Extended Data Fig. 6: Gene expression differences between embryonic and extraembryonic gut cells.

a) Bright-field and fluorescence microscopy images of an E9.5 embryo generated via the two-colour lineage tracing. The embryo was manually split into anterior and posterior halves (upper row). The posterior half, containing a large fraction of mCherry+ cells, was used for manually isolating the midgut and the tailbud contains the hindgut (lower row). These were used for sorting, then RNA-seq and WGBS (n = 16, one representative embryo is shown, corresponding tissues from four embryos were pooled). b) Flow cytometry dot plot of the epithelial fraction (EPCAM+) from the pooled hindgut tissues (n = 4). mCherry and GFP intensities were used to sort mCherry+ extraembryonic gut cells and dual+ cells with low mCherry intensity as embryonic hindgut. Our single-cell RNA-seq experiment (Fig. 1) confirmed that epithelial dual+ cells are gut endoderm of embryonic origin and, therefore, ideal to utilize as a stage-matched embryonic comparison. c) Log2-transformed expression of origin and lineage marker genes for E6.5 epiblast and extraembryonic endoderm as well as E9.5 gut and yolk sac endoderm in single replicates. d) Scatterplot comparing the log2 fold change between exGut and emGut samples with the average log2-transformed expression in emMidgut, emHindgut, exMidgut and exHindgut. e) Overrepresentation analysis of exGut low and high genes in cellular components. f) Log2-transformed enrichment of the chromosomal ___location of exGut low and high genes compared to the genomic background distribution of all genes (=0 equals no difference, > 0 implies enrichment, < 0 implies depletion). exGut high genes are enriched on the X chromosome. g) Overlap of exGut high genes with genes known to escape X chromosome inactivation⁴⁶. The small overlap suggests that the expression of exGut high genes is not caused by sex differences between emGut and exGut or the effect of double dosage from X chromosome inactivation escapees. h) Average log-normalized expression of endoderm marker genes, axonogenesis-associated exGut low genes and germline-associated exGut high genes across embryonic (emGut) and extraembryonic (exGut) gut cells from E8.75 (ref. ⁴) and E9.5 (ref. ¹⁹) embryos using published datasets.