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1Murdoch Children's Research Institute, Royal Children's Hospital, Parkville, 3052, Victoria, ... Pediatric and Developmental Pathology 5, 224–247, 2002.
Pediatric and Developmental Pathology 5, 224 –247, 2002 DOI: 10.1007/s10024-001-0142-y © 2002 Society for Pediatric Pathology

PERSPECTIVES IN PEDIATRIC PATHOLOGY

Enteric Nervous System: Development and Developmental Disturbances—Part 1 DONALD NEWGREEN1* AND HEATHER M. YOUNG2 1

Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, 3052, Victoria, Australia Department of Anatomy and Cell Biology, University of Melbourne, 3010, Victoria, Australia

2

Received March 1, 2001; accepted August 1, 2001.

ABSTRACT This review, which is presented in two parts, summarizes and synthesizes current views on the genetic, molecular, and cell biological underpinnings of the early embryonic phases of enteric nervous system (ENS) formation and its defects. In the first part, we describe the critical features of two principal abnormalities of ENS development: Hirschsprung’s disease (HSCR) and intestinal neuronal dysplasia type B (INDB) in humans, and the similar abnormalities in animals. These represent the extremes of the diagnostic spectrum: HSCR has agreed and unequivocal diagnostic criteria, whereas the diagnosis and even existence of INDB as a clinical entity is highly controversial. The difficulties in diagnosis and treatment of both these conditions are discussed. We then review the genes now known which, when mutated or deleted, may cause defects of ENS development. Many of these genetic abnormalities in animal models give a phenotype similar or identical to HSCR, and were discovered by studies of humans and of mouse mutants with similar defects. The most important of these genes are those coding for molecules in the GDNF intercellular signaling system, and those coding for molecules in the ET-3 signaling system. However, a range of other genes for different signaling systems and for transcription factors also disturb ENS formation when they are deleted or mutated. In addition, a large proportion of HSCR cases have not been ascribed to the currently known *Corresponding author

genes, suggesting that additional genes for ENS development await discovery. Key words: Hirschsprung’s disease, intestinal neuronal dysplasia type B, aganglionosis, intestinal motility, enteric nervous system, neural crest

INTRODUCTION: STRUCTURE, FUNCTION, AND DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM (ENS) The autonomic nervous system consists of three unequally sized divisions, the sympathetic, the parasympathetic, and the enteric nervous systems (ENS). The ENS is defined as the system of neurons and their supporting cells that is present within the wall of the gastrointestinal tract. In terms of cell numbers, the ENS is the largest division of the autonomic nervous system, with neuron numbers comparable to that of the spinal cord [1]. This division of the autonomic nervous system is very extensive, being spread over the entire oroanal extent of the gastrointestinal tract. The ENS is also by far the most complex division in terms of the number of different functional types of neurons present and their connectivity [2– 4]. The range of neurotransmitters expressed by enteric neurons is also very broad, and most of the substances that act as neurotransmitters in the central nervous system (CNS) are also found in the ENS

[2,5,6]. Enteric glia, while less investigated, appear to share more properties with CNS glial cells than with support cells associated with sympathetic and parasympathetic ganglia [7,8]. The ENS mediates motility reflexes and plays a major role in controlling water and electrolyte transport by the mucosal epithelium [9] and also regulates intestinal blood supply [10]. Many neurons of the ENS receive input from the hindbrain, and from ganglia of the sympathetic and parasympathetic nervous systems [11–13]. Peripheral processes of sensory neurons in the nodose and dorsal root ganglia are also present in the ENS. Despite the presence of these inputs, it is noteworthy that, in some intestinal regions, the ENS shows great functional autonomy [14]. This autonomy is permitted by complete reflex circuits within the ENS, comprising intrinsic sensory neurons, interneurons, and motor neurons [2,15–18]. For example, in the guinea pig small intestine, motility reflexes can be elicited after complete extrinsic denervation, in which all neural connections with the CNS, sympathetic ganglia, and dorsal root ganglia are cut and have degenerated [14]. In addition, like the CNS, the ENS may show considerable adaptive plasticity to preserve overall gastrointestinal tract function even when challenged by pathological or experimental insult [19]. Despite these similarities between the CNS and ENS, the organization of the ENS differs radically from that of the CNS. The ENS is a distributed system laid out as numerous ganglia spaced regularly in two dimensions as nodes connected by bundles of nerve fiber, called internodal strands, in the myenteric (or Auerbach’s) plexus, between the circular and longitudinal muscle coats of the gut and in the submucous (or Meissner’s) plexus, internal to the circular muscle layer (Fig. 1). Myenteric ganglia are present throughout the length of the gastrointestinal tract, but submucosal ganglia are absent from the esophagus and stomach. The size of ENS ganglia is variable, between myenteric and submucosal ganglia, between gut regions and, to a lesser extent, between species. Typically, myenteric ganglia are considerably larger than submucosal ganglia in the same region. The ENS neurons, although clustered into ganglia, do not form nuclei of morphologically, functionally, and biochemically similar neuron types as occurs, for ex-

ample, in the brain stem. Instead, each enteric ganglion contains many different neuron types, and neighboring ganglia will contain similar types of neurons although not always in identical proportions. The ENS therefore forms repeating units of neuronal circuitry along the gastrointestinal tract. However, the ganglia of the ENS also show systematic differences according to the region of intestine where they are found. These differences include variations in ganglion size, types of neurons present, projection patterns, and connectivity (Fig. 1). Because of its critical role in intestinal motility, absorption, and secretion, the ENS is absolutely essential for all stages of postnatal life. Mice lacking enteric neurons throughout the gastrointestinal tract [20,21] or from particular regions such as the esophagus [22] or small and large intestines [23] usually die within 24 h of birth. In contrast, the ENS is not essential at earlier embryonic stages. Consequently, abnormalities that affect ENS development alone will not contribute to prenatal mortality or morbidity but will have powerful effects immediately following birth. The neurons and glia of the ENS are all derived from precursor cells from the CNS primordium, as described in detail in the second part of this review [24]. These cells, termed neural crest cells, also produce the cells of the sympathetic and parasympathetic nervous systems, dorsal root ganglia, and many other cell types in specific locations [25]. Neural crest cells are produced from the entire length of the neural axis but only certain tightly defined regions of the neural crest give rise to the ENS. These precursor cells first migrate from the CNS primordium into the oral and anal ends of the intestinal tract early in embryonic life, then migrate along the gut to colonize the entire intestine, which is itself carrying out its own growth, morphogenesis, and differentiation programs. Once in the intestine, these precursor cells assemble into groups, which come to occupy particular positions in relation to the intestinal tissue layers that are forming at the same time. The ENS precursor cells differentiate into a range of neuron types and glial cells, and form the complex circuitry necessary for ENS function. As well as using clinical genetic studies of human ENS dysmorphologies to understand ENS ENTERIC NERVOUS SYSTEM

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Figure 1. Whole-mount preparations of myenteric (A,B) and submucosal (C,D) plexus from the ileum (A,C) and colon (B,D) of a guinea pig. The tissue was processed for immunohistochemistry using antisera to the nitric oxide synthetic enzyme, nitric oxide synthase (NOS, red), and the calcium binding protein, calretinin (green). Myenteric ganglia in the colon tend to be larger than those in the ileum, but ganglia in both regions contain numerous NOS-immunoreactive (filled arrows) and calretinin-immunoreactive (open arrows) cell bodies (A,B). In both the ileum and colon, neurons containing NOS are descending interneurons and inhibitory motor neurons, and neurons containing calretinin are interneurons and excitatory motor neurons. In the submucosal ganglia of the ileum (C), there are many NOS-immunoreactive nerve terminals (filled arrows) that arise from cell bodies in the myenteric plexus, but no NOS-immunoreactive cell bodies. In contrast, there are numerous NOS-immunoreactive cell bodies (filled arrows) in the submucosal ganglia of the colon (D). Unlike submucosal ganglia in the ileum of the guinea pig, some neurons in the submucosal ganglia of the colon, such as the NOS neurons, innervate the circular muscle. Scale bar ⫽ 50 ␮m.

