11.1.1

Contribution of Postotic Hindbrain (Vagal) Neural Crest Cells to the ENS

Surgical removal of premigratory neural crest cells from different levels of the neural axis of chick embryos and chick-quail chimera studies showed that the most important source of enteric neurons is “vagal” neural crest cells, which emigrate from the caudal hindbrain adjacent to somites 1–7. Vagal neural crest cells enter the foregut ( Fig. 11.1 ), and then migrate caudally within the gut mesenchyme ( Fig. 11.2 ). In mice, when vagal neural crest-derived cells enter the midgut, the midgut and hindgut are transiently closely apposed, and a subpopulation of cells leaves the midgut, takes a short-cut through the mesentery and enters the colon without passing through the caecum. These “transmesenteric” neural crest-derived cells are then the front-runners to colonize the colon.

Embryonic day (E)9.5 and E10.5 mice following processing for the immunohistochemical localization of Sox10, which is expressed by all neural crest cells. At E9.5, vagal neural crest cells (between dotted lines ) are migrating ventrally (arrow) towards the foregut. In the E10.5 mouse, the ectoderm was removed to reveal the gut. A stream of vagal neural crest cells (arrow) extends along the gut; the most caudal cell (open arrow) is in the midgut. BA1 , branchial arch 1; BA3 , branchial arch 3; OV , otic vesicle.

Vagal ENCCs migrate from rostral-to-caudal along the gut in close association with neurites. (A) Wholemount preparation of small intestine and colon from an E11.5 mouse processed for immunohistochemistry using antibodies against p75, which label all ENCCs. The most caudal vagal ENCC is entering the caecum (open arrow) . (B) Wholemount preparation of the colon from an E12.5 mouse in which RET-expressing ENCCs also express green fluorescent protein (GFP). The most caudal vagal ENCC is now midway along the colon (open arrow) . Sacral neural crest ( nc ) cells are present in the mesentery immediately adjacent to the distal hindgut, but have not yet entered the gut. (C) E12.5 colon in which ENCCs express GFP that has been immunostained with an antibody to neurofilament-M, which labels neurites (red; arrows) . Neurites are in close association with most ENCCs.

In human embryos, it takes around 3 weeks from when vagal enteric neural crest-derived cells (ENCCs) enter the foregut until they reach the anal end of the gastrointestinal tract , and in mice and chicks it takes 5 days.

11.1.2

Contribution of Sacral Neural Crest Cells to the ENS

Sacral level neural crest cells have been shown to give rise to some enteric neurons in mice, chick and quail. In mice, around 12% of all neural crest-derived cells in the distal hindgut are of sacral origin, while in the chick, sacral cells give rise to around 17% of neurons.

Studies in chick have shown that following surgical ablation of vagal neural crest cells, sacral crest cells cannot compensate for their loss, probably due to lower levels of expression of the receptor tyrosine kinase, RET, by sacral crest cells. In both mouse and chick, sacral neural crest cells aggregrate in the connective tissue adjacent to the hindgut, but then undergo a 2–3 day waiting period before they enter the hindgut ( Fig. 11.2 B). Although sacral neural crest cells do not enter the hindgut until after the hindgut has been colonized by vagal neural crest-derived cells, sacral crest cells do not require the presence of vagal neural crest cells to enter the hindgut, and the delay in the entry of sacral cells is due to the expression of inhibitory cues in the hindgut mesenchyme (see Section 11.2.10 ).

11.1.3

Contribution of Schwann Cell Precursors to the ENS

Sensory, visceromotor, and sympathetic neurons project their nerve fibers to the gut after the arrival of vagal ENCCs. Neural crest-derived Schwann cell precursors (SCPs) migrate along these extrinsic nerves (mesenteric nerves) and invade the gut. SCPs are derived from neural crest cells at all axial levels and associate with all nerves that project to peripheral tissues. Most SCPs in the mesenteric nerves of rodents differentiate into non-myelin-forming Schwann cells, which form bundles by surrounding several axons, and ensheath each axon in a pocket of cytoplasm without forming compact myelin. In contrast, enteric glial cells display distinct morphological features: (1) process extension over nerve fibers, (2) a partial sheath to nerve fibers, (3) long laminar processes between nerve processes and wrapping multi-axonal bundles. A fate-tracing study of SCPs revealed that a subset of SCPs generate enteric neurons postnatally, and contribute to a small proportion (< 5%) of submucosal neurons in the small intestine and < 20% of both myenteric and submucosal neurons in the large intestine. Given that SCPs are derived from all neural axial levels, SCP-derived enteric neurogenesis suggests the contribution of trunk neural crest to enteric neurons.

