Genes and lineages in the formation of the enteric nervous system

101

Genes and lineages in the formation of the enteric nervous system Michael D Gershon The enteric nervous system is large, complex, and independent of the CNS. Its neural-crest-derived precursors migrate along defined pathways to colonize the bowel. Recent studies of the sequential actions of essential growth and transcription factors have revealed that enteric neuronal development involves a complex interaction of lineage-determined

and microenvironmental

elements.

Addresses Department of Anatomy and Cell Biology, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA; e-mail: [email protected] Abbreviations CGRP calcitonin gene related peptide ciliary neurotrophic factor CNTF CNTFR CNTF receptor embryonic day E endothelin 3 EDN-3 EDNRB endothelin-B receptor enteric nervous system ENS glial cell line derived neurotrophic factor GDNF leukemia inhibitory factor LIF LIF receptor IJFR nerve growth factor NGF neurotrophin 3 NT-3 NT-415 neurotrophin 415 common neurotrophin receptor pW’TR peripheral nervous system PNS transient catecholaminergic (cell) TC receptor tyrosine kinase Trk

Current Opinion in Neurobiology

1997, 7:101-109

Electronic identifier: 0959-4388-007-00101 0 Current Biology Ltd ISSN 0959-4388

Introduction The enteric nervous system (ENS) is composed of a collection of autonomic ganglia and associated neural connectives in the wall of the bowel [1,2]. Even though the ENS is a component of the peripheral nervous system (PNS), it is unlike any other. In contrast to extra-enteric peripheral ganglia, myenteric ganglia lack collagen and receive their support not from Schwann cells but from asrrocyte-like enteric glia. In fact, the ultrastructure of the ENS resembles that of the CNS more than that of the rest of the PNS. A more striking peculiarity of the ENS, which distinguishes it from the other two autonomic divisions (the sympathetic and the parasympathetic), is that the majority of enteric neurons are not directly innervated by the brain or spinal cord. This unique independence of the

ENS enables the bowel to manifest reflex activity in the absence of CNS input. This ability is made possible by the presence in the gut of intrinsic sensory neurons and interneurons (as well as the moror neurons that excite or inhibit smooth muscle, glands, and blood vessels). The ENS, moreover, not only functions autonomously, but it can also influence other organs. Neurons within the gut project out of the bowel to innervate pre-vertebral sympathetic ganglia, as well as ganglia in the gall bladder and pancreas. The ENS is thus an independent nervous system that structurally resembles the brain. It is the only such system in vertebrates other than the CNS itself. The phenotypic diversity of its component neurons transcends that found in other ganglia and includes every class of neurotransmitter found in the CNS; moreover, the number of neurons in the ENS rivals that of the spinal cord and probably exceeds the combined total number of neurons in extra-enteric peripheral ganglia. The special nature of the ENS requires for its maturation the participation of a variety of early- and late-acting factors that may not be needed by simpler ganglia. As a result, the ENS has often unexpectedly been found to be defective in transgenic knock-out mice constructed for the study of other systems. The focus of this review will be on the roles played by microenvironmental factors, the lineages of progenitor cells, and the interaction between the two in enteric neuronal development.

The ENS is a derivative

of the neural crest

All enteric neurons and glia are the progeny of neural crest immigrants. The cellular imigris that give rise to the ENS migrate to the gut from three regions of the neural crest (Table 1; Figure 1). Two of these regions, vagal and sacral [3-91, have been appreciated for years, although the vagal source of enteric progenitors has recently been found to be much smaller than previously believed and is limited to the levels of somites 3-6 [lo]. The third, newly discovered, region lies within the truncal crest and colonizes only the rostra1 foregut (presumptive esophagus and cardiac stomach) [ 111. The sources of enteric neuronal precursors appear to be similar in avian and mammalian embryos [9,11]. Despite the restriction of enteric neuronal progenitors to specific regions of the crest, premigratory crest cells do not appear to be endowed with ‘homing’ information that enables them to ‘find’ their correct destination in the bowel. For example, truncal crest, which would not, if left undisturbed, contribute precursors to the bowel below the foregut, will do so if transplanted to the vagal or sacral regions of a recipient embryo [12]; moreover,

102

Development

Table 1 Lineage of the crest-derived ceils that form the ENS. Source of crest ceils

Regions colonized

Dependencies

Analogous lineage in other systems

Truncal

Rostra1 foregut: presumptive esophagus and cardiac stomach

Ret/GDNF-independent Mash-l-dependent

Caudai sympathetic ganglia: Ret-independent

Vagal

Caudai foregut, midgut, and hindgut Presumptive caudai stomach, small intestine, cecum, colon

RetIGDNF-dependent At least two lineages: one that is Mash-l-dependent one that is Mash-l-independent

Superior cervical ganglion: affected by RetlGDNF knock-out, but Mash-l-independent Thyroid parafollicuiar ceils: express, but do not depend on Mash-7

Sacrai

Post-umbilical bowel Presumptive ileum, cecum, colon

Ret/GDNF-dependent Mash-l-dependent?

7

within the host’s bowel, the donor cells will express phenotypes that are normally found in the ENS, but which may be inappropriate for the level of the neural crest from which the grafted precursors were obtained [l&14]. These observations suggest that crest-derived cells reach the gut because they migrate along defined pathways that lead to the bowel from particular levels of the neural crest. The studies also suggest that the fate of the population of crest cells that sets out to colonize the gut is not predetermined, but that crest-derived cells are influenced by the microenvironments of the migratory pathway and/or the bowel. Clones of crest cells [15-221, moreover, and even crest-derived cells that have entered the gut are multipotent [23,24”]. In fact, when crest-derived cells that have colonized the bowel are back-transplanted to younger embryos, they re-migrate in their new host [ZS]. The targets reached by the re-migrating donor crest-derived cells and their ultimate phenotype are determined by the location of the grafts and not by their having previously migrated to the gut [Z&26]. Donor crest-derived cells will not enter the host’s bowel unless the transplants replace the host’s vagal or sacral crest. Crest-derived cells, therefore, remain multipotent, even after they have colonized the gut, although they appear to have lost the ability to give rise to ectomesenchyme or melanocytes [25,26].

