THEMES
Lessons From Genetically Engineered Animal Models
II. Disorders of enteric neuronal development: insights from transgenic mice*

Michael D. Gershon

Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York 10032


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Understanding the development of congenital defects of the enteric nervous system, such as Hirschsprung's disease, was, until recently, an intractable problem. The analysis of transgenic mice, however, has now led to the discovery of a number of genetic abnormalities that give rise to aganglionic congenital megacolon or neuronal intestinal dysplasia. The identification of the responsible genes has enabled the developmental actions of their protein products to be investigated, which, in turn, has made it possible to determine the causes of aganglionoses. Two models of pathogenesis have emerged. One, associated with mutations in genes encoding endothelin-3 or its receptor, endothelin B, posits the premature differentiation of migrating neural crest-derived progenitors, causing the precursor pool to become depleted before the bowel has been fully colonized. The second, associated with mutations in genes encoding glial cell line-derived neurotrophic factor (GDNF), its preferred receptor GFRalpha 1, or their signaling component, Ret, appears to deprive a GDNF-dependent common progenitor of adequate support and/or mitogenic drive. In both cases, the terminal bowel becomes aganglionic when the number of colonizing neuronal precursors is inadequate.

Hirschsprung's disease; endothelin-3; endothelin B receptor; glial cell-derived neurotrophic factor; GFRalpha 1; Ret; aganglionosis; neural crest; neuronal development


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INTRACTABILITY IN biomedical problems is rarely permanent. In fact, the perception of intractability frequently seems remarkably quaint when yesterday's insoluble issue becomes today's discovery and tomorrow's classic observation. Tractability in biomedical problems is often a simple matter of technology. The questions themselves are not so difficult, but the tools that are needed to solve them are not available. Once a critical technical advance provides these tools, many previously insoluble problems get solved. The development of transgenic mice is an example of the kind of technical breakthrough that converts intractable problems into new insights. The ability to create novel animals, almost at will, that carry extra copies or no copies of the gene of one's choice, or which express a gene at particular times and places during development, is extraordinarily powerful. Transgenic mice are also becoming a routine component of a developmental biologist's experimental repertoire. The standard workup of virtually any newly discovered mammalian gene now includes, in addition to determining its sequence and expression pattern, overexpression and knockout in transgenic mice. Transgenic mice have also begun to bring tractability to problems of the bowel.

Hirschsprung's disease, or congenital megacolon, is a vexing condition that nicely illustrates how useful transgenic mice can be. Hirschsprung's disease is neither rare nor benign. It occurs with an incidence of 1 in 5,000 births and is a cause of significant pediatric morbidity and mortality (30). Although Hirschsprung's disease can be treated, treatment is surgical, with concomitant risks and an outcome that is not always ideal. In a superficial sense, it is not difficult to understand why the gut behaves as it does in Hirschsprung's disease. A variable length of the terminal colon in affected individuals is congenitally deprived of ganglia. This segment of bowel acts as an obstructive lesion so that the gut proximal to the aganglionic zone dilates, sometimes massively. A hypoganglionic region usually borders the aganglionic segment, and both have to be removed to relieve the megacolon.

The bowel is the only organ that is able to control its own behavior in the absence of input from the brain or spinal cord (12). The gut can do this because its intrinsic innervation, the enteric nervous system (ENS), contains microcircuits that include all of the components of reflex arcs, including not only the motoneurons that excite or inhibit gastrointestinal effectors but also intrinsic sensory receptors, primary afferent, and interneurons. When this neural apparatus is missing, as it is in Hirschsprung's disease (30), pseuodobstruction occurs. Megacolon thus arises because normal motility requires an intact ENS, and the ENS is not intact in the Hirschsprung's gut. The important issue, however, is not to explain why the bowel dilates proximal to a segment that is aganglionic but why the gut becomes aganglionic in the first place. Before the advent of transgenic mice, that question was as intractable as any question in biology or medicine.

