The ENS cells are derived from precursor cells from three axial levels of the neural crest.9 These include the vagal,10 rostral-truncal,11 and lumbo-sacral10,12 levels. The enteric neurones mainly arise from the vagal neural crest of the developing hindbrain and colonize the gut by migration in a rostro-caudal direction. Vagal crest cells are not restricted to a particular intestinal region. Some enteric neurones arrive in the hindgut from the lumbosacral level via a caudo-rostral wave of colonization. In rare cases, the migrating cells do not reach the entire gut; usually this affects the terminal portion of the bowel, as in classical forms of Hirschsprung’s disease. Even more rarely, there may be a zonal form of Hirschsprung’s disease (Fig. 2), which is postulated to result from incomplete caudo-rostral migration of neuroblasts during embyonal development.13 The neural crest cells that migrate and colonize the gut become neuroblasts or neuronal support cells, glioblasts. However, differentiation into neurones and glial cells seems not to take place until they have reached their final destinations in the gut. Movement through the gut mesenchyme, survival in the gut and differentiation into mature cells are influenced by contacts of precursor cells with the microenvironment.
Figure 2. Zonal adult Hirschsprung’s disease in an adult. Narrowed segment of the sigmoid colon on barium enema and gross pathological examination. Reproduced from Fu et al., Gut 1996; 39: 765–7.
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The microenvironment consists of other cells in the mesenchyme, neural crest-derived cells, and the extracellular matrix. The extracellular matrix components provide directional signals to migrating neural crest cells and, together with neighbouring cells, provide signals for neural crest cell differentiation. For example, the appearance of neural crest cells in the gut is preceded by expression of extracellular matrix molecules,14 and other factors, such as glial derived neurotropic factor (GDNF), ensure survival of committed neuroblasts.15
A subpopulation of sacral neural crest cells appears predetermined to function in the hindgut. They do not require the presence of vagal-derived enteric precursors in order to colonize the hindgut, nor are they capable of dramatically altering their proliferation or differentiation.16 On the other hand, the environment at the sacral level allows neural crest cells from other levels of the axial region of the developing nervous system to enter the mesentery and gut mesenchyme. At least two environmental conditions at the sacral level enhance ventral migration of the sacral neural crest cells. Firstly, sacral neural crest cells take a ventral rather than a medial-to-lateral path through the somites and arrive near the gut mesenchyme many hours earlier than their counterparts at the thoracic level. There is only a narrow window of opportunity to invade the mesenchyme of the mesentery and the gut, therefore, their earlier arrival assures the sacral neural crest cells of gaining access to the gut. Secondly, the gut endoderm is more dorsally situated (i.e. closer to the crest with its migrating cells) at the sacral level than at the thoracic level. As a result, sacral neural crest cells preferentially populate the colo-rectum. In addition, a barrier to migration at the thoracic level prevents neural crest cells at that axial level from migrating to the gut.17 The barrier also prevents lumbo-sacral crest neurones and glial cells from migrating to the nearby small intestinal tissue.18
Defects of the neural crest cells themselves or alterations of the microenvironment of the pathway through which the neural crest cells migrate may result in maldevelopment of the ENS. In humans, this disordered development results in congenital enteric neuromuscular diseases.
Differentiation of neurones
Migrating crest cells are multipotent.19 The gut wall is itself a critical site where terminal differentiation of enteric neurones and glia occurs, and determines what kind of nervous system arises within the bowel.20 Enteric growth factor–receptor combinations influence differentiation. Combinations that enhance differentiation include: glial cell line-derived neurotrophic factor (GFR)-1-Ret, neurotrophin-3 (NT-3)-TrkC, and serotonin (5-HT)-2B. ret is a proto-oncogene that encodes for a tyrosine kinase receptor necessary for the development of the ENS. Because ret is a proto-oncogene, a single ‘activating’ mutation on one allele is sufficient to cause neoplastic transformation. Receptor tyrosine kinases transduce diverse processes, including cell growth, cell differentiation, survival and programmed cell death (apoptosis). A receptor tyrosine kinase is a 28-amino-acid single peptide that has an extracellular domain (rich in cadherin and cysteine), a transmembrane domain and an intracellular domain. Binding of ligands (e.g. GDNF, neurotrophins) to the receptor results in its activation leading to auto-phosphorylation of tyrosine residues and signal transduction. Figure 3 shows examples of mutations in the tyrosine kinase receptor that are associated with specific genetic disorders.
Figure 3. Tyrosine kinase receptor: genetic disorders and dysmotility. Tyrosine kinase receptor with examples of mutations associated with specific genetic isorders. ([F]MTC , [familial] medullary carcinoma of the thyroid; MEN,multiple endocrine neoplasia). Drawn from Edery et al., Nature 1994; 367: 378–80, and Romeo et al., Nature 2000; 367: 377–8.
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A qualitatively different effect on enteric nervous system development is shown by the peptide–receptor combination of endothelin-3 and the endothelin B receptor, ET-3/ETB, which prevents the premature differentiation of enteric neurones before colonization of the GI tract has been completed.
The first molecule found to affect the development of enteric neurones and glia was neurotrophin-3 (NT-3).21 Crest-derived cells in the fetal bowel express TrkC, the high-affinity receptor for NT-3. Overexpression of NT-3 in transgenic mice causes an increase in the size of developing ganglia and neurones in the myenteric plexus.20 The ENS, however, is relatively normal in the bowel of mice following the knockout of NT-3,22 suggesting that NT-3 affects the development of only a subset of enteric neurones or glia.19,23
Stimulation by glial cell line-derived neurotrophic factor (GDNF) is absolutely necessary for the survival of the vagal and sacral crest-derived cells that colonize the gut. If either GDNF24,25 or its signalling receptor, Ret,26 are knocked out in developing mice, the gut becomes totally aganglionic in the vagal and sacral domains of the bowel and persists only in the small region of the gut that is colonized by cells from the truncal crest. GDNF potently promotes neuronal development in vitro27,28 and acts as a mitogen in early development,27 greatly expanding the numbers of enteric crest-derived neural precursors. The initial GDNF-dependent crest-derived precursor that colonizes the bowel gives rise to multiple cell lineages that require particular growth or transcription factors. For example, the truncal crest depends on GDNF, not on Ret,11 but it also needs Mash-1. From this GDNF- and Mash-1-dependent lineage, all of the serotonergic neurones (which develop early in ontogeny), and many excitatory and inhibitory motor neurones, will develop. Later, GDNF loses its ability to promote proliferation, and it acts only as a growth-differentiation factor for enteric neurones, not for glia. All enteric neurones that contain CGRP are derived from Mash-1-independent neurones and differentiate late in ontogeny, after the last serotonergic neurone has become postmitotic (i.e. terminally differentiated).
Both crest- and noncrest-derived cells of the enteric mesenchyme also contain GFR-1,27 a peripheral glycosylphospho-inositol-anchored molecule, which binds GDNF and is necessary for the activation of Ret.29 Neural crest cells anchor GFR-1 to their plasma membranes (perhaps in a complex with Ret), where GDNF can bind to it and ensure survival of the crest-derived cells that colonize most of the bowel.20
Enteric serotonergic neurones appear so early that they coexist in primordial enteric ganglia with still-dividing neural precursors. 5-HT may not only be a neurotransmitter; the 5-HT2B receptor in the fetal bowel is regulated, and optimal at specific times, thus, 5-HT strongly promotes development of neurones at specific times and affects the development of late-arising enteric neurones.30
These growth-differentiation factors affect virtually the entire bowel. The peptide ET-3, and its receptor, ETB, play a critical, localized role in ENS development.31,32