THEME
Physiology and Pathophysiology of the Interstitial Cell of Cajal: From Bench to Bedside
I. Functional development and
plasticity of interstitial cells of Cajal networks
Sean M.
Ward and
Kenton M.
Sanders
Department of Physiology and Cell Biology, University of Nevada
School of Medicine, Reno, Nevada 89557
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ABSTRACT |
Interstitial cells
of Cajal (ICC) are the pacemaker cells in gastrointestinal (GI)
muscles. They also mediate or transduce inputs from enteric motor
nerves to the smooth muscle syncytium. What is known about functional
roles of ICC comes from developmental studies based on the discovery
that ICC express c-kit. Functional development of ICC
networks depends on signaling via the Kit receptor pathway.
Immunohistochemical studies using Kit antibodies have expanded our
knowledge about the ICC phenotype, the structure of ICC networks, the
interactions of ICC with other cells within the tunica
muscularis, and the loss of ICC in some motility disorders. Manipulating Kit signaling with reagents to block the receptor or
downstream signaling pathways or by using mutant mice in which Kit or
its ligand, stem cell factor, are defective has allowed novel studies
of the development of these cells within the tunica muscularis
and also allowed the study of specific functions of different
classes of ICC in several regions of the GI tract. This article
examines the role of ICC in GI motility, focusing on the functional
development and maintenance of ICC networks in the GI tract and the
phenotypic changes that can occur when the Kit signaling pathway is disrupted.
pacemakers; enteric nervous system; gastrointestinal motility; c-kit; stem cell factor; electrical slow waves; development
of rhythmicity
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INTRODUCTION |
INTERSTITIAL CELLS OF CAJAL (ICC) were identified in
the gastrointestinal (GI) tract over a century ago, and several
possible functions were ascribed to these cells on the basis of their
morphology and close anatomic relationships with smooth muscle cells
and neurons (for review, see Ref. 16). Until recently,
however, the function of ICC has been speculative and not based on
physiological tests. The use of murine models that have developmental
lesions in ICC networks and new reagents that show specificity for
labeling and affecting the development of ICC have recently led to the conclusions that ICC are pacemaker cells, responsible for the generation and propagation of slow waves, and mediators of inputs from
the enteric nervous system in GI smooth muscles (17, 23).
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MORPHOLOGICAL IDENTIFICATION OF ICC |
The original studies describing the existence of a specialized
cell type in the tunica muscularis were those of Cajal
(3). On the basis of the staining characteristics of ICC
with methylene blue and silver chromate, Cajal believed ICC were a type
of primitive neuron. Taxi (18), however, using light and
electron microscopy, distinguished ICC from neurons, macrophages, and
Schwann cells. Although these cells were distinguishable from other
cell types in the tunica muscularis, considerable debate
about their origin continued for many years. Many of the
ultrastructural features of ICC, including a basal lamina (which may be
discontinuous), smooth and rough endoplasmic reticulum, dense
filaments, cell-to-cell contacts with other ICC and smooth muscle
cells, and close contacts with nerve endings, seemed to exclude a nerve
or connective tissue nature and suggested to some investigators that
ICC are specialized or primitive smooth muscle cells (6,
9), whereas others thought that ICC possessed the
ultrastructural characteristics of fibroblasts (10).
But the debate about whether ICC are smooth muscle cells or fibroblasts
could probably never be settled by anatomic location and
ultrastructural analysis alone, because the morphological features of
ICC are shared with several cell types within the tunica
muscularis (19). Work on ICC was hampered by the lack of specific reagents to label these cells in GI tissues. Daniel and
Berezin (4) noted this problem in 1992 and stated: "A
major problem, delaying our full understanding of ICC function, arises from the fact that definitive recognition of these cells still depends
on ultrastructural identification of their characteristic structures.
This requires that sample sizes for study are small, that 3-dimensional
analysis of their organization into networks is so tedious as to be
daunting, and that the number of laboratories studying them directly is
limited." Recently, immunochemical and molecular biology techniques
have provided new ways to label and study the three-dimensional
structures of ICC networks. A "chemical coding" of ICC has begun
that unequivocally separates these cells into a distinct class.
