GDNF regenerates the missing enteric nervous system of Hirschsprung mice via non-canonical signaling in diverse subtypes of tissue-resident progenitors

Hirschsprung disease (HSCR) is a deadly congenital disorder where the enteric nervous system (ENS) is absent from the distal bowel. Current surgical treatment is generally life-saving but is often accompanied by long-term bowel problems and comorbidities. As alternative, we are developing a regenerative therapy based on rectal administration of Glial cell-derived neurotrophic factor (GDNF). We previously showed that administering GDNF enemas to HSCR mice soon after birth is sufficient to permanently induce a new ENS from tissue-resident neural progenitors. Here, we elucidate the underlying mechanism using single-cell transcriptomics, signal transduction inhibitors and genetic cell lineage tracing tools. We found that the neurogenic effect of GDNF is mediated by NCAM1 (Neural cell adhesion molecule 1), rather than by its canonical signaling receptor RET (Rearranged during transfection). We also unveiled the existence of multiple neuronal differentiation pathways that involve a larger than expected repertoire of tissue-resident neural progenitors, including a surprising one not derived from the usual neural crest. These data support feasibility of GDNF-based therapy in most human patients, even those bearing a RET variant. This work also has far-reaching implications for the choice of ENS progenitor source to use when developing cell transplantation-based therapeutic approaches.