These data indicate that the signals provided by the microenvironment of the bowel are critical determinants of the phenotypes expressed by crest-derived cells within the gut. This conclusion has been supported by in vitn, experiments that have demonstrated directly that the wall of the bowel promotes the expression of ENS-appropriate phenotypes by crest-derived precursors [27,28]. To understand the formation and acquisition of the special properties of the ENS, it is therefore necessary to identify the molecular signals provided to crest-derived cells by the enteric microenvironment.

Molecules that affect neuronal development have been identified in the fetal gut Some of the developmental signals that are critical in the formation of enteric ganglia have been identified by studying natural and targeted mutations in mice (Table 2). One of the most dramatic (and probably early-acting) of these is a knock-out of glial cell line derived neurotrophic factor (GDNF) [29*,30’,31”] or of its functional receptor, Ret [32]. Ret is encoded by the c-ret proto-oncogene, which is transiently expressed by enteric neural precursors [24**,33]. When c-ret is knocked out, the bowel contains no neurons or glia caudal to the esophagus and cardiac stomach [ 11,321. The gut is similarly aganglionic when the gene encoding GDNF is knocked out [34”-370.1.

Interestingly, GDNF does not appear to bind directly to Ret, but to an associated ~1 protein that, when bound by the ligand, forms a complex with Ret [30*,31**]. Recent experiments (M Gershon, A Chalazonitis, TP Rothman, J Chen, unpublished data) have revealed that GDNF dramatically promotes the development of enteric neurons in vitro and that its expression is developmentally regulated.

In c-RV knock-out mice, the enteric neurons of the esophagus and cardiac stomach remain; however, the enteric neurons of the majority of the bowel are absent [ll]. The superior cervical ganglion resembles the caudal ENS in that it too fails to develop in c-rerl- mice; however, the more caudal sympathetic ganglia, like the ENS of the esophagus and cardiac stomach, are independent of Ret. These data have led to the suggestion that neurons of the rostra1 foregut are lineally related to the neurons of caudal sympathetic ganglia, whereas the neurons of the remainder of the bowel are lineally related to the superior cervical ganglion (Table 1). This hypothesis may be correct, at least in part, but the Ret-dependent lineage of the neurons

Genes and lineages in the formation

of the enteric

nervous system

Gershon

103

Figure 1

W

a) Pluripotent crest-derived progenitor cell

0

Truncal

Vagal

0

_

Rostra1

rl

foregut

es-’ _

_

_ . -’ 0,

-. - -1 a

RetlGDNFdependent

0

l

1 #//Ret/GDNF

Mash-l

-

dependent Sympathoenteric)

0

I!3

l

Post-umbrlrcal

\

bowel

5-HT neurons

Motor neurons

?

UI\

CGRP-containing neurons

0 1997 Current Opinion I” Neurobiologj

The formation of the ENS from crest-derived precursors. Premigratory crest cells are thought to be pluripotent. Precursors of the ENS migrate to the gut from vagal, truncal, and sacral crest. Vagal-crest-derived cells give rise to multiple progenitor lineages and colonize the entire bowel caudal to the rostra1 foregut. (a) The vagal crest family tree. A common progenitor requires activation of the Ret receptor tyrosine kinase by GDNF. As a result, when either Ret or GDNF are knocked out (line l), no enteric neurons develop in the gut below the rostra1 foregut. The common progenitor gives rise to at least two successor lineages, one of which, the sympathoenteric, is derived from a progenitor that must express Mash-l. Cells that develop from this progenitor are ablated when Mash-7 is knocked out (line 2), whereas the Mash-f-independent cells persist. A later lineage has been recognized

by knock-outs

of CNTFRu

(line 3), which eliminates motor neurons. Cells in this group

have been postulated (without, as yet, direct evidence) to be in the Mash-l-dependent superlineage because of the early birthdates of motor neurons. (b) The truncal crest colonizes only the rostra1 foregut. Cells in this lineage differ from vagal-crest-derived cells in that they are independent of RetlGDNF; however, they evidently must express Mash-7 as neurons of the rostra1 foregut do not develop in Mash-l-lmice. The sacral-crest-derived cells ascend in the bowel and join precursors of vagal crest origin to colonize the post-umbilical bowel. As the region colonized by the sacral crest is without neurons in Ret knock-out mice, the sacral crest is probably dependent or EDNRB

are lost (line 4), no enteric neuronal progenitors

on Ret/GDNF

migrate into the terminal colon. This defect is not crest-autonomous

other factors, such as the excess of laminin that develops in the abnormal bowel, that combine with the loss of EDN-3 migration of neural precursor into the presumptive aganglionic bowel. 5-HT, 5-hydroxytryptamine.

When

EDN-3

and involves

stimulation to prevent the

that populate the ENS caudal to the rostra1 foregut itself comprises more than a single lineage of precursor cells.

thoadrenal cells, and thyroid parafollicular cells, each of which is derived from neural crest [39,40,41*].

The ENS is formed by multiple lineages of crest-derived precursor cells

Targeted mutations of Mash-2 result in the near-total loss of sympathetic neurons (except for the superior cervical ganglion) and what was originally thought to be delayed development of the ENS [42]. What actually occurs in the bowel, however, is not a developmental delay, but the complete loss of the early-developing subset of enteric neurons (43**]. This absence creates an apparent delay that is followed by the development, on time, of late-developing subsets of enteric neurons.