Probably the most important advance in interstitial cell biology came
when it was recognized that ICC express the gene product of
c-kit, a protooncogene that encodes a receptor tyrosine
kinase, Kit (8, 14, 20, 26). Labeling of Kit receptors or
c-kit mRNA has provided an efficient means of identifying
ICC throughout the GI tracts of several species, including human,
guinea pig, mouse, rat, and birds, by using light microscopy (Fig.
1). The availability of this technique
has brought several investigators, including clinical investigators,
into the field of interstitial cell biology. Immunohistochemistry using Kit labeling has improved our understanding of the structure and distribution of ICC networks and enhanced our perception of the anatomic relationships between ICC and enteric neurons, smooth muscle
cells, and other resident cells in the tunica muscularis, such as macrophages. A second, ultimately more important development has been the use of Kit immunohistochemistry by pathologists to determine the status of ICC in a variety of GI motility disorders (17). ICC networks are disrupted in several motility
disorders of heretofore unclear etiology, and new hypotheses, including the loss of function that occurs when ICC are lost, have emerged.

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Fig. 1.
Confocal composite images showing labeling of interstitial cells of
Cajal (ICC) networks with anti-Kit antibody. ICC can be labeled
throughout the gastrointestinal (GI) tract by using antibodies to Kit.
A: intramuscular ICC (IC-IM) in the gastric fundus. IC-IM
are within the circular (arrows) and longitudinal muscle layers
(arrowheads) and run parallel to the muscle fibers. These cells are
also closely associated with varicose endings of enteric motor neurons
and mediate neurotransmission (2, 23). B: IC-IM
(arrows) and myenteric ICC (IC-MY; arrowheads) in the gastric antrum.
IC-MY are pacemaker cells and have been shown to generate electrical
slow waves (26). C: IC-MY (arrowheads) and ICC
at the level of the deep muscular plexus (IC-DMP; arrows) in the murine
small intestine. D: ICC in the proximal colon. ICC are
located at the level of the myenteric plexus (arrowheads), within the
circular and longitudinal muscle layers, and at the submucosal surface
of the circular muscle layer (arrows). Images are from 1-µm
z-steps taken through 10 µm in A and
C and 20 µm in B and D.
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Another important advance was the realization that cell signaling via
Kit receptors is essential for the development and maintenance of ICC
networks (20, 26) (Fig. 2).
Blocking Kit function with neutralizing antibodies impairs the
development of ICC (20), and several studies have shown
that animals carrying nonlethal mutations in c-kit
(2, 8, 26) or stem cell factor (SCF; Ref.
25), the natural ligand for Kit, have defects in ICC
networks. Experiments on these animal models, which lack specific
populations of ICC but do not appear to have neural or smooth muscle
defects, have provided important insights into the function of ICC.

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Fig. 2.
Role of Kit in development and
maintenance of ICC phenotype. a: Gross morphological
features of the normal murine GI tract from a 10-day-old (D10) animal.
St, stomach; Si, small intestine. b: An image of a cryostat
cross section through the tunica muscularis of the ileum
from a D10 animal. ICC are located at the level of the myenteric plexus
(arrows), between the longitudinal (lm) and circular (cm) muscle
layers, and at the level of the deep muscular plexus near the
submucosal surface of the circular muscle layer (arrowheads).
c: GI tract of a mouse that was injected with
Kit-neutralizing antibody between D0 and D8. The stomach, small
intestine, and colon were greatly distended after Kit was blocked.
d: Loss of IC-MY and IC-DMP after treatment with
Kit-neutralizing antibody. Redrawn with permission from Ref.
20.
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DEVELOPMENT OF ICC WITHIN THE GI TRACT |
Before the use of Kit antibodies as a marker for ICC,
developmental studies were difficult to perform because definitive
identification of these cells required ultrastructural analysis.
