Extended Data Fig. 2: Benchmarking different complementation strategies.

a) Schematic illustrating the conventional tetraploid complementation by morula aggregation, with selective and stable lineage labelling (similar to the two-colour lineage tracing strategy used throughout the study) with the caveat that extraembryonic cells are tetraploid. 2-cell-stage mCherry+ embryos are electrofused. Then, two tetraploid mCherry+ pre-compaction morulas are aggregated with a GFP+ mESC colony (2N indicates diploid, 4N indicates tetraploid). At E9.5, the embryo overall consists of diploid GFP+ cells, where only the gut contains tetraploid mCherry+ extraembryonic cells. At E9.5, embryos without visible malformations were collected and counted: 1) mCherry signal specific to the region resembling the gut tube or 2) fully mCherry+ embryos or 3) chimaeras where both mCherry+ and GFP+ cells are broadly distributed (data provided in the table). b) Bright-field and fluorescence microscopy images of an E9.5 embryo generated via tetraploid complementation with morula aggregation, which is overall diploid and GFP+, while tetraploid mCherry+ extraembryonic cells are present only in a distinct area resembling the gut tube (n = 22, one representative embryo is shown). c) Bright-field and fluorescence microscopy images of a yolk sac corresponding to the embryo shown in b (n = 22, one representative yolk sac is shown). d) Schematic illustrating the diploid complementation by blastocyst injection, which leads to fully chimeric embryos because embryonic and extraembryonic lineages are not distinctly labelled (in contrast to the two-colour lineage tracing strategy used throughout the study). GFP+ mESCs are injected into blastocyst-stage mCherry+ embryo (2N indicates diploid). As the blastocyst already has a defined ICM, mCherry+ cells contribute to the embryo proper as well, resulting in chimeras. At E9.5, embryos without visible malformations were collected and counted as described in a (data provided in the table). e) Bright-field and fluorescence microscopy images of an E9.5 embryo generated via diploid complementation by blastocyst injection (n = 26/32, one representative chimeric embryo is shown). f) Bright-field and fluorescence microscopy images of a yolk sac corresponding to the embryo shown in e (n = 26/32, one representative yolk sac is shown).