The ENS is Derived from the Neural Crest

The first clear demonstration that the ENS is derived from the neural crest was made by Yntema and Hammond who noted that enteric ganglia fail to appear when the "anterior" neural crest is deleted in chick embryos [38, 39]. Their work was confirmed, and levels of the crest that contribute to the ENS were more precisely identified by Le Douarin and her colleagues [40, 41]. These investigators took advantage of the distinctive nucleolar-associ-ated heterochromatin of quail cells, which allows these cells to be readily identified following their transplantation into embryos of other species. Le Douarin and her co-workers replaced segments of the chick neural crest with those of quail (or the reverse) and traced the migration of crest-derived cells in the resulting interspe-cies chimeras by identifying cells of the donor (chick or quail, depending on the particular experiment). These studies suggested that the ENS is derived from both the vagal (somites 1-7) and the sacral (caudal to somite 28) crest. The vagal crest colonizes the entire bowel, while the sacral crest colonizes only the postumbilical gut.

The conclusion that there are two sites of origin of enteric neuronal precursors was soon challenged, because other investigators could recognize only a single proxi-modistal progression of cells thought to be "neuroblasts" in the avian gut [42]. This progression was believed to imply that neuronal precursors in the bowel only descend, as would be expected of vagal progenitors. No ascent, of the kind predicted for precursors from the sacral crest, could be found. These observations led to the suggestion that the data derived from experiments with interspecies chimeras could have been obtained if crest-derived cells were to be more invasive in a foreign embryo than they are when they migrate in embryos of their own species. If so, then quail cells might reach ectopic destinations in a chick embryo and chick cells might behave in a similarly abnormal manner in a quail embryo. There are, however, reasons why only a single proximodistal progression of cells that can be recognized as belonging to a neuronal lineage can be detected, even though multiple levels of the crest contribute precursors to the bowel. Neuronal progenitors have been shown to colonize various levels of the gut before they actually give rise to progeny that express recognizable neural properties [43]; thus, neurons develop in vitro in segments of gut that appear to be aneuronal at the time of explantation, thereby demonstrating that otherwise unrecognizable neural precursor cells were present in the explants. The delay, however short it might be, between the arrival of progenitors and their differentiation into neurons provides an opportunity for crest-derived precursors to interact with, and be influenced by, the enteric microenvironment. In fact, the enteric microenvironment has been demonstrated to play a critical role in the development of enteric neurons and glia [44-46]. The observed proximodistal progression of perceived "neuroblasts" (which is not found in all species), therefore, may be due to a proximodistal gradient in the maturation of the enteric microenvironment, rather than to the timing of the descent of the neuronal precursors.

More recent studies, in which endogenous crest cells have been traced by labeling them with a vital dye or a replication-deficient retrovirus, have confirmed that both the avian and murine gut are each colonized by cells from both vagal and sacral levels of the neural crest [47, 48]. The human bowel, like that of mice, appears to be colonized by sacral as well as vagal crest cells [49, 50]. In the mouse, studies with labeled crest-derived cells have also revealed that a third site, truncal crest, contributes to the rostral-most foregut (esophagus and adjacent stomach) [51]. Retroviral tracing in avian embryos has suggested that the entire vagal crest does not contribute to the formation of the ENS; instead, the bulk of the enteric neuronal progenitors evidently originate from only the portion of the vagal crest lying between somites 3 and 6

[52]. The specificity of vagal and sacral regions as sources of enteric neuronal progenitors is well illustrated by back-transplantation experiments. Back-transplantation consists of grafting a developing organ or piece of tissue from an older to a younger host embryo. It is a technique that provides insight into whether cells in the older tissue retain and can manifest, in a suitably permissive environment, properties associated with earlier stages of development. Crest-derived cells that have colonized the bowel will leave segments of gut that are back-grafted into a younger embryo and remigrate in their new host

[53]. These cells will only reach the bowel of their host if the graft is situated so as to replace the host's vagal or sacral crest [54].

