Extended Data Fig. 1: Identification of non-ENS cells, glial and plausible myenteric cells in the P24 submucosal scRNA-seq dataset.

a, Uniform manifold approximation and projection (UMAP) of level 1 clustering of P24 submucosal cells. See Supplementary Table 1 for differentially expressed genes of the clusters. Jagged boxes are further analyzed in b–f. b, Feature plots indicating epithelial cells based on Cdh1 and various subclass markers and immune-related cells (H2-Aa). c, Analysis of Baf53b-Cre x R26RtdTom mice revealed TOM⁺/5-HT⁺ double-positive cells, showing transgene expression in serotonergic enterochromaffin cells. Image represents observations from 10 fields of view from 2 mice. Feature plots displaying marker genes of clusters representing mesenchymal-derived lineages, including smooth muscle cells (d) and lymphatic epithelial cells (LECs; e). f, Feature plot of Sox10 indicating an enteric glia cell cluster. g, UMAP of cluster level 2, displaying putative contaminating glial and myenteric classes as indicated in feature plots. h,i, Representative submucosa peels showing lack or little ENK (h) and NDUFA4L2 (i) expression in PGP9.5⁺ neurons. 0/2583 neurons expressed ENK; n = 3 mice. 2/7615 neurons expressed NDUFA4L2; n = 5 mice. j, CCK expression was investigated in Cck-IRES-Cre mice injected with AAV-DIO-EYFP or AAV-DIO-tdTOM. Only 1 of 2353 submucosal neurons expressed a fluorescent reporter; n = 4 mice (age, 8–12 weeks). Scale bars, 20 µm.