Extended Data Fig. 10: Persisting p53-mutant extraembryonic cells in vitro and in vivo.

a) Single channel bright-field and fluorescence microscopy images, showing time points of representative in vitro cultured gut cell assemblies over 5 days, sorted from the posterior part of wild type lineage-traced embryos (top) or the posterior part of embryos with extraembryonic p53 KO (bottom) at E9.5 (n = 3, one representative gut cell assembly is shown for each condition). Embryonic gut cells show substantial growth and WT extraembryonic gut cells do not show signs of proliferative capacity, while p53 KO extraembryonic gut cells show substantial and comparable growth to the embryonic gut cells. b) Merged bright-field and fluorescence microscopy images from the in vitro culture experiment at day 1 and day 5 from a. c) Growth quantification of in vitro cultured gut cell assemblies (represented in a,b) as determined by the relative area calculated by normalizing the assembly area to the average area on day 1 (n = 3). Central line denotes the mean, whiskers denote standard deviation. d) Boxplots showing the exGut low and high gene groups separated by promoter methylation in the E6.5 exEndo 1 compared to the epiblast, for E9.5 and E13.5 tissues (RRBS). The distinct promoter methylation patterns of cells with embryonic and extraembryonic origin are still present at E13.5. Lines denote the median, edges denote the IQR, whiskers denote 1.5× IQR, and outliers are represented by dots (n = 3–4 biological replicates). e) Z-score-transformed expression across E9.5 gut and E13.5 intestine samples of all E9.5 exGut low and high genes split using k-means clustering. Germline- and axonogenesis-associated genes shown in Fig. 3e are indicated next to their assigned cluster.