A subset of the vagal crest-derived cells that colonize the gut can be visually identified in transgenic mice directed to express lacZ by the promoter for dopamine ^-hydroxylase (DBH) [55]. The DBH-lacZ transgene is permanently expressed in these mice by neurons that are not catecholaminergic in the adult gut. The colonization of the bowel by the transgenically labeled cells has been studied in detail in both normal mice and in murine models of Hirschsprung's disease [56, 57]; however, it is important to note that the DBH-lacZ transgene probably demonstrates only a subset of vagal crest-derived cells and does not reveal those of sacral origin. Some enteric neurons develop from precursors that are transiently catecholaminergic (TC) [58-61]. DBH is one of the enzymes that participate in the formation of norepinephrine (NE) and thus its presence is a component of the catecholaminergic phenotype. Even in normal mice, and especially in rats, the genes encoding DBH are not completely repressed in the noncatecholaminergic neurons that develop from TC cell progenitors. Neurons derived from TC cells continue to express DBH, although they inactivate other elements of the catecholaminergic phe-notype [59]. It is likely that the cells that are marked by the expression of the DBH-lacZ transgene are members of this lineage, that is they are cells that originate from catecholaminergic progenitors. Unfortunately, not every enteric neuron originates from a TC cell precursor. In fact, the subset of neurons that arises from progenitors that never exhibit catecholaminergic properties is larger than that which is TC cell-derived [61]. As a result, many enteric neuronal precursors are not subject to surveillance by the DBH-lacZ transgene tracing technique.

However cells are traced, it is now apparent that in both fetal mice and in avian embryos, the ENS arises from multiple regions of the neural crest, not just one. Although the number of sources of enteric neurons in the neural crest is limited, it is necessary to take account of this multiplicity in attempting to explain the abnormal colonization of the gut that arises in Hirschsprung's disease and other dysganglionoses.

3.5 The Crest-Derived Cells that Colonize the Gut are Originally Pluripotent and Migrate to the Bowel Along Defined Pathways in the Embryo

The restriction of the levels of the premigratory crest that contribute precursors to the ENS raises the possibility that the crest cells in these regions might be predetermined to migrate to the bowel and give rise to enteric neurons and/or glia. Such a predestination, however, is not supported by experimental evidence, which indicates instead that premigratory crest cells are pluripotent. For example, when levels of the crest are interchanged so as to replace a region that normally colonizes the gut with one that does not, the heterotopic crest cells still migrate to the bowel and there give rise to neurons the pheno-types of which are ENS-appropriate, not level of origin-appropriate [62, 63]. An analogous process, moreover, is seen when the interchange of crest cells is reversed. Vagal and sacral crest cells give rise to non-enteric neurons in ectopic locations, such as sympathetic ganglia, when they are grafted so as to replace crest cells at other axial levels. Clones derived from single crest cells, furthermore, give rise, both in vitro [64-68] and in vivo [69-71], to progeny that may express many different phenotypes. A single cell that gives rise to a clone containing many phe-notypes has to be pluripotent. The crest-derived cells that colonize the gut, moreover, remain multipotent with respect to their ability to give rise to neurons and glia, even after they have completed their migration to the bowel. This potency is well demonstrated by back-transplantation experiments. When segments of gut are back-transplanted into a neural crest migration pathway at a trun-cal level, which normally colonizes sympathetic ganglia and the adrenal gland, donor crest-derived cells leave the graft, but they do not migrate to the host's gut. Instead, they migrate to the host's sympathetic ganglia, adrenal gland and peripheral nerves; moreover, instead of giving rise to enteric neurons and glia, the donor crest cells, despite their previous migration to and residence in the bowel, now form catecholaminergic neurons in the ganglia, chromaffin cells in the adrenals, and Schwann cells in the nerves [53].

Analogous results have been obtained from in vitro studies of cells developing from cloned crest-derived cells of enteric origin. The progeny found in these clones express a variety of different phenotypes, including some that are not present in the normal ENS [72]. Despite their multipotent nature, however, the developmental potential of enteric crest-derived cells in vivo [53] and in clonal culture is not as great as that of their progenitors in the premigratory crest [72, 73]. The pluripotency of the crest-derived cells that colonize the gut, revealed by studies of clones and the behavior of cells emigrating from back-transplants [54], indicates that the bowel does not become colonized by precursors from restricted regions of the neural crest because only these regions contain crest cells endowed with homing information that programs them to migrate to the gut. Instead, these regions are the only levels of the crest from which there are defined migratory pathways that lead to the bowel. The pathway from the vagal crest conveys the largest cohort of crest-derived émigrés to the gut and in avian embryos leads crest-derived cells to the entire bowel between the pro-ventriculus and the cloaca. In mammals the equivalent region would extend from the corpus of the stomach to the rectum. The cohort that follows the sacral pathway is much smaller and leads crest-derived émigrés only into the postumbilical bowel. The cohort following the trun-cal pathway is still smaller and leads crest-derived cells only to the presumptive esophagus and the most rostral portion of the stomach.

