The timing of enteric neural crest cell colonisation of the chick embryo cloaca.

Neural crest cell (NCC) migration and formation of the enteric nervous system (ENS) is an essential process in the development of the normal human gut. Abnormalities of the ENS lead to a number of neurochristopathies. In avian embryos, the cloaca acts as a common chamber into which gastrointestinal, urinary and genital tracts emerge. Previous studies have elucidated the specific timeframes at which NCCs reach the various regions of the developing chick gut but, to date, none have looked at NCC colonisation of the cloaca. The aim of our study was to investigate the exact timing of the appearance of NCCs in the cloaca of chick embryos. Chicken embryos were harvested on embryonic days (E) 8-12. Whole embryos were fixed, embedded in paraffin and sectioned. Fluorescent immunohistochemistry, using an anti-HNK-1/N-CAM monoclonal antibody, was performed and images were obtained by confocal microscopy. There was no evidence of NCCs in the cloaca of embryos from E8 to E11. Intense immunoreactivity to HNK-1 first appeared in the cloaca of E12 embryos, demonstrating a profuse circumferential colonisation by NCCs at this time. Our study is the first to show the exact timing of enteric NCC colonisation of the chick embryo cloaca. Further studies, involving quail-chick chimeras, are required to establish the true origin of cloacal NCCs and to establish the relationship between NCCs and persistent cloaca.