An abnormality of the ENS that is less dramatic than that seen in GDNF or Ret knock-out mice, but no less lethal, occurs in mice with a targeted mutation in the Mash-2 gene (Table 2). Mash-I encodes a basic helix-loop-helix transcription factor and is the mammalian homologue of adaete-sscute in IkosopWa [38-40]. During development, Mad-f is expressed by enteric neural precursors, sympa-

104

Development

Table 2 Gene knock-outs and the development of the ENS. Gene/product

Defect(s) after knock-out

c-ret

Complete failure of enteric neurons and glia to develop in the entire bowel below the rostra1 foregut

Encodes Ret, a receptor tyrosine kinase expressed by crest-derived cells that colonize the gut Ret is the functional receptor for GDNF

GDNF

Similar to knock-out of c-ret

Growth factor essential for development and/or survival of early crest-derived precursors

Mash-l

Aganglionosis of the rostra1 foregut (i.e. esophagus and cardiac stomach) Absence of the early-born TC lineage of enteric neurons in the rest of the bowel

Encodes a transcription factor required by the precursors of serotonergic, but not CGRP-containing, neurons

CNTFRa or LIFRP

Loss of neurons that express substance P or nitric oxide synthase (smooth muscle motor neurons)

Newborn lethal, not mimicked by knock-out of genes encoding the ligands, CNTF or LIF

Endothelin 3 (edn-3)

Aganglionosis of the terminal colon Spotted coat color

Selectively activates the EDNRB Naturally mutated in lethal spotted (/s//s) mice Effects are not crest-autonomous

Endothelin B receptor (ednrb)

Aganglionosis of the terminal colon and spotted coat color similar to that seen in edn-3 knock-out, but even more severe

Activated with equal potency by endothelins 1, 2, and 3 Naturally mutated in piebald lethal (s7/s1) mice

Comments

Knock-outs with global effects

Knock-outs with limited effects

Effects expected, but not yet detected NT-3

Expressed in ENS, but not yet studied in detail Causes hyperganglionosis when overexpressed in the gut of transgenic mice

Selectively promotes development and survival of enteric neurons and glia Stimulates neurite extension The preferred ligand for TrkC

TrkC

Expressed in ENS, but not yet studied in detail

The only high affinity neurotrophin receptor expressed by crest-derived neural and glial precursors in the developing bowel

Mm/i-Z expression

is developmentally regulated in enteric neurons and sympathoadrenal cells [39,40], but is permanent in parafollicular cells, which are also lost in Mash-l-lmice [41*]. Parafollicular cells, like some enteric neurons, share an origin in the vagal crest and are serotonergic [44]. In fact, parafollicular cells neuralize when they are exposed to nerve growth factor (NGF) in vifm or co-cultured with gut; they actually join the ENS when co-cultured with explants of bowel. The cells in the gut that express Mash-l resemble sympathetic neurons in that both are catecholaminergic, although the Ma&l-positive enteric cells are catecholaminergic only transiently [43”]. Both sympathetic neurons and transient catecholaminergic (TC) cells also transiently express the same differentiation antigens that are recognized by monoclonal antibodies SA, and then BZ, at the same times during development [43”,4547]. These observations led to the proposal that enteric neurons are derived from the sympathoadrenal lineage [46]; however, given the above discussion of Ret/GDNFand Mash-Z-defined lineages, this hypothesis cannot be entirely correct.

The neurons of the rostra1 foregut are different from their caudal counterparts, in that the rostra1 enteric neurons are independent of Ret and dependent on Mash-l, whereas caudal enteric neurons, like the superior cervical ganglion, are not totally eliminated by a Mar/l-Z knock-out, but are ablated when Ret is mutated [ll]. Two lineages have thus been proposed, a sympathoenteric lineage, which gives rise to the majority of the ENS and the superior cervical ganglion, and a sympathoadrenal lineage, which forms the caudal sympathetic nervous system, the adrenal medulla, and the ENS of the rostra1 foregut. Actually, even the existence of the sympathoenteric lineage for the caudal gut has now been shown to be only partially correct, in that a subset of enteric neurons (and possibly parafollicular cells), rather than the whole set, appears to be derived from a common sympathoenteric progenitor. The neuronal precursors in the crest-derived population that initially colonizes the bowel (in mice, at embryonic days E9-ElO; in rats, at ElO-Eli) [48,49] have been shown to be TC cells that express p7SNTR, the common neurotrophin receptor [SO’].

Genes and lineages in the formation of the enteric nervous system Gershon

Although TC cells share with mature sympathetic neurons the expression of every known characteristic of the catecholaminergic phenotype [Sl-561, including neuronal markers (e.g. neurofilament proteins and peripherin) [48,49], they proliferate. TC cells, therefore, cannot be neurons, which are postmitotic [48,49,51,57]. Rather, they are neural precursors that disappear because their progeny develop as neurons that are not catecholaminergic. The effects of the absence of TC cells have been studied using two approaches. One approach has been to examine ENS development in the gut of M&z-l knock-out mice in which TC cells fail to appear [43”]. The other has been to ablate rat TC cells in vitro by complement-mediated lysis using B2 antibodies. In neither approach does the complete elimination of the entire TC/M&-l-dependent cell population prevent the development of enteric neurons. The set of enteric neurons that develop after TC cells are ablated, however, is not the same as that which arises when TC cells are intact: enteric serotonergic neurons are absent, whereas neurons that contain calcitonin gene related peptide (CGRP) are present. These two markers identify neurons with very different birthdates. The sequence in which enteric neurons are born reflects their phenotype [58]. Serotonergic neurons are among the earliest-born of enteric neurons (ES.%E14); some of these neurons are already postmitotic before crest-derived cells begin to colonize the bowel (E9) [59]. In contrast, the precursors of CGRP-containing neurons do not start to become postmitotic until two days after the birth of the last serotonergic neuron. The ENS, therefore, must be derived from at least two precursor lineages. One of these could be considered sympathoenteric. This lineage is comprised of early-born neurons that are derived from progenitors that express M&-Z and are dependent on Mm?-f. In the gut, these cells are only transiently catecholaminergic, whereas in sympathetic ganglia, catecholaminergic expression becomes permanent. All enteric serotonergic neurons (among other cells) and parafollicular cells are derived from precursors in this lineage. A second enteric lineage that also populates the bowel caudal to the rostra1 foregut is born late, is independent of Mask-l, is never catecholaminergic, and gives rise to CGRP-containing neurons (among other cells) [43”].