The possibility that crest-derived cells of different origins are not identical exists and has some experimental support. It is also conceivable that the crest-derived émigrés from different levels interact with one another during the formation of the ENS. The molecular nature of the migratory pathways and the nature of the mechanisms that guide progenitors to their correct destinations within the gut itself have yet to be identified. Chemoat-tractant or repellent molecules for growing axons have been identified in the vertebrate CNS [74]. These molecules include netrins [74-77] and semaphorins [78-80]. The directional growth of migrating crest-derived cells is a property also shown by path-finding axonal growth cones [81, 82]. Both netrins 1 and 2 (2 > 1) are expressed in the developing bowel [75] and mice with a targeted mutation in netrin-1 die at birth with a bloated bowel and no milk in their stomach (Tessier-Lavigne, personal communication). It is thus conceivable, although there is as yet absolutely no direct supporting evidence, that netrins play a role in the guidance of crest-derived progenitors and/or axons to their proper destinations in the gut. The roles, if any, of the netrins or semaphorins in the formation of the ENS are thus intriguing possibilities that remain to be investigated.

3.6 Enteric Neurons are Derived from More Than One Progenitor Lineage

The developmental potential of the originally pluripotent population of premigratory crest cells becomes progressively restricted as development proceeds. This restriction is accompanied by the sorting of crest-derived progeny into recognizable lineages [83-85]. A lineage restriction has occurred in the crest-derived population that colonizes the bowel [61]. At least two lineages of enteric neuronal progenitor have been distinguished. Recognition of these lineages is significant, because the fate of the neuronal precursors in the bowel depends, not just on the enteric microenvironment, but also on the lineages of the crest-derived cells. Lineages, as much as environmental factors, determine patterns of phenotypic expression. In order for any progenitor to respond to a microenviron-mental signal (whether that signal is a growth factor or a molecule of the extracellular matrix) the responding cell first has to have expressed receptors capable of being stimulated by the microenvironmental signal. The expression of these receptors is lineage-dependent. Lineage thus establishes which developmental options are open to precursor cells and which are not, and which growth factors can affect the cells and which cannot. The development of the ENS can thus be understood as a symphony in which lineage-determined properties provide the themes and environmentally provided factors provide the counterpoint.

Perhaps the earliest indication of the multiplicity of the lineages of crest-derived precursors contributing to the formation of the ENS was the discovery in the developing mammalian bowel [86, 87] and vagal crest migration pathway [58, 60] of TC cells. These remarkable cells mimic all of the known properties of sympathetic neurons except one. TC cells express tyrosine hydroxylase (TH) and DBH, and take up and store NE [88-90]. The one property of sympathetic neurons that TC cells do not mimic is that sympathetic neurons, like every other neuron, are postmitotic cells, while TC cells proliferate [58, 59, 91, 92]. TC cells, therefore, cannot by definition be neurons, which are postmitotic. Still, TC cells do express neural markers, including neurofilament proteins and pe-ripherin [58, 59]; moreover, TC cells give rise to neurons in vitro [60]. TC cells thus are crest-derived neural precursors [59, 60]. In fact, the persistence of DBH after the cessation of transcription of TH made it possible to demonstrate that TC cells are the ancestors of at least some mature enteric neurons [59]. The persistence of DBH in a subset of enteric neurons probably explains the ability, discussed above, of a transgene driven by the DBH promoter to label these cells and their precursors [55].

TC cells have more in common with sympathoadrenal progenitor cells than just their catecholaminergic characteristics. Both TC cells and sympathoadrenal progenitors express the same cell surface differentiation antigens and each changes these antigens at the same time of development. The first common antigens to be expressed are "SA" proteins, recognized by a series of monoclonal antibodies [93, 94]. The SA antigens disappear at the time another transiently expressed antigen, recognized by "B2" antibodies, appears. The sharing of characteristics by enteric and sympathetic neuronal precursors led to the suggestion that there is a common sympathoadrenal-en-teric precursor lineage from which both the sympathetic nervous system and ENS are derived [93]. Studies of catecholamine expression in clonal cultures of chicken enteric crest-derived cells led investigators to conclude that there is a also a common sympathoadrenal-enteric precursor lineage in avians [72].

