Extended Data Fig. 5: Full developmental potential of mCherry+ cells.

a) Schematic illustrating the generation of a chimaeric embryo at E13.5 by diploid complementation with blastocyst injection (as described in Extended Data Fig. 2d,e). This served as a control to show that mCherry+ cells, when also contributing via the embryonic lineage, have full developmental potential. b) Bright-field and fluorescence microscopy images of an E13.5 chimaeric embryo and its corresponding yolk sac, generated by diploid complementation with blastocyst injection. The intestine was manually separated into the colon and small intestine, indicated by the black line (n = 6, one representative embryo is shown). c) Representative flow cytometry dot plots of the corresponding colon and small intestine from an E13.5 chimeric embryo showing the endoderm (EPCAM+) and the non-endoderm (EPCAM−) fractions (n = 6). mCherry+ cells are present in significant proportion confirming that mCherry+ cells are eliminated from the embryo only if they originate from the extraembryonic lineage, such as in our two-colour lineage tracing. d) Boxplot showing the abundance of epithelial endoderm cells (EPCAM+), the abundance of mCherry+ cells in the endoderm (EPCAM+) and non-endoderm (EPCAM−) populations in the organs isolated from E13.5 chimeras. The single embryo replicates are indicated by colour-coded dots. Lines denote the median, edges denote the IQR, whiskers denote 1.5× IQR, and minima/maxima are defined by dots. e) Schematic illustrating the generation of a fully mCherry+ embryo at E13.5 via natural mating. This served as a control to exclude that silencing of the mCherry transgene is the reason why mCherry+ extraembryonic cells are not detected in E13.5 embryos generated by the two-colour lineage tracing (Fig. 2). f) Bright-field and fluorescence microscopy images of an mCherry-only E13.5 embryo and its corresponding yolk sac generated via natural mating (n = 1). The intestine was manually separated into the colon and small intestine, indicated by the black line. g) Flow cytometry dot plots of the colon and small intestine from an E13.5 mCherry-only embryo showing the endoderm (EPCAM+) and the non-endoderm (EPCAM−) fractions (n = 1). All the cells are mCherry+, and no silencing of the mCherry transgene occurs.