11.1.4

Hirschsprung’s Disease

The embryonic gut grows considerably in length while it is being colonized by ENCCs. For example, in mice, the post-caecal gut increases in length about fivefold between when ENCC first enter the post-caecal gut and when ENCC reach the anal end of the gut at E14.5. ENCC therefore migrate further than any other neural crest cell population. Failure of ENCC to colonize the entire length of the gastrointestinal tract results in distal aganglionosis, which in humans is called Hirschsprung’s disease. Because the ENS is essential for propulsive gut motility, infants with Hirschsprung’s disease suffer from intractable constipation and develop a megacolon, which is treated by surgical removal of the affected bowel region.

11.1.5

Migratory Behavior of Enteric Neural Crest-Derived Cells

After emigrating from the hindbrain neural tube, vagal neural crest cells migrate through paraxial tissues to the foregut. Retinoic acid is produced by paraxial tissues and induces the vagal cells to migrate in chains within the gut mesenchyme. ENCC chains migrate in close association with the neurites of caudally projecting, early enteric neurons.

While some vagal ENCCs must advance caudally towards the distal hindgut, a subpopulation of ENCCs must remain behind in each location to ensure that there are neurons present along the entire bowel. It had been assumed that the ENCCs that get left behind in each gut region had ceased migrating, however, time-lapse imaging studies have shown that the cells that remain behind continue to migrate for at least 24 h, but they migrate circumferentially, rather than caudally.

Sacral neural crest cells enter the hindgut along the axons of extrinsic pelvic plexus neurons and then migrate rostrally within the gut. Schwann cell precursors also enter the gut along the axons of extrinsic neurons.

11.2.1

Proliferation/Cell Number Is Essential for Migration

There are more neurons in the ENS than there are in the spinal cord. The many millions of enteric neurons and glial cells arise from a small population of neural crest-derived cells (estimated to be around 1000 cells) that enter the foregut. Hence, immense proliferation of ENCCs must occur during development of the ENS.

ENCC number plays a vital role in the colonization of the gut. For example, surgical reduction of the number of vagal neural crest cells in chick embryos reduces the distance that ENCCs migrate along the gut and therefore results in aganglionosis of the most distal regions of the gut, similar to Hirschsprung’s disease. Proliferation of ENCCs is essential both en route to the gut and while migrating within the gut. In contrast to neural crest-derived cells in sympathetic and dorsal root ganglia that undergo a short, but rapid, proliferative period, many ENCC remain proliferative throughout embryonic development in mice. The role of cell death in ENS development is discussed later in this chapter ( Section 11.2.6 ).

11.2.2

Effects of Neuronal Differentiation on ENCC Migration

While ENCCs are migrating along the developing gut, a subpopulation starts to differentiate into neurons and expresses pan-neuronal markers. Inhibition of endothelin receptor B (EDNRB) signaling results in both premature neuronal differentiation and delayed migration of ENCCs (see Section 11.2.8 ), which lead to the idea that early or excessive neuronal differentiation of ENCCs directly or indirectly retards their migration. ENCCs expressing pan-neuronal markers continue to migrate, but at a slower rate than the ENCC population. Neuronal differentiation is likely to retard ENCC migration indirectly by reducing the number of proliferating progenitors. However, neuronal differentiation might also promote the colonization of the gut by ENCCs, as time-lapse imaging studies have shown that ENCCs use neurites as a migratory substrate.