Neurotrophin 3, cytokines, and endothelin affect enteric neuronal development

3

GDNF is not the only growth factor that affects the development of enteric neurons (Table 2). Others have also been identified, and their effects were appreciated before those of GDNF were known. Probably the first growth factor demonstrated to promote the development of enteric neurons in vitro was neurotrophin 3 (NT-3) [60]. The experiments that demonstrated this effect utilized crest-derived cells immunoselected from the fetal rat

105

gut with NC-l/HNK-1 monoclonal antibodies [61]. This reagent, in fetal rats, as well as avian embryos, recognizes migrating crest-derived cells and those developing as neurons and glia [49,62]. Neurons and glia develop almost exclusively in cultures of immunoselected cells and almost none appear in cultures of residual cells, which are those that remain after the crest-derived cells have been removed by immunoselection [60,61]. Upon exposure to NT-3 in serum-free defined media, cells in the immunoselected, but not the residual cultures, transiently express the proto-oncogene c-fos. After 24 hours, c-fos is no longer expressed; however, in the continued presence of NT-3, the number of cells that exhibit neuronal or glial markers increases rapidly. This NT-3-induced increase in neurons and glia is not attributable to the proliferation of their precursors, because in pulse-labeling experiments, NT-3 does not increase the number of cells that take up bromodeoxyuridine. To increase the number of neurons in the cultures, NT-3 must act on postmitotic cells to promote their expression of neuronal characteristics and/or survival. NT-3 also enhances neurite outgrowth. In contrast to NT-3, other neurotrophins, including NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin 4/S (NT-4/S) do not affect the in vitro development of enteric neurons [60]. Promotion of enteric neuronal development by neurotrophins, therefore, is specific to NT-3. As would be predicted from studies of the in vitro efficacy of NT-3 in promoting enteric neuronal development, mRNA encoding TrkC (the receptor tyrosine kinase that is the primary receptor for NT-3) is expressed by the crest-derived cells that colonize the gut [60]. TrkC transcripts are highly enriched in the population of cells immunoselected from the fetal rat bowel with NC-l/HNK-1 or with antibodies to p7WTR, which is expressed only by crest-derived cells in the fetal rat gut [49]. TrkC transcripts have been detected in subsets of developing and adult enteric neurons by in situ hybridization [63*]. As TrkC mRNA is not expressed by all enteric neurons, it is likely that only some enteric neuronal precursors are sensitive to NT-3. The lineage of these precursors remains to be established; however, since sympathetic neurons respond to NT-3, enteric NT-3-sensitive cells may be dependent on M&z-2. Neither TrkA nor TrkB mRNA can be detected in either the adult or developing rat gut [63*], supporting the idea that NT-3 is the only neurotrophin that affects the development of the ENS. Although trkC is the only trk to be expressed by intrinsic components of the ENS, it seems likely that the extrinsic sympathetic neurons that grow into the bowel express TrkA. As axons lack ribosomes, they would contain the TrkA receptor protein, but not mRNA. In contrast to in situ hybridization, which labels only intrinsic enteric neurons, immunocytochemical studies that utilize a pan-Trk antibody would not be able to discern which immunoreactive neurites are intrinsic and which are extrinsic. TrkA on sympathetic axons would

106

Development

be difficult to distinguish from TrkC on intrinsic enteric neurites. This problem, however, would not extend to nerve cell bodies. Given that TrkC is the only Trk expressed in the fetal gut, any Trk immunoreactivity detectable in nerve cell bodies is probably TrkC. Enteric neurons in the fetal rat bowel become Trk immunoreactive as early as El4 [63*]. In the fetal human gut, TrkC immunoreactivity has been found on developing enteric neurons, whereas both TrkA and TrkB immunoreactivities have been reported to be present on both neurons and glia [64]. It is not clear why TrkA and TrkB should be expressed by intrinsic enteric neurons in humans and not in rats. There may be a true species difference in the neurotrophin requirements of developing enteric neurons; alternatively, antibodies thought to be human T&A- or TrkB-specific might also recognize TrkC. Enteric neurons develop in mice that lack NT-3 [65,66], TrkC [67] (Table Z), or p7SNTR [68]; however, the presence of enteric neurons does not establish that the ENS is normal in any of these knock-out animals. Although the crest-derived precursors that colonize the ENS are relatively pluripotent, a progressive decrease in the developmental potential of their successors occurs as a function of time during ontogeny [24**]. Cells become sorted into developmental lineages that can be defined by common requirements for growth/differentiation factors or expression of certain genes. Defects in the ENS are, thus, likely to be much more massive if they are attributable to the absence of factors or genes that are required earlier than later in ontogeny. For example, the total failure of the ENS to develop in the entire bowel below the rostra1 foregut in mice lacking GDNF/Ret probably occurs because early precursors are dependent on GDNF (Figure 1). The GDNF/Ret-dependent lineage is thus large and gives rise to smaller sublineages. The ENS defect in Mad-ZJmice, which involves the loss of neurons in the rostra1 foregut and the sympathoenteric subset of cells in the remainder of the bowel, is much smaller than that of mice lacking GDNF or Ret. Presumably, therefore, at least in the bowel below the rostra1 foregut, the Mar/z-Z-dependent set of cells is a later sublineage that arises from that defined by its dependence on GDNF/Ret. Since all enteric neurons in this region of the gut depend on GDNF/Ret, but only some depend on Mad-l, two separate lineages, Mash-l-dependent and Ma&Z-independent arise from precursors that require GDNF/Ret stimulation before they reach the age when some cells must express Mad-l. Cytokines appear to be needed for a still more limited sublineage of enteric neurons and are, therefore, probably required still later in development. Knock-out mice that do not express a or B components of the tripartite receptor for ciliary neurotrophic factor (CNTFRa or LIFRB) die at birth and appear to lack motor neurons to smooth

