Extended Data Fig. 1: Two-colour lineage tracing by stage-specific diploid complementation.

a) Schematic illustrating the two-colour lineage tracing strategy used throughout the study to selectively and stably label embryonic and extraembryonic lineages. An mCherry+ pre-compaction morula is aggregated with a GFP+ ESC colony (2N indicates that both are diploid). Representative experiments at E9.5 are summarized in the table, where in the majority of the cases (more than 95%, 54/56), embryos were GFP+, and only the gut region contained diploid mCherry+ cells, while in rare cases, embryos were fully mCherry+ without detectable GFP+ cells (less than 5%, 2/56). b) Bright-field and fluorescence microscopy images of a yolk sac (corresponding to the embryo shown in Fig. 1a) generated via the two-colour lineage tracing (n = 54, one representative yolk sac is shown). c) Confocal laser scanning microscopy images showing an expanded blastocyst, where the GFP signal is present in the region indicating the early epiblast, while the mCherry signal is present in the region indicating the extraembryonic lineages. Nuclei were stained with DAPI (n = 10, one representative embryo is shown). d) Bright-field and fluorescence microscopy images of an E9.5 embryo, which contains only diploid mCherry+ cells (n = 2, one representative embryo is shown) as a likely outcome of failed aggregation and mESC incorporation. Such fully mCherry+ embryos were excluded from further experiments. e) Bright-field and fluorescence microscopy images of a yolk sac (corresponding to the embryo shown in Extended Data Fig. 1d), which contains only diploid mCherry+ cells as a likely outcome of failed aggregation and mESC incorporation (n = 2, one representative yolk sac is shown). f) Maximum-intensity projection of optical sections acquired by confocal laser scanning microscopy showing a lineage-traced E9.5 embryo and confirming the presence of mCherry+ extraembryonic cells specifically in the gut tube. E-CADHERIN, a surface marker of epithelial cells, is present not only in the gut endoderm but also in the surface ectoderm, where no mCherry+ cells are located. Nuclei were stained with DAPI, and immunofluorescence was used for mCherry and E-CAD (n = 3, one representative embryo is shown).