Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
themes Neural Injury, Repair, and Adaptation in the GI Tract V. Genes, lineages, and tissue interactions in the development of the enteric nervous system* MICHAEL D. GERSHON Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York 10032 growth factors; glial cell line-derived neurotrophic factor; Ret; endothelin-3; endothelin B; serotonin; Hirschsprung’s disease EVER SINCE BAYLISS AND Starling published the results of their pioneering studies of motility in dog intestine (see Ref. 13 for references), it has been (or should have been) clear to anyone who thought about the innervation of the bowel that the enteric nervous system (ENS) cannot be like any other part of the peripheral nervous system (PNS). Bayliss and Starling’s experiments, dramatically confirmed 18 years later by Trendelenburg, who demonstrated that the peristaltic reflex could actually be elicited in vitro (a situation in which the brain, spinal cord, dorsal root, and cranial ganglia have all been discarded), established that the bowel can manifest neurally mediated reflex activity in the absence of input from the central nervous system (CNS). No other component of the PNS can act similarly, that is, as an independent center of integrated neuronal * Fifth in a series of invited articles on Neural Injury, Repair, and Adaptation in the GI Tract. activity. To carry out its unique ‘‘brainlike’’ functions, the ENS acquires, during its development, complex microcircuits, which include not only the excitatory and inhibitory motoneurons that innervate smooth muscle, glands, and blood vessels but intrinsic primary afferent neurons and interneurons as well. The structure of the ENS is as unusual as is its functional capability. Instead of Schwann cells, enteric neurons are supported by glia, which resemble astrocytes, and enteric neurons live in a milieu free of collagen, just like their CNS counterparts. The neurons found in enteric ganglia also exhibit an extensive array of neurotransmitters and neuromodulators, in abundance exceeding those found in the ganglia of any extraenteric portion of the PNS. In both ultrastructural and neurochemical senses, therefore, the ENS is as brainlike as it is in its ability to serve as an independent center of integrative neural activity. The special nature of the ENS implies that the story of its development cannot be a banal recapitulation of the ontogeny of other sets of peripheral ganglia. Clearly, something has to occur during embryonic or fetal life that sets the ENS apart from the other divisions of the PNS. Given the resemblance of the ENS to the CNS, it is reasonable that lessons learned in analyzing ENS development may prove to be more applicable to understanding the ontogeny of the brain than similar studies of admittedly simpler autonomic relay ganglia. This potential for the provision of insight relevant to the brain has accounted, in part, for a recent flowering of investigations into ENS development. The use of the ENS as a model nervous system, however, does not fully account for the recent burgeoning of enthusiasm for enteric developmental neurobiology. An additional factor has been the unwitting recruitment to gastrointestinal research of investigators who have been surprised to find major defects in the ENS following the knockout of murine genes not previously known to affect the gut, such as those encoding endothelin-3 (ET-3) or its receptor, endothelin B (ETB ) (1, 15). The obvious clinical significance of ENS development has also acted as a catalyst for basic research on this subject. Congenital neuromuscular disorders of the bowel are both common and serious. Understanding 0193-1857/98 $5.00 Copyright r 1998 the American Physiological Society G869 Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on June 17, 2017 Gershon, Michael D. Neural Injury, Repair, and Adaptation in the GI Tract. V. Genes, lineages, and tissue interactions in the development of the enteric nervous system. Am. J. Physiol. 275 (Gastrointest. Liver Physiol. 38): G869–G873, 1998.