Two recent lines of evidence have shown that the hypothesis that the ENS arises from a single sympathoad-renal-enteric progenitor lineage is only partially correct

[61]. Dissociated cells of the fetal rat gut were repeatedly exposed to B2 antibodies in vitro in the presence of complement. This treatment causes all of the crest-derived cells that express surface antigens in common with sym-pathoadrenal progenitors to lyse, thereby eliminating the putative common sympathoadrenal-enteric progenitor. If such a precursor were to be the sole source of enteric neurons, its destruction by complement-mediated lysis would be expected to prevent the in vitro development of neurons in the treated cultures of cells from the dissociated fetal bowel. In fact, complement-mediated lysis reduces the number of neurons differentiating in the cultures and eliminates all that express TH, DBH, or B2; nevertheless neurons that express none of these antigens continue to arise in the cultures. These findings suggest that at least two precursor lineages contribute to the development of the ENS. Only one of these lineages can be ablated by destroying cells that express sympathoadrenal markers.

The second line of evidence showing that multiple precursor lineages contribute to the development of the ENS has come from studies of mice with a homozygous targeted mutation in a gene encoding a transcription factor, mash-1. This gene is a mammalian analog of achaete-scute of Drosophila [95]. Mash-1 is expressed by both sympathetic and enteric neural precursors [96, 97] and thus its expression is one more shared property that implies a sympathoadrenal-enteric commonality. The sympathetic nervous system fails to develop in mash-1-/-mice [98], indicating that sympathetic neurons are mash-1-dependent. The common precursor idea suggests that enteric neurons should also be mash-1 -dependent. The ENS, however, is not absent in mash-1-/- animals. Instead, enteric neurons are lacking only in the esophagus. In the remainder of the bowel, neurons develop, but there is a delay of about two days in the timing of their appearance. In the absence of additional evidence, it was initially impossible to know whether this delay was due to the elimination of a mash-1 -dependent set of early-developing neurons, or to the slower development of all neurons. Subsequent studies, however, revealed that the delay was caused by the interference in mash-1-/- mice with the development of a mash-1-dependent set of early-developing neurons.

The birth dates of enteric neurons vary in relationship to their phenotype [99]. Enteric serotonergic neurons are among the first to be born, some becoming postmitotic (at E8.5), even before they colonize the gut. Others, such as neurons containing calcitonin gene-related peptide (CGRP) originate quite late and continue to be born postnatally. The first CGRP neuron (E16) is not born until about two days after the last serotonergic neuron has become postmitotic. Mash-1 and TH immunoreactivities are colocalized, indicating that mash-1 is expressed in TC cells [61]; moreover, TC cells do not develop in mash-1-/- mice. The ENS of mash-1-/-animals, furthermore, contains no serotonergic neurons, although it does contain neurons that express CGRP. These findings suggest that TC cells are the postulated mash-1 -dependent common sympathoadrenal-enteric progenitor; moreover, serotonergic neurons would appear to be an example of an enteric neuron derived from this lineage. CGRP-containing neurons, which arise later, and are mash-1-independent could not be derived from such a common lineage. These suggestions have been confirmed by examining which types of neuron do or do not develop in cultures of dissociated rat intestine that have been subjected to complement-mediated lysis with antibodies to common sympathoadrenal-enteric antigens (B2). Serotonergic neuronal development is prevented, but neurons that contain CGRP continue to appear.

These findings confirm that the ENS is derived from at least two progenitor lineages. One of these is related to sympathoadrenal precursors. This lineage expresses and depends on mash-1, arises early in ontogeny, is transiently catecholaminergic, and gives rise to limited subsets of enteric neurons, including all of those that populate the esophagus and all of the remainder that express a serotonergic phenotype. The second lineage, which may not be homogeneous, is unrelated to sympathoadrenal cells, develops late, does not express or depend on mash-1, is not catecholaminergic, and gives rise to gastric and intestinal neurons, some of which contain CGRP.