Immature ICC possess few of the ultrastructural features associated
with mature ICC. For example, the numerous mitochondria, endoplasmic
reticulum, caveolae, myofilaments, basal lamina, and structural
junctions with smooth muscle cells are not apparent during development. Thus it is next to impossible to identify these cells in embryos and
neonatal animals with electron microscopy. With
c-kit expression as a label for ICC, it is possible to
follow ICC to an earlier point in their development, and it was found
that Kit-positive cells develop much earlier than had been suggested
from ultrastructural studies. In situ hybridization studies showed
transcriptional expression of c-kit from about embryonic
day 9 (E9; Ref. 15) in the murine GI tract, at
E7 in chick embryos, and as early as E6 in quail embryos
(12). Immunoreactivity for Kit was clearly abundant in the
murine intestinal wall at E12, suggesting that ICC or precursors of ICC
are present approximately midway through embryonic development
(21).
Embryological source of ICC precursors.
Several biological systems have been used to determine the
embryological source of ICC in the gut. The quail/chick chimeric model
was used to determine whether ICC were of neural crest or mesenchymal
in origin. Chimeric bowels were constructed by isotopically grafting
quail vagal neural crest into chick embryos at E2. Enteric nerves of
these animals were derived from quail precursors; however, in situ
hybridization experiments showed that all c-kit-positive cells were of chick origin. Therefore, the ICC came from the gut mesenchyme and were not derived from neural crest cells
(12). These investigators also tested whether the
development of ICC was dependent on the presence of an enteric nervous
system by culturing aneural chick gut on chorioallantoic membranes.
Ultrastructurally typical ICC that expressed Kit developed in the
absence of enteric innervation. Similar conclusions were reached in
studies on the developing mammalian colon. Segments of murine colon
removed from embryos as young as E9.5, before colonization of neural
crest cells, were cultured under the renal capsule of host adult mice. After 18-41 days, Kit-immunoreactive ICC were present in the
aneuronal explants (31). It has also been shown that
Kit-positive ICC precursor cells do not express c-ret, a
ubiquitous marker of neural crest cells.
At the time of first expression of Kit protein (E12) and up through
E15, the longitudinal muscle layer in the murine small intestine has
not developed (21, 30). By E15, the circular smooth muscle
layer has developed and expresses smooth muscle antigens such as
-enteric actin, smooth muscle myosin, and desmin. At this stage,
cells expressing Kit aggregate into clusters at the outer aspect of the
small intestine, peripheral to the developing myenteric plexus.
Kit-positive cells are also immunopositive for vimentin, and some cells
were found to express desmin, a marker for smooth muscle. This
observation showed that some Kit-positive cells develop smooth muscle
characteristics, suggesting that both ICC and longitudinal smooth
muscle cells may develop from the same Kit-positive mesenchymal
precursors. Additional support for this hypothesis was provided by in
situ hybridization studies showing coexpression of c-kit and
smooth muscle myosin heavy-chain mRNAs in cells at the periphery of the
murine small intestine at about E15.
Between E15 and E18, cells along the outer serosal aspect of the
Kit-positive layer develop ultrastructural features reminiscent of
smooth muscle cells and lose expression of Kit. By E18, a distinct layer of cells that express desmin and not Kit are found peripheral to
the Kit-positive cells adjacent to the myenteric plexus. Thus myenteric
interstitial cells (IC-MY) and longitudinal muscle cells diverge from a
common precursor late in gestation (Fig.
3). At E18, Kit-positive cells were
organized into anastomosing networks like IC-MY in adult animals.
Ultrastructural studies, however, showed that these cells still had an
undifferentiated appearance.

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Fig. 3.
Cartoon depicting the
development and plasticity of ICC. Mesenchymal cells at the periphery
of the small bowel develop Kit expression by embryonic day 12 (E12).
Neuroblasts, in close proximity to the Kit-positive cells, express stem
cell factor (Kit ligand) and might provide early signaling; however,
functional ICC develop in the absence of neurons. Smooth muscle cells
on which Kit-expressing cells develop also express stem cell factor.