—The enteric nervous system is derived from the vagal, rostral-truncal, and sacral levels of the neural crest. Because the crest-derived population that colonizes the bowel contains multipotent cells, terminal differentiation occurs in the gut and is influenced by both the enteric microenvironment and the responsivity of multiple lineages of precursors. Enteric growth factor-receptor combinations, which promote the development of enteric neurons and/or glia in most of the gastrointestinal (GI) tract, include glial cell line-derived neurotrophic factor-GFRa-1-Ret, NT-3-TrkC, a still-to-be-identified neuropoietic cytokine-ciliary neurotrophic factor receptor-a, serotonin (5-HT)-5-HT2B, and LBP110, a 110-kDa laminin-1 binding protein. A qualitatively different effect is shown by the peptide-receptor combination ET-3-ETB, which inhibits neuronal differentiation and appears to prevent the premature differentiation of enteric neurons before colonization of the GI tract has been completed (resulting in aganglionosis of the terminal colon). G870 ENTERIC NERVOUS SYSTEM DEVELOPMENT: GENES AND LINEAGES tify specific molecules in the wall of the gut that influence the development of enteric neurons and/or glia. Historically, the first molecule found to affect the development of enteric neurons and glia was a neurotrophin. Although the development of the ENS had clearly been shown to be independent of nerve growth factor, the more recently discovered neurotrophin-3 (NT-3) turned out to be a potent and specific promoter of the development of both enteric neurons and glia (4). Moreover, cells in the fetal bowel were found to express TrkC, the high-affinity receptor for NT-3, and these cells were found to be crest derived. Overexpression of NT-3, targeted by the dopamine-b-hydroxylase promoter to developing enteric neurons (see Refs. 17 and 18 for references), causes an increase in the size of developing ganglia in the myenteric plexus of transgenic mice as well as an increase in the size of the neurons they contain (unpublished data). The ENS, however, is relatively normal in the bowel of mice following the knockout of NT-3 (10), suggesting either that compensatory mechanisms can substitute for the loss of NT-3 during development or, more likely, that NT-3 affects the development of only a relatively small subset of enteric neurons or glia. More recently, stimulation by glial cell line-derived neurotrophic factor (GDNF) has been demonstrated to be an absolute requirement for the survival of the vagal and sacral crest-derived cells that colonize the gut. If either GDNF (20, 27) or its signaling receptor, Ret (26), are knocked out in developing mice, the gut becomes totally aganglionic below the level of the rostral foregut. ENS development, therefore, completely fails in the vagal and sacral domains of the bowel and persists only in the small region of the gut that is colonized by cells from the truncal crest. GDNF, as one would expect, has been found to be a potent promoter of neuronal development in vitro (6, 14). Early in development [through embryonic day 12 (E12) in rats], GDNF acts as a mitogen (6), greatly expanding the numbers of enteric crest-derived neural precursors, but, later, GDNF loses its ability to promote proliferation and acts only as a growth-differentiation factor, supporting enteric neuronal but not glial development. In addition to GDNF, which is supplied to developing crest-derived precursors by the mesodermally derived cells of the enteric mesenchyme, the developing gut contains GFRa-1, a peripheral glycosylphosphoinositol-anchored molecule that binds GDNF and is necessary for the activation of Ret (30). mRNA encoding GFRa-1 is found both in crest- and non-crest-derived cells of the enteric mesenchyme, but GFRa-1 immunoreactivity can be demonstrated only on crest-derived cells (6). These observations suggest that GFRa-1 may be produced by both crest- and non-crest-derived cells in the wall of the gut, but only cells from the neural crest anchor it to their plasma membranes (perhaps in a complex with Ret). If so, then the gut may have evolved a fail-safe mechanism for the provision of adequate amounts of GFRa-1, which, as much as GDNF, is critical for the survival of the crest-derived cells that colonize most of the bowel. GFRa-1 can be produced by the Retexpressing cells that require it (an interaction called ‘‘in Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on June 17, 2017 their pathogenesis and devising means to prevent them are thus important investigative goals. The unique nature of the function, structure, and chemistry of the ENS is not matched by the source of its neural and glial precursors. These cells, like the majority of other peripheral neurons and Schwann cells, are the descendants of émigrés from the neural crest (19). To be sure, only three axial levels of the crest have been shown to contribute precursor cells to the ENS. These include the vagal (19), rostral-truncal (8), and sacral (19, 22, 28) levels. The vagal crest is the most significant of the three because it colonizes the entire gut. In contrast, the rostral-truncal crest colonizes only the esophagus and adjacent stomach, whereas the