11.2.3

Cell/Cell Contact Between ENCCs Influences Migration

Time-lapse imaging studies have shown that ENCCs migrate mostly in chains. A key role for cell-cell contact in ENCC migration is suggested by studies that have shown that reducing ENCC number retards ENCC migration (see Section 11.2.1 ). Inhibition of L1CAM (L1 cell adhesion molecule) signaling or complement anaphylatoxin C3a signaling in gut explants in vitro increases the number of solitary ENCCs and retards the rate of migration. Moreover, when a small number of the most caudal ENCCs were surgically isolated from other ENCCs, they migrated slower, reinforcing the notion that cell-cell contact is important for ENCC migration.

11.2.4

Role of Vascularization in ENCC Migration

In chick embryos and zebrafish larvae, ENCCs migrate in close association with endothelial cells, which already exist as a network in the gut wall. Furthermore, when endothelial cell development is pharmacologically inhibited in chick embryos, ENCC migration is perturbed, suggesting that blood vessels act as a substrate to promote ENCC migration. However, while there is a temporal and spatial correlation between ENCC migration and primary vascularization in the embryonic mouse gut, ENCC migration is unaffected when gut vascular development is genetically impaired. Thus, there might be species differences in the role of vascularization in ENS development.

11.2.5

The Extracellular Matrix (ECM) Influences ENCC Migration

Numerous ECM molecules are present within the developing gut. The principle ECM receptors for ECM molecules are integrins. Neural crest-specific knock out of the β1-integrin, a monomer common to most ECM receptors at embryonic stages, results in slowed and incomplete colonisation of the hindgut in the mouse. In addition, even in the region that is colonised by ENCC, the ganglion size, shape, and geometry are perturbed. This points strongly to the importance of interactions between ENCCs and the ECM in establishing the normal ENS.

A variety of ECM molecules have been shown to influence ENCC migration, and some of these ECM components are produced by the ENCCs themselves. The ECM glycoprotein, tenascin-C, promotes ENCC migration and is produced by both ENCCs as well as other cell types in the developing gut. Collagen VI is also produced by ENCC, but excess collagen VI production is inhibitory to ENCC migration. Thus, production of the correct amounts of ECM molecules by ENCCs and other cell types is essential for normal ENCC migration.

11.2.6

Apoptosis in ENS Development

In some parts of the developing nervous system, up to 50% of young neurons die. Although perturbations to RET signaling result in dramatic increases in cell death in the developing ENS, apoptosis was originally thought to be a relatively rare event during normal ENS development. Later studies demonstrated apoptosis in vagal neural crest cells in chick embryos prior to their entry into the gut, and showed that inhibition of caspase-9 in vagal crest cells resulted in hyperganglionosis. Thus apoptosis does appear to play a role early in normal ENS development by regulating the number of ENS progenitors prior to their entry into the gut.

11.2.7

GDNF Family Ligands/GFRα/RET Signaling

GFLs (GDNF Family Ligands) are a neurotrophic factor family that includes GDNF, neurturin (NRTN), artemin (ARTN), and persephin (PSPN). GFLs bind to the GDNF family receptor alphas (GFRαs), which are GPI-anchored cell surface proteins, and activate the RET tyrosine kinase.

GDNF is expressed in the gut mesenchyme whereas RET and GFRα1 are expressed in immature ENCCs as early as they invade the gut wall. Gdnf -, Gfra 1-null, and Ret -null mice lack enteric ganglia in the gut below the stomach. This phenotype is already evident soon after ENCCs enter the foregut and is likely to be caused by compound deficits in migration, proliferation and survival of ENCCs. Conditional inactivation of RET or GFRα1 after gut colonization by ENCCs causes massive death of ENCCs specifically in the colon, suggesting that GDNF signaling via RET/GFRα1 is essential for the survival of colonic ENCCs.

RET deficiency also affects expression of neuronal nitric oxide synthase (nNOS), which catalyzes the production of nitric oxide, a neurotransmitter involved in the relaxation of gastrointestinal smooth muscle. The exact ligand and GFRα receptor involved in nNOS expression are unclear.