muscle [69”]. Enteric ganglia are present in these animals, and the defect, as detected by immunocytochemistry, is strategically devastating but affects few neurons. This has been demonstrated by the specific loss of motor neurons containing substance P and nitric oxide synthase that innervate intestinal smooth muscle (Table 2). The two cytokines that activate this receptor complex, ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF), promote the in v&o development of enteric neurons from crest-derived precursors immunoselected from the developing bowel with antibodies to p7SNTR (A Chalazonitis, TP Rothman, MD Gershon, Sot Neurosci Abstr 1995, 25:1545). Even though CNTF and LIF are each effective alone, their effect is much greater when they are given together with NT-3 (CNTF>LIF). Mice with targeted knock-outs of genes encoding CNTF [70] or LIF [69”], however, unlike those lacking CNTFRa, are viable (Table 2); therefore, it seems likely that the cytokine that acts on CNTFRa to affect enteric neuronal development is not CNTE This cytokine has not yet been identified, but it is not one of the other known members of the family of ligands for the tripartite cytokine receptor, such as oncostatin M or cardiotrophin-1 [69**]. A final factor that critically affects the development of only a limited subset of enteric neurons is endothelin 3 (EDN3). When this factor is knocked out either in transgenic mice [71] or as the result of a natural mutation, /edza/spotted (L+!s) (72-741, the ENS fails to develop in the terminal colon, which is innervated, but aganglionic (Table 2). The terminal colon becomes similarly aganglionic when the endothelin B receptor (EDNRB), for which EDN3 is a ligand, is deleted by gene targeting or natural mutation [72,75,76]. Considerable evidence suggests that both the EDN3 and the EDNRB mutations prevent migrating crest-derived cells from completing the task of colonizing the bowel [74,76-791. The effect of these mutations, and that of a similar dominant aganglionosis (Dom), which is not related to EDN3 or the EDNRB [80], is not crest-autonomous and may involve both the subset of crest cells that attempt to colonize the terminal colon and the extracellular matrix of this region of the gut [81*]. The terminal colon is probably the last region of the bowel to be colonized. The development of the ENS is thus likely to be affected far more dramatically by mutations (such as GDNF/Ret) that disrupt genes encodipg factors that are critical early in development and/or are required by the pluripotent precursors of multiple lineages of enteric neurons than by mutations (such as NT-3/TrkC, CNTFRa, LIFRB or EDN3/EDNRB) that affect the expression of factors expressed later and/or are required by more restricted sublineages of neurons or their precursors. The development of the ENS is, therefore, clearly not an all or none phenomenon. Investigators could easily be misled into thinking that knocking out a particular gene has no effect on the ENS because they detect

Genes and lineages in the formation of the enteric

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Sextier-Sainte-Claire Deville F, Ziller C, Le Douarin NM: Developmental potentialities of cells derived from the truncal neural crest in clonal culhrres. Dev Brain Res 1992, 66:1-l 0.

Papers of particular interest, published within the annual period of review, have been highlighted as: . l

1.

*

of special interest of outstanding interest Gershon MD, Kirchgessner AL, Wade PR: Functional anatomy of the enteric nervous system. In fhysiolo9y of the Gastrointestinal Tract, edn 3, vol 1. Edited by Johnson LR, Alpers DH, Jacobson ED, Walsh JH. New York: Raven Press; 1994:381-422.

2.

Fumess JB, Costa M: The Enteric Nervous System. New York: Churchill Livingstone; 198X

3.

Le Douarin NM, Teillet MA: The migration of neural crest cells to the wall of the digestive tract in avian embryo. J Embryo/ .&p Morpholl973, 30:31-40.

107

Le Douarin NM, Teillet MA: Experimental analysis of the mJq@iQtU3nrldi&Xer&a&Xdoa&KQt&%st%Q~tthe~~ ne?vous-sys&wrenbol?mur’~mm’ de&&t-. usiqp a bio@&cat QE# rna&qg tach&gue. Dev Bioc’ 1974, 4I:162-184.

Acknowledgements This work was supported by grant NS15547 from the National Institutes of Health.

Gershon

4.

stomach.

“The early crest-derived e’migre’s appear to be relatively pluripotent and their fate is influenced by microenvironmental factors encountered within the bowel itself; however, the developmental potential of enteric neuronal precursors decreases during ontogeny. Lineages can be recognized by a common dependence of members on particular growth/differentiation factors or genes that must be expressed. These lineages include a large one that is defined by a common dependence on the stimulation of the Ret receptor by GDNF. This lineage gives rise to all of the neurons of the bowel except those of the rostral’foregut. A second lineage is dependent on Mash-l. These cells are transiently catecholaminergic, born early, and give rise to the entire set of serotonergic. neurons. Ma&Z-dependent neurons include all of those of the rostra1 foregut and a subset of cells in the remainder of the bowel. A third lineage is independent of Mash-l; these neurons are never catecholaminergic, born late, and give rise to peptidergic neurons, such as those that contain CGRF? Other, more limited sublineages of enteric neurons are affected by, or dependent on, NT-3, EDN-3, and a still unknown cytokine ligand for CNTFRa. The development of the ENS can thus be thought of as a highly sophisticated symphony, in which the point of lineage dependency is played against the counterpoint of actions exerted by environmental factors.

nervous system

24. ..

Lo L, Anderson DJ: Postmigratory neural crest cells expressing c-RET display restricted developmental and proliferative capacities. Neuron 1995, 15:527-539. A fascinatmg paper that clearly demonstrates that the colonizing crest-derived cells that arrive in the mammalian bowel are not the same as the cells

106

Development

of the premigratory crest, in that the developmental potential of the gmigr& has been reduced: fewer options are open to them and they proliferate less. The authors take advantage of the transient expression of the Ret receptor by enteric neuronal progenitors early in ontogeny to immunoselect these cells and isolate them from their neighbors in the enteric mesenchyme. They use an elegant technique of clonal analysis to study the developmental potential of individual progenitors. 25.