development, embryological evidence from nonhuman mammalian sources, particularly rodents, comes to the fore, particularly when techniques of molecular genetics and transgenesis are used. In addition, the evidence discussed below, particularly in the second part of this review [24], will stem in major part from bird embryos, especially when the techniques of investigation are the classic embryological techniques of transplantation, abla-

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tion, and grafting. In addition, data from developmental studies exist for the ENS of fish and amphibians. There are so many points of concordance between all these vertebrate classes that we will take the view that species differences are in general minimal, and thus these animal models can be used with confidence, yet with appropriate caution, as models of human ENS development, normal and abnormal. The applicability of convenient

animal models is an enormous advantage in seeking to understand events concealed in the early part of intrauterine life. Also, we can infer that the complex developmental program for establishing the ENS is ancient since it is conserved in detail among fish, amphibians, birds, and placental mammals, groups that last shared a common ancestor well over 300 million years ago. In the last decade, enormous strides have been made in understanding the genetics and cell biology of the early stages of ENS formation in normal embryos, and hand in hand with this, in understanding how one of the major common defects of the ENS, Hirschsprung’s disease (HSCR), arises. The deeply interdependent relationship between clinically driven research and basic developmental studies is perhaps nowhere as well illustrated as in this example, and this forms the basis of this review. In contrast, relatively little progress has been made in understanding other putative developmental defects of the ENS. Nontrivial reasons for this include the difficulty in clinically defining singular disease entities; this difficulty of diagnosis is often not appreciated fully by basic scientists. Also, establishing that a disease detected postnatally arises as a disturbance of development in the strict sense is not straightforward. We will use intestinal neuronal dysplasia type B (INDB) as an example of a disease emerging as a possible developmental defect of ENS formation to illustrate these difficulties. This review summarizes and synthesizes current views on the genetic, molecular, and cell biological underpinnings of the early phases ENS formation and its defects in the following order. The first part, presented here, gives a brief description of the critical features of HSCR and INDB in humans, and the similar abnormalities in animals. The difficulties in diagnosis and treatment of both these conditions are also discussed. This part also presents an introduction to the relatively small number of genes now known which, when mutated or deleted, may cause defects of ENS development. Most, but not all, of these genetic abnormalities give a phenotype similar or identical to HSCR, and were discovered by studies of humans and of mouse mutants with similar defects. The second part of this review, to be presented in a following issue [24], gives a description of the development

of the ENS, concentrating mainly on the origin of the ENS precursor cells, and on the cell migration by which they become distributed throughout the gastrointestinal tract. This section also includes experimental evidence on the controls of ENS formation. In addition, for reasons of completeness, we also briefly describe the origins of the interstitial cells of Cajal, a cell population closely related anatomically and functionally to the ENS. Finally, a brief sketch is presented of current notions on the developmental processes between the genes and the morphogenesis of the ENS, and of how the known genetic abnormalities might result in the ENS phenotype observed in HSCR.

HIRSCHSPRUNG’S DISEASE, INTESTINAL NEURONAL DYSPLASIA, AND THEIR COUNTERPARTS IN ANIMALS Hirschsprung’s disease (HSCR) HSCR is a birth defect affecting the bowel [26]. Usually this involves just a short segment, the rectum and sigmoid colon, but occasionally there is much greater involvement of the colon and, even more rarely, involvement extending to the proximal ileum (so-called short- and long-segment HSCR) [27]. The functional characteristic of the disease is intestinal obstruction, or severe constipation, caused by the localized inability of the gut to transmit a motile relaxation and contraction (peristaltic) wave; this section of the bowel is typically contracted and devoid of contents. In contrast, the segment of intestine proximal to this becomes grossly distended by fecal accumulation, and this is termed megacolon. The disease is due to an absence, or near absence, of ENS ganglia in the contracted terminal region. It is important to note that the grossly abnormal-looking, distended region contains a relatively normal ENS, and the distension is an indirect effect of the blockage in the more distal regions lacking enteric neurons. In addition, there is a marked overabundance and increased size of nerve fiber tracts, arising from extrinsic neurons, in the aganglionic segment of bowel [28 –30] and also in the internal anal sphincter [31]. These nerves include noradrenergic nerve fibers that are likely to arise from the pelvic plexus. In a significant proportion of HSCR cases, abnormalities of ENS structure or function are not restricted to the aganglionic region; proximal to this ENTERIC NERVOUS SYSTEM

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there may be a region of variable extent termed the transition zone with hypoganglionosis, or conversely of hyperganglionosis with an INDB-like character [32,33]. The diagnosis of HSCR is unequivocal [34], being based on a lack of ENS neurons in biopsy specimens of submucous tissue that are processed for enzyme histochemistry for acetylcholinesterase. The chief difficulty encountered is precisely gauging from the ENS distribution the proximal extent of the functional abnormality. Contributing to this is the uneven density of distribution of neurons in the normal colon. HSCR occurs in around 1/5000 live births, with a male:female ratio of about 4:1 (see [35] for historical review). HSCR also occurs in association with other syndromes and anomalies such as congenital central hypoventilation syndrome (Ondine’s curse), 22q11 deletion syndromes, and in a variant of Shah-Waardenburg (WS4) syndrome [36]. These conditions primarily involve abnormalities in systems developmentally related to the neural crest. Thus HSCR is regarded as a neurocristopathy [37]. HSCR clearly has a strong genetic component, with increased intrafamilial risk (4% for siblings versus ⬃0.02% in the general population). Moreover, the risk increases with the length of affected segment, and the sex ratio discordance decreases [27]. Classic genetic analysis [27] was consistent with a dominant inheritance with incomplete penetrance (long segment) and autosomal recessive or multifactorial profile (short segment), although it is now known that both longand short-segment HSCR can be caused by the same mutation [38]. Mutations in a number of genes (discussed in [36]) are involved in HSCR, but it should be noted that all of the known genes account for ⬍50% of distal aganglionosis cases [30,36,39,40]. This suggests that there are still unknown genes that, when mutated, predispose to HSCR and/or that some forms of intestinal aganglionosis are not strictly genetic. In any case, the advancing knowledge of the etiology of HSCR makes it a model for complex multigenic congenital dysmorphology syndromes. Similar conditions of distal gut aganglionosis occur in animal models, such as lethal spotting (ls), piebald lethal (Sl) [41], and dominant megacolon

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(Dom) [42] in mice, spotting lethal (sl) in the rat [43], and lethal white foal syndrome in horses [44]. All of these mutations involve prominent abnormalities in another neural crest derivative, the melanocytes, since all have major areas of unpigmented skin and hair. In chickens, aganglionosis of the most distal gut has also been produced by partial ablation during early embryogenesis of a specific section of the neural crest [45– 47].

