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The development of the enteric nervous system and its role in Hirschsprung’s disease

The enteric nervous system is the independently operating part of the PNS that is responsible for coordinating the complex and often multifaceted behaviours of the gut. The ENS acts as a monitoring device, ensuring a steady state in the lumen and walls of the gut, responding to appropriate stimuli and initiating responses that lead to peristaltic movements and altered blood flow and electrolyte secretions. The ganglia present in the ENS contain distinct neuronal and glial subtypes, the quantity of which is a close match to the number of neurones found in the spinal cord.



Figure 1 | Illustration portraying the organisation of the enteric neurons in ganglia as seen through a transverse section of the small intestine. The outer myenteric plexus is formed first and is situated in between the longitudinal and circular muscle layers. Later in gestation the submucosa harbours the inner submucosal plexus (Adapted from Furness & Costa 1980)

The connections between the enteric ganglia give rise to two plexuses that occupy various lengths of the bowel. These are the outer myenteric and inner submucosal plexus (FIG. 1). The former spans the entire length of the gut and the latter is predominantly found in the large and small intestines. Normal ENS function is dependent on a synchronised interplay between various neurons present in the ganglion of both plexuses. Furthermore, to ensure harmony in ENS regulation of smooth muscle movement in the gut, the actions of the enteric neurons must be in concert with other cells type resident in the organ, including the interstitial cells of Cajal, which are responsible for providing pacemaker activity to the gut wall and facilitate the interactions between motor neurones and smooth muscle cells.

For decades human geneticists and developmental biologists have aspired to understand normal human biology as a way of gaining better insight into disease states. The ENS can serve as an exemplary system for such endeavours, whereby understanding the cellular and molecular events involved in normal ENS development can enable researched to investigate and explore novel therapeutic avenues, which could lead to therapies for treating disorders of human ENS. In addition, recent advances in stem cell research has meant that such studies can potentially utilise the promise of stem cell therapies for countering human genetic disease such as Hirschsprung’s disease (HSCR).

By using examples from both human and model organisms, here will try to capture the classic principles and recent advances in our understanding of the basic biology of ENS development and highlight the importance of neural crest cells in the formation of this system. We will also attempt to delineate the signalling mechanisms involved in normal ENS function, impairments of which can give rise to the most prevalent congenital condition that affects the ENS, Hirschsprung’s disease.

Neural crest cells and development of the enteric nervous system


Figure 2 | Diagram showing the gene expression, source, and migratory routes of the neural crest cells that give rise to the ENS. Embryonic Day (E) 8.5-9 marks the entry of the vagal neural crest cells (orange dots) into the anterior foregut. Subsequently they start a rostral caudal migration pattern to populate the foregut (FG), midgut (MG), caecum and hindgut (HG) forming a large proportion of the enteric nervous system (red dots). The most caudal vagal neural crest cell which arise from a overlapping region with the anterior trunk neural crest cells contribute a small fraction to the ENS of the oesophagus and the anterior stomach (blue dots). Sacral neural crest cells are the last to begin migrating (E13.5), and make a small contribution to the ENS of the colon (yellow dots). The RET ligand glial cell-lined-derived (GDNF) is highly expressed in the foregut, while the EDNRB ligand endothelin 3 (EDN3) is expressed in the midgut and hindgut (Adapted from Heanue & Pachnis 2007)

The enteric nervous system (ENS) is principally formed from migratory vagal neural crest cells. These cells originate from the embryo’s precursor to the central nervous system (CNS), the neural tube, at the levels of somites 1-7 (FIG. 2). Vagal neural crest cells have different subsets, with each contributing to distinctive regions of the gut. In addition, the migratory anterior trunk neural crest cells (in the posterior vagal region) are also implicated in contributing a small amount to the development of the foregut ENS(Burns et al. 2000; Durbec et al. 1996).

In humans, the 4th week of gestation marks the entry of the neural crest cells into the foregut(Gershon 1997), after which they are termed enteric neural crest-derived cells (ENCCs) and regarded as the progenitors of the enteric nervous system. Soon after entry, ENCCs begin the process of colonising the foregut, midgut and the hindgut, by sequentially migrating in a rostro – caudal direction along the body axis until the process is complete at approximately after 7 weeks gestation (Fu et al. 2003). Furthermore, in the distal midgut and hindgut a small fraction of enteric neurons and glia are formed from the migration of sacral neural crest cells at the later stages of neural crest cell migration (Burns & Le Douarin 1998).