Rothman TP, Le Douarin NM, Fontaine-P&us JC, Gershon MD: Developmental potential of neural crest-derived cells migrating from segments of developing quail bowel back-grafted into younger chick host embryos. Development 1990, 109:41 l-423.

26.

Rothman TP, Le Colonization of migrating from regions of host

27.

Coulter HD, Gershon MD, Rothman TP: Neural end glial phenotypic expression by neural crest cells in culture: effects of control and presumptive aganglionic bowel from /s//s mice. J Neurobiol 1966, 19:507-531.

28.

Douarin NM, Fontaine-P&us JC, Gershon MD: the bowel by neural crest-derived cells reforegut backtransplanted to vagal or sacral embryos. Dev Dyn 1993, 196:217-233.

Mackey HM, Payette RF, Gershon MD: Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches. Development 1966, 104:205-217.

29. .

Trupp M, Arenas E, Fainzilber M, Nilsson A-S, Sieber B-A, Grigoriou M, Kilkenny C, Salazar-Grueso E, Pachnis V, ArumPe U et a/.: Functional receptor for GDNF encoded by the c-ret proto-oncogene. Nature 1996, 381:765-769. _ One of an important series of papers [30’,31**,34”,37**1 that identlfles GDNF as the functional ligand of the Ret receptor, which had previously been an orphan receptor known to play a critical role in the formation of the ENS.

Treanor JJS, Goodman L, De Sauvage F, Stone DM, Poulsen KT, Beck CD, Gray C, Armanini MP, Pollock RA, Hefti F et al.: Characterization of a multicomponent receptor for GDNF. Nature 1996, 36260-63. An important paper that helped to establish that Ret is the functional receptor for GDNF. This report and the ones that follow [31**] show that activation of Ret by GDNF requires the interaction of multiple receptor components.

not only influences the development of the ENS, but its action is critical for the development of any ENS at all. 37. ..

Durbec P, Marcos-Gutierrez CV, Kilkenny C, Grigoriou M, Wartiowaara K, Suvanto P, Smith D, Ponder B, Costantini F, Saarma M et al: GDNF signalling through the Ret receptor tyrosine kinase. Nature 1996, 361:769-793. A very important study from the laboratory of Pachnis, who discovered the critical role played by Ret in the development of the ENS. This study clearly shows that GDNF is the physiological ligand responsible for activating Ret. 38.

Johnson JE, Birren SJ, Saito T, Anderson DJ: DNA binding and transcriptional regulatory activity of mammalian achaete-scute homologous (MASH) proteins revealed by interaction with a muscle-specific enhancer. Proc Nat/ Acad Sci USA 1992, 69:3596-3600.

39.

Lo L, Guillemot F, Joyner AL, Anderson DJ: MASH-I: a marker and a mutation for mammalian neural crest development. Perspect Dev Neurobioll994, 2:191-201.

40.

Guillemot F, Joyner AL: Dynamic expression of the murine echeete-scute homolog (MASH-l) in the developing nervous system. Mech Dev 1993,42:171-l 65.

41. .

Clark MS, Lanigan TM, Page NM, Russo AF: Induction of a serotonergic and neuronal phenotype in thyroid C-cells. J Neurosci 1995, 156167-6176. An excellent investigation and review of the literature that follows up the original observations of Barasch er al. (see [441), who were the first to show that the crest-derived parafollicular cells of the thyroid are capable of becoming neurons. This study both confirms those findings and documents the neuralization of the serotonergic parafollicular cells in molecular terms. The observations support the idea that parafollicular cells, which like enteric neurons express Mash-l, are members of a common developmental lineage. 42.

30. .

31. ..

Jing S, Wen D, Yu Y, Hoist PL, Luo Y, Fang M, Tamir R, Antonio L, Hu Z, Cupples R et a/.: GDNF-induced activation of the Ret protein tyrosine kinase is mediated by GDNFR-a. a novel receptor for GDNF. Cell 1996, 66:1113-l 124. Reveals that the receptor for GDNF is not, strictly speaking, the Ret receptor itself. GDNF evidently binds to an a component that, in turn, forms a complex with Ret. The multicomponent nature of this receptor is analogous to that for CNTF, where, again, there is an a component that actually combines the ligand and contributes to the active multicomponent receptor complex. 32.

Schuchardt A, D’Agati V, Larsson-Blomberg L, Costantini F, Pachnis V: Defect in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret. Nature 1994, 367:3ao-383.

33.

Pachnis V, Mankoo 8, Costantini F: Expression of the c-ret proto-oncogene during mouse embryogenesis. Development 1993,119:1005-1017.

34. ..

Moore MW, Klein RD, Farias I, Sauer H, Armanini M, Phillips H, Reichardt LF, Ryan AM, Carver-Moore K, Rosenthal A: Renal and neuronal abnormalities in mice lacking GDNF. Nature 1996, 362:76-79. Reports some of the first evidence that GDNF is vital for the development of the ENS and the kidneys. The observations reveal that the targeted deletion of the ligand, GDNF, induces a syndrome that is similar to the one previously characterized in knock-outs of the receptor, Ret. 35. ..

Pichel JG, Shen L, Sheng HZ, Granholm A-C, Drago J, Grinberg A, Lee El, Huang SB, Saarma M, Hoffer BJ et al.: Defects in enteric innervation and kidney development in mice lacking GDNF. Nature 1996, 362:73-76. Like the preceding paper [34”l, this study establishes that the knock-out of GDNF leads to the same developmental defects as the knock-out of Ret. In addition to the contribution that these observations make toward demonstrating that Ret is the functional receptor for GDNF, they also show that GDNF plays a previously unsuspected critical role in the development of the ENS. 36. ..

