Scale bars, 50 μm (low-magnification images), 20 μm (high-magnification images). g, Laser-scanning confocal images of two E5.5 wild-type 4n embryo <-> H2B-tdTomato embryonic stem cell (ESC) chimaeras. Top two rows and bottom two rows represent low- and high-magnification 2D images, respectively (n = 9/19 embryo chimaeras showed tdTomato-positive cells in the visceral endoderm). Yellow arrowheads indicate EPI cells intercalating into the visceral endoderm layer. Embryos are counterstained with Hoechst to label nuclei, and phalloidin to label F-actin. Scale bars, 20 μm (low-magnification images), 10 μm (high-magnification images).