sacral crest is restricted in its colonizing territory to the postumbilical bowel. One might imagine that ENS precursors could be derived from specific regions of the crest because premigratory cells in these zones are already predetermined to ‘‘find’’ the gut and differentiate as enteric neurons or glia. That expectation, however, has turned out not to be the case. Regions of the premigratory crest can be interchanged in avian embryos without interfering with the formation of an ENS. For example, cells from axial levels of the crest that do not (if left undisturbed) migrate to the bowel have been demonstrated to colonize the gut and form an ostensibly normal ENS when they are transplanted to the vagal region of a host embryo (19). The populations of premigratory and migrating crest cells, moreover, contain precursors that are multipotent (see Ref. 12 for references). Even more surprising, the population of crest-derived cells destined to colonize the bowel still contains pluripotent precursor cells. These observations imply that 1) the gut wall is itself a critical site where terminal differentiation of enteric neurons and glia occurs and 2) the enteric microenvironment has an opportunity to play a vital role in determining what kind of nervous system arises within the bowel. Work on the role of the enteric microenvironment in determining the fates of crest-derived cells was greatly enhanced by the development of an effective means (immunoselection) of isolating crest-derived cells from the developing bowel (21). Immunoselection and the subsequent culture of the isolated enteric crest-derived cells in defined media make it possible to determine the direct effects of putative growth factors on the precursors of neurons and glia. Immunoselection of enteric crest-derived cells also provides relatively pure populations of such cells for the analysis of their receptors, transcription factors, or other developmentally relevant molecules. Isolation of crest-derived precursors and culture in defined media is needed because experiments carried out with mixed populations of cells, or with cells cultured in serum-containing media, cannot be interpreted. The analyses of data obtained under those conditions are confounded by the uncontrolled interactions crest-derived cells may have with their non-crest-derived neighbors and by the potential effects of unknown substances present in complex media. The immunoisolation of crest-derived cells from the fetal enteric mesenchyme has now made it possible to iden- ENTERIC NERVOUS SYSTEM DEVELOPMENT: GENES AND LINEAGES enteric neurons can be detected in the mouse foregut as early as E12, new neurons continue to be added at least through the first 3 wk of postnatal life (see Ref. 12 for references). Early and late-developing neurons, however, are not the same kind of cell. Although serotonergic neurons are all born very early (before E15), the first calcitonin gene-related peptide-containing neurons do not begin to be born until after the last serotonergic neuron has become postmitotic. Because of their precocious appearance, enteric serotonergic neurons coexist in primordial enteric ganglia with still-dividing neural precursors. In fact, electron micrographs have even revealed that synapses are present on the surfaces of dividing neuroblasts. For this reason, it has long been speculated that serotonin (5-HT) might not just be a neurotransmitter but, also in the primitive ENS, might be a growth factor that affects the development of late-arising enteric neurons. Recent observations have provided considerable evidence that this may well be the case (11). The 5-HT2B receptor has been found to be developmentally regulated in the fetal bowel. 5-HT2B expression can first be detected in the fetal mouse gut at E14. It peaks at E15–E16, when 5-HT2B expression can be detected in virtually all myenteric ganglia, and declines to adult levels (expression in fewer than one neuron in every four ganglia) by E18. When added to cells isolated at E15, the peak time of 5-HT2B expression, 5-HT strongly promotes the in vitro development of enteric neurons. This effect is blocked by the nonselective 5-HT2 antagonist, ritanserin, and mimicked by the 5-HT2 agonist, 2,5-dimethoxy-O-iodoamphetamine. The selective 5-HT2A antagonist, ketanserin, does not inhibit the 5-HT-induced development of enteric neurons. 