Extended Data Fig. 5: Endoderm trajectories.

a, Top, graph abstraction of the endoderm landscape after fine sub-clustering as an alternative method to resolve which cells should be part of the visceral endoderm (VE)–hindgut 1 trajectory or the definitive endoderm (DE)–hindgut 2 trajectory (supporting transport maps; see Methods). Edges along the VE–hindgut 1 trajectory are highlighted in yellow (nodes 1–4; yellow numbers). Edges along the DE–hindgut 2 trajectory are highlighted in orange (nodes 1–12; orange numbers). Bottom, graph abstraction with the subset of nodes related to the DE–hindgut 2 trajectory to resolve the origin of cluster 4 (between 5 and 6 in the top panel). Resulting DE–hindgut 2 trajectory includes clusters 1–4 and 6–9. The right-hand panels overlay information about the composition of each cluster by developmental stage. b, Force-directed graph coloured by partition-based graph abstraction (PAGA) trajectories. Note that this independent approach for trajectory identification reaches very similar results to those inferred by the transport maps in Fig. 2h. HG1, hindgut 1; HG2, hindgut 2. c, Gene-normalized dynamics of all clusters found along the VE–hindgut 1 and the DE–hindgut 2 trajectories (x axis: DPT along the trajectory; y axis: normalized expression levels). The black line is the mean fitted expression level across all genes in each cluster; the grey shading indicates the standard deviation along the trend across all genes in the cluster; the pink area highlights intercalation process; and the blue area highlights gut maturation steps. Vertical dashed lines correspond to additional stages in the process, deduced from the changes in gene expression trends. Points below the plots are the DPT coordinates of cells from each time point coloured according to time point as in Fig. 2b (from E6.5 in red to E8.5 in blue). d, Gene-normalized dynamics of VE genes along the VE–hindgut 1 trajectory, indicating VE maturation before the intercalation stage. Plot design is as in c. e, Left, Venn diagram of genes that were upregulated during the intercalation process in both VE–hindgut 1 (in clusters 3, 5, 8, and 11) and DE–hindgut 2 (in clusters 4, 6, 7, 8, and 9) trajectories. The overlapping fraction was enriched in genes that are a signature of epithelial remodelling (top 20 genes are listed). Right, gene-normalized dynamics of illustrative genes (Pcna, Epcam, Slc2a1, Vim, Crb3, and Cadm1) along the trajectories. f, Left, Venn diagram of genes that were upregulated after the intercalation process in both trajectories (VE–hindgut 1: clusters 1, 2, 5, and 10; DE–hindgut 2: clusters 1, 3, 5, and 10). The overlapping fraction was enriched in genes that encode transcription factors (TFs), including a large subset of homeodomain proteins (genes are listed). Right, gene-normalized dynamics of Hox and Cdx genes along the trajectories. g, Gene-normalized dynamics of transcription factors that were upregulated specifically in the VE–hindgut 1 trajectory during endoderm intercalation. Points below the x axis in d–g are as in c.