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Keywords:

  • hirschsprung disease;
  • intestinal dysmotility;
  • enteric neuropathy;
  • pseudo-obstruction;
  • submucosal plexus

Abstract

  1. Top of page
  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References

Normal intestinal motility requires orderly development of the complex nerve plexuses and smooth muscular layers in the gut wall. Organization of these structures results, in part, from cell autonomous programmes directed by transcription factors, which orchestrate appropriate temporal and spatial expression of specific target genes. Hox proteins appear to function in combination to dictate regional codes that establish major structural landmarks in the gut such as sphincters and muscle layers. These codes are translated in part by intercellular signals, which allow populations of cells in the embryonic gut wall to alter the developmental fate of their neighbours. Some of the best characterized intercellular signalling pathways involved in enteric neurodevelopment are mediated by GDNF/GFRa1/RET, EDN3/ENDRB, and NETRINS/DCC. These signals affect enteric neural precursors as they colonize the gut, and perturbations of these molecules are associated with various types of intestinal neuropathology.


Embryonic patterning of the gut wall by hox genes

  1. Top of page
  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References

Homeobox genes are groups of developmental control genes implicated in the positioning and patterning of organs in the embryo.1 Along the rostro-caudal body axis, including within the gastrointestinal tract, individual Hox genes are generally expressed with discrete rostral limits that coincide with either existing or emergent anatomical landmarks (e.g. caecum). It has therefore been suggested that they serve to specify component parts of the vertebrate body plan and this is particularly clear in segmented structures, such as the branchial arches and the vertebral column.2 Significantly, at least in the axial mesoderm, the rostro-caudal limits of Hox gene expression correlate precisely with their 3′−5′ order within their respective cluster and with the temporal order in which these genes are activated, a phenomenon known as spatial and temporal colinearity.3

It is also clear that non-overtly segmented structures such as limbs or internal organs are also specified by Hox genes and thus Hox genes could be upstream regulatory genes for the morphogenesis of the embryonal gut for the migration and maturation of neural crest cells and, possibly, splanchnic mesoderm. For example, Hox genes from the 5′ paralogous groups, 12 and 13, are expressed in the most caudal splanchnic mesoderm and are involved in patterning of the hindgut.4

In segmented structures such as the branchial arches, the ‘Hox code’ defined by the patterns of combinatorial Hox gene expression has been interpreted as a developmental strategy whereby positional specification made axially within the neural tube is transmitted to the periphery via the migrating neural crest. This process is viewed as integral to the mechanisms whereby the embryo develops an organ such as a head and face or a gut.5 In an analogous manner, Hox genes from paralogous groups 4 and 5 seem to be particularly good candidates as regulators of enteric neuromusculature as they are expressed in the developing hindbrain, at the level of rhombomeres 6–8, from where a proportion of vagal neural crest cells migrate through the branchial arches into the intestine and differentiate into enteric ganglia.4

Expression of 3′Hox genes in the guts of developing mouse embryos shows nested domains along the length of the gut, comparable to that observed in the neural tube and axial mesoderm.6 Within these domains, Hox transcripts are initially present throughout most of the gut mesoderm and are progressively restricted to the developing longitudinal muscle layer as gestation progresses. A few isolated observations of transgenic mouse models suggest that Hox genes do indeed play an important role in gut morphogenesis. Overexpression of Hoxa4 is associated with disorganization of the muscular and neural elements in the gut wall and megacolon.7,8 Disruption of Hoxc4, Hoxa59 and Hoxd1310 severely affect the morphologies of oesophageal smooth muscle, stomach and anal sphincter, respectively, while coincident mutations in Hoxa13 and Hoxd13 cause anal stenosis.11 Mice engineered to lack five members of the HoxD cluster fail to form ileocecal sphincters.12 It is clear that Hox genes are important within the genetic hierarchy of gut morphogenesis and delineation of those genetic or environmental perturbations that comprise the enteric HOX code will be an integral part of understanding the molecular events underlying gut dysmorphogenesis in humans.