In culture, GDNF acts not only as a neuronal survival factor, but also as a mitogen for immature ENCCs. This mitogenic activity is thought to be a crucial driving force for ENCCs to undergo caudal migration as a whole (see Section 11.2.1 ). In addition to promoting ENCC migration indirectly by promoting proliferation, GDNF probably also promotes the migration of ENCCs directly. .

ENS development is sensitive to the levels of RET signaling. In humans, RET haploinsufficiency can result in Hirschsprung’s disease, while homozygous RET mutations have been associated with total intestinal aganglionosis. In mice, a reduction in Ret gene expression to around one-third of normal levels impairs migration and survival of ENCCs. Gdnf ^+/− mice have hypoganglionosis (a reduction in around 50% in the number of enteric neurons) and abnormal motility, whereas mice with augmented GDNF availability to the ENS have increased numbers of enteric neurons. Negative regulators of RET signaling are also important in the developing ENS. Mice lacking Kinesin superfamily protein 26A (KIF26A) or the tyrosine kinase inhibitor, Sprouty2, have hyperganglionosis (an increase of around 50% in the number of enteric neurons) and motility disorders due to overactivated GDNF-RET signaling. Thus, both under- and overactivation of the RET signaling pathway result in defects in ENS development and consequent motilty disorders.

In summary, GDNF signaling via RET/GFRα receptors is pleiotropic in ENS development, regulating multiple vital processes including migration, proliferation, survival, and differentiation of ENCCs.

NRTN signaling via GFRα2 plays a more limited role in ENS development. The ENS is formed in adult Nrtn – and Gfr α 2 -null mice, but myenteric neurons exhibit reduced cell soma size and fewer excitatory nerve fibers are observed in the myenteric plexus. Nrtn -null mice also display a partial loss of submucosal neurons.

11.2.8

Endothelin-3/EDNRB (Endothelin Receptor Type B) Signaling

Endothelin-3 is produced by the mesenchyme of the developing gut, with the highest expression in the caecum. . Endothelin-3 activates a G protein-coupled molecule, EDNRB, which is expressed by neural crest-derived cells as well as the gut mesenchyme. Mice, rats, and horses that lack EDNRB or endothelin-3, and humans with mutations in the genes encoding these molecules, lack enteric neurons in the most distal regions of the bowel. Endothelin-3 appears to promote the migration of ENCCs both directly, and also indirectly by promoting proliferation and inhibiting neuronal differentation. One mechanism by which endothelin-3 influences ENCC migration is by stimulating cell adhesion. In mice lacking EDNRB, the delay in migration of ENCCs is exacerbated by changes in the hindgut microenvironment with developmental age that make it less permissive to ENCC migration.

11.2.9

Transcriptional Regulation of ENS Development

A network of transcription factors, some of which are common to all peripheral autonomic neurons, regulates ENS development. Here, we briefly discuss the roles of SOX10 and PHOX2b. Other transcription factors that have also been shown to play a role in ENS development are listed in Table 11.1 .

Sox10 is expressed in all premigratory neural crest cells where it plays a key role in inducing migration. SOX10 is also a pivotal transcriptional regulator of ENS development because it is necessary for the induction of both Ret and Phox2b and because it regulates Ednrb expression in cooperation with another transcription factor, ZEB2. Loss of SOX10 in mice results in the death of vagal neural crest cells prior to their entry into the gut. SOX10 is also required at later stages of ENS development for the differentiation of enteric glial cells.

Like Sox10 null mutants, mice lacking PHOX2B do not have enteric neurons in any region of the gastrointestinal tract. There is no RET expression in ENCCs in Phox2b ^−/− mice and hence PHOX2B plays a key role in ENS development because it is required for Ret expression. PHOX2B can also repress Sox10 , which might be important for neuronal differentiation.