In retrospect, it seems clear that the first real progress made in understanding the pathogenesis of Hirschsprung's disease came from the discovery of two strains of mutant mouse in which congenital megacolon arises. The chance discovery of these mice provided the fortunate early investigators with, what were in essence, naturally occurring transgenic animals before the development of the transgenic technology. These particular strains, "lethal spotted" (ls/ls) (1, 27, 33) and "piebald lethal" (sl/sl) (16, 18), are born with an aganglionic terminal bowel that is remarkably similar to that of patients with Hirschsprung's disease (22). Because the ls/ls mice survive longer than the sl/sl animals, much more work has been done on the ls/ls animals. Homozygous ls/ls mice can be mated, assuring that all of the offspring exhibit the trait. This makes it possible to examine the development of the ENS in fetal ls/ls animals at stages that precede recognizable manifestations of the disease and enabled experiments to be carried out even before the genetic defect responsible for the condition was known.

Two different hypotheses emerged from early analyses of the development of the ENS in the ls/ls gut. One, which we can call "tardy migration," postulated that the neural precursors from the vagal region of the neural crest migrate too slowly (33). This theory held that there is a critical and limited time period available for crest-derived cells to complete their migration to the end of the bowel. If the cells do not finish the job within the allotted time, the remaining gut becomes refractory to colonization. The terminal bowel, which is last to be colonized by vagal precursors migrating proximodistally within the gut, thus remains devoid of neuronal precursors. The assumption, now known to be wrong, was made that the ENS is formed by émigrés derived only from the vagal crest. The tardy migration concept does not take into account the more recent observations that the postumbilical bowel is colonized by cells from the sacral as well as the vagal crest (3). Sacral precursors migrate in a distal to proximal direction.

The second hypothesis posited that the terminal bowel of the ls/ls mouse is itself abnormal and cannot be colonized by crest-derived cells (27). This concept arose from experiments in which neuronal development was assessed in vitro in small explants of fetal bowel. Each of these explants is, in essence, an assay for neural precursors because neurons can develop in vitro only if their precursors are present in the bowel at the time of explantation. Because neurons never appeared in explants of the terminal gut of ls/ls mice, it was suggested that crest-derived precursors do not enter it. That suggestion was supported by coculture experiments that revealed that, although crest-derived cells from a variety of sources enter the terminal colon of wild-type mice, no source provides neuronal progenitors that enter the presumptive aganglionic gut of ls/ls animals (17). A second set of experiments revealed that quail crest-derived cells migrate into and through grafts of control mouse colon but are stopped from migrating when they reach a transplanted segment of ls/ls terminal bowel (28). Third, crest-derived cells with either an ls/ls (19, 28) or an sl/sl (18) genotype colonize the entire gut of aggregation chimeric mice.

The implication that the colon of ls/ls or sl/sl mice is abnormal was dramatically supported by studies with transgenic mice expressing bacterial beta -galactosidase (lacZ) under the control of the dopamine-beta -hydroxylase (DBH) promoter (7, 18, 19). This gene is expressed in a set of vagal crest-derived precursors of enteric neurons, probably because these cells are transiently catecholaminergic during development (2). Their selective expression of lacZ makes these cells readily demonstrable by a simple histochemical procedure. The transgenic labeling revealed that vagal crest-derived cells migrate normally through the proximal bowel as far as the cecum but that, when they reach the colon, the further progression of these cells becomes distinctly abnormal and ultimately ceases (7). A direct demonstration of an intrinsic defect in the ls/ls colon came with the demonstration that components of the extracellular matrix, including laminin, accumulate and are abnormally distributed in the aganglionic zone (23). Similar matrix abnormalities are found in human patients with Hirschsprung's disease (22).