The combinatorial activities of Hox genes not only pattern enteric mesoderm, but also have indirect effects on adjacent nonmesodermal components. For example, ectopic expression of Hoxd13 in midgut mesoderm indirectly induces overlying endoderm to adopt hindgut characteristics.13 Similarly, the effect of Hoxa4 overexpression on enteric ganglia in transgenic embryos is thought to be secondary to changes in the microenvironment of neural precursors as they colonize the gut.14 These inductive influences are presumably mediated by intercellular signalling pathways, possibly one or more of those known to regulate enteric neurodevelopment.

Regulation of enteric neurodevelopment by intercellular signalling pathways

  1. Top of page
  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References

The beginning of the enteric nervous system (ENS) is found in the colonization of the gut by émigrés from the neural crest.15 These incoming cells arrive from defined regions of the crest. The vagal crest (somite levels 3–7) provides the bulk of the precursors of enteric neurones and glia and colonizes the whole gut,16–19 but the postumbilical bowel also receives a later-arriving contribution from the sacral crest,20 and the rostral foregut (primordial oesophagus and adjacent stomach) receives cells from the nearby truncal crest.21 Crest-derived cells probably do not migrate as a uniform array of committed and uncommitted precursors, but appear to constitute a heterogeneous population that changes progressively as a function of developmental stage, both as the cells migrate and after they arrive in the target bowel.22–24 Along their route of travel the crest-derived precursors have ample opportunity to interact with microenvironmental signalling factors, which include growth factors and elements of extracellular matrix that irreversibly change the precursors and contribute to the determination of their fates. Two intercellular signalling pathways that are absolutely necessary for complete colonization of the gut by neural precursors are those mediated by RET and endothelin receptor B (EDNRB).

The GDNF/GFRα1/RET-mediated pathway

  1. Top of page
  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References

RET is the signalling component of multisubunit receptor complexes for the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs), which include four distant members of the transforming growth factor TGF-β superfamily: GDNF, neurturin, artemin and persephin.25 Interactions between each of the known GFLs and RET are mediated by one of four GPI-linked cell surface glycoproteins, GFRα1–4. GDNF signals preferentially through GFRα1.26 Heterozygous loss-of-function mutations of RET in humans are a cause of Hirschsprung disease (HSCR), which is characterized by the absence of enteric ganglia from the distal colon, with concomitant impairment of intestinal motility and congenital megacolon.27 Similarly, mice homozygous for null mutations of ret have aganglionosis of the entire small and large intestines.28 As expected, null mutations in gfrα1 or gdnf produce enteric phenotypes identical to that of RET-deficient animals.29,30

Gdnf is expressed by non-neural crest cells in distinct centres in advance of the progressive ‘wavefront’ of enteric neural progenitors.31,32 It appears that GDNF activation of RET through GFRa1 has both chemoattractive, mitogenic and possibly cytotrophic effects on enteric neural precursors. Apoptosis of neural crest cells is observed between the neural tube and foregut of ret -/- embryos21 and exogenous GDNF promotes the survival and replication of isolated neural progenitors in vitro.33 Enteric neural crest cells, as opposed to other Ret-expressing neural crest derivatives, migrate preferentially toward a source of GDNF in vitro.32 Impairment of any, or all, of these cellular responses to GDNF is presumably the pathogenesis of aganglionosis in ret-, gdnf- and gfra1-null embryos. The same signalling molecules may play more subtle roles in enteric neurobiology, given that heterozygous ret and gfra1 mutations produce motility defects, but no major neuroanatomical alterations.34