SBnchez M, Silos-Santiago I, F&n J, He B, Lira S, Barbacid M: Renal agenesis and the absence of enteric neurons in mice lacking GDNF. Nature 1996, 362:70-73. One of the series of manuscripts that appeared at about the same time [34”-37*‘] that demonstrate the potent in situ developmental actions of GDNF. The series, taken as a whole, elevates GDNF from a crowd of natural factors of unknown significance to one of transcendent importance. GDNF

Guillemot F, Lo L-C, Johnson JE, Auerbach A, Anderson DJ, Joyner AL: Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Ceil 1993, 75~463-476.

43. ..

Blaugrund E, Pham TD, Tennyson VM, Lo L, Sommer L, Anderson DJ, Gershon MD: Distinct subpopulations of enteric neuronal progenitors defined by time of development, sympathoadrenal lineage markers, and Mash-l-dependence. Development 1996, 122:309-320. The first study to show that the ENS is foned by multiple lineages of precursor cells. Previous investigations had suggested that there might be a common sympathoadrenallsympathoenteric neuronal progenitor 1461. This investigation, however, indicates that there are at least two progenitor lineages giving rise to the ENS of the bowel below the rostra1 foregut. One of these, which is like sympathetic neurons (albeit only transiently), is catecholaminergic and shares the Mash-7 dependence of sympathetic neurons as well as the expression of common cell-surface differentiation antigens. Neurons derived from these cells are born early and give rise to all enteric serotonergic neurons. Another lineage of enteric neurons is completely unlike sympathetic neurons in that they are Mash-7 independent and never catecholaminergic. Neurons derived from precursors in this lineage are born late and include CGRP-containing cells. 44.

Barasch JM, Mackey H, Tamir H, Nunez EA, Gershon MD: Induction of a neural phenotype in a serotonergic endocrine cell derived from the neural crest J Neurosci 1967, 7:2674-2663.

45.

Anderson DJ, Car&an JF, Michelsohn A, Patterson PH: Antibody markers identify a common progenitor to sympathetic neurons and chromaffin cells in viva and reveal the timing of commitment to neuronal differentiation in the sympathoadrenal lineage. J Neurosci 1991,11:3507-3519.

46.

Carnahan JF, Anderson DJ, Patterson PH: Evidence that enteric neurons may derive from the sympathoadrenal lineage. Dev Bioll991, 146:552-561.

47.

Camahan JF, Patterson PH: The generation of monoclonal antibodies that bind preferentially to adrenal chromaffln cells and the cells of embryonic sympathetic ganglia. J Neurosci 1991, 11:3493-3506.

48.

Baetge G, Gershon MD: Transient catecholaminergic (TC) cells in the vagus nerves and bowel of fetal mice: relationship to the development of enteric neurons. Dev Bioll969, 132:169-211.

49.

Baetge G, Pintar JE, Gershon MD: Transientfy catecholaminergic CrC) cells in the bowel of fetal rats and mice: precursors of non-catecholaminergic enteric neurons. Dev Bioll990, 141:353-380.

50. .

Bothwell M: p75NTR: a receptor after all. Science 272:506-507.

1996,

Genes

and lineages

A perceptive review of the history and evolution of ideas concerning the role played by the common neurotrophin receptor, p75Nr8. The author recounts the initial enthusiasm that greeted the discovery of p75Nrn, the decline in interest in it that accompanied the rush to attribute all neurotrophin actions to Trk receptors, and the current realization that p75Nrn cao itself mediate responses. 51.

Teitelmah G, Gershon MD, Rothman TP, Joh TH, Reis DJ: Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats. Dev Biol1981,88:348-355.

52.

Gershon MD, Rothman TP, Joh TH, Teitelman GN: Transient and differential expression of aspects of the catecholaminergic phenotype during development of the fetal bowel of rats and mice. J Neurosci 1984, 4:2269-2280.

53.

Cochard P, Goldstein M, Black IB: Ontogenetic appearance and disappearance of tyrosine hydroxylase and cetecholamines. Proc Nat/ Acad Sci USA 1978. 75:2986-2990.

54.

Teitelman G, Joh TH, Reis DJ: Transient expression of a noredrenergic phenotype in cells of the rat embryonic gut Brain Res 1978, 158:229-234.

55.

Jonakait GM, Wolf J, Cochard P, Goldstein M, Black IB: Selective loss of noradrenergic phenotypic characters in neuroblasts of the rat embryo. Proc Nat/ Acad Sci USA 1979, 76:4683-4686.

56.

Jonakait GM, Rosenthal M, Morrell JI: Regulation of tyrosine hydroxylase mRNA in the catecholaminergic cells of embryonic ret: analysis by in situ hybridization. J Histochem Cytochem 1989, 37:1-5.

57.

Baetge G, Schneider KA, Gershon MD: Development and persistence of cetecholaminergic neurons in cultured explants of fete1 murine vegus nerves and bowel. Development 1990, 110:689-701.

56.

F’hamTD,

Gershon MD, Rothman TP: Time of origin of neurons in the murine enteric nervous system. J Comp Nemo/ 1991, 314~789-798.

59.

Rothman TP, Gershon MD: Phenotypic expression in the developing murine enteric nervous system. J Neurosci 1982, 2:381-393.

60.

Chalazonitis A, Rothmao TP, Chen J, Lambatle F, Batbacid M, Gershon MD: Neurotrophin-3 induces neural crest-derived cells from fetal rat gut to develop in vitro as neurons or glia. J Neurosci 1994, 1416571-6584.

61.

Pomeranz HD, Rothman TP, Chalazonitis A, Tennyson VM, Gershon MD: Neural crest-derived cells isolated from the gut by immunoselection develop neuronal and glial phenotypes when cultured on laminin. Dew Biol1993, 156:341-361.

62.

Erickson CA, Loring JF, Lester SM: Migratory pathways of HNK-1 -immunoreactive neural crest cells in the rat embryo. Dev Biol1989, 134:112-l 18.

63. .