Cells that are signaled via Kit develop into functional IC-MY, and
these cells become pacemaker cells of the intestine. Cells not signaled
via Kit develop smooth muscle antigens and may become the longitudinal
smooth muscle layer. After development of IC-MY, blocking Kit signaling
causes a phenotypic transition toward a smooth muscle-like phenotype.
This is accomplished within days in newborn animals but requires weeks
in mature animals. Pathophysiological conditions, which are not fully
understood, also cause a phenotypic transition that leads to loss of
IC-MY and pacemaker activity. Hybrid cells appear that may be similar
to the cells that develop when Kit signaling is blocked. An important
question is whether the hybrid cells can redifferentiate into
functional ICC if normal conditions in the intestinal microenvironment
are restored.
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At the time of birth, only one layer of Kit-positive cells was found in
the tunica muscularis of the small intestine. These cells
are located in the region of the myenteric plexus, between the circular
and longitudinal muscle layers. We have referred to these cells as
IC-MY, and these cells, at about E18, begin to provide pacemaker
activity to the proximal (including jejunum) small intestine. A second
layer of Kit-positive cells emerges at about the time of birth at the
level of the deep muscular plexus. These cells appear to come from
undifferentiated cells along the submucosal surface of the circular
muscle layer. Since this population of Kit-positive cells eventually
produces a network of ICC in the region of the deep muscular plexus, we
have referred to these cells as IC-DMP.
By birth, smooth muscle cells lose expression of vimentin, and they
become strongly desmin positive as the adult phenotype develops. ICC
retain vimentin expression into adulthood. It is possible IC-DMP and
the thin layer of circular muscle between the DMP and the submucosa
share a common origin, just as IC-MY and longitudinal muscle cells
appear to come from the same progenitor cells.
The factors in the microenvironment that determine whether a
Kit-positive precursor develops into an ICC or a smooth muscle cell are
not fully understood. The developmental fate of a given precursor cell
is likely to be determined by its physical location and whether or not
it lies in close proximity to cells that express SCF. The opportunity
for SCF/Kit signaling would favor the development of an ICC, whereas a
lack of signaling would favor development of a smooth muscle phenotype.
This hypothesis is supported by the fact that IC-MY development is
impaired in W and steel mutants but the
longitudinal and circular muscle layers appear to develop normally in
these animals. Smooth muscle cells have been reported to express SCF
mRNA and are immunopositive for SCF, but other cells within the
myenteric region may contribute to stimulation of Kit-positive cells.
The composition of the microenvironment and the trophic factors that
are responsible for development (and maintenance) of the
nerve/ICC/smooth muscle motor complexes that exist within the GI tract
are of great importance for future investigations.
Molecular interactions between ICC and enteric neurons during
development of the GI tract.
In adulthood, ICC form close contacts with enteric neurons. This led
Cajal, in early descriptions, to suggest that these cells may be
involved in peripheral neurotransmission. The close association between
enteric neurons and ICC could also indicate a role for neurons in the
development of ICC or vice versa. Appearance of Kit-positive cells
follows closely after colonization of the gut by cells from the neural
crest. Kit-positive cells that become IC-MY are closely associated with
neuroblasts before they develop into functional ICC. It is possible
that neuroblasts provide an early source of SCF that initiates Kit
signaling and the development of Kit-positive cells into IC-MY. Enteric
neurons throughout the GI tract have been shown to express SCF mRNA by
in situ hybridization. The role of enteric neurons in development of
Kit-positive cells has been tested with a transgenic animal in which
the promoter for SCF was linked to lacZ. LacZ is
subsequently expressed in the same cells that normally express SCF. A
subpopulation of enteric neurons was positive for
-galactosidase,
the gene product of lacZ, and these neurons projected to
myenteric ganglia and the circular smooth muscle (22).