Intestinal neuronal dysplasia type B (INDB) In children with grossly slowed intestinal transit time and intractable constipation, a subgroup has been correlated with megacolon and ENS abnormalities [48 –50]. This is now generally termed intestinal neuronal dysplasia type B (INDB), though the older abbreviation, NID (neuronal intestinal dysplasia) is often encountered. These abnormalities do not include a region of aganglionosis, in absolute contrast to HSCR, but they are reported to sometimes show increased extrinsic nerve fibers in the affected gut as in HSCR [48]. In descriptions of INDB there are larger than normal ENS ganglia (“giant ganglia”) in the submucous plexus that are suggested as definitive [48], often a hyperplasia of the ENS, and ectopic ganglia especially in the muscularis mucosae or lamina propria. As noted above, there is a connection between these two ENS abnormalities because HSCR often includes a region of INDB-like character proximal to the aganglionic segment [33,48,51]. Despite the increasing number of published studies on INDB, doubts have been raised as to whether INDB is a distinct clinical, i.e., diagnosable, entity [52,53]. These doubts were substantiated by the failure of three pathologists, who had previously participated in a consensus meeting on diagnostic guidelines, to achieve agreement on a diagnosis of INDB when presented with a series of 377 biopsy specimens from 108 children in a blind trial [34]. In contrast, the same three pathologists agreed completely with respect to the diagnosis of HSCR [34]. The diagnosis of HSCR is made on a single agreed, objective, and qualitative criterion. Unfortunately, unequivocal diagnosis is still problematic for INDB, partially because at present diagnostic criteria are not agreed upon or are subjective [52,53], and because the differences from the nor-

mal ENS are quantitative rather than qualitative. This quantitative requirement for diagnosis is placed on a background of variability of the ENS between normal individuals in the postnatal period [34], and on variability in ENS density from place to place in the colon. Also, there is disagreement as to whether this diagnosis can be extended to a subgroup of chronic constipation in adults. For examples of these difficulties, in INDB the socalled giant ganglia in the submucosal plexus, defined as containing seven or more neurons [48], are not dramatically larger than the normal ganglia. Moreover, not all ganglia in INDB patients are “giant” and some ganglia in functionally normal ENS must be classed as “giant” [52]. In addition, some or all variables used to differentiate the ENS in INDB from the normal ENS show age-related changes in both normal and INDB cases, so diagnosis may have to take patient age into account [54]. To further complicate recognition, other characteristics may indicate INDB. For example alterations in ratios of ENS neurotransmitters such as decreased substance P and increased vasoactive intestinal peptide VIP [55,56], hyperplasia of interstitial cells of Cajal [57], or possible defects of the neuromuscular junction markers [58], have all been described in patients diagnosed with INDB. However these may represent subgroups of INDB or be shared with other conditions that are not INDB. It is also possible that several underlying abnormalities are being lumped under the INDB heading, contributing to the uncertainties of diagnosis. Despite the questionable, if not impossible, nature of diagnosis in any individual patient [52,53], INDB is regarded as a real clinical entity by some clinicians [50], with a developmental genetic component deduced from a twin study [59]. The fact that INDB has been recognized in newborns does not necessarily qualify it as a direct developmental defect. Abnormalities of the ENS such as achalasia appear in adult life and are therefore not developmental, and there is no reason a priori why nondevelopmental problems could not occur at earlier stages. While achalasia is a neuronal deficit disorder, neural disorders of the opposite type could also occur. As an example of a possible mechanism, inflammatory cells have been described in the intestinal plexuses in INDB. In

CNS injury responses, such cells and, in particular, activated macrophages secrete the neurotrophic factors brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF; see The GDNF Gene and Protein Family, below), which are active in promoting nerve growth in the region of inflammation [60,61]. A similar expression of factors by inflammatory cells in the gastrointestinal tract could account for hyperplasia of the ENS. One relatively strong piece of evidence that INDB, or at least some form of intestinal neuronal hyperplasia, might be a real entity stems from animal models. Mice with mutations of Hox11L1 (see Hox11L1, below) show an ENS pathology resembling INDB in that neuron numbers in the colon are increased over normal [62,63]. The human homologue of this gene is known [64] but at present seems not to have been linked to any case diagnosed as INDB or HSCR in humans [65]. Moreover, the most commonly mutated gene in HSCR has not been found to be mutated in the few cases of INDB studied [66]. Despite this, future knowledge of the pathways in which Hox11L1 is involved, for example, the genes modulated by Hox11L1 protein, would produce candidate genes for involvement in INDB, and if so, possible diagnostic markers. Judging from the presence of other disorders of morphogenesis that occur in individuals with INDB, at least some of the causes of INDB-like abnormalities to the ENS may be due not to disturbance of the primary ENS developmental processes, but may be secondary responses to a range of bowel and abdominal disorders, some of which may be developmental [51,,67]. In any case, this absence of an agreed upon clinical definition and etiology for INDB is a handicap for any attempt to probe the condition genetically.

Treatment for HSCR and INDB HSCR status is confirmed by suction rectal biopsies or muscular biopsy [68]. Usually two pieces are taken— one is frozen and processed for acetylcholinesterase histochemistry and counterstained with hematoxylin, and the other is embedded in paraffin and the sections stained for hematoxylin and eosin to examine for the presence of ganglion cells. Current treatment of HSCR involves surgical ENTERIC NERVOUS SYSTEM

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resection of the aganglionic segment (ascertained by biopsy) and reanastomosis, in a one- or twostage procedure [69]. Problems with this, aside from the loss of distal bowel, frequently include continuing functional abnormalities. One of the causes is when, in some individuals, the most distal retained region possesses ganglia but is the so-called transition zone. Therefore the most distal ganglionated segment retained after resection of the HSCR gut in these patients may structurally and functionally resemble the abnormal situation in INDB [33,70,71]. After resection, functional recovery of patients with HSCR with associated INDB is slower than in simple aganglionosis, and is more likely to require secondary intervention [33]. Current treatment for INDB is in the first instance conservative, consisting of laxatives and enemas [50]. In many cases the clinical problem resolves or is manageable in this way [33]. Continued, long-term, severe bowel problems may be treated by resection, but the length of resection seems much more difficult to assess. Long-term follow-up is likely in any case because there is unpredictable variability in outcome [33,49] that is not closely related to the initial histological assessment of the severity of the INDB abnormality [49]. For the surgical approach to HSCR, one immediate goal is to predict more precisely the optimum level for resection, so that the minimum amount of bowel tissue is removed while ensuring that the remaining bowel is neurophysiologically competent. A more distant goal is to eliminate the need for surgery by building an ENS in the aneural segment, perhaps by seeding of the region of bowel with neural progenitors able to form a functional ENS. Human stem cells capable of neural differentiation are obtainable from postnatal stages [72]. Experiments with avian tissues showed that it is possible for early migratory ENS precursor cells to migrate into older and more differentiated intestine and, in turn, differentiate into appropriately placed neurons, if this recipient gut lacks its own ENS [73]. The observation that considerable variability and developmental changes in the normal human ENS are still occurring postnatally, as well as the plasticity of the ENS, suggests that such manipulations might be possible in human fetuses with intervention in utero, or even at postnatal

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stages. But to do this will require great expansion of our ability to isolate, expand, and manipulate stem cells and a great increase in our knowledge of the molecular genetics and cell biology of normal development of the ENS.

Origins of Hirschsprung’s disease The unifying feature of the HSCR-like ENS deficiency syndromes in humans and animals is that the affected region involves the distalmost region of the gut. The early undifferentiated gut tube is prepatterned along its oro-anal axis, and this is marked by the differential expression of various Hox genes [74]. This could suggest that the regionality of the ENS deficit is related to the prepatterned regionality of the gut. However, the extent of ENS absence along the intestine is highly variable. In humans, short-segment and long-segment HSCR have been identified but recent genetic evidence [38] shows that they do not necessarily represent separate clinical entities, as was previously thought [27]. In ls neonatal mice, the aganglionosis involves only the distal few millimeters of the colon; in Sl mice, about half the colon; in the sl rat, the entire colon and sometimes a variable extent of the ileocecal region. In contrast, Ret, gdnf, and gfr␣1 homozygous knockout mice show total absence of the ENS in the entire intestine distal to the stomach. This suggests that, whatever causes the aganglionosis to be distal, it is unrelated to the regional prepatterning that defines the various segments of the gastrointestinal tract. This pattern of intestinal aganglionosis immediately points to disturbance in a rostrocaudal pattern of normal early development in the ENS. This focuses attention on the striking rostrocaudal migration of neural crest cells through the gastrointestinal tract as the candidate process disturbed in HSCR [75]; (see [24]). Given the interactive nature of neural crest development, defects that interfere with cell migration could arise in the neural crest cells themselves, or in their local environment, either at the site of origin at the CNS primordium, in the migration pathways before reaching the gut, in the gut in general, or in the gut localized to the aganglionic zone. The mutations could affect the ability of neural crest– derived cells to migrate, divide, survive, or differentiate. Understanding the pathways between genotype and phe-

Table 1.