Although the results of investigations of the naturally occurring murine models of Hirschsprung's disease were consistent with the idea that aganglionosis arises because crest-derived cells fail to colonize the terminal colon, they revealed very little about why that failure occurs. To get beyond mere speculation, it is necessary to know which gene(s) is involved and to understand the actions of the relevant gene products. Identification of the genetic defects seemed like an intractable problem, however, until an elegant and totally unexpected series of experiments was carried out with transgenic knockout mice. Congenital megacolon and coat spotting were found to develop in mice carrying either targeted disruptions of genes encoding endothelin-3 (ET-3) (1) or its preferred receptor, endothelin B (ETB) (16). These observations led to the discovery that ls/ls mice carry a mutation in the gene encoding ET-3 (1) and sl/sl mice carry a mutation in the gene encoding ETB (16). The same series of studies went on to reveal that a subset of patients with Hirschsprung's disease also carry ETB mutations (24). Since the original observations, ET-3 mutations have been found in humans with congenital megacolon (Shah-Waardenburg syndrome) (9). ETB mutations, moreover, have been shown to give rise to megacolon in the spotting lethal rat (10) and the lethal white foal (D. Cass and M. Yanagisawa, personal communication). As would be predicted from knowledge of the results of deleting ET-3, targeted mutations in endothelin-converting enzyme-1, which converts the inactive big ET-3 to the active ET-3 peptide, cause the terminal bowel to become aganglionic (35). The association of ET-3/ETB defects with congenital megacolon thus seems to occur with some frequency in a variety of mammals.

The discovery that genes encoding ET-3 or ETB are mutated in animals and humans with congenital megacolon did not itself reveal the function of ET-3/ETB in the development of the ENS, nor did it reveal why the colon becomes aganglionic when ETB stimulation is lacking. Nevertheless, ET-3, which had not previously even been suspected of playing a role in ENS development, was firmly established as a major participant, and the effect of ET-3/ETB on enteric neuronal development suddenly became a high-priority target of investigation. As a result, the accepted concept of how the ENS and melanocyte development is regulated has permanently been changed.

The original hypothesis, prompted by the observations that defects in ET-3 or ETB caused aganglionosis and coat spotting, was that ET-3 is an autocrine growth factor essential for the development of enteric neurons and melanocytes (1). This concept, however, implies that the defect in the ENS is neural crest autonomous, which evidently is not the case (see above). The autocrine hypothesis, moreover, was offered before the identity of the cells that express ET-3 and ETB was known. ET-3 turned out not to be produced by crest-derived cells but by their mesenchymal neighbors (21). ETB, moreover, is expressed not only by crest-derived cells but also by cells in the smooth muscle lineage (34). ET-3 is thus not an autocrine growth factor. Surprisingly, it is also not essential for the development of survival of enteric neurons (14, 34), however counterintuitive that conclusion may be.

Although ET-3 is a potent mitogen for primary crest cells (20), it does not cause the postmigratory crest-derived cells within the gut wall to proliferate (34). Crest-derived cells immunoselected from within the bowel survive and develop as neurons perfectly well in the absence of ET-3 and even in the presence of the ETB inhibitor, BQ-788. ET-3 actually exerts a concentration-dependent inhibitory effect on enteric neuronal development (14, 34). At the same time, ET-3 promotes the development of smooth muscle in cultures depleted of crest-derived cells by negative immunoselection and downregulates their expression of the alpha 1-subunit of laminin-1 (34).

Certainly, ET-3/ETB is a critical factor in enabling the bowel to become colonized by cells from the neural crest. Any remaining doubts about how critical a role ET-3 plays were removed by extremely clever experiments in which the ability of the DBH promoter to direct expression of transgenes to developing enteric neurons was harnessed to "rescue" the ENS from aganglionosis in ET-3-deficient animals (R. Kapur, personal communication) or ETB-deficient animals (11). In essence, these experiments put the missing gene back into the gut, establishing that the bowel is indeed the site of action of ET-3 in the prevention of aganglionosis. This site of action has been directly confirmed by in vitro experiments in which crest-derived cells from the proximal colon or cocultured explants of small intestine have been "enabled" to colonize explants of the ET-3-deficient (ls/ls) terminal bowel by adding ET-3 to the culture medium (34).