Projections of these neurons may reach the deep muscular plexus. The
studies described above, however, showed that Kit-positive cells
develop in aneural segments of gut from bird and mammalian models taken
for organ culture before colonization by neural crest cells. A recent
study has also shown development of functionally competent ICC in the
gastric antrum and small intestines of glial-derived neurotropic factor
knockout animals that fail to develop an enteric nervous system
(29). Other studies, however, have demonstrated
normal development of enteric ganglia and functional varicose processes
of motor neurons in tissues from which specific populations of ICC are
absent. Together, these findings suggest that, although certain enteric neurons express SCF, other cellular sources within the microenvironment can provide adequate stimulation of Kit receptors for ICC development. More recently, it has been shown that isolated smooth muscle cells and
ICC from mature animals both express SCF but that the expression of
soluble and membrane-bound factors of SCF varied depending on the
region of the GI tract from which these cells were isolated. Smooth
muscle cells of the gastric fundus expressed only the membrane-bound form of SCF, whereas ICC in the fundus (IC-IM) expressed only the
soluble form. Because membrane-bound SCF expression is required for
proper development of ICC, it is possible that cell-to-cell contact
between ICC and smooth muscle cells is needed for the development of
ICC in the gastric fundus. Interestingly, in the small intestine, ICC
and smooth muscle expressed only the soluble form. It is therefore
possible that other cells express the membrane-bound form or that the
membrane-bound form is expressed during early stages of development and
becomes switched off in mature smooth muscle cells and ICC
(5).
Postembryonic neural development and outgrowth of motor neuron
processes do not appear to depend on the presence of ICC, even though
these cells may well be eventual targets for enteric neurotransmitters. The interaction between ICC and nerves in the gut are not just limited
to enteric motor nerves. Vagal afferent nerve fibers ending in the
muscle layers of the stomach and duodenum as intramuscular arrays have
been reported to form close morphological relationships with both ICC
and smooth muscle cells (7). At present, it appears that
the tight relationship between ICC and enteric motor neurons may be a
refinement formed after independent development of these cells. It is
currently not known if there is a developmental dependence of vagal
afferent fibers on ICC.
Stage of development when Kit signaling is important.
Expression of Kit in the gut begins midway through embryogenesis and
continues through adulthood, but the point during development at which
Kit is critical for development of the ICC phenotype is controversial.
Most studies that have evaluated distributions of Kit-positive cells
have examined tissues after birth and concluded that Kit function is
necessary for the survival and maintenance of ICC. A couple of studies
have tried to address the question of when Kit becomes important for
the ICC phenotype. In one study (1), Kit-expressing cells
were examined in transgenic mice in which lacZ was
introduced by gene targeting at the W/kit locus in murine
embryonic stem cells. Insertion of lacZ in exon 1 of c-kit resulted in a null allele
(WlacZ). Heterozygous animals
(WlacZ/+) expressed lacZ (determined
by
-galactosidase histochemistry) in cells that normally express
c-kit. Homozygotes for the null allele
(WlacZ/WlacZ) failed to
express functional Kit. ICC were distributed equally in
WlacZ/WlacZ and
WlacZ/+ mice during embryogenesis, and the
authors concluded that Kit is only required for the postnatal
development of ICC. More recent work, however, has suggested
that the lacZ-positive cells observed in this study could be
ICC or the undifferentiated precursors of the longitudinal muscle layer
(21). Therefore, it is only possible to say that the
proliferation of c-kit/lacZ precursors did not
require Kit during embryogenesis. A second study (11) examined the distribution of ICC in Wbanded
(Wbd) murine mutants. Wbd
is a 2.5-megabase genomic rearrangement of chromosome 5 that inverts the c-kit gene and causes ectopic expression of Kit.
At 5 days of age (D5),
Wbd/Wbd mice displayed normal
networks of ICC and normal circular and longitudinal muscle layers, but
adult Wbd/Wbd mice lacked functional
ICC networks. This study suggested that Kit is necessary for normal
postnatal development and survival of ICC, but Kit is not involved in
the lineage decision of mesenchymal precursors that become ICC or
smooth muscle cells.
We have addressed this question by testing whether blockade of native
Kit interferes with ICC development in the late embryonic period.
