Analysis of the Sacral Neural Crest Cell Contribution to the Hindgut Enteric Nervous System in the Mouse Embryo

BACKGROUND & AIMS: The majority of the enteric nervous system is derived from the vagal neural crest, with a second contribution, which is restricted to the post-umbilical gut, originating from the sacral neural crest. In mammals, although sacral neural crest cells (NCCs) have been shown to enter the hindgut, information on their development and role remains scant. Our aim was to determine the migratory routes of sacral NCCs to the hindgut, their timing and site of entry into the gut, and their migratory behaviors and differentiation within the hindgut. METHODS: We used in situ cell labeling, whole embryo culture, immunofluorescence, organotypic culture, and time-lapse live-cell imaging in mouse embryos. RESULTS: Sacral NCCs emigrated from the neural tube at embryonic day 9.5, accumulated bilateral to the hindgut to form prospective pelvic ganglia at embryonic day 11.5, and from there entered the distal hindgut through its ventrolateral side at embryonic day 13.5. They then migrated along nerve fibers extending from the pelvic ganglia toward the proximal hindgut, intermingling with rostrocaudally migrating vagal NCCs to differentiate into neurons and glia. In organotypic culture, genetically labeled sacral and vagal NCCs displayed different capabilities of entering the hindgut, implying differences in their intrinsic migratory properties. Time-lapse live-cell imaging on explants ex vivo showed that sacral NCCs migrated along nerve fibers and exhibited different migratory behaviors from vagal NCCs. CONCLUSIONS: Murine sacral NCCs are a distinct group of cells that migrate along defined pathways from neural tube to hindgut. They exhibit discrete migratory behaviors within the gut mesenchyme and contribute neurons and glial cells to the hindgut enteric nervous system.