5-HT appears to act by stimulating the phosphorylation and consequent nuclear translocation of mitogen-activated protein (MAP) kinase. The effect of 5-HT on MAP kinase in enteric neural precursors is also blocked by ritanserin. These observations imply that 5-HT can indeed act as a growth factor as well as a conventional neurotransmitter and that its developmental action is mediated by the transient and developmentally regulated expression of 5-HT2B receptors. An exciting potential implication of the discovery that 5-HT is a growth factor is that it might explain how the early experience-related activity of the ENS can sculpt its subsequent development. Put another way, the observation might provide a molecular basis for taking seriously, and understanding, the often-told anecdotes that ‘‘colicky’’ babies grow up to be adults with irritable bowel syndrome. All of the growth-differentiation factors discussed to this point have in common that they are generally active agents. That is, they affect virtually the entire bowel. Another type of agent has recently been discovered. The peptide, ET-3, and its cognate receptor, ETB, have been found to play a critical role in ENS development (1, 15, 23); however, ET-3 is uniquely important, not to the development of ganglia in the whole bowel but to gangliogenesis in the terminal colon. The terminal colon is the sole region of the gut that becomes aganglionic when ET-3 or ETB are knocked out in mice or mutated in humans. In humans, this condition, Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on June 17, 2017 cis’’) or by neighboring cells that by secreting the molecule also make it available to be anchored to the membranes of the crest-derived cells that express Ret (an interaction called ‘‘in trans’’) (30). Thus far, GDNF has been the only growth factor observed to be globally required for the development of all enteric neurons and glia. Other growth factors appear to be more like NT-3 in that they are needed only for the development and/or survival of restricted subsets of enteric neurons and/or glia (2, 12). In fact, the initial GDNF-dependent precursor that colonizes the bowel gives rise to multiple lineages of crestderived successors that can be defined by their requirements for particular growth or transcription factors. For example, the targeted mutation of mash-1, a proneural gene that is the mammalian homologue of the achaete-scute complex of Drosophila, causes the esophagus to become aganglionic and leads also to the loss of about one-third of the neurons in the remainder of the bowel. The truncal crest, therefore, which as noted above is Ret independent (8), is mash-1 dependent. The Ret-dependent vagal and sacral crest-derived cells, which fail to develop in mash-1 (2/2) animals, comprise a set of neural precursors, which are all transiently catecholaminergic and give rise to the earliest born of enteric neurons. This set includes all of the serotonergic neurons of the gut [which are missing in mash-1 (2/2) mice] and probably also excitatory and inhibitory motoneurons. In contrast, the neurons that are mash-1 independent are never catecholaminergic and are not born until relatively late in ontogeny. All enteric neurons that contain calcitonin gene-related peptide are members of this set. Additional enteric neuronal precursor lineages can be defined by growth factors required only by still smaller subsets of the neurons that develop from mash-1-dependent precursors. For example, enteric motoneurons appear to be derived from precursors in the mash-1 lineage that require stimulation of a still-tobe-identified neuropoietic cytokine that interacts with the a-component of the ciliary neurotrophic factor receptor (CNTFRa) (see Ref. 5 for references). The circular muscle layer lacks both nitric oxide synthase (NOS)- and substance P-immunoreactive nerve fibers when either the a or the b component (leukemia inhibitory factor receptor-b) of the CNTFR is knocked out in transgenic mice. Substance P is a marker for excitatory and NOS for inhibitory motoneurons. Because the knockouts of CNTF or leukemia inhibitory factor do not themselves produce the lethal effects of deletion of the neuropoietic cytokine receptor, it is apparent that neither is the endogenous ligand that is critical for the development of enteric neurons. Both CNTF and leukemia inhibitory factor stimulate the Janus kinase-signal transducer and activator of transcription signal transduction pathway and promote the development in vitro of neurons and glia from crestderived cells immunoisolated from the fetal gut. The effects of these factors on neuronal development are additive with those of NT-3. The addition of enteric neurons to the developing bowel persists for a surprisingly long time. Although G871 G872 ENTERIC NERVOUS SYSTEM DEVELOPMENT: GENES AND LINEAGES The observations on the effects of ET-3 on enteric nerve and muscle development suggest that ET-3 might not affect the precursors of enteric neurons as a conventional growth factor but might act instead as a regulator of the timing of enteric neuronal development. The function of ET-3 might thus be to prevent the premature differentiation of neurons. When ET-3-ETB is lacking, conditions favor neuronal differentiation. A brake, ET-3, is missing and an accelerator, laminin-1, is present in excess. The last part of the bowel to be colonized is the terminal colon (19). Clearly, differentiation of neurons must be prevented from depleting the precursor pool before the entire bowel has been colonized. Crest-derived cells are motile and migratory; neurons are not. In addition, crest-derived cells multiply as they migrate, constantly expanding the precursor pool. Differentiation of neurons thus brings this expansion, as well as crest-derived cell migration, to a complete halt because neurons are postmitotic cells. To colonize the terminal bowel, vagal crest-derived cells have to migrate all the way down the gut, and sacral crest-derived cells, which do not start to migrate until long after the departure of their vagal counterparts from the neural crest, have to reach the colon. The ET-3-ETB deficiency-induced premature differentiation of neurons would thus be predicted to leave the terminal colon uncolonized and thus aganglionic. In fact, ectopic ganglia are found outside the terminal colon of ET-3-deficient mice (24). These ganglia are probably produced by sacral crest-derived cells that have differentiated and stopped migrating before their time. Hirschsprung’s disease is often associated with mutations in genes encoding GDNF-Ret (3, 16) or ET-3-ETB (9, 23). Mutations in Ret and ETB are much more frequently encountered than mutations in the two ligands. Because Ret and ETB are so fundamentally different in their actions on developing enteric neurons, it seems likely that, from the viewpoint of pathogenesis, there is not one Hirschsprung’s disease but at least two forms of the condition. The final phenotype of each, congenital megacolon, is the same because the gut has only one way to manifest aganglionosis; nevertheless, two different mechanisms can be envisioned to cause the terminal colon to become aganglionic. One of these might be the result of a deficiency of GDNF-Ret that is not so severe as to cause the entire bowel to become aganglionic (as it is in GDNF or Ret knockout mice). Such a defect might lead to a precursor pool of crest-derived cells that is too small to colonize the whole gut. Whereas the complete loss of GDNF-Ret causes neurogenesis to fail totally in the entire bowel (below the rostral foregut), a submaximal deficiency of GDNF-Ret might cause the crest-derived precursor pool to expand insufficiently, so that the numbers of available cells produced from this pool are inadequate. The ability of GDNF to serve as a mitogen for crestderived enteric neuronal precursors supports this idea (6). This mechanism for causing the terminal colon to become aganglionic is very different from that postulated to result from a deficiency of ET-3-ETB. In that case, as explained above, crest-derived cells are thought to differentiate prematurely, causing crest-derived pre- Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on June 17, 2017 known as Hirschsprung’s disease or congenital megacolon, occurs in about 1 in 5,000 live births. Strains of mice also carry natural mutations in genes encoding ET-3 (lethal spotted, ls/ls) and ETB (piebald lethal sl/sl ). When the relationship of ET-3-ETB to aganglionosis was first discovered, ET-3 was postulated to be an autocrine growth factor essential for the development of enteric neurons and melanocytes (mice with ET-3 or ETB defects are spotted). Unfortunately, this hypothesis, which treats ET-3 like any of the other growth factors that affect ENS development, does not account for the striking geographical limitation of the lesion that results from the loss of ET-3-ETB. Clearly, if ET-3-ETB were really essential for the development of enteric neurons (as a set), these cells would not be able to develop normally in the proximal bowel of ET-3-ETBdeficient individuals. The resulting disease would be more global, like that which results from deletions of GDNF or Ret, not a lesion that is restricted to the terminal colon. Another problem with the autocrine growth factor hypothesis for the action of ET-3 is that the aganglionosis that results from ET-3-ETB mutations is not neural crest autonomous. For example, although crest-derived cells from exogenous sources can enter the normal mouse colon in vitro, no