Despite clear evidence implicating the RET signalling pathways in mammalian ENS development, the cellular targets of the receptor and the intracellular mechanisms that potentially integrate its activity are largely unknown. RET encodes two major isoforms, RET9 and RET51, which are generated by alternative splicing and differ only at their C-terminal tails; RET9 has a 9-amino acid tail, which in RET51 is replaced by an unrelated sequence of 51 amino acids. To address functional differences between the two RET isoforms, de Graaff et al.35 used homologous recombination in embryonic stem cells to replace the wild-type ret with alleles that encode only a single RET isoform, RET9 or RET51. The study of mice that derived from these embryonic stem cells showed that RET9 is sufficient to support normal embryogenesis and postnatal life. However, mice that expressed only RET51 had severe defects in gut innervation, suggesting that the C-terminal sequences specific to RET9 and RET51 are critical for their developmental functions. Consistent with this idea, it has been shown recently that RET9 and RET51 have different patterns of autophosphorylation and associate with different complexes of proteins.36

The EDN3/EDNRB-mediated pathway

  1. Top of page
  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References

Endothelin receptor B (EDNRB) is a heptihelical, G-protein-coupled receptor that resides in the plasma membrane of enteric neural and melanocytic precursors.37 ENDRB can be activated by any of the three established endothelins (EDN1, EDN2 or EDN3), but only EDN3 is known to be required for normal enteric neurodevelopment.38 EDN3 is produced by mesenchymal cells adjacent to neural crest cells as they colonize the gut and skin, and is expressed at particularly high levels in the ileocecum.39,40 The mature EDN3 peptide is 21 amino acids, which derive from a much longer preprotein that is sequentially cleaved, first by ubiquitous furin enzyme and secondarily by one of two ‘endothelin converting enzymes’.

Deficiency of EDNRB, EDN3 or endothelin converting enzyme-1 causes aganglionosis of the terminal colon and cutaneous pigment defects (piebaldism).41 The embryological roles of this intercellular signalling pathway are conserved, as similar phenotypes are associated with genetic or pharmacological perturbation of EDNRB-mediated signals in a variety of vertebrates. Mutations in EDNRB or EDN3 are estimated to account for 5–10% of cases of sporadic HSCR in humans, and are responsible for high rates of HSCR observed in some genetically isolated populations.27 In many HSCR patients and most animal models of the disorder, mutations that affect the ENDRB pathway also produce deafness and piebaldism. This trio of clinical findings (HSCR, deafness, piebaldism) is known as the Waardenburg–Shah syndrome. Another rare condition associated with HSCR and mutations in the same genes is congenital central hypoventilation (‘Ondine's curse'), although respiratory defects have not been reported in animal models.42

In vitro studies of cell cultures of enteric neural precursors suggest that EDNRB activation inhibits differentiation.33,43 A similar phenomenon is believed to occur in vivo to maintain a critical mass of mitotically active crest cells, which is required to colonize the entire length of the large intestine. In mouse embryos, EDNRB-mediated signals are necessary in the finite time period, when neural crest cells transit the small intestine and enter the proximal large intestine.44,45 In the absence of EDNRB or EDN3, colonization of the small intestine is slightly retarded and spread of neural precursors from the distal ileum into the caecum is severely impaired. The nature of the apparent impediment to colonization of the cecum is incompletely understood, but coincides with the location of maximal EDN3 expression in the embryonic gut.40

Most published data are consistent with a simple paracrine model in which EDN3 produced by gut mesenchyme binds to EDNRB on enteric neural crest cells to directly modulate cellular behaviour. However, an alternative or complementary mechanism has been proposed based on the results of in vitro and in vivo studies that show EDNRB mRNA is expressed by non-neural crest cells in the gut wall, the latter cells produce excess laminin when EDN3 is deficient and laminin stimulates neural differentiation in vitro.43 These support the hypothesis that a non-neural crest, laminin-mediated, indirect effect of EDN3/EDNRB signalling promotes premature differentiation of neural precursors. Although the ‘indirect signalling pathway’ has not been disproven its relative importance is questionable, given that a reporter gene inserted into the ednrb locus is only expressed in enteric neural precursors, not surrounding mesoderm, and that transgenic expression of EDNRB selectively in neural precursors is sufficient for complete colonization of the gut.46,47

