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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
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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
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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,
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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
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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-
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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.
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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.
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