Genes involved in enteric nervous system (ENS) development

Gene

Human chromosomal location

Phenotype of ENS in mice in which gene is homozygously inactivated

RET

10q11.2

Absence of neurons from small and large intestine

GDNF

5p12–13.1

Absence of neurons from small and large intestine

GFR␣1

10q25

Absence of neurons from small and large intestine

ETB

13q22

Absence of neurons from distalmost large intestine

ET-3

20q13.2–13.3

Absence of neurons from distalmost large intestine

ECE-1

1p36.1

Absence of neurons from distalmost large intestine

Phox2b

4p12

Absence of neurons from entire gastrointestinal tract

SOX10

22q13

Absence of neurons from entire gastrointestinal tract

PAX3

2q37

Absence of neurons from small and large intestine

HASH1 (Mash1)

12

Absence of neurons from esophagus

IHH (Indian hedgehog)

2q333q35

Absence of neurons from parts of the small intestine and colon

SHH (Sonic hedgehog)

7q36

Ectopic nerve cell bodies within mucosa

HOX11L1

2p13.1

ENS hyperplasia in colon and hypoplasia in small intestine

SIP1

2q22

Not reported

notype in these ENS disorders has made great progress recently, and continues as a subject of strong interest (see next section and [24]).

GENES AND MOLECULES INVOLVED IN ENS DEVELOPMENT AND DYSPLASIAS Over the last decade, a number of genes have been identified that, when mutated or deleted, interfere with ENS development. The molecules these genes encode are therefore involved in normal ENS formation. Many of these have been identified by clinically based molecular genetic approaches directly investigating HSCR, although some are known from basic research on development. Of principal importance are genes that code for elements of two cell– cell signaling systems (GDNF-Ret/GFR␣1 and ET-3-ETB), whose functions have been the subject of intensive recent study in relation to HSCR. The genes for elements of the Hedgehog signaling pathway, known from basic research on development, are also described, since these influence ENS formation in model systems. Several transcription factors (Mash1, Phox 2a and 2b, Pax 3, Sox10, and Hox11L1) are also introduced, which are clearly important for ENS development, but their precise functions are less well understood. These are summarized in Table 1, and the hypothesised interac-

Figure 2. Interactions between the transcription factors Mash1, Phox2a, Phox2b, Pax3, and Sox10 and Ret (which is part of the receptor complex for GDNF) and the catecholamine synthetic enzymes, tyrosine hydroxylase (TH) and dopamine ␤-hydroxylase (D␤H). The expression of Ret is positively regulated, directly or indirectly, by all of the transcription factors shown in this figure. See text for references. GDNF: glial-derived neurotrophic factor.

tions between several of the genes mentioned are diagrammed in Figure 2. A caveat must be raised with regard to the interpretation of nearly all the experimental genetic studies on early ENS formation and, indeed, to all the manipulations involving the developing ENS, in which the outcome is not simple absence of ENS. This caveat is that the presence of neurons ENTERIC NERVOUS SYSTEM

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in the intestinal plexuses in approximately normal numbers is almost always interpreted as comprising a normal ENS. In reality, in almost no case is it known whether this is actually so; abnormalities of the neurotransmitters, the neurite projection patterns, electrophysiological properties, or the glial cells could exist with grossly normal neuron distribution and number. Thus some genes may be wrongly dismissed as having no crucial role in ENS formation; more detailed assessment of ENS phenotype must be a feature of future studies. We will follow the convention that the gene (or mRNA) for any molecule is written in italics and the protein in standard font. Human versions of the gene or molecule are always written entirely in uppercase while non-human homologues are entirely or in part in lower case.

GDNF–RET–GFR␣1 signaling system This signaling pathway is of importance for subpopulations of both peripheral and central neurons, having been shown by in vitro and in vivo assays to promote survival of neurons, mitosis of neural progenitor cells, differentiation of neurons, and neurite extension [76 – 80]. In addition, this signaling system is vital for early kidney development, at least in mice [23,81– 83]. RET gene and protein Short- and long-segment HSCR were linked first to defects in chromosome 10q11.2 [84,85] and specifically to the RET gene [86,87]. It is likely that about 50% of familial and some sporadic HSCR arise from mutations in RET [36,39]. Sequence analysis of RET showed that it codes for a single membrane pass cell surface molecule with a cytoplasmic domain containing a tyrosine kinase. At the time of discovery, RET was an orphan receptor [88]. The striking similarities between the phenotypes of Ret⫺/⫺ and gdnf⫺/⫺ mice (both exhibit renal agenesis plus lack of ENS in the small and large intestines) first led to the suggestion that Ret and glial-derived neurotrophic factor (GDNF; see GDNF Gene and Protein Family, below) are involved in the same signaling pathway [81– 83]. Shortly afterwards it was shown that GDNF does act through Ret, but an accessory molecule, termed GFR␣, to which GDNF binds, is also required for signaling [89,90]. The ligands for RET

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are now known to be more complex than just GDNF, and include other members of the GDNF family (see The GDNF Gene and Protein Family, below). The numerous, diverse, and scattered mutations in RET gene that cause HSCR (premature stop codons and deletions in the kinase domain) inactivate the kinase. The resultant aganglionosis and functional impairment is variable in that the same mutation in the one family can result in long-segment or short-segment HSCR with severe intestinal motility problems, through milder constipation to functionally normal ENS [86,91]. However, infants with long-segment HSCR appear to exhibit a higher percentage of RET mutations, particularly in the tyrosine kinase domain, than those with short-segment HSCR [92]. There is a rare form of HSCR, known as total intestinal aganglionosis, in which enteric neurons are absent from the entire small and large intestines. One patient suffering from this was shown to be homozygous for a particular RET mutated allele (codon 969), whereas a patient heterozygous for the same mutated allele showed aganglionosis extending distally from the jejunum [92]. In contrast to humans, mice with heterozygous mutations in Ret do not have an aganglionic segment of gut nor do they show signs of megacolon [23]. Homozygous Ret knockout mice show total absence of the ENS from all regions of the gastrointestinal tract except the esophagus. This suggests that normal ENS development is quantitatively dependent on the RET gene product, with humans being essentially on the borderline of haploinsufficiency—that is, the protein production from only one functional RET gene is close to the minimum required to perform the developmental task fully. The variability in outcome with heterozygous RET mutation is consistent with there being other genes that are involved in the same process of ENS formation, the severity of aganglionosis being determined by allele variation of these other genes. The effects of mutation of Ret are not confined to the ENS. The mice also lack the superior cervical sympathetic ganglia and show kidney agenesis [23]. Interestingly, although RET is expressed in the developing kidney of humans [93], HSCR patients, even the total intestinal aganglionosis patient, only very rarely have renal abnormalities [94].

In chicken embryos [95], Ret mRNA can be detected by in situ hybridization in the vagal level neural crest even before cell migration commences, but in the mouse, Ret mRNA has not been detected until later stages, as the cells are entering the foregut [88,96]. Whether effective amounts of Ret protein are present at these early stages in birds is not known. In any case, in both bird and mouse, the neural crest cells seem not to respond to the Ret ligand, GDNF, as they are initially emigrating from the neural tube [97,98], but they may become responsive shortly prior to or shortly after entering the foregut. The Ret gene and protein is expressed in the ENS cells as they migrate through the gut mesenchyme of the developing mouse [88,96,99 –101], rat [102,103], chick [104,105], and human [93], and later in differentiated enteric neurons of these species [102,106]. Enteric neural crest– derived cells are responsive to GDNF in cell culture assays, showing increased growth, survival, differentiation, and chemotactic migration [97,98,107–111]. It seems that virtually all neural crest cells (both neural and glial precursors) within the gut initially express Ret, and although the expression of Ret is maintained in differentiating neurons, it is down-regulated in differentiating glial cells [101,106,112]. One of the most interesting aspects of RET is that different mutations, specifically those that cause the kinase to be constitutively active even in the absence of GDNF, do not usually cause HSCR, but instead cause familial medullary thyroid carcinoma (FMTC) and multiple endocrine neoplasias (MEN) 2A and 2B [79,113–115]. Since these conditions are initiated as hyperplasias, it is possible that the kinase-inactivating mutations cause hypoplasia of the ENS-producing cell population, and that this generates the HSCR phenotype. However, further complexity may be expected since up to 5% of HSCR patients also have MEN 2A or FMTC [116,117]. At least some of these patients, while having a putative activating mutation of RET, express less RET protein than normal [118]. It may be that the constitutive activity is sufficient to drive endocrine neoplasia, although total active RET protein is not sufficient to support normal ENS morphogenesis. There are also some reports of achalasia (death of enteric neurons, typically in the

esophagus) forming as a secondary disorder in patients with MEN 2B [119,120]. GDNF gene and protein family Genetic and biochemical studies have identified that the ligand for the Ret receptor is GDNF [39,121]. The GDNF gene is localized to human chromosome 5p12–13.1. Heterozygous mutations to this gene, alone, have not been found to be responsible for HSCR [122]. However, it is possible that GDNF gene mutations may contribute to the severity of HSCR if the mutation coincides with other HSCR genes [123,124]. A preliminary report [125] has described hypoganglionosis in gdnf⫹/⫺ mice. Homozygous mutations of GDNF have not been reported in humans, but gdnf⫺/⫺ mice have an intestinal phenotype identical to Ret⫺/⫺ mice, namely an absence of the ENS caudal to the stomach, renal agenesis, and neonatal death [81– 83]. The GDNF gene codes for a secreted growth factor, which exists as a dimer linked by disulphide bonds between cysteine residues. This dimerization device, the so-called cysteine knot, is characteristic of the transforming growth factor ␤ (TGF-␤) family of growth factors [126]. In terms of sequence, GDNF is a distant member of this family [76] and functionally shows very great divergence from other TGF-␤s in that it signals through a tyrosine kinase receptor (Ret; see The Ret Gene and Protein and [127]) whereas all other members use a conserved group of serine–threonine kinase receptors. In addition, the signal transduction pathway initiated by GDNF via RET is not carried by the SMAD family used by other TGF-␤ receptors. More recently still, other members of the GDNF family, neurturin [128] (on human chromosome 19p13.3), artemin [129], and persephin [130], have been identified, and while each has their own ligand-binding coreceptor (GFR␣), they also all utilize Ret as receptor (see [131,132]). Mutation of the gene encoding NEURTURIN alone does not result in HSCR [133], although it is possible that, like GDNF, mutations in GDNF-related factors could contribute to the severity of HSCR due to other mutations. In mice, homozygous knockout of the genes encoding either neurturin [134] or its ligand-binding molecule, gfr␣2 [135], results in a decrease in the density of cholinergic ENTERIC NERVOUS SYSTEM

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neurons in the ENS, but no renal abnormalities, and the mice survive and breed. Mutations to ARTEMIN and PERSEPHIN do not contribute to HSCR in humans or mice [80]. In situ hybridization reveals that gdnf mRNA is expressed in the embryonic gut mesenchyme [83,98,136]. This can be detected at low levels in mice at around embryonic day 10.5 (E10.5; roughly equivalent to human age 30 days from fertilization). This is shortly after the first ENS precursors, vagal neural crest cells, enter the foregut, and gdnf mRNA levels increase rapidly throughout the entire length of the gastrointestinal tract from this time. There is scant evidence for a proximodistal wave of expression [98] in advance of, or congruent with, the migration wave of the vagal neural crest population. Nor is there convincing quantitative evidence for stronger expression of the gene in some segments of the gastrointestinal tract. Protein levels for GDNF, over time and in different gut segments, have not been described. GFR coreceptors Signaling via Ret requires coreceptors (ligandbinding molecules) of the GFR␣ family (for reviews see [131,137,138], which are glycosylinositolphosphate linked to the cell surface membrane in the same cells of neural crest lineage that express Ret [105]. All four of the known GDNF-related ligands (GDNF, neurturin, artemin, and persephin) have their own preferred coreceptor; GDNF signals preferentially via GFR␣1 (human chromosome 10q26). The relationship between Ret and the GFR␣s in the absence of any GDNF-related ligand is still unclear. Either GDNF or its related ligands first bind to the appropriate GFR␣, and this complex then binds to and stimulates autophosphorylation of RET, or the GFR␣s form a preassociated complex with Ret which acts as the binding site for GDNF or its related ligands (see [127]). GFR␣s are localized to lipid rafts in the cell membrane; binding of GDNF to GFR␣1 appears to recruit Ret to the lipid rafts and initiates an association with the signal transduction adaptor motif Src, which is necessary for downstream signaling leading to neuronal survival and differentiation [139]. Mutations in GFR␣1 have not been associated with any case of HSCR, although a number of polymorphisms occur [140]. Nevertheless, ho-

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mozygous GFR␣1 knockout mice have almost total intestinal aganglionosis distal to the esophagus, similar to Ret and GDNF knockout mice [141–143]. There is, however, a small population of ENS neurons present in the distalmost colon in these mice [141], which may be the sacral neural crest– derived ENS cells (see [24]). Mutations in GFR␣3, the gene encoding the ligand-binding molecule for artemin, also are not associated with HSCR [144]. Functions of the RET–GDNF–GFR␣1 system Neural crest– derived cells such as those in the embryonic sensory and sympathetic ganglia express the RET–GFR␣ receptor complex and GDNF promotes their survival and neurite outgrowth [78,145,146]. Studies on avian, mouse, and rat embryonic cells in vitro have shown that GDNF acts on early ENS precursors as a mitogen, and also stimulates differentiation into neurons and neurite growth (Fig. 3A,B) [97,107–111]. In addition, using organ cultures of mouse intestinal tissues containing ENS cells, a recent study [98] has shown that GDNF can directionally attract the migrating ENS precursor cells (Fig. 4). Interestingly, other neural crest– derived cells lacked this directed migration response. This suggests that those response modalities (survival, proliferation, differentiation, attraction) to GDNF that are downstream of the Ret/ GFR␣ receptor complex can be differentially expressed in a cell type-specific manner. These same sets of experiments suggested that the vagal neural crest cells destined to form the ENS, but tested at an earlier stage as they were emigrating from the neural tube, were unresponsive to GDNF. Thus it seems that developmental changes occur in these vagal neural crest cells either just before they enter foregut or after they have been exposed to the gut microenvironment, such that they become GDNF-responsive.

Endothelin– endothelin receptor system The endothelins (ET–1, ET–2, and ET–3) are intercellular local messengers that act via cell surface receptors (ETA and ETB). The endothelin system in adults is important in the regulation of blood pressure by controlling tone in the microvasculature. During early embryogenesis, however, they have important additional roles in regulating morphogenesis, which have been revealed by gene knock-

Figure 3. Dissociated enteric precursor cells from E4.7 quail gut grown for 4 days in culture and then fixed and processed for immunohistochemistry using antibodies to neurofilament. In control (A) cultures there is a low density of neurons (arrows), whereas in the presence of GDNF there is a very high density of neurons (B). Et-3 alone has little effect (C), but Et-3 markedly reduces neuronal differentiation induced by GDNF (D). Scale bar ⫽ 50 ␮m. Micrographs courtesy of Dr. Catherine Hearn; see [97] for more details.

