Embryology and development of the enteric nervous system.

Origin and migration of neural crest derived cells in the gut The neurones and glial cells of the enteric nervous system (ENS) are derived from the neural crest (fig 1). While they are migrating, neural crest cells are morphologically indistinguishable from mesenchymal cells through which they migrate, and thus a variety of experimental approaches have been used to examine colonisation of the gut by neural crest derived cells. Yntema and Hammond (1954) ablated defined rostrocaudal regions of the neural crest of chicken embryos and examined the eVects of the ablations on the ENS. They found that ablation of vagal level (somites 1–7) neural crest resulted in absence of enteric neurones throughout the gut, and therefore concluded that vagal level neural crest is the sole source of the ENS. Following transplantation of vagal level neural tubes from quail embryos into chicken embryos, in which the equivalent region of neural tube had been removed, quail cells were found throughout the gut of the chimera. This also supported the idea that vagal level neural crest cells give rise to enteric neurones throughout the gut. However, when the quail neural tube was transplanted into the sacral level (caudal to somite 28) of chicken embryos, quail cells were found within the myenteric plexus of the hindgut of the chimeras, indicating that sacral level neural crest cells also contribute to the ENS in the hindgut. Since then, the contribution of sacral level neural crest to the ENS in the hindgut has been controversial. Studies in which explants of chicken gut were removed and grown on the chorioallantoic membrane of host embryos, or in which the midgut of chicken embryos was transected prior to the arrival of vagal neural crest cells, suggested that either the vagal neural crest cells are the sole source of enteric neurones throughout the gut or that sacral level neural crest cells do not give rise to enteric neurones in the hindgut unless the vagal level neural crest cells are present. In contrast, studies in which pre-migratory cells had been labelled with a lipophilic dye or retroviruses have shown that some cells that populate the hindgut arise from sacral level neural crest, and these cells colonise the hindgut well before the arrival of vagal level neural crest cells. 6 While they are migrating through the gut, neural crest derived cells do not express a neuronal or glial cell phenotype. Recently, a number of markers of neural crest derived cells within the mouse gut have been identified, and thus colonisation of the mouse gut by neural crest cells can be observed directly. These markers include the transgene DBH-nlacZ, receptors for neurotrophic factors including p75, 9 RET, GFRá1, 12 and endothelin receptor B, and the transcription factors MASH1, Phox2a, Phox2b, and SOX10. 18 We have examined colonisation of the embryonic mouse gut using antibodies to Phox2b, p75, and RET. Most of the above markers of neural crest derived cells within the gut are downregulated during development. However, expression of Phox2b is maintained in adult mice and all diVerentiated enteric neurones show Phox2b immunoreactivity. Thus it would seem likely that Phox2b is expressed by all enteric neurone precursors. Double label studies revealed that Phox2b, p75, and RET are expressed by identical populations of neural crest derived cells in embryonic day 12–14 (E12–E14) mice, and it appears likely that these three molecules are expressed by all migratory, or immediate post-migratory, neural crest cells in the gut. We mapped the appearance of Phox2b, p75, and RET immunoreactive cells in the embryonic mouse gut. At E9.5-E10, labelled cells were present only in the stomach, and during subsequent development Phox2b, p75, and RET positive cells appeared as a unidirectional, rostral to caudal wave along the gastrointestinal tract; the entire gut was colo-