Segments of the small intestine were removed from animals between E15
and E17 and placed in organ culture. We picked E15-E17 because
this is the point during development when mesenchymal precursors decide
whether to become IC-MY or longitudinal muscle cells. IC-MY networks
developed normally, and pacemaker activity was recorded from control
tissues after 3-5 days in culture (i.e., approximate time of
birth). Inclusion of neutralizing Kit antibody in the cultures,
however, caused disappearance of Kit-positive cells and blocked the
development of rhythmicity (Fig. 4).
These experiments provide direct evidence that Kit signaling is
critical for the emergence of the functional ICC during embryogenesis.

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Fig. 4.
Development of functional ICC networks (arrows) before birth in the
murine jejunum depends on Kit signaling. Jejunal muscles were removed
from an E17 fetus (A) and placed in organ culture for 2.5 days. At E19.5, extensive Kit-positive ICC networks are present in the
murine jejunum (B, arrows). Parallel culturing of jejunal
muscle from a sibling in the presence of neutralizing Kit antibody
caused loss of ICC networks. Kit immunoreactivity was only observed in
occasional cells (arrows; C). Electrical recordings showed
that slow waves were present in the tissue cultured from E17 to E19.5
in normal medium (D), but the muscle cultured with the
neutralizing Kit antibody lost ICC and was electrically quiescent
(E). These data demonstrate the importance of Kit signaling
during late gestation when functional IC-MY develop.
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Development of electrical rhythmicity.
ICC networks develop throughout the GI tract before the onset of
electrical slow waves. Gastric antral muscles are electrically quiescent at E15 and E16, and resting membrane potentials are relatively depolarized (e.g., about
40 mV). By E19, resting
potentials that are more negative develop and slow waves are present.
The amplitude and frequency of these slow waves are enhanced within 24 h of birth, and adult-like activity was observed by D10.
Jejunal circular muscle was electrically quiescent on E16 and E17, and resting membrane potential was ~10 mV less negative than the resting potential of mature small intestinal muscles. Two days before birth
(E18), irregular fluctuations were observed, and at D19 slow waves can
be recorded (21) (Fig. 4). Although the frequency of this
activity was less than in adult tissues, slow wave activity in these
preparations was not sensitive to the L-type calcium channel blocker
nifedipine. Resting membrane potential and the frequency and amplitude
of slow waves continued to develop in the postnatal period and reached
a maximum ~1 wk after birth. Interestingly, the ileum and colon are
electrically quiescent at birth. The ileum starts to develop
rhythmicity ~3 days after birth and by ~1 wk of age in the proximal
colon, revealing a rostral-caudal gradient in the functional
development of ICC (21, 27).
Spindle-shaped IC-IM are well developed before birth in the stomach
(E17), and neurally mediated responses could be recorded from preterm
tissues (unpublished observations). IC-DMP continued to develop after
birth in the small bowel, and neural responses in the small bowel
developed during the same time period. From a functional viewpoint, ICC
networks are laid down throughout the gut before rhythmicity develops.
Functional elements within the proximal gut are ready to generate
motility patterns at birth, whereas the distal gut must wait several
days after birth before ICC and mature electrical and mechanical
patterns develop.
Kit-mutant mice, such as the W/Wv, fail to
develop IC-IM throughout the stomach and in sphincters, such as the
lower esophageal sphincter and pylorus (2, 23, 28).
Without IC-IM, neural transmission from enteric motor neurons is
significantly compromised. In the gastric fundus, for example, only
IC-IM can be found in wild-type animals, and these cells form close,
synaptic-like relationships with the varicose processes of
excitatory and inhibitory motor neurons. In fundus
muscles of W/Wv animals, cholinergic
and nitrergic neurotransmission is greatly reduced (2,
23). Enteric neurons, varicose processes, and the ability to
release neurotransmitter are not reduced in these tissues, and smooth
muscle cells demonstrate responsiveness to exogenous transmitters.
These findings suggest that IC-IM play a critical role in mediating
neural inputs. Loss of this class of ICC during development or in
pathophysiological conditions would significantly compromise the
ability of GI muscles to generate typical motor reflexes.