source of crest-derived cells can provide émigrés that enter the colons of ET-3-deficient mice (see Ref. 24 for references). Moreover, when segments of mouse gut are back transplanted between the neural tube and somites of quail embryos, quail crest-derived cells cannot enter the presumptive aganglionic bowel of ET-3-deficient mouse donors (25). In contrast, quail crest-derived cells have no difficulty in entering and passing through segments of normal mouse bowel. Finally, crest-derived cells that are genotypically ET-3- or ETB-defective will colonize the terminal bowel of aggregation chimeric mice as long as substantial numbers of the surrounding cells are normal (17, 18, 25). That means that the gut wall, as well as the crest-derived cells themselves, is probably abnormal in ET-3-ETB-defective individuals. Recent studies have suggested that the role of ET-3ETB is not to promote but to inhibit enteric neuronal development (14, 29). Certainly, that is the effect of ET-3 and ETB agonists on crest-derived cells immunoselected from the fetal mouse gut (29). The ability of ET-3 to inhibit the in vitro development of enteric neurons can be blocked by BQ-788, a specific ETB antagonist. In contrast to its effect on the development of enteric neurons, ET-3 enhances the development of smooth muscle in cultures depleted of crest-derived cells by negative immunoselection. By promoting smooth muscle development and maturation, ET-3 downregulates the secretion of laminin by smooth muscle precursors. This effect is important because laminin-1, through an interaction of its a1-subunit with a receptor [110-kDa laminin-1 binding protein (LBP110)] expressed by crest-derived cells, is itself a powerful promoter of enteric neuronal development (7). Furthermore, laminin-1 biosynthesis is upregulated and laminin-1 accumulates in the terminal bowel of ET-3deficient mice (24). ENTERIC NERVOUS SYSTEM DEVELOPMENT: GENES AND LINEAGES cursors to cease dividing and migrating before the gut has been entirely colonized. As far as the terminal colon is concerned, the result is the same: it becomes aganglionic because the terminal colon is the last part of the bowel to be colonized by cells from the neural crest. In summary, the development of the ENS can be understood as a symphony, complete with point and counterpoint. The point in the enteric music is the genetic background and lineages of crest-derived precursor cells. This theme plays out against the counterpoint of the effects of the microenvironment of the bowel wall. The result is the ‘‘multicultural’’ mélange of the mature ENS, an entity with neurons that displays extraordinary phenotypic diversity, complex microcircuits, and a brainlike ability to function independently of CNS control. REFERENCES 1. Baynash, A. G., K. Hosoda, A. Giaid, J. A. Richardson, N. Emoto, R. E. Hammer, and M. Yanagisawa. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 79: 1277–1285, 1994. 2. Blaugrund, E., T. D. Pham, V. M. Tennyson, L. Lo, L. Sommer, D. J. Anderson, and M. D. Gershon. Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers, and Mash-1dependence. Development 122: 309–320, 1996. 3. Carlomagno, F., G. De Vita, M. T. Berlingieri, V. de Franciscis, R. M. Melillo, V. Colantuoni, M. H. Kraus, P. P. Di Fiore, A. Fusco, and M. Santoro. Molecular heterogeneity of RET loss of function in Hirschsprung’s disease. EMBO J. 15: 2717–2725, 1996. 4. Chalazonitis, A., T. P. Rothman, J. Chen, F. Lamballe, M. Barbacid, and M. D. Gershon. Neurotrophin-3 induces neural crest-derived cells from fetal rat gut to develop in vitro as neurons or glia. J. Neurosci. 14: 6571–6584, 1994. 5. Chalazonitis, A., T. P. Rothman, J. Chen, E. N. Vinson, A. J. MacLennan, and M. D. Gershon. Promotion of the development of enteric neurons and glia by neuropoietic cytokines: interactions with neurotrophin-3. Dev. Biol. 198: 343–365, 1998. 6. Chalazonitis, A., T. P. Rothman, and M. D. Gershon. Ageand cell type-dependence of the responses of crest-derived cells immunoselected from the fetal rat gut to GDNF and NT-3 (Abstract). Neuroscience 23: 1430, 1997. 7. Chalazonitis, A., V. M. Tennyson, M. C. Kibbey, T. P. Rothman, and M. D. Gershon. The a-1 subunit of laminin-1 promotes the development of neurons by interacting with LBP110 expressed by neural crest-derived cells immunoselected from the fetal mouse gut. J. Neurobiol. 33: 118–138, 1997. 8. Durbec, P. L., L. B. Larsson-Blomberg, A. Schuchardt, F. Costantini, and V. Pachnis. Common origin and developmental dependence on c-ret of subsets of enteric and sympathetic neuroblasts. Development 122: 349–358, 1996. 