Fig. 1: Two-colour lineage tracing identifies dual-labelled embryonic cells.

a, Schematic of the two-colour lineage labelling strategy for lineage tracing (2N indicates that cells are diploid). Embryonic versus extraembryonic lineage segregation can be seen at the blastocyst stage (Extended Data Fig. 1c). At E9.5, embryos are GFP⁺ and only the gut contains a small fraction of mCherry⁺ extraembryonic cells (see b). b, Bright-field (left) and fluorescence (right) microscopy images of an E9.5 embryo generated via the two-colour lineage tracing (n = 54; one representative embryo is shown). c, Maximum-intensity projection of optical sections acquired by confocal laser scanning microscopy showing an E9.5 embryo and confirming the presence of mCherry⁺ extraembryonic cells specifically in the gut, which is positive for FOXA2 (additionally expressed in the notochord and floor plate). Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) and immunofluorescence was used for mCherry and FOXA2 (n = 3; one representative embryo is shown). d, Percentage of dual⁺ and mCherry⁺ cells (left) as well as the ratio of these two populations (right) in E9.5 embryos analysed by flow cytometry. Individual embryos are indicated by colour-coded dots (n = 9). Boxplots: the lines denote the median, the edges denote the interquartile range (IQR), whiskers denote 1.5× the IQR and minima/maxima are defined by dots. e, Transversal optical section of an E9.5 embryo acquired by light-sheet imaging (the dashed line in the schematic depicts the axial position, bottom left). E-CAD marks the surface ectoderm and gut endoderm. Magnified views of the gut (yellow box) are shown; mCherry foci are highlighted (white arrows). Nuclei were stained with DAPI and immunofluorescence was used for mCherry and E-CAD (n = 3; a section from one representative embryo is shown). f, Uniform manifold approximation and projection (UMAP) coloured by the assigned cell states showing dual⁺ (left) and mCherry⁺ (right) cells subjected to scRNA-seq (the dual⁺ population is indicated in grey on the right). The fractions of cells belonging to the individual cell states are indicated with the bars. g, Average log-normalized scRNA-seq expression of reporter transgenes and known marker genes of the indicated cell types. Expression is shown separately for embryonic dual⁺ endoderm and non-endoderm as well as mCherry⁺ extraembryonic cells.