Once enteric NC-derived cells begin to colonize the gut, they differentiate into glial cells and diverse subsets of neurones that differ in their axon projection patterns, dendritic morphology, soma size and complements of neurotransmitters, ion channels and receptors. All enteric neurones express pan-neuronal proteins (e.g. neurofilaments, Hu antigens, SCG10, PGP9.5), and different subsets of neurones express particular type-specific markers such as neurotransmitter synthetic enzymes. During the differentiation of enteric neurones, panneuronal markers seem to be expressed prior to any of the neurotransmitter synthetic enzymes that present in mature enteric neurones. Some vagal NCC begin to express pan-neuronal markers prior to their entry into the foregut,10 and the proportion of crest-derived cells expressing pan-neuronal markers then increases after their entry into the gut (Fig. 2).11 Various types of enteric neurones and glia are generated at different developmental stages12,13 and are produced in precise ratios with respect to one another. The first neurone type-specific marker expressed by subpopulations of enteric crest-derived cells is nitric oxide synthase (NOS) [the synthetic enzyme for nitric oxide (NO)], which in embryonic mice is expressed over 2 days after the arrival of vagal crest-derived cells into the gut.14 5-HT neurones also develop early, but cholinergic and CGRP neurones develop later and are not present in significant numbers until late embryonic and early newborn stages.
What regulates the generation of different types of enteric neurones and glial cells? In all parts of the nervous system investigated to date, the generation of the correct types of neurones in the appropriate location (called ‘cell fate determination’) is regulated by a combination of intrinsic and extrinsic cues. Intrinsic cues that influence neural fate determination include: (i) the expression of combinations of transcription factors that specify particular neurone types or glial cells; (ii) the expression of cell surface receptors that enable some precursors, but not others, to respond to environmental signals; and (iii) asymmetric cell division in which cytoplasmic cell fate determinants are segregated during mitosis and inherited by only one daughter cell. Extrinsic cues can be in the form of diffusible molecules, cell surface proteins or molecules in the extracellular matrix. Thus far, the only intrinsic factors identified that are associated with cell fate determination of enteric neural crest-derived cells are the transcription factors, Mash1 and Sox10. Mash1 appears to be involved in the development of enteric 5-HT neurones,15 and Sox10 is required for enteric glial cell differentiation.16 Transplantation and in vitro studies have shown that molecules within the gut environment can influence the types of neurones that develop from NC-derived cells.17,18 GDNF, neurotrophin-3 and BMP2, which are expressed by the gut mesenchyme, promote the differentiation of NCC isolated from the gut6,19,20 but it is unknown whether they promote the differentiation of specific classes of enteric neurones. Neurturin, a member of the GDNF family of neurotrophic factors, is also expressed by the gut mesenchyme, and appears to play a role in the development of cholinergic neurones.21,22 As different types of enteric neurones develop at different stages, it is possible that environmental cues within the gut change over time, and/or that the intrinsic cues expressed by enteric neural progenitors vary with developmental age, so that the cells are only competent to differentiate into a particular cell type at a particular stage of development. Recent data suggest that, like the CNS, not all NCC within the gut differentiate during development, and that a small pool of enteric neural crest stem cells persist in adult gut.23 Should such stem cells, or cells from other locations with similar developmental potency, be isolated and manipulated, they may have therapeutic potential in addressing conditions where there is a lack, or reduction in number, of enteric neurones.