Figure 2—figure supplement 1.

Figure 2—figure supplement 1.

(A) A representative FACS plot showing nuclei from the muscularis externa of adult Tg(sox10:Cre;Cherry) zebrafish guts gated on single intact DAPI⁺ nuclei. mCherry⁺ nuclei were collected, representing less than 1% of the starting population. An equivalent number of mCherry^- nuclei were also collected. (B) Principal component analysis of the adult gut transcriptomes reveals segregation of the samples by Cherry⁺ vs. Cherry^- expression (30% of variability explained in PC1, 13% in PC2). Five biological replicates of each condition. (C–H) Analysis of the adult gut Cherry⁺ vs Cherry^- transcriptomic data by comparison to previously published data and publicly available reference data. The adult gut Cherry⁺ vs Cherry^- transcriptomic data (Supplementary file 1) was filtered to select those genes with log fold-change >0 (in Cherry⁺ vs Cherry^-) and with p-value<0.05. The resulting set is enriched for statistically significant zebrafish ENS-associated genes. (C) Gene set enrichment analysis shows that GO Biological Processes enriched in the Cherry⁺ population include nervous system associated terms. (D–E) Enrichment plots of representative gene sets (D) Synaptic Signalling and (E) Neuron cell-cell adhesion shows enrichment in Cherry⁺ samples. (F) Clustered heat map showing expression of a list of genes enriched in zebrafish larval ENS neurons (from Roy-Carson et al., 2017) that is analysed in our adult zebrafish gut transcriptomic data. We observe that > 750 of these neural expressed genes are enriched in the Cherry⁺ samples relative to Cherry^- samples (Supplementary file 2), and these are candidate adult ENS neuron-associated genes. These include phox2bb, phox2a, ret, elavl3, elavl4, vip, and nmu. (G) Clustered heat map showing the top 25 genes identified as enriched in mammalian Plp1⁺ glial cells (Rao et al., 2015) that have zebrafish orthologues and which are upregulated in Cherry⁺ vs Cherry^- samples, revealing nine candidate zebrafish ENS glial cell-associated genes. Cross-species comparisons (zebrafish to mouse) utilise publicly available homology assigning resources (see methods). (H) Clustered heat map showing expression of genes in the adult zebrafish ENS transcriptome after removing genes associated with zebrafish ENS neurons (from C, above). Over 600 unique genes are identified (Supplementary file 6), which are candidate adult ENS non-neuronal or ENS glial cell-associated genes. These include sox10, foxd3, tfap2a, sox2, col28a1b, plp1b, ptprz1a and ptprz1b.