Others have presented contrasting data concerning the timing of the
development of rhythmicity in the small intestine. It has been reported
that in unfed neonates 50% of tissues were electrically quiescent
(13). In other tissues, action potentials of irregular frequency were present and these events were sensitive to verapamil. In
these studies, ICC networks and pacemaker activity were not observed
before birth, and it was only 6 h after birth that ICC developed
in association with slow waves. Unfortunately, this study employed
methylene blue as a marker for ICC, and this technique can give false
impressions about the state of IC-MY development at birth. ICC are
clearly present at birth in the mouse, and these cells can be
visualized with Kit immunoreactivity. In fact, a well-developed network
of IC-MY appears as early as E18 (21, 27). In the jejunum,
ICC are functional and generate nifedipine-insensitive slow waves
before birth (21) (Fig. 5).

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Fig. 5.
ICC networks and slow waves present at birth in the murine jejunum.
A: confocal reconstruction (4 × 0.56 µm) revealing
Kit-immunoreactive IC-MY in the jejunum of an unfed neonate. IC-MY
networks (arrows) are well developed at the level of the myenteric
plexus in the small intestine by birth. B: slow wave
activity recorded with intracellular microelectrode techniques from the
circular muscle of the jejunum of an unfed neonate. These events were
insensitive to nifedipine (trace recorded in the presence of
10 6 M nifedipine).
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Electrical rhythmicity in the murine small intestine preceded the
development of IC-DMP by several days, giving additional support to the
earlier conclusion that IC-DMP are not involved in generation of slow
waves in the murine small bowel. Concomitant with the development of
IC-DMP, however, was development of enteric inhibitory
neurotransmission, suggesting a role for these cells as mediators of
neural inputs.
Development of ICC and rhythmicity is encoded in the tunica
muscularis.
Immunohistochemical studies have shown that ICC networks, already
developed by birth (Fig. 5), can be maintained in organ culture and
that IC-DMP, which are in the process of developing at birth, continue
to develop in an apparently normal manner in organ culture. The
structural integrity of ICC networks is matched by function. In organ
culture, gastric antrum and jejunal muscles, electrically rhythmic at
birth, continue to develop and slow wave activity grows in amplitude
and frequency. After 10 days in culture, the electrical activity was
equivalent to that of muscles taken from 10-day-old animals in which
ICC networks were allowed to develop in vivo. Ileal muscles,
electrically quiescent at birth, developed normal electrical activity
in organ culture. Furthermore, mature tissues can also be maintained in
culture. Jejunal muscles isolated from a 30-day-old mouse can be
maintained for periods up to 35 days (24).
Having a stable model of electrical rhythmicity allows in vitro
manipulation of ICC, and this facilitates studies of ICC that are not
affected in W or steel mutants. Addition of
neutralizing Kit antibody to culture media causes loss of IC-MY from
jejunal and ileal muscles cultured from birth. Loss of IC-MY is
associated with a loss of slow waves in jejunal muscles, and ileal
muscles failed to develop slow wave activity. Thus the Kit signaling
pathway, which is intrinsic to the tunica muscularis, is
required for postnatal development and maintenance of the ICC
phenotype. It should also be noted that when muscles are removed from
the embryonic bowel (E15-E17), IC-MY networks develop normally in
organ culture and so does electrical rhythmicity, but inclusion of
Kit-neutralizing antibody blocked IC-MY development and slow waves were
inhibited (Fig. 4). These observations suggest that the elements
necessary for Kit signaling (expression of SCF, Kit, and downstream
signaling molecules) are in place by E15-E17 and are not dependent
on external stimuli such as blood-borne factors or the presence of a
mucosa. It also appears that Kit signaling is important during the late embryonic period when IC-MY are developing into networks and
establishing function. These findings argue against studies suggesting
that Kit signaling is not required for embryonic development of ICC but
is only required during the postnatal period for maintenance and
maturation of the ICC phenotype.
Dependence of ICC networks on the Kit receptor.