Figure 4. Explant of a small piece of midgut from an E11.5 mouse grown on collagen culture with an agarose bead impregnated with GDNF at one side and a control agarose bead at the opposite side. The explant was grown for 4 days and then fixed and processed for immunohistochemistry to reveal neurons. The GDNF bead is surrounded by neurons and neurites. Scale bar ⫽ 100 ␮m.

out studies in mice. For example, knocking out the Et-1 gene in mice leads to severe craniofacial and cardiac defects; significantly, this involves dysmorphogenesis of multiple neural crest– derived structures [147,148]. Endothelin receptor B (ETB) Studies of familial HSCR in a Mennonite community led to the identification of ETB receptor as a

gene which, when inactivated, could cause HSCR [149]. In humans about 3%–7% of HSCR appears to stem from ETB mutations [150 –152]. Conventionally this would be described as a dominant effect with reduced penetrance, since heterozygous mutations result in 21% affected individuals, and up to a quarter of individuals with homozygous mutations are unaffected [153]. The ETB receptor gene lies on human chromosome 13q22 [149], and codes for a G protein– coupled receptor with seventransmembrane domains. EtB receptor is expressed in neural crest and crest-derived cells of various lineages, including the cells that produce the ENS. It is expressed very early, in the neural folds prior to neural crest cell emigration, and expression is maintained as the neural crest cells migrate to their target tissues [154 –156]. Targeted deletion of the EtB receptor gene caused a similar HSCR-like condition, as well as pigmentation defects, in mice [157]. The Piebald lethal (Sl) mutant in the mouse and the spotting lethal (sl) mutant in the rat are also due to loss of ET function from inactivation of the ETB receptor gene [157,158]. In an elegant study using an inducible system to modulate the expression of EtB in transgenic mice, Shin et al. [159] showed that to prevent distal aganglionosis, EtB needed to be expressed from E10, close in time to when ENS precursors enter the gut, to E12.5. The latter stage is interesting because complete colonization is not ENTERIC NERVOUS SYSTEM

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achieved until E14.5 [160,161]. This suggests that either EtB mRNA and/or protein persist for approximately 2 days or that the last part of colonization of the colon is relatively unreliant on EtB activity. Endothelins (ET) ET-1, -2 and -3 are secreted molecules produced as large inactive precursor molecules. These must be cleaved at two positions to generate a small 21 amino acid active peptide. Even though EtB binds Et-1, -2, and -3 with equal affinity, the principal ligand for the EtB receptor of the ENS precursor cells is Et-3, since this is the only ET produced in significant amounts by the gastrointestinal tract mesenchyme cells [155,162–164]. Mutations in the ET-3 gene in humans, located at chromosome 20q13.2–13.3, are responsible for about 5% of HSCR [165]. Mutation of the mouse Et-3 gene, as in the lethal spotting mouse (ls), produces a pigmentation and ENS phenotype similar to that of EtB mutations, but is less severe in that the length of the aganglionic segment of gut is shorter. This is believed to be due to partial compensation by low levels of Et-1, to which the receptor is equally responsive. Endothelin converting enzyme-1 (ECE-1) The protease that performs the second cleavage to generate functional ET is endothelin converting enzyme (ECE-1). The gene for this enzyme, on chromosome 1p36.1, has been found to be mutated in a patient with a group of complex neurocristopathies involving cardiac lesions, craniofacial defects, autonomic dysfunction, and intestinal aganglionosis [166]. The multiple abnormalities are presumed to stem from impaired function of ET-1 as well as of ET-3, as ECE-1 appears to act as an activating protease for both big ET-1 and big ET-3 [167]. Mice lacking Ece-1 have craniofacial and cardiac abnormalities very similar to those seen in Et-1 and EtA knockout mice, and also lack epidermal melanocytes and enteric neurons in the distal gut, which are similar abnormalities to those found in mice lacking Et-3 or EtB [167]. Functions of the ET-3–ETB system in ENS cells The developmental effects of signaling via the endothelin system are subtle. ENS precursor cells have been grown in cell culture after immunoselection from the gut of early avian embryos using an

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antibody to the neural crest marker HNK-1. The addition of ET-3 alone to these cells had little effect, but ET-3 dramatically decreased the differentiation of neurons that is driven by GDNF [97] (Fig. 3C,D). The survival and mitogenic effects of GDNF apparently were not impaired. Similarly, in ENS precursor cells immunoselected from the mouse gut, ET-3 decreases the number of neurons that develop in culture [110]. This differentiation-inhibiting or retarding effect of ET-3 signaling appears to be nonspecific in that it is not restricted to affecting differentiation driven by a particular factor such as GDNF. For example, neuronal differentiation of rat and avian ENS precursor cells in vitro can be stimulated by ciliary neurotrophic factor (CNTF) [168], and the CNTF-induced differentiation is also decreased by ET-3 (C. Hearn, personal communication). In addition, the effect of ET-3 is not restricted to inhibiting a single line of differentiation, such as enteric neurogenesis. For example, thoracic neural crest cells from avian embryos cultured in the presence of chick embryo extract differentiate as melanocytes because of the presence of an uncharacterized inducer of differentiation in the embryo extract [169]; addition of ET-3 markedly delays but does not prevent the appearance of melanocytes. This has the paradoxical effect of increasing the number of melanocytes eventually present in the cultures [170]. The reason for this is that the mitotic rate of the undifferentiated crest cells is much higher than that of the melanocytes [171], therefore maintaining the cells in this undifferentiated state for a longer period of time increases the number of cells eventually capable of melanocyte differentiation. A conceptually similar result is seen in cell culture of rat embryo Schwann cell precursors, a cell lineage also derived from the neural crest. Here, differentiation into mature Schwann cells is driven by ␤-neuregulins augmented by fibroblast growth factor-2 (FGF-2), but this differentiation is retarded by addition of ET-3 acting through the EtB receptor [172]. Taken together, these results indicate that ET-3 acts on cells of the neural crest lineage to prevent or retard differentiation in general, whether to melanocyte, Schwann cell, or ENS neuron. In addition, since the known differentiationinducing factors, ␤-neuregulin, FGF-2, GDNF, and CNTF, act via unrelated receptors, the damping

effect of ET-3 on differentiation may be at a common point downstream of these receptors. Such a common point may be the activity of the transcription factor Sox10 and perhaps Pax3 (see [24]). These factors acting synergistically favor differentiation of melanocytes, Schwann cells, and ENS neurons; it is possible that, directly or indirectly, ET-3 inhibits the expression or activity of these transcription regulating molecules. This would be predicted to alter the levels of expression of a range of downstream genes, including down-regulation of Ret [173]. Two groups [97,110] have independently proposed that, as the EtB–Et-3 signaling pathway appears to inhibit or retard enteric neuron differentiation, in the absence of signaling via this pathway, the enteric neuron precursor population differentiates too early and too completely into neurons, prior to colonizing the distal part of the hindgut. The result of this would be to cause a deficit of proliferative and migratory cells to complete the colonization of the distal gut. This would explain the absence of neurons in the distal hindgut in mice or humans with mutations in the genes encoding either EtB or Et-3. However, the theory assumes that the migratory ability of enteric neuron precursors is inhibited once neuron differentiation has commenced, which has yet to be directly demonstrated. This theory is consistent with the results of recent experiments in which explants of mouse gut were removed and grown in organ culture when enteric neuron precursors were present in the terminal ileum, but not in the hindgut. Colonization of the hindgut occurred in control cultures, but in the presence of an EtB antagonist to block Et-3–EtB signaling, terminal aganglionosis resulted [156]. However, the relevance of these results must be weighed against a recent study [159] in which manipulation of expression of EtB gene in mouse embryos indicated that continued expression of EtB is unnecessary for complete colonization of the distal hindgut. The apparent contradiction of the two studies may rest partly on the different techniques, but especially on the precise stage of colonization at which ETB function was interfered with: the function of EtB peptide was inhibited from E11.5 [156], whereas the transgenic mice [159] were re-

calcitrant to EtB gene expression loss at E12.5 or later.

