Sacral neural crest-independent origin of the enteric nervous system in mouse

BACKGROUND & AIMS The enteric nervous system (ENS), the gut’s intrinsic nervous system critical for gastrointestinal function and gut-brain communication, is believed to mainly originate from vagal neural crest cells (vNCCs) and partially from sacral NCCs (sNCCs). Resolving the exact origins of the ENS is critical for understanding congenital ENS diseases but has been confounded by the inability to distinguish between both NCC populations in situ. Here, we aimed to resolve the exact origins of the mammalian ENS. METHODS We genetically engineered mouse embryos facilitating comparative lineage-tracing of either all (pan-) NCCs including vNCCs or caudal trunk and sNCCs (s/tNCCs) excluding vNCCs. This was combined with dual-lineage tracing and 3D-reconstruction of pelvic plexus and hindgut to precisely pinpoint sNCC and vNCC contributions. We further employed co-culture assays to determine the specificity of cell migration from different neural tissues into the hindgut. RESULTS Both pan-NCCs and s/tNCCs contributed to established NCC derivatives but only pan-NCCs contributed to the ENS. Dual lineage-tracing combined with 3D-reconstruction revealed that s/tNCCs settle in complex patterns in pelvic plexus and hindgut-surrounding tissues, explaining previous confusion regarding their contributions. Co-culture experiments revealed unspecific cell migration from autonomic, sensory, and neural tube explants into the hindgut. Lineage-tracing of ENS precursors lastly provided complimentary evidence for an exclusive vNCC origin of the murine ENS. CONCLUSIONS sNCCs do not contribute to the murine ENS, suggesting that the mammalian ENS exclusively originates from vNCCs. These results have immediate implications for comprehending (and devising treatments for) congenital ENS disorders, including Hirschsprung’s disease.