11.2.10

Axon/Neuronal Guidance Molecules—Slit, Netrins, Semaphorin 3A

Although some Schwann cell precursors derived from trunk neural crest give rise to enteric neurons during late embryonic development (see Section 11.1.3 ), trunk level neural crest cells do not enter the gut immediately after emigrating from the neural tube. Slits are expressed in the splanchnic mesoderm dorsal to the gut, and Robo receptors are expressed by trunk neural crest cells. It has been proposed that Slit-Robo signaling prevents trunk neural crest cells from entering the gut. However, even when cocultured in direct apposition with explants of embryonic gut, trunk neural crest cells give rise to few enteric neurons, indicating that Slit-Robo signaling is not the only mechanism precluding trunk neural crest cells from colonizing the gut after leaving the neural tube.

In mammals, the first enteric ganglia to develop are in the myenteric region, immediately outside of where the circular muscle layer forms. Submucosal ganglia form later following the centripetal migration of a subpopulation of ENCCs. Netrin is expressed by gut epithelial cells, and its receptor, deleted in colonic cancer (DCC), is expressed by ENCCs. Netrin appears to act as a chemoattractant to induce the centripetal migration of neurons to form submucosal ganglia, as submucosal ganglia do not form in DCC null mutant mice.

Sacral neural crest cells undergo a waiting period prior to entering the distal hindgut (see Section 11.1.2 ). In both chick and mouse embryos, Semaphorin 3A is initially expressed by all mesenchymal of the distal hindgut. The outer mesenchymal cells subsequently downregulate Semaphorin 3A expression, which coincides with the entry of sacral neural crest cells into the hindgut. There is premature entry of extrinsic axons and sacral neural crest cells into the hindgut of mice lacking Semaphorin 3A, confirming that Semaphorin 3A acts as an inhibitory cue to regulate entry of extrinsic axons and sacral neural crest cells into the gut.

11.2.11

Cell Adhesion Molecules—L1CAM

L1CAM is expressed by many ENCCs. Perturbing L1CAM activity retards the migration of ENCCs along gut explants, and there is also a delay in the migration of ENCCs along the gut of L1cam null mutant mice, although the entire length of the gut is eventually colonized. Human infants with mutations in L1CAM have severe CNS defects, and some also have Hirschsprung’s disease. Studies using gut explants have shown that L1CAM is probably important for ENCC migration because it promotes cell-cell contact between ENCCs, particularly close to the migratory wavefront.

11.2.12

Dietary Factors—Vitamin A and Medicines

Retinoic acid, a metabolite of Vitamin A, influences the proliferation and differentiation of ENCCs in vitro. Mice lacking serum retinol-binding-protein (Rbp4), which is required for the transport of hepatic retinol to peripheral tissues, can be depleted of peripheral tissue retinoids by eliminating vitamin A from the diet. Vitamin A depletion of pregnant female Rbp4 ^−/− mice at a stage prior to the entry of ENCCs into the gut of their embryos resulted in a delay in ENCC migration and distal aganglionosis.

The commonly used anti-inflammatory medicine, ibuprofen, reduces the speed of ENCC migration in zebrafish and chick embryos, and in mice genetically predisposed to Hirschsprung disease. Mycophenolate, an immunosuppressant, has also been shown to inhibit ENCC migration in zebrafish and mice. Thus, dietary factors and some drugs might increase the risk of Hirschsprung disease in genetically susceptible infants.

11.2.13

Interactions Between Signaling Pathways

ENS development can be influenced by interactions between signaling pathways. Interactions between RET and EDNRB were first revealed in studies of humans with Hirschsprung’s disease. . Studies in mice verified that interactions between RET and EDNRB influence the severity and penetrance of aganglionosis. . Subsequent studies have reported additional interactions that can influence ENS development; between RET and endothelin-3, SOX10 and EDNRB, SOX10 and SOX8, SOX10 and endothelin-3, SOX10 and L1CAM, EDNRB and L1CAM, endothelin-3 and L1CAM, SOX10 and β1-integrins, PAX3 and CDX, N-cadherin and β 1-integrins, endothelin-3 and β 1-integrins, and SOX10 and ZFHX1b. Some of the interactions are direct, whereas others are indirect. For example, Sox10 directly regulates the expression of Ednrb and L1cam , whereas at least some of the interactions between the RET- and EDNRB-signaling pathways are likely to be due to common downstream signaling molecules, such as protein kinase A.