Hedgehog signaling system Members of the hedgehog family are secreted proteins that participate in vital developmental processes during development. Recent evidence suggests that two members of this family, Indian hedgehog (Ihh) and Sonic hedgehog (Shh), may influence ENS development. Both Ihh and Shh bind to the transmembrane protein, Patched (Ptc). Ptc normally represses signaling from the cell surface molecule Smoothened (Smo); binding of hedgehog inhibits Ptc and derepresses Smo signaling. Signaling via Ihh or Shh activates the transcription factor Gli1, and also induces expression of the TGF-␤ family member, bone morphogenetic protein 4 (BMP4) [174 –177]. The genes for Shh and Ihh are expressed in the gut endoderm [178,179], while Ptc, Gli, and Bmp4 are expressed by the gut mesenchyme. Homozygous Ihh or Shh loss-of-function mice die during early embryonic stages, but heterozygous mice survive until shortly after birth [180]. Late fetal Ihh⫹/⫺ mice exhibit a dilated colon with an abnormally thin wall, and enteric neurons are often missing from parts of the small intestine and from the dilated regions of the colon [180]. The presence of an ENS in nondilated parts of the colon of Ihh⫹/⫺ mice suggests that enteric neuron precursors migrate into the gut, but fail to survive and/or differentiate in the absence of Ihh. Further evidence for a role for hedgehog signaling in ENS development comes from study in which the human GLI gene was overexpressed in mice; these transgenic mice also had a dilated colon and an absence of enteric neurons in parts of the gut [181]. However, it is not clear why gli1 overexpression mimics Ihh underexpression. Shh⫹/⫺ mice do not lack an ENS in any part of the gut, but nerve cell bodies are present within the mucosa, under the endodermal epithelium, and in the lamina propria of the villi, which are locations where enteric neurons are not normally found [180]. Shh secreted by the gut endoderm has been shown to activate expression of Ptc and Bmp4 in the neighboring non-muscle mesenchyme, and to inhibit neuronal and smooth muscle differentiation [182]. Thus, signaling via Shh is thought to be important for the radial patterning of the gut ENTERIC NERVOUS SYSTEM

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tube so that smooth muscle cells and neurons differentiate only in the outer layers distant from the endoderm. The presence of enteric neurons close to the lumen in Shh⫹/⫺ is compatible with this hypothesis, although it remains to be explained which mechanisms induce enteric neuron precursors to migrate close to the lumenal surface in Shh⫹/⫺ mice, since in wild-type animals, migratory and postmigratory undifferentiated neural crest– derived cells are not present in the mesenchyme close to the endoderm [99,183]. Both IHH and SHH are possible candidate genes for ENS defects in humans, but given the widespread use of this signaling system in the craniofacial region, CNS, and limb buds, these may be part of much more complex abnormalities.

Neurotrophin signaling systems Over the past 50 years, members of the neurotrophin (NT) family of neurotrophic factors (nerve growth factor, BDNF, NT-3, and NT4/5) have been shown to play a prominent role in the differentiation, growth, and survival of many parts of the nervous system, including sympathetic neurons and dorsal root ganglion neurons. The actions of neurotrophins are mediated largely through Trk receptor tyrosine kinases. Although all neurotrophins, except NT4/5, and all three Trk receptors (TrkA, TrkB, and TrkC) are present in the developing ENS of some species, including humans [184], there is no evidence that neurotrophins play a role in early ENS development, apart from NT-3. A number of in vitro studies have shown that NT-3 promotes the differentiation of ENS precursors immunoselected from the embryonic gut [107,185], and it also promotes neurite outgrowth and neuron differentiation in dissociated ganglia from postnatal rats [186]. Furthermore, mice lacking NT-3 or its receptor TrkC have reduced numbers of both myenteric and submucosal neurons, and mice overexpressing NT-3 have increased numbers of myenteric, but not submucosal, neurons [187]. It therefore seems likely that NT-3 is required for the development of subpopulations of enteric neurons [187].

Transcription factors Transcription factors are proteins that regulate the expression of genes by binding to regulatory ele-

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ments of DNA, thereby promoting or repressing transcription of the gene itself. Transcription factors alone often bind to DNA sequences relatively nonspecifically, but normally they combine with other transcription factors to form a molecular complex that targets the regulatory elements of specific genes or specific sets of genes. Phox2b The genes encoding Phox2a and Phox2b are very similar, but are unlinked in both human and mouse genomes [188]. The gene encoding PHOX2a is located on human chromosome 11q13, and that of PHOX2b, on chromosome 4p12 [188]. Phox2a and Phox2b are paired box homeodomain transcription factors expressed by many differentiating autonomic neurons as well as some cranial ganglia and hindbrain nuclei [189 –191]. Both proteins are expressed by all noradrenaline-producing neurons, but they are also expressed by some non-noradrenergic neurons, including enteric neurons and parasympathetic neurons [190,191]. The Phox2a gene, but not Phox2b, appears to be a downstream target of another transcription factor, Mash1 [192,193] [see Mash1 (Mammalian Achaete-Suite Homologue 1), below], but both Phox2a and Phox2b regulate the expression of Ret (part of the receptor for GDNE; see The Ret Gene and Protein, above) and the expression of the noradrenaline synthetic enzymes, tyrosine hydroxylase and dopamine ␤-hydroxylase [21,193,194]. Phox2a⫺/⫺ mice have no obvious defects in their ENS, but Phox2b⫺/⫺ mice have no ENS in any region of the gastrointestinal tract [21]. In fact, Phox2b⫺/⫺ mice completely lack a peripheral autonomic nervous system. In Phox2b knockout mice, neural crest cells arrive at the foregut, but fail to migrate further, probably because of the absence of expression of Ret [21]. To date, there have been no published studies linking mutations in PHOX2a or PHOX2b with defects in humans. Sox10 (dominant megacolon gene) The genes of the large Sox (Sry-box) family code for transcription factors defined by a common sequence termed the high mobility group (HMG) box (for review see [195]). This is central to the DNA binding capacity of the genes of this family. Despite all Sox genes possessing a conserved HMG domain, each has a set of unique, downstream

gene targets, judging from the range of processes (from sex determination to skeletal growth) that are disrupted when various Sox genes are knocked out. This specificity of DNA recognition is related to the Sox protein acting as part of a transcriptional complex. As with other transcription factors, the identification of the genes whose activity the Sox proteins control has been very difficult. In addition, Sox genes are turned on at specific times in development in specific cell types, and therefore they have precise upstream regulators, but these are also mostly not known. There are four types of Waardenburg syndrome (WS), which show a range of developmental defects in cells and structures of neural and neural crest lineage. Mutations in SOX10 (human chromosome 22q13) can cause Waardenburg-Shah syndrome (WS4 [196,197]) in humans, and dominant megacolon in mice [20,198,199], both with defects in neural crest– derived melanocytes and ENS cells. WS4 is also caused by mutations in ET-3 and ETB genes. Other Waardenburg syndrome variants can be caused by mutation of genes for two other transcription factors, microphthalmiaassociated transcription factor (MITF) in WS2 [200] and paired box 3 (Pax3; see section below) in WS1 and WS3 [201]. There is also one report of a patient with a heterozygous frameshift mutation in SOX10, who suffers from chronic intestinal pseudo-obstruction and deafness and has a peripheral neuropathy associated with peripheral hypomyelination; she does not, however, have any pigment abnormalities [202]. Biopsies of the colon revealed that nerve cell bodies and fibers were present in both the myenteric and submucosal plexuses [202], but it is unknown whether specific subpopulations of enteric neurons are missing or whether enteric neurons and glial cells are present at normal numbers. In the neural crest– derived Schwann cell lineage, Sox10 positively regulates the promoter of the gene for myelin protein zero (P0) [203], a major protein in peripheral myelin. It was therefore suggested that many of the abnormalities in the patient with the heterozygous frameshift mutation in SOX10, such as the peripheral neuropathy, may result from defects in P0 protein [202]. It is unclear how defects in P0 protein could cause chronic intestinal pseudo-obstruction as intrinsic enteric neurons