It seems likely that the key to understanding why ET-3/ETB defects lead to aganglionosis in the terminal colon is the observation that ET-3 inhibits the genesis of enteric neurons. Crest-derived neuronal precursors migrate but enteric neurons do not; therefore, if the pool of migrating precursors is depleted by the premature differentiation of enteric neurons, then the last part of the bowel to be colonized will never receive its aliquot of crest-derived cells and will become aganglionic. The expression of ETB by cells in the smooth muscle lineage and the consequent ability of ET-3 to promote smooth muscle differentiation (34) are likely to exaggerate this effect. As smooth muscle cells differentiate, they evidently become less secretory and express less laminin-1; thus, when ET-3 is deficient, smooth muscle development is retarded and laminin-1 accumulates in the bowel (23). Laminin-1 promotes the development of enteric neurons through its alpha 1-subunit (6). Loss of ET-3/ETB, therefore, both removes a restraint on premature neuronal development (the direct effect of ET-3 on crest-derived neuronal precursors) and adds an accelerant (the indirect effect of laminin-1 accumulation). As the public relations people emphasize repeatedly, timing is everything. For the ENS to be completed, enteric neurons must make their appearance on schedule and not before. A debut made too soon may deplete the supply of progenitors, causing the distal bowel to become aganglionic.

Transgenic mice have helped to make clear that more than one class of genetic defect and mechanism can cause enteric neuronal development to fail and lead to aganglionosis. In fact, the first mutation found to be associated with Hirschsprung's disease was not in the gene encoding ETB but in RET (4). RET encodes a receptor tyrosine kinase that is the transducing element responsible for the actions of glial cell line-derived neurotrophic factor (GDNF) (25). The causal relationship of these mutations to the aganglionosis of Hirschsprung's disease was immediately suspected because studies with transgenic knockout mice had already demonstrated that c-ret expression is essential for the development of all enteric neurons below the level of the esophagus and the immediately adjacent stomach (8).

Ret is actually not the receptor for GDNF. It is the common signaling component utilized by four known members of a ligand family that includes GDNF, neurturin (NTN), persephin, and artemin (25). These ligands bind to one of four glycosyl-phosphatidyl inositol-linked proteins, GFRalpha 1-GFRalpha 4, that enable a Ret-activating functional complex to form (Fig. 1). In the absence of Ret, each GFRalpha binds only to one ligand, but, in its presence, they interact with two or three. Mice that lack GDNF acquire essentially the same defect in the ENS as mice that lack Ret, suggesting that GFRalpha 1 is the actual binding receptor for GDNF in vivo and is the physiological receptor responsible for Ret activation. Because GFRalpha 1 is a receptor that can interact with a number of the GDNF family of ligands (Fig. 1), the question arises as to what roles the other GFRalpha receptors play. It now appears, on the basis of information derived from studies of mice lacking GFRalpha 2, that GFRalpha 2 is essential for the survival not like GFRalpha 1 of all enteric neurons but of a limited subset (26). The ENS of knockout mice lacking NTN is virtually identical to that of animals lacking GFRalpha 2 (15), indicating that NTN is the physiological ligand for GFRalpha 2 (25) and functions as such in the development of the ENS (15). The limited nature of the defects in the ENS of GFRalpha 2- or NTN-deficient mice is paralleled by a much more limited number of enteric neurons that bind GFRalpha 2 than GFRalpha 1 (5). GFRalpha 1 binds in vitro to almost all crest-derived cells immunoselected from the fetal gut that are developing along a neuronal lineage.


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Fig. 1.   The 4 ligands of the glial cell line-derived neurotrophic factor (GDNF) family [GDNF, neurturin (NTN), artemin (ART), and persephin (PSP)] interact with 4 glycosyl-phosphatidyl inositol linked proteins, GFRalpha 1, GFRalpha 2, GFRalpha 3, and GFRalpha 4, respectively, to form complexes that can interact with Ret. Dotted lines show interactions between proteins that occur in the presence of Ret. Dashed line depicts an interaction for which only binding has been measured (modified after Ref. 25).

GDNF is a strong mitogen for early crest-derived cells of the avian or mammalian bowel (5, 14). In contrast, as the neurons mature, GDNF loses its mitogenic action but still promotes the development and/or survival of enteric neurons. Interestingly, GDNF does not appear to promote the development of glia when it is applied to relatively pure populations of enteric crest-derived cells after its mitogenic period (5). In contrast, GDNF does promote the development of glia in unselected mixed populations of cells from the fetal murine bowel (15). It is thus conceivable that GDNF exerts an indirect effect on glial development, possibly by causing another cell to secrete a factor that causes glial progenitors to respond.