3.7 Dependence of Enteric Neuronal Subsets on Different Microenvironmental Signals (Growth/Differentiation Factors) Defines Sublineages of Precursor Cells: RET and Glial Cell Line-Derived Neurotrophic Factor

The c-ret protooncogene is a gene upon which most enteric neurons are critically dependent for survival [51, 100, 101]. This gene encodes a receptor tyrosine kinase, for which glial cell line-derived growth factor (GDNF) has recently been identified as the functional ligand [102104]. GDNF was first identified as a factor, produced by a glial cell line (B49), that promotes the survival of midbrain dopaminergic neurons [105]. GDNF was later observed to enhance the survival of spinal motor neurons [106]. GDNF is a distant relative of transforming growth factor-^ (TGF-P). It is a homodimer, consisting of two peptide chains of 134 amino acids linked by a disulfide bridge. A larger precursor of 211 amino acids is synthesized first. This big molecule is proteolytically cleaved in-tracellularly to produce mature GDNF, which is secreted. During development, GDNF is not restricted to the brain, but rather is very highly expressed in the gut and other peripheral organs [106, 107]. In keeping with its peripheral distribution, GDNF is not just a survival factor for central CNS neurons [103], but also enhances the in vitro survival of peripheral sensory and sympathetic neurons, and also promotes their extension of neurites [106].

The observation that GDNF affects sympathetic neurons suggests that it should also affect at least some neurons of the ENS. In fact, both enteric and sympathetic neurons express c-ret, at least transiently [100, 108]. When c-ret is knocked out in transgenic mice, the ENS totally fails to develop in the entire bowel, with the exception of the rostral foregut [51, 101]. Since Ret is the functional receptor for GDNF, the fact that a similar lesion has recently been found to occur in the bowel of knockout mice lacking GDNF [109-111] is not surprising. Neither is the observation surprising that, in contrast to the trophic effects GDNF exerts on autonomic neuroblasts from control mice, GDNF fails to exert trophic effects on analogous cells from c-ret-/- animals [104]. Activation of the Ret receptor by GDNF is thus a critical event in the formation of the ENS. Actually, GDNF does not appear to bind directly to the Ret receptor itself. Instead, GDNF binds to a glycosylphosphatidylinositol-linked cell surface protein called GDNFR-a, which then complexes with Ret to trigger the autophosphorylation and other actions of Ret [102, 112].

Despite the fact that most of the bowel is aganglionic in c-ret-/- mice [101], there are neurons in the portions of the gut that develop from the rostral foregut of these animals [51]. Although the superior cervical ganglion is missing in c-ret-/- mice, most other sympathetic ganglia do develop. The crest-derived cells that colonize the rostral foregut and the superior cervical ganglion have been traced by injecting a fluorescent dye (Dil) that intercalates into the lipid of the plasma membrane. The Dil-la-beled cells that colonize the presumptive esophagus and rostral stomach originate from the same pool of truncal crest cells that gives rise to the sympathetic chain ganglia below the superior cervical ganglion. In contrast, the post-otic vagal crest cells that colonize the entire bowel distal to the rostral foregut also contribute the crest-derived cells that form the superior cervical ganglion. There thus appears to be not one but two common sympa-thoadrenal-enteric lineages. One of these is c-ret- and GDNF-dependent, while the other is c-ret- and GDNF-independent. The bulk of the ENS is constructed of cells in the c-ret/GDNF-dependent sympathoadrenal-enteric lineage, which evidently also gives rise to the superior cervical ganglion. The c-ret/GDNF-independent lineage forms the ENS of the rostral foregut and the entire sympathetic chain, except for the superior cervical ganglion.

The mash-1 -dependent and c-ret-dependent lineages seem superficially to be opposite sides of a single coin [51]. For example, the ENS of the esophagus, which is totally mash-1 -dependent, happens to be the region of the gut that is c-ret-independent. In contrast, the ENS of the bowel below the proximal stomach is totally c-ret-dependent, yet it contains neurons in mash-1 knockout mice. Still, as noted above, there is no region of the ENS that is completely mash-1-independent. Although there are neurons in the intestines of mash-1 knockout mice, TC cells and all the neurons derived from TC cells are missing. Still to be explained as well is why the presumably c-ret-independent crest-derived cells of the rostral foregut do not migrate distally in the bowel of c-ret-/-mice (or mice lacking GDNF). Possibly, the evident inability of the c-ret-independent cells of the rostral fore-gut to expand their territory in c-ret-/- mice is due to an inhibition of their migration, or possibly proliferation. Alternatively, all enteric neurons may be GDNF/Ret-de-pendent but able to survive in the rostral foregut, despite the absence of GDNF or Ret, because a compensatory factor (currently unknown) is expressed only in this region of the bowel.

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