Studies focusing on mouse embryos have meticulously analysed the migration of vagal and sacral neural crest cells within the gut. The use of fluorescent proteins for genetically labeling ENCCs has enabled researchers to witness the migration pattern of these populations of cells using time-lapse fluorescence video microscopy. In these observations, cells leading the migratory stream of vagal neural crest cells (the ‘wavefront’) tend to travel in an interconnected network of cells in the shape of chains or strands, while extending towards the lower end of the body within the strands(Druckenbrod & Epstein 2005; Young et al. n.d.). In addition, a second group of isolated cells, known as ‘advance cells’, are seen to travel in front of the wavefront (Young et al. n.d.).

The arrival of ENCCs in the caecum is marked by a prominent pause in migration, lasting several hours. During this period, the organised strands of cells become fragmented, increasing the number of advance cells, which appear to be more exploratory in their movement, albeit displaying a relatively restricted movement in comparison to the wavefront (Druckenbrod & Epstein 2007). Such specific changes of behavior in this region may be a testament to the idea that the caecum possesses unique properties, acting as a signalling center guiding the migration of cells; this observation highlight the modularity of the developing gut, whereby various parts are believed to contain distinct tissue microenvironments, a topic which will be discussed later on in greater detail. Immediately after leaving the caecum, the cells continue their migration caudally through the hindgut, thus resuming their journey as a network of migratory ENCCs (Druckenbrod & Epstein 2005).

Proliferation in the embryonic ENS

Due to the relatively small pool of progenitors that invade the foregut, there is an essential need for active proliferation of ENCCs to yield the large number of enteric neurons and glia that go on integrating into the adult intestine(Young et al. 2005). There is an equivalent rate of proliferation amongst all regions of ENS, regardless of the relative position that the migratory wavefront assumes(Gianino et al. 2003). At the wavefront, proliferation is necessary for providing progenitor cells that go on to colonise distal gut regions; meanwhile, the progressively expanding intestine means that cells behind the wavefront also have to proliferate to increase ENS cell numbers in order to fully populate the gut(Sidebotham et al. 2002).

Experimental studies in chicks have shown that a deliberate reduction in the pool of pre-migratory vagal neural crest cells results in an incomplete colonization of the gut, leading to aganglionosis of the terminal gut(Burns et al. 2000). On the other hand, similar studies on mutant mice, wherein enteric progenitors have declined in numbers, show that there is a reduction in the quantity of neurons throughout the entire length of the gut, and in certain mutants this leads to aganglionosis of the terminal gut(Stanchina et al. 2006).

The reason as to why such reductions in progenitor numbers leads to such distinctive phenotypes is yet unclear, this may be due to additional effects of the mutated genes on regulation of cell migration. Considering these outcomes, it is clear that the numbers of ENCCs that enter the gut are critical for normal and complete colonization, whilst also highlighting the important of proliferation in development of the ENS(Heanue & Pachnis 2007).

Phenotypically distinct neuronal and glial subtypes are the building blocks of the mature ENS, where in contrast to other part of the PNS, most of the neurons do not receive direct innervation from the central nervous system(Gershon 1997). Variances in function and cell morphology as well as specific molecular markers and patterns of axonal projections allow for distinguishing individual neuronal types within the ENS.

Further investigations have utilized markers of neuronal and glial differential such as pan neuronal protein Hu and brain fatty acid binding protein (B-FABP) respectively to analyse patterns of ENCCs migration and subsequent glial and neuronal differentiation in mouse gut. It is shown that Glial precursors (expressing B-FABP) migrate behind the ENCC wavefront with a significant lag, echoing the fact the neuronal differentiation precedes glial differentiation(Young et al. 2005). Another distinguishing factor in the development of the ENS and CNS is the observation that some of the neuronal and glial precursors in the ENS continue to proliferate while progressively differentiating(Baetge & Gershon 1989); thus further differentiating the development of the two systems.

While some enteric precursors continue proliferating during migration, there are also terminally differentiated neurons that reach their mature form during ENCC migration stages(Pham et al. 1991). Neuropeptide Y is one such neuron, reflecting a niche population of neurons that form during this stage, in contrast to other subpopulation where they’re only developed during postnatal stages, several days after completion of ENCC migration. In addition, terminally differentiated glial cells only appear at or after embryonic day 16.5 (E16.5) in the mouse(Rothman et al. 1986).