One of the most exciting areas of ongoing investigation concerns potential genetic and molecular interactions between the GDNF/RET/GFRα1 and EDN3/ENDRB pathways. Parallel studies of human and murine populations at risk for aganglionosis have demonstrated clearly that coexistent alterations in genes from both pathways confer a significantly greater risk of enteric aganglionosis than associated with the same genetic alterations in isolation.48,49 For example, ret± heterozygotes do not exhibit aganglionosis, in contrast with ret -/- homozygotes, which lack ganglion cells throughout their entire intestines. Similarly, ednrbsl/slmice have ganglion cells throughout their gastrointestinal tracts. However, aganglionosis is observed in 100% of ret ± : ednrbsl/sl, indicating that mutations at one locus will modify the phenotype associated with mutations at the other locus. At present, the molecular and cellular events that underlie this genetic interaction are unknown. However, the observation raises the intriguing possibility that the RET and EDNRB pathways may converge at some level in neural progenitors.

The NETRIN/DCC-mediated pathway

  1. Top of page
  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References

In addition to the acquisition of correct phenotypes, the neurones and glia of the ENS must also assume correct locations within the bowel. Not a great deal is known about how the crest-derived precursor find the gut as they migrate through the developing embryo; however, there is evidence that crest-derived cells migrate along specific pathways that overlap initially with neural crest cells destined for other sites.15,50 Within the gut, the predominant direction of migration for the big population of émigrés from the vagal crest has to be in the proximo-distal direction to accomplish colonization. For the most part, crest-derived cells migrate in the outer gut mesenchyme. In avian embryos, there is evidence that this stream of migrants divides when it reaches the caecum and colorectum,20,51 so that one stream passes near the mucosa while the other remains peripheral. In mammals, no such division has been seen and the cells migrate in the hindgut, as they do in the foregut and midgut, in the outer mesenchyme of the bowel.52 This position raises the question of how certain cells of the population, at the correct moments in time, are induced to take critical perpendicular turns in their migration and enter the submucosa to give rise to the submucosal plexus and upon passing the pancreatic diverticulae, to enter them and colonize the pancreas, give rise to pancreatic ganglia.

Evidence is strong that the crest-derived cells that form the submucosal plexus and pancreas both make their critical perpendicular moves in response to the influence of netrins secreted, respectively, by the gastrointestinal mucosa and primordial pancreatic acinar cells.53 These cells, in both mammals and avians, express netrins from early in development. Crest-derived cells, moreover, express a number of potential netrin receptors, including deleted in colorectal cancer (DCC), which is sharply regulated during development. The expression of DCC by enteric crest-derived cells is maximal when they make their turn to colonize the submucosal plexus and when others enter the pancreas. Physiological studies carried out in vitro establish that the DCC-expressing crest-derived cells are attracted to sources of netrins and that this attraction can be abolished by antibodies to DCC or by drugs that interfere with signal transduction for DCC receptors. Transgenic mice that lack DCC lack both a submucosal plexus in the bowel and ganglia in the pancreas. It thus appears likely that netrins interact with DCC expressed by subsets of crest-derived precursors and guide these cells to the submucosa and pancreas.

Conclusions

  1. Top of page
  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References

Development of the neuromuscular tissues in the gut is a complex process that spans much of embryogenesis and continues in fetal and early postnatal life. Important early patterns of regional differentiation in the gut wall are dictated by transcription factors, including HOX proteins, and intercellular signalling systems. Perturbations of these systems produce gross or subtle anatomic and/or physiological alterations in the gut wall, some of which are recognized forms of intestinal dysmotility in humans. Ongoing research into the developmental events that are controlled by some of the molecules discussed in this review is likely to improve our understanding of these conditions and hopefully will lead to better diagnosis and treatment of affected individuals.

References

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  2. Abstract
  3. Embryonic patterning of the gut wall by hox genes
  4. Regulation of enteric neurodevelopment by intercellular signalling pathways
  5. The GDNF/GFRα1/RET-mediated pathway
  6. The EDN3/EDNRB-mediated pathway
  7. The NETRIN/DCC-mediated pathway
  8. Conclusions
  9. Acknowledgment
  10. References
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