are not myelinated, and although some vagal preganglionic fibres are myelinated, bilateral vagotomy has not been reported to cause intestinal pseudo-obstruction. As well as positively regulating the promoter of the P0 gene, recent molecular analysis suggests that Sox10 also dramatically activates the MITF promoter and that this is increased further by Pax3 protein [204,205]. Likewise, Sox10 and Pax3 also activate Ret [173], which is of central importance for ENS formation. Like Ret⫺/⫺ mice, vagal neural crest– derived cells also die just prior to entering the foregut in mice lacking Sox10 [20,199]. Sox10 is expressed in migrating neural crest cells and their derivatives in human [206], mouse [20], and chick [207]. It is first expressed in birds following the expression of the zinc-finger transcription factor Slug, but it is not known whether Slug protein activates Sox10. Slug is one of the genes instrumental in instigating the migration of neural crest cells [208]. Pax3 Pax3 is a member of the paired-box-containing family of transcription factors [209]. Patients with WS without HSCR usually have mutations in the PAX3 gene [210,211]. Affected individuals are heterozygous for mutations in PAX3, but homozygous loss of function of PAX3 is lethal [212]. In mice, Pax3 is expressed by many neural crest– derived cells, including enteric neuron and melanocyte precursors [173,209]. Mice that are heterozygous for Pax3 mutations are characterized by a white belly spot (Splotch phenotype) and have no reported ENS defects, but homozygous loss-of-function mice die during midgestation with neural tube and cardiac defects, and an absence of enteric neurons caudal to the stomach [173]. It appears that Pax3 is essential for the initiation of Ret expression by enteric neuron precursors (Fig. 2), and thus there is no expression of Ret caudal to the stomach in Pax3⫺/⫺ mice [173]. Mash1 (mammalian achaete-scute homologue 1) Mash1 encodes a transcription factor that belongs to the basic helix-loop-helix (bHLH) family. In mice, it is expressed transiently during embryogenesis in the CNS and in many neural crest– derived cells, including those that colonize the gut [213– 215]. Mash1 is a mammalian homologue of the ENTERIC NERVOUS SYSTEM

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achaete-scute complex of Drosophila, which is involved in neurogenesis in both the central and peripheral nervous systems [216]. Mash1⫺/⫺ mice die within 48 h of birth and they lack sympathetic neurons and enteric neurons in the esophagus [22]. Nerve fibers that mediate the relaxation of the lower esophageal sphincter are also absent, but the extrinsic innervation of the esophagus, arising from the brain stem, is present in the knockout mice [217]. Enteric neurons are present in the stomach and small and large intestines, although some types of neurons, for example, the serotonincontaining neurons, are absent in these regions [218]. During the development of Mash1⫺/⫺ mice, neural crest cells migrate to their correct locations, but fail to differentiate into neurons [22]. It appears, therefore, that Mash1 is required for the differentiation of neuronal precursors [219], but is not required for neural crest cell migration. Mash1⫺/⫺ mice have a complementary phenotype to Ret⫺/⫺, gdnf⫺/⫺, and gfr␣1⫺/⫺ mice (see The GDNF–RET–GFR␣1 signaling system, above), in that mice lacking Mash1 lack enteric neurons in the esophagus only, whereas mice lacking members of the GDNF signaling pathway possess neurons in the esophagus, but lack neurons in the small and large intestines. The regional differences between the phenotypes is surprising since there is evidence that Mash1 and Ret belong to the same signaling cascade [192,193] (Fig. 2), and it also appears that most or all enteric neuron precursors, regardless of gut region, express both Mash1 and Ret [88,214]. It has been proposed [96] that the regional differences in the presence of neurons in the different knockout mice may be related to the different rostrocaudal level of origin of esophageal neurons from enteric neurons that are present in the more caudal regions of the gastrointestinal tract (see [24]). A human homologue of Mash1, which is 95% homologous to rat Mash1, has been isolated and termed HASH1 (human achaete-scute homologue 1) [220]. HASH1 is expressed by sympathetic neuron precursors early in human embryonic development (weeks 6 –7) [221], and in fetal pulmonary neuroendocrine cells [222]. It is also highly expressed in tumors of neuroendocrine cells such as medullary thyroid cancer (MTC), small cell lung cancer, and pheochromocytomas [223], and ap-

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pears to be necessary for progression of lung epithelial cells through a neuroendocrine differentiation pathway characteristic of small cell lung cancer [222]. Interestingly, the HASH1 gene contains multiple, repeated copies of the trinucleotide CAG, which is unique to the human gene and is not conserved in the rodent homologue [220]. Repeats of the triplet CAG can exhibit meiotic instability, such as in the FMR-1 gene in fragile X syndrome [224] and in the human androgen receptor in Kennedy’s disease [225]. However, to date, structural alterations in the HASH1 gene have not been linked to any human disease. In lacking nerve fibers in the lower esophageal sphincter, Mash1⫺/⫺ mice show features similar to those of humans with achalasia of the esophagus. Achalasia is a disorder in which the lower esophageal sphincter does not relax in response to the advance of a food bolus, and it is characterized by a dearth of nerve fibers in the muscle of the sphincter; extrinsic nerve fibers do not appear to be affected [226,227]. Achalasia is primarily manifest in adults. Since Mash1 is expressed only transiently during embryonic development and appears to be required for neuronal differentiation, it is very unlikely that defects in Mash1 function underlie achalasia of the esophagus. Hox11L1 The Hox11 family (Hox11, Hox11L1, Hox11L2) consists of genes containing the 180 base pair homeobox, which characterizes them as transcription factors. They are expressed in non-overlapping ways in the developing nervous system and elsewhere in the mouse embryo [174]. The HOX11L1 gene resides on human chromosome 2p13.1 [64] and is expressed in the neurons of the developing ENS and in other neural crest– derived neurons. Mice with mutation of Hox11L1 gene (also known as Tlx2, Enx, and Ncx) develop an INDB-like condition—that is, megacolon with ENS hyperplasia in the colon and hypoplasia in the ileum [62,63], followed by death of some of these neurons [62]. These defects of the development and maintenance of the ENS are not likely to be secondary to other abnormalities, since the gene is expressed in the neurons of the ENS [63]. However, at present, although some of the regulatory

elements for this gene have been defined [228], there is no information on the genes that regulate it nor on the transcriptional targets of HOX11L1. Such genes would be candidates for involvement in INDB in humans. SIP1 Some HSCR patients who also suffer from microencephaly, submucous cleft palate, and short stature have been recently shown to have mutations in SIP1 at chromosome 2q22 [229,230]. The defects are thought to result from haploinsufficiency of SIP1 caused by null mutations in one allele. Studies in mice have shown that Sip1 is a Smad1-, 2-, 3-, and 5-interacting protein [231] that is a member of the ␦EF1/Zfh-1 family of zinc finger/homeodomain proteins. The Smad proteins are central for the transduction of TGF-␤ (including Bmp-4) signals to the nucleus [232]. Experiments in several systems suggest that Sip1 is a repressor of transcription, and can oppose the transcriptional activation produced via the Smad pathway [233]. To our knowledge, there have been no reports of the phenotype of mice lacking this protein, and the pathway from SIP1 mutation to HSCR is at present obscure.

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