The earliest ENS neurons that are detectable have a remarkable patterns of axonal projection, whereby the axons extend caudally, pointing towards the same direction as the vagal ENCC progenitors migration path(Young et al. 2002). This is remarkable since it suggests that there may be a common guidance mechanism that controls certain attributes of both axonal outgrowth and cell migration. Further studies are required to identify underlying mechanisms involved in other projection patterns including rostra and circumferential.

In terms of neuronal distribution throughout the gut, neurons of specific classes tend to assume a relatively even distribution. The underlying cause of this characteristically even allocation is yet ambiguous. In the CNS, neuronal diversity is achieved by specification of a distinct set of neurones in one region and is followed by their subsequent migration towards their functional destination for fulfilling their roles(Xu et al. 2004); although this mechanism doesn’t seem to be responsible for ENS neuronal differentiation. It’s been proposed that the ganglia in the ENS can themselves function as self-regulating controllers of neuronal diversity, this is in part because the generation of functional microcircuits requires the ganglia to posses the correct cohort of distinct neurons. This model highlights the presence and importance of cell-cell communication between the migrating and differentiating ENCCs, whereby in the later stages the microenvironment of the developing ganglia serves as a critical influence on neuronal organisation.

Hirschsprung’s disease



Figure 3 | Summary of the phenotypes observed in the ENS of mouse mutant strains commonly used as models of Hirschsprung’s disease. The diagrams displays the various states of ganglionosis in the gut wherein the red dots depict the density of the ENS within the corresponding regions of the gut (Adapted from Heanue & Pachnis 2007)

The peristaltic activity of the gut wall is dependent on the accurate colonisation of the gut by the appropriate numbers of diverse sets of glial and neuronal subtypes. Amongst the many congenital gut motility disorders identified in humans, HSCR is the most prevalent and most investigated of these abnormalities, affecting 1 in 5000 live births(Scriver 2001; Brooks, Oostra, et al. 2005). The main characteristic of HSCR is the absence of enteric neurones in the terminal regions of the gut, leading to obstruction of the intestine and significant enlargement of the proximal bowel (megacolon).

The extent of aganagligenesis is another criteria, which further divides HSCR into two classes. Short-segment HSCR (S-HSCR) is the most commonly occurring type, which affects the rectum and short segments of the large intestine. On the other hand long-segment HSCR (L-HSCR) disturbs lengthier parts of the large intestine. There are also rare cases whereby there is a total intestinal or colonic aganglionosis. Sporadic cases of HSCR are more common, although it can also be inherited. The mode of inheritance in the familial cases of HSCR is non-Mendelian, suggesting the presence of a multifactorial complex in aetiology of HSCR. Low penetrance, variability in the degree of aganagligenesis and higher ratio of transmission in males (4:1) are characteristics of the familial forms of HSCR(Brooks, Oostra, et al. 2005). Although 70% of HSCR cases occur sporadically, often occurring as a part of a syndrome e accompanied with other congenital abnormalities. In addition, chromosomal abnormalities such as trisomy 21 (Down’s syndrome) are also associated with HSCR in approximately 10% of HSCR cases(Amiel et al. 2008).

The diagnosis of HSCR is usually confined within a 48-hour window after birth, characterised by the infant’s inability to pass stool, often accompanied by vomiting and an abnormally large abdomen. Gut biopsies are required for definitive diagnosis of HSCR via histological investigations, showing the extent of aganglionosis (FIG. 3). Surgical interventions are critical for survival and have become a routine therapy for HSCR affected infants. Although such procedures may manifest in a lifetime of gastrointestinal complications(Baillie et al. 1999); therefore there has been increased interest and alternative modes of therapy.

Human geneticist can utilise the high incidence rate of familial HSCR as an opportunity for identifying HSCR associated genes and susceptibility loci. Meanwhile, animal models can prove invaluable in the quest for identifying and manipulating genes that lead to HSCR-like phenotypes. The discovery of mice that exhibit similar phenotypes to HSCR has lead researchers to investigate possible mutations in the corresponding loci in HSCR patients. On the other hand, as new HSCR loci are discovered in humans, there is an increased interest in the development of genetically modified mice models that harbour mutations at corresponding loci, which can enhance our understanding of HSCR’s pathogenesis.