The difference between the actions of ET-3/ETB on the one hand and GDNF/GFRalpha 1/Ret on the other suggests that at least two different mechanisms can give rise to aganglionosis of the terminal bowel. In the case of ET-3/ETB, as noted above, it seems likely that the problem is one of regulating the timing of the differentiation of enteric neurons. When ET-3/ETB is deficient, enteric neuronal precursors lose control of themselves and develop as neurons before they have finished colonizing the bowel (34). In contrast, the loss of GDNF/GFRalpha 1/Ret appears to deprive an early progenitor of critical support and a mitogenic drive (5). This early GDNF-dependent progenitor is evidently responsible for the development of all enteric neurons except those of the esophagus and immediately adjacent stomach (8). The GDNF-dependent progenitor ultimately gives rise to a heterogeneous set of sublineages with different growth factor dependencies (2). One of these sublineages, as discussed above, is evidently dependent on NTN/GFRalpha 2 (15, 26). In mice, the complete loss of GDNF/GFRalpha 1/Ret seems to be necessary for the development of aganglionosis; moreover, the aganglionosis, when it occurs, involves the entire intestine and most of the stomach. In contrast, although the aganglionosis-associated RETs in humans also involve a loss of function, they are heterozygous and probably leave some residual RET activity (4). As a result, the aganglionosis is geographically limited to the terminal bowel, although RET mutations are likely to be found in individuals with long-segment Hirschsprung's disease. Given the known actions of GDNF, it seems reasonable to propose that the terminal aganglionosis in individuals with mutations of GDNF, GFRalpha 1, or RET results from an inadequate mitogenic expansion of both the vagal- and sacral crest-derived progenitor populations and perhaps also from the death of a subset of the GDNF-dependent precursors of enteric neurons and glia. Depletion of the enteric neuronal precursor pool because of cell death, insufficient proliferation, or premature differentiation would all seem likely to lead to varying degrees of the same result, aganglionosis of the terminal bowel.

Further insights about the development of the ENS and its congenital defects have come from studies of transgenic mice. Additional genes that give rise to aganglionosis have been identified, including sox10 (which causes a dominant form of megacolon to develop in mice) (31) and, of equal significance, the condition known as intestinal neuronal dysplasia, which has been shown to be a real entity, mimicked by a targeted mutation in mice (13, 29). Until the transgenic studies were carried out, the diagnosis of intestinal neuronal dysplasia was controversial because it cannot, like Hirschsprung's disease, be simply diagnosed. It requires a quantitative diagnosis. The observation that neuronal intestinal dysplasia occurs reproducibly in mice carrying a targeted mutation in a member of the Hox11 homeobox gene family (known variously as Enx, Hox11L.1, or Ncx) strongly suggests that the disease is real and provides an experimental model of the human condition (13, 29). Congenital megacolon also occurs in transgenic mice that overexpress hoxa-4 (32), although, in this case, the problem is not typically intestinal neuronal dysplasia but one in which the terminal colon is hypoganglionic and contains ganglia with the structure of extraenteric peripheral nerves.

Transgenic mice have provided a cornucopia of discoveries that have revolutionized the study of the development of the ENS. The number of genes that are known to influence ENS development is already large, and it is growing. Probably the greatest benefit of research on transgenic and knockout animals is yet to be realized. That benefit will be to help sort out real diseases of the ENS, those with genetic bases, from indirect actions of other nonenteric conditions that indirectly cause gastrointestinal malfunction. It is difficult to underestimate how exciting a transgenic model of the irritable bowel syndrome would be.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institute of Neurological Disorders and Stroke Grants NS-12969 and NS-15547.


    FOOTNOTES

* Second in a series of invited articles on Lessons From Genetically Engineered Animal Models.

Address for reprint requests and other correspondence: M. D. Gershon, Dept. of Anatomy & Cell Biology, Columbia University College of Physicians and Surgeons, 630 West 168th St., New York, NY 10032.


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Am J Physiol Gastroint Liver Physiol 277(2):G262-G267
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society