Figure 3. Trajectory reconstruction for cells of the interstitial lineage suggests a cell state common to neurogenesis and gland cell differentiation.

A) t-SNE representation of interstitial cells with clusters labeled by cell state. Solid arrow: neurogenesis/gland cell differentiation. Dashed arrow: nematogenesis. B) HvSoxC expression in progenitor cells. Arrow indicates putative ISC population, which is negative for HvSoxC and positive for biomarker Hy-icell1 expression (C). D) URD differentiation tree of the interstitial lineage. Color represents URD segments and do not correspond to the colors in the t-SNE (see fig. S19. E)) Myb (green) is expressed in the neuron/gland cell progenitor state and during early neurogenesis/gland cell differentiation. Expression of Myb (green, > 0) partially overlaps with high expression of the neuronal gene NDA-1 (magenta, > 3) and the gland cell gene COMA (t2163) (magenta, > 0); COMA is also expressed in a subset of endodermal neurons. Co-expressing cells are black. Star and close-up highlights cell states with co-expression. F) Double labeling using fluorescent RNA in situ hybridization is consistent with neuron differentiation in the endodermal and ectodermal epithelial layers and demonstrates the existence of transition states observed in the trajectory analysis. Additionally, endodermal gland cell differentiation transition states were observed in the endodermal epithelial layer (see also fig. S22). gc: gland cell, gp: gland cell progenitor, n: neuron, np: neuron progenitor, p: Myb positive progenitor. G) Model for progenitor specification. Ectodermal ISCs give rise to a progenitor that can give rise to ectodermal neurons. Progenitors that translocate to the endoderm are able to give rise to glands or neurons. ec: ectoderm, en: endoderm, gmgc: granular mucous gland cell, gc: gland cell, hyp: hypostome, ISC: interstitial multipotent stem cell, mgc: mucous gland cell, nb: nematoblast, smgc: spumous mucous gland cell, prog: progenitor, zmg: zymogen gland cell.