The majority of the enteric nervous system is derived from vagal neural crest cells (NCC), which migrate to the developing gut, proliferate, form plexuses and differentiate into neurons and glia. However, for some time, controversy has existed as to whether cells from the sacral region of the neural crest also contribute to the enteric nervous system. The aim of this study was to investigate the spatiotemporal migration of vagal and sacral NCC within the developing gut and to determine whether the sacral neural crest contributes neurons and glia to the ENS. We utilised quail-chick chimeric grafting in conjunction with antibody labelling to identify graft-derived cells, neurons and glia. We found that vagal NCC migrated ventrally within the embryo and accumulated in the caudal branchial arches before entering the pharyngeal region and colonising the entire length of the gut in a proximodistal direction. During migration, vagal crest cells followed different pathways depending on the region of the gut being colonised. In the pre-umbilical intestine, NCC were evenly distributed throughout the splanchnopleural mesenchyme while, in the post-umbilical intestine, they occurred adjacent to the serosal epithelium. Behind this migration front, NCC became organised into the presumptive Auerbach's and Meissner's plexuses situated on either side of the developing circular muscle layer. The colorectum was found to be colonised in a complex manner. Vagal NCC initially migrated within the submucosa, internal to the circular muscle layer, before migrating outwards, adjacent to blood vessels, towards the myenteric plexus region. In contrast, sacral NCC, which also formed the entire nerve of Remak, were primarily located in the presumptive myenteric plexus region and subsequently migrated inwards towards the submucosal ganglia. Although present throughout the post-umbilical gut, sacral NCC were most numerous in the distal colorectum where they constituted up to 17% of enteric neurons, as identified by double antibody labelling using the quail-cell-specific marker, QCPN and the neuron-specific marker, ANNA-1. Sacral NCC were also immunopositive for the glial-specific antibody, GFAP, thus demonstrating that this region of the neural crest contributes neurons and glia to the enteric nervous system.

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