The dependence of ICC on Kit signaling changes as a function of the
developmental state. For example, administration of neutralizing Kit
antibody immediately after or during the first couple of days following
birth disrupted ICC populations within 2-4 days. However, delaying
administration of the antibody until animals were 20-30 days old
required a much longer administration time before ICC were affected (up
to 35 days). The relative resistance to blockade of Kit signaling in
more mature tissues may be similar to variable resistance of different
types of ICC to W mutations. For example, IC-MY of the small
intestine and IC-IM of sphincters and stomach are lost in
W/WV mutants, but IC-MY of the stomach and colon
and IC-DMP of the small intestine are not greatly affected. We found
that IC-MY can also be removed from gastric muscles in organ culture by
treatment with neutralizing Kit antibody, but 2-3 wk of exposure
to the antibody was required vs. 2-4 days of exposure to remove
IC-MY in the small intestine. We have recently observed that
neutralizing antibody treatment reduces IC-MY in small intestinal
muscles removed from adult animals, but several weeks of exposure to
the antibody are necessary. Thus small intestinal IC-MY may develop
resistance to loss of Kit signaling with age, but gastric IC-MY may
inherently possess this resistance at birth. Regardless of the
increased resistance with age, Kit signaling plays an important role in the long-term maintenance of the ICC phenotype in adult tissues, and
chronic loss or defects in this signaling pathway may result in loss or
disruption in ICC networks with concomitant functional losses.
Downstream signaling pathways activated by Kit.
Specific elements of downstream signaling activated by Kit have also
been tested in organ-cultured muscles. Binding of SCF to Kit activates
its tyrosine kinase function. Kit is autophosphorylated, creating
high-affinity sites for intracellular signaling molecules such as
phosphatidylinositol 3-kinase (PI3 kinase), phospholipase C
, and
p21ras GTPase-activating protein in the kinase insert sequence of Kit.
Inhibitors of PI3 kinase, wortmannin and LY-294002, have been tested on
the development of small intestinal IC-MY and electrical rhythmicity.
Addition of wortmannin or LY-294002 to organ-cultured muscle strips
caused disappearance of IC-MY and inhibited the development of
electrical slow waves. Acute exposure of muscles to wortmannin or
LY-294002 had no effect on slow waves. Treatment of adult small
intestinal muscles with inhibitors of PI3 kinase also resulted in loss
of ICC, but much longer exposure times were necessary than with muscles
treated with these agents from birth (24). These
experiments demonstrate that secondary signaling via PI3 kinase is an
essential arm of the signal transduction cascade in ICC that is
activated by Kit. It may be possible to test other elements of the
signaling cascade with a similar approach or with tissues of transgenic
animals lacking specific signaling molecules.
 |
SUMMARY AND CONCLUSIONS |
In conclusion, the use of antibodies against the Kit receptor have
given us new methods to study the development of ICC in the GI tract
during embryogenesis. Within the last few years, we have developed a
good understanding about the time course of development, the molecular
markers that are expressed during development of ICC, and the
rostral-to-caudal development in morphology and function of ICC in GI
tissues. We also recognize that, although the dependence on Kit
signaling is universal to ICC, there is variability among ICC in
resistance to partial loss-of-function mutations in Kit. Sensitivity to
blockade of Kit signaling also changes with age, but adult tissues
continue to require Kit signaling to maintain the ICC phenotype. Loss
of ICC in many motility disorders, therefore, could result from loss of
Kit receptors, loss of SCF (a natural ligand for Kit) expression by
cells in the microenvironment of ICC, or loss or desensitization of
downstream signaling pathways. (The loss of ICC in pathophysiological
conditions and the ability of ICC to regenerate will be the topic of a
later article in this series.)
Many questions remain unanswered regarding the development and
plasticity of ICC. When the Kit signaling pathway is blocked, ICC
appear to differentiate toward a smooth muscle-like phenotype (Fig. 3).
However, information is lacking about whether these smooth muscle-like
cells retain the ability to regenerate the ICC phenotype if Kit
signaling is restored. Furthermore, we know little of the interaction
of ICC and nerve cells within the developing gut. Nerves and ICC
develop from different cellular lineages and can survive independently
of one another. However, when both cell types are present in the GI
tract they form a very specