Fig. 3: Transcriptional signatures of gut cells reflect their lineage origins.

a, Schematic of the origin and composition of gut endoderm: emGut cells (E9.5) originating from the embryonic epiblast (Epi, E6.5) and exGut cells (E9.5) originating from the distal part of the extraembryonic endoderm at E6.5 (exEndo 1) both contribute to the formation of gut endoderm. The proximal part of the extraembryonic endoderm (exEndo 2) gives rise to the E9.5 YsEndo. E6.5 Epi and E9.5 emGut cells are positive for GFP, whereas E6.5 exEndo 1 and 2, E9.5 exGut and YsEndo cells are positive for mCherry in lineage-traced embryos. E9.5 emGut cells can also be positive for mCherry in the case of dual⁺ cells that have taken up remnants of dying mCherry⁺ cells (not illustrated). b, Principal component analysis (PCA) of E6.5 and E9.5 RNA-seq samples based on the 5,000 most variably expressed genes. Samples are largely separated by tissue, with exGut cells grouping close but distinguishable from their embryonic counterparts. c, Genes that are differentially expressed (two-sided Wald test; P values were adjusted for multiple testing using false discovery rate (FDR)) between E9.5 exGut and emGut (including both midgut and hindgut). Genes that are expressed at significantly higher levels in exGut cells compared with emGut cells (termed exGut high) include known extraembryonic marker genes as well as the mCherry transgene, whereas GFP is expressed at significantly higher levels in emGut cells (termed exGut low). Vertical and horizontal lines denote log₂ fold change and adjusted P-value boundaries used for differential expression calling. d, Overrepresentation analysis of exGut low and high genes in biological processes. The exGut low genes are enriched in axonogenesis-related processes, whereas exGut high genes are enriched in germline- and meiosis-associated processes. e, Expression (log₂-transformed) of a selection of exGut low (top) and high (bottom) genes associated with the terms shown in d in E6.5 progenitor and E9.5 gut cells. The selected exGut low genes are not expressed in the E6.5 exEndo 1 and are insufficiently activated in the E9.5 exGut cells. In contrast, exGut high genes are generally not expressed, or are expressed at low levels, in the E6.5 Epi and E9.5 emGut samples, whereas both the extraembryonic progenitor and exGut cells express them. TPM, transcripts per million.