Stemini C, Su D, Arakawa J, DeGiorQio R, Rickman DW, Davis BM, Albers KM, Brecha NC: Cellular localization of pantr& immunoreacttvtty and fr&c mRNA in the enteric nervous system. J Comp Nemo/ 1996, 368:597-607 Presents an excellent morphologtcal descriptton of cells that express TrkC in the developing and mature ENS and demonstrates that TrkC is probably the only Trk expressed by intrinsic components of the rat ENS. 64.

Hoehner JC, Wester.T, P&hlman S, Olsen L: Localization of neurotrophin and their high affinity receptors during human enterlc nervous system development Gastroenterology 1996, 110:756-767.

65.

Fariaa I, Jones KR, Backus C, Wang X-Y, Reichardt LF: Severe sensory and sympathetic deficits in mice lacking neurotrophin-3. Nature 1994, 369:658-661.

66.

Tojo H, Kaisho Y, Nakata M, Matsuoka K, Kitagawa Takami K, Yamamoto M, Shino A, lgarashi K et a/.: disruption of the neurotrophin-3 gene with IacZ of bkC-positive neurons in sensory ganglia but cords. Brain Res 1995, 669:163-l 75.

87.

Klein R, Silos-Santiago I, Smeyne RJ, Lira SA, Brambilla R, Bryant S, Zhang L, Snider WD, Barbacid M: Disruption of the neurotrophin-3 receptor gene trkC eliminates la muscle efferents and results in abnormal movements. Nature 1994, 368:249-251.

M, Abe T, Targeted induces loss not in spinal

68.

in the formation

of the enteric

nervous

system

Gershon

109

Lee K-F, Li E, Huber LJ, Landis S, Sharpe AH, Chao MV, Jaenisch R: Targeted mutation of the gene encoding the low affinity NGF receptor ~75 leads to deficits in the peripheral sensory nervous system. Cell 1992,69:737-749.

69. ..

DeChiara TM, Vejsada R, Poueymirou WT, Acheson A, Suri C, Conover JC, Friedman B, McClain J, Pan L, Stahl N et aL: Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth. Cell 1995, 83:313-322. A landmark study that establishes the critical role played by CNTFRa in development. The investigation also provides compelling evidence for the existence of a (cytokine) tigand for the CNTFRa receptor that is not CNTF, LIF, or any of the other currently known cytokines. 70.

Masu Y, Wold E, Holtmann B, Sendtner M, Brem G, Thoenen H: Disruption of the CNTF gene results in motor neuron degeneration. Nature 1993, 36527-32.

71.

Baynash AG, Hosoda K, Giaid A, Richardson JA, Emoto N, Hammer RE, Yanagisawa M: Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons. Cell 1994, 79:1277-l 285.

72.

Lane PW: Association of megacolon with two recessive spotting genes in the mouse. J Hered 1966,57:29-31.

73.

Rothman TP, Gershon MD: Regionally defective colonization of the terminal bowel by the precursors of enteric neurons in /et/w/ spotted mutant mice. Neuroscience 1984, 12:1293-l 311.

74.

Kapur RP, Yost C, Palmiter RD: Aggregation chimeras demonstrate that the primary defect responsible for aganglionic megacolon in lethal spotted mice is not neuroblast autonomous. Development 1993, 117:993-999.

75.

Webster W: Embryogenesis of the enteric ganglia in normal mice and in mice that develop congenital aganglionic megacolon. J Embvol Exp Morpholl973, 30:573-585.

76.

Kapur RP, Sweetser DA, Doggett B, Siebert JR, Palmiter RD: Intercellular signals downstream of endotbelin receptor-B mediate colonization of the large intestine by enteric neuroblasts. Development 1995, 121:3787-3795.

77.

Coventry S, Yost C, Palmiter RD, Kapur RP: Migration of ganglion cell precursors in the ileoceca of normal and lethal spotted embryos, a murine model for Hirschsprung disease. Lab /west 1994, 71:82-93.

78.

Jacobs-Cohen RJ, Payette RF, Gershon MD, Rothmao TP: Inability of neural crest cells to colonize the presumptive aganglionic bowel of /s//s mutant mice: requirement for a permissive microenvironment J Comp Neural 1987, 255:425-438.

79.

Rothman TP, Goldowitz D, Gershon MD: Inhibition of migration of neural crest-derived Cells by the abnormal mesenchyme of the presumptive aganglionic bowel of /s//s mice: analysis with aggregation and interspecies chimeras. Dev Biol 1993, 159:559-573.

80.

Kapur RP, Livingston R, Doggett B, Sweetser DA, Siebert JR, Palmiter RD: Abnormal microenvironmentel signals underlie intestinal aganglionosis in Dominent megecolon mutant mice. Dev Biol1996, 1741360-369.

81. .

Rothman TP, Chen J, Howard MJ, Costantini FD, Pachnis V, Gershon MD: Increased expression of laminin-1 and collagen (Iv) subunits in the aganglionic bowel of /s//s, but not c-ret-Jmice. Dev Bioll996, 178:498-513. An important investigation that suggests that the EDN-3 mutation in lethal spotted (/s//s) mice affects the extracellular matrix (causing an increase in the biosynthesis of laminin-1). as well as neurons. The observations support the idea that lack of EDN-3 stimulation exerts two effects on crest-derived enteric neuronal precursors that are attempting to colonize the terminal bowel. One effect may be on the crest-derived cells themselves, but the other is on the extracellular matrix. It seems likely that the effect of EDN-3 deprivation on crest-derived cells is insufficient, by itself, to cause aganglionosis; however, when combined with the excess of laminin- I, the improperly supported crestderived precursors may differentiate prematurely and fail to complete their colonization of the hindgut. These data are in agreement with observations derived from chimeric mice, which also indicate that the lesion (aganglionosis of the terminal colon) in Is//s mice (and animals lacking EDN-3 or the EDNRB) is not crest-autonomous [74,761. The great interest in Is//s mice and the other EDNlEDNRB knock-out animals is the close resemblance of their developmental defect to that of humans with Hirachsprung’s disease 1711.