9. Edery, P., T. Attie, J. Amiel, A. Pelet, C. Eng, R. M. Hofstra, H. Martelli, C. Bidaud, A. Munnich, and S. Lyonnet. Mutation of the endothelin-3 gene in the Waardenburg-Hirschsprung disease (Shah-Waardenburg syndrome). Nat. Genet. 12: 442– 444, 1996. 10. Fariñas, I., K. R. Jones, C. Backus, X.-Y. Wang, and L. F. Reichardt. Severe sensory and sympathetic deficits in mice lacking neurotrophin-3. Nature 369: 658–661, 1994. 11. Fiorica-Howells, E., L. Maroteaux, and M. D. Gershon. 5-HT2B receptors mediate the 5-HT-induced development of enteric neurons. Neuroscience 23: 1686, 1997. 12. Gershon, M. D. Genes and lineages in the formation of the enteric nervous system. Curr. Opin. Neurobiol. 7: 101–109, 1997. 13. Gershon, M. D., A. L. Kirchgessner, and P. R. Wade. Functional anatomy of the enteric nervous system. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 381–422. 14. Hearn, C. J., M. Murphy, and D. Newgreen. GDNF and ET-3 differentially modulate the numbers of avian enteric neural crest cells and enteric neurons in vitro. Dev. Biol. 197: 93–105, 1998. 15. Hosoda, K., R. E. Hammer, J. A. Richardson, A. G. Baynash, J. C. Cheung, A. Giaid, and M. Yanagisawa. Targeted and natural (piebald-lethal) mutation of endothelin-B receptor produce megacolon associated with spotted coat color in mice. Cell 79: 1267–1276, 1994. 16. Ivanchuk, S. M., S. M. Myers, C. Eng, and L. M. Mulligan. De novo mutation of GDNF, ligand for the RET/GDNF-alpha receptor complex in Hirschsprung’s disease. Hum. Mol. Genet. 5: 2020–2026, 1996. 17. Kapur, R. P., D. A. Sweetser, B. Doggett, J. R. Siebert, and R. D. Palmiter. Intercellular signals downstream of endothelin receptor-B mediate colonization of the large intestine by enteric neuroblasts. Development 121: 3787–3795, 1995. 18. Kapur, R. P., C. Yost, and R. D. Palmiter. Aggregation chimeras demonstrate that the primary defect responsible for aganglionic megacolon in lethal spotted mice is not neuroblast autonomous. Development 117: 993–999, 1993. 19. Le Douarin, N. M. The Neural Crest. Cambridge, UK: Cambridge University Press, 1982. 20. Pichel, J. G., L. Shen, H. Z. Sheng, A.-C. Granholm, J. Drago, A. Grinberg, E. J. Lee, S. B. Huang, M. Saarma, B. J. Hoffer, H. Sariola, and H. Westphal. Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 382: 73–76, 1996. 21. Pomeranz, H. D., T. P. Rothman, A. Chalazonitis, V. M. Tennyson, and M. D. Gershon. Neural crest-derived cells isolated from the gut by immunoselection develop neuronal and glial phenotypes when cultured on laminin. Dev. Biol. 156: 341–361, 1993. 22. Pomeranz, H. D., T. P. Rothman, and M. D. Gershon. Colonization of the post-umbilical bowel by cells derived from the sacral neural crest: direct tracing of cell migration using an intercalating probe and a replication-deficient retrovirus. Development 111: 647–655, 1991. 23. Puffenberger, E. G., K. Hosoda, S. S. Washington, K. Nakao, D. deWit, M. Yanagisawa, and A. Chakravarti. A missense mutation of the endothelin-receptor gene in mutagenic Hirschsprung’s disease. Cell 79: 1257–1266, 1994. 24. Rothman, T. P., J. Chen, M. J. Howard, F. D. Costantini, V. Pachnis, and M. D. Gershon. Increased expression of laminin-1 and collagen (IV) subunits in the aganglionic bowel of ls/ls, but not c-ret2/2 mice. Dev. Biol. 178: 498–513, 1996. 25. Rothman, T. P., D. Goldowitz, and M. D. Gershon. Inhibition of migration of neural crest-derived cells by the abnormal mesenchyme of the presumptive aganglionic bowel of ls/ls mice: analysis with aggregation and interspecies chimeras. Dev. Biol. 159: 559–573, 1993. 26. Schuchardt, A., V. D’Agati, L. Larsson-Blomberg, F. Costantini, and V. Pachnis. Defect in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 367: 380–383, 1994. 27. Sénchez, M., I. Silos-Santiago, J. Frisén, B. He, S. Lira, and M. Barbacid. Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 382: 70–73, 1996. 28. Serbedzija, G. N., S. Burgan, S. E. Fraser, and M. BronnerFraser. Vital dye labeling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos. Development 111: 857–866, 1991. 29. Wu, J. J., T. P. Rothman, and M. D. Gershon. Inhibition of enteric neuronal development by endothelin-3 (ET-3) (Abstract). Neuroscience 23: 24, 1997. 30. Yu, T., S. Scully, Y. Yu, G. M. Fox, S. Jing, and R. Zhou. Expression of GDNF family receptor components during development: implications in the mechanisms of interaction. J. Neurosci. 18: 4684–4696, 1998. Downloaded from http://ajpgi.physiology.org/ by 10.220.33.6 on June 17, 2017 This work was supported by National Institute of Neurological Disorders and Stroke Grants NS-12969 and NS-15547. Address reprint requests to M. D. Gershon. G873