Genetics of Hirschsprung’s disease

GDNF, RET and GFRα1 signalling

Approximately 15-35% of all sporadic cases of HSCR and 50% of familial cases are linked to heterozygous mutation in the RET receptor tyrosine kinase(Scriver 2001). Meanwhile, non-coding RET mutations are thought to affect susceptibility of other cases of HSCR(Griseri et al. 2007). Mouse models lacking the tyrosine kinase receptor Ret exhibit complete intestinal aganglionosis(Schuchardt et al. 1994). Similar experiments in zebrafish show that knockdown of Ret results in a gut devoid of enteric neurons(Shepherd et al. 2004). Ret signalling is activated upon dimerization; this is achieved by formation of a ligand/receptor complex including glial cell line derived neurotrophic factor (GDNF) and glycosylphosphatidylinositol (GPI) anchored coreceptor GDNF family receptor α1 (GFRα1) respectively. Absence or malfunction of either components of this signalling pathway results in phenotypically equivalent aganglionosis in mice and zebrafish. More recently there have been HSCR patients identified with heterozygous mutations in GDNF(Angrist et al. 1996).

ENCC express Ret and Gfrα1 as they enter the gut tube, meanwhile Gdnf expression is detected in the gut mesoderm (FIG 2). Mouse models harbouring mutations in Ret, Gdnf or Gfrα1 exhibit failure in ENCCs colonisation of the caudal parts of their gastrointestinal tract, where all gut structure beyond the oesophagus are compromised. This regional deficiency in ENS is perhaps a mixture of failure in migration and cell apoptosis(Taraviras et al. 1999). GDNF has been discovered to possess chemoattractive properties. In vitro studies suggest that GDNF attracts migrating ENCCs, which are expressing Ret and Gfrα1, and by doing so guides them to their destined region of the developing gut(Young et al. 2001). Further proof for this suggestion come from the observation that when ENCC wavefront approaches the rudimentary parts of the oesophagus, Gdnf is expressed in the stomach at the same time, with the caecum experiencing a rise in Gdnf expression as cells continue migration towards the end parts of the gut (FIG. 2)(Natarajan et al. 2002).

Furthermore, Gdnf heterozygous mouse mutant (Gdnf +/ ) exhibit an estimated 50% decrease in ENCC number(Gianino et al. 2003). This reduction is probably due to a combination of declined ENCC proliferation and delayed ENCC migration. The consensus amongst similar studies is that GDNF-GFRα1-RET signalling has an indispensible role in proliferation, survival and migration of ENCCs. There has also been suggestion about the later functions of GDNF, whereby its thought to be involved in axonal outgrowth(Young et al. 2001).

Biochemical examinations of intracellular signalling pathways downstream of GDNF-GFRα1-RET have shed light on certain aspects of signalling that are critical for normal ENS development(Takahashi 2001). Alternative splicing gives rise to various RET configurations, with RET9 and RET51 being the main isoforms. RET9 and RET51 are distinguished from each other by the differences in their carboxyl (C)-terminal intercellular regions and sequence differences in the multi-docking site (tyrosine 1062) for intracellular adaptor and effector signalling molecules, which go on to activate mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)(Takahashi 2001).

Such variations in sequence can alter the binding properties of adapter proteins associated with RET51 or RET9(Takahashi 2001). Expression of RET9 isoform in absence of RET51 in genetically engineered mice leads to complete colonisation of the gut. Whereas the sole expression of RET51 isoform in similarly engineered mice lead to failure if enteric neurons in colonisation of the distal gut. These studies show that RET9 is sufficient for mediating GDNF-GFRα1-RET signalling. The phenotype observed in mice lacking the RET9 isoform closely mimics the colonic aganglionosis that is characteristically seen in HSCR patients; this is believed to be due to faulty ENCC migration and/or proliferation(Natarajan et al. 2002). Although, some studies have shown that are no extreme ENS phenotypes in mice that express only one of the Ret9 or Ret51 isoforms(Jain et al. 2006). This may partially be due to difference in mice strain and disparity between human RET transgene and chimeric mouse-human Ret transgene, thus possibly affecting the receptors signalling properties(de Graaff et al. 2001).

In a bid to better elucidate RET’s role during ENS development, potential signalling sites within RET have been subjected to mutational analysis in mice. For instance, mice carrying a mutation in the Y1062 multi-docking site of RET9, which leads to its conversion into phenylalanine, show total intestinal aganglionosis. This highlights the important of normal Y1062 signalling for ENS development. Furthermore, a mutation in serine 697 of RET, causing it to change to alanine, leads to absence of enteric neurons in the distal portion of the colon(Jain et al. 2006). Serine 697 of RET is a putative protein kinase A phosphorylation site.

One of the most common RET mutations seen in HSCR patients involves the extracellular cysteine residue 620. Mice that have been genetically engineered to express an equivalent of this mutation (RetC620R) display total intestinal aganglionosis(Plaza-Menacho et al. 2006). Furthermore, heterozygous RetC620R mice display a similar phenotype to Gdnf +/– mice thus exhibiting hypoganglionosis of the gut(Carniti et al. 2006).

The notion of gene dosage doesn’t follow the same correlation in humans and mice when it comes down to ENS phenotype. While heterozygous mutations in RET leads to HSCR in humans(Brooks, Oostra, et al. 2005), the same form of mutation in Ret leads to hypo-ganglionosis in mice with minimal disturbance to their intestinal function. The reasons for these variations are largely speculative; therefore it may be more suitable to use better representing mice models such as Ret51/51, which display total intestinal aganglionosis (FIG. 3) (de Graaff et al. 2001).

NTN and GFRα2 

Neurturin (NTN), artemin and persephin are the other members of the GDNF family of ligands that can activate RET by binding their corresponding GPI-linked co-receptors GFRα2, GFRα3, and GFRα4. With regards to ENS development, NTN and GFRα2 are thought to be involved. This is partly because mice engineered to express mutations at these loci display slowed gut motility, which here is associated with reduction in density of excitatory cholinergic neurones, particularly of their projections(Heuckeroth et al. 1999). A very small proportion of HSCR patients are known to carry mutation in NTN(Doray et al. 1998).


Figure 4 | Signalling through the RET and EDNRB receptors is an important regulatory force in the development of the ENS. The expression of RET receptor tyrosine kinase and Endothelin receptor B (EDNRB) on the enteric neural crest derived cells (ENCCs), highlights the profound effects of GDNF and EDN3 signalling on ENCCs survival, proliferation, migration and differentiation (Adapted from Heanue & Pachnis 2007)

EDN3/EDNRB signalling

Another highly relevant signalling pathway in relation to ENS development is the pathway of endothelin 3 (EDN3) ligand and its G-protein coupled receptor EDNRB (FIG. 4). Approximately 5% of human cases of HSCR are understood to carry heterozygous mutations in EDN3, EDNRB and endothelin converting enzyme ECE1 (responsible for activating EDN3). Such cases are often associated with Waardenburg syndrome, patients of which present with colonic aganglionosis, pigmentation defects and hearing loss (Brooks, Oostra, et al. 2005). Similar defects are observed in mice carrying mutations in Edn3, Ednrt or Ece1 where they also display defects in pigmentation and hearing loss(Amiel et al. 2008). 

In contrast to Ednrb, which is primarily expressed by migrating ENCCs, Edn3 is expressed in the mesoderm of the midgut and hindgut during initial phases of ENCC migration. There are high levels of Edn3 expression in the caecum and proximal colon, a peak that occurs simultaneously with ENCC colonisation of the gut(Yanagisawa et al. 1998). This expression pattern could be an indicator of the involvement of EDN3-EDNRB signalling in regulation of normal ENCC migration. This is consistent with data from Edn3-Ednrb mutant mice where ENCC migration is delayed(Lee et al. 2003). Similar studies have suggested that EDN3-EDNRB signalling is important for sustaining enteric progenitors in a proliferate state, since Edn3 mutant mice exhibit reduced ENCC numbers. In support of this proposition is the ability of Edn3 to inhibit ENCC differentiation (Bondurand et al. 2006), thus further highlighting the ability of EDN3 to maintain ENCCs progenitor state. Therefore, EDN3-EDNRB signalling appears to be an important aspect of various stages of ENS development and similar to GDNF–GFRα1–RET it is also involved in harmonising ENCCs colonisation of the gut.


SOX10 is an SRY related high mobility group (HMG)-box transcription factor, haploinsufficiency of which is associated with HSCR in Waardenburg syndrome(Brooks, Oostra, et al. 2005), and it’s thought to account for less than 5% of standalone cases of HSCR(McCallion et al. 2003). Homozygous mutations in Sox10 in mice and zebrafish exert a similar impairment on neural crest derived cell lineages manifesting in colonic aganglionosis and impaired pigmentation(Herbarth et al. 1998). Neural crest cells begin expressing SOX10 as soon as they leave the neural tube, hence SOX 10 can be used as a marker of ENS progenitors. More evidence of the importance of SOX for maintaining the progenitor state of ENCCs comes from Sox10 mutant mice, which have a significantly smaller pool of ENS progenitors(Paratore et al. 2002). On the other hand, in vitro studies show that overexpression of Sox10 in ENCCs results in inhibition of neuronal and glial differentiation(Bondurand et al. 2006). SOX10 is shown to be important in cell fate specification and glial cell differentiation, most evidently at later stages of ENS development where high levels of SOX10 expression is detected in glial cells(Kelsh 2006).

Other human HSCR loci

Studies focusing on the less investigated HSCR disease loci have discovered additional mutation in transcription factors paired like homeobox 2b (PHOX2b) and zinc finger homeobox 1b (Zfhx1b, also referred to as SIP1)(Benailly et al. 2003). Under normal conditions, migrating ENCCs in mice and zebrafish express Phoxb2; however, the absence of this expression manifests in aganglionosis in both species because of ENCC’s failure to colonise the gut(Elworthy et al. 2005). On the other hand, both pre-migratory and migratory neural crest cells express Zfhx1b. Vagal neural crest cell precursors are non-existent in Mice with mutation in Zfhx1b(Brooks, Bertoli-Avella, et al. 2005).

Signalling pathway interactions


The intricate interconnections of pathways involved in the development of the ENS have captured the interests of researchers and clinicians alike. The discovery of associations between EDNRB mutations and specific RET allele in HSCR patient using genome wise studies suggests the presence of a genetic link between these two loci(Carrasquillo et al. 2002). Animal studies show that mice carrying both the recessive hypomorphic allele of Ednrb and heterozygous allele of Ret null mutation (Ret+/–) display total intestinal aganglionosis, a phenotype that is rarely seen in mice which carry only one of the Ednrb or Ret mutations(Barlow et al. 2003) (FIG. 3).

The combination of these studies, all hint at the presence of genetic interactions between GDNF–GFRα1–RET and EDN3–EDNRB signaling pathways (FIG. 4). EDN3 and GDNF have been both implicated in proliferation of ENS progenitors(Kruger et al. 2003). This relationship appears to be of an antagonistic nature, whereby each has a different role in the chemoattraction of ENCCs(Barlow et al. 2003). Remarkably, PKA inhibitors can replicate the effects of EDN3 on ENCC chemoattraction and proliferation, this hints to an integrative role for PKA in EDN3–EDNRB and GDNF–GFRα1–RET signalling(Barlow et al. 2003). PKA’s importance in ENCC migration is further highlighted by the observation that mutations of the putative PKA phosphorylation site of RET, serine 697 leads to phenotypes of aganglionosis(Barlow et al. 2003).

On the other hand, the underlying molecular mechanisms of other ENCC culture results have proven more difficult to elucidate. For instance, migratory ENCCs are encouraged by GDNF to proliferate and differentiate(Hearn et al. 1998). Such findings have lead to a model being devised whereby EDN3 acts as a balancer of GDNF effects, thus preventing precocious ENCC differentiation, this allows ENCCs to maintain a proliferative state so that an appropriate number is reached for complete colonisation of the gut(Wu et al. 1999). Thus according to this model, in absence of Edn3, GDNF results in premature ENNC differentiation and consequently a small pol of progenitor cells(Hearn et al. 1998). Although, the exact mechanisms of how such integrations between RET and EDNRB signalling results in influences that regulate migration, proliferation and differentiation of ENCCs is a topic that necessitates further research.

Interactions between SOX10 and EDN3-EDNRB signalling are currently well documented. However, our understanding of the underlying molecular mechanisms of these interactions is yet negligible. The background dependent variance in severity and penetration of aganglionosis displayed by Sox10 mutant mice is thought to be due to a genetic interaction between Sox 10 and Ednrb loci(Cantrell et al. 2004). Sox10 mutant mice carrying compound mutations of this gene with either of Edn3 or Ednrb shows an exaggerated phenotype of aganglionosis, in comparison to mice carrying only a single mutant allele (FIG. 3). Furthermore, Ednrb enhancer region contains binding sites for SOX10, which is essential for appropriate spatiotemporal Ednrb expression in the ENS(Zhu et al. 2004). Ednrb’s direct regulation by SOX10 could rationalize co-expression of the two genes, but it falls short in clarifying the genetic interactions between Sox10 and Ednrb, there has been no association acknowledged in animal studies between Sox10 and the SOX10 binding sites within the Ednrb enhancer(Cantrell et al. 2004).

Uncovering novel HSCR loci

Despite numerous genetic discoveries concerning HSCR disease loci, these identified genes only account for approximately 55% of familial cases of HSCR and a smaller percentage of sporadic cases. There is accumulating evidence suggesting the HSCR susceptibility may be linked to non-coding mutation at the RET locus (Brooks, Oostra, et al. 2005), that could affect the levels and spatiotemporal attributes of RET expression(Emison et al. 2005). It is most likely that HSCR inheritance is multigenic; non-coding mutations mentioned earlier are additional risk factors that when coupled with various HSCR coding mutations, lead to variable extents of the disease.

HSCR families with multigenerational inheritance of the disease have been subjected to linkage studies. These studies show that different RET mutations can potentially result in different disease phenotypes.

The Old Order Mennonites population have a ten times increased HSCR incidence. This fact has enticed researchers to carry out a genetic evaluation of this population in the hope of discovering new genetic modifiers. Genome wide association studies in these families have shown that they carry missense mutations in EDNRB and that it has associations at 16q23(Carrasquillo et al. 2002). This susceptibility loci may harbour many genes that may lead to increased susceptibility to HSCR, an area that remains to be further explored.

High-resolution genetic analysis can be instrumental in mapping the susceptibly loci to specific genes. In addition, relatively novel techniques such as real time PCR and DNA microarrays can be used in profiling of gene expression in the ENS, whereby gene expressions within a specific locus can be identified and marked as susceptibility loci candidates(Vohra et al. 2006). For instance, many studies have discovered numerous ENS expressed genes that map to chromosome 21, within chromosome 21 lie many ENS related genes, in which dose variations might be the reason for high incidence rate of HSCR in Down’s syndrome.

Mouse and zebra fish models have been invaluable sources of information for HSCR disease loci. There is a new wave of mouse models emerging that display ENS phenotypes. Pax3 mutant mice are one example, which show intestinal aganglionosis in absence of this transcription factor. In addition, deleted in colorectal carcinoma mutant mice (DCC), with impairments in netrin receptor mediated transmission display submucosal ganglia deficit(Jiang et al. 2003). Deficits in either the indian or sonic hedgehog secreted proteins leads to incomplete intestinal aganglionosis in mice; this is often coupled with megacolon (in absence of indian hedgehog) or ectopic ganglia formation (in absence of sonic hedgehog). Mutations in the neurotrophic factor neurotrophin 3 or its receptor, tyrosine kinase C, leads to a reduction in the number of enteric neurones in mice (Bates et al. 2006). Further examples include, mutant mice with defects in Hlx1 transcription factor, which often exhibit hypoganglionosis, whereas in terms of migration defects, mutations in L1 cell adhesion molecule, is linked to defects in ENS migration in mice. Mice that are mutant for Sall4 transcription factor and those that lack the extracellular matrix receptor β1-integrin in the neural crest lineage exhibit colonic aganglionosis(Breau et al. 2006).


Genome-wide phenotype driven screens are a promising frontier for discovering new HSCR loci. Such screens in zebrafish have been instrumental in discovering mutant alleles affecting ENS, particularly information about the number and distribution of the enteric neurons. In addition, the transparency of zebrafish has allowed researchers to easily analyse gut motility. The simplicity of cell organisation and diversity in zebrafish may distance them as a convincing representation of the enteric organisation in mice or humans, however there are various genes that display conservation of function. One example of such a gene is the trap100 (subunit of the TRAP transcriptional regulation complex), mutations of which lead to decrease in number of enteric neurons(Pietsch et al. 2006). Identification of such genes that previously have not been even considered to be associated with ENS development signify the value of genetic screens in paving the way for future discoveries of regulators in development of the ENS.

Cite this article as: Milad Golsharifi, "The development of the enteric nervous system and its role in Hirschsprung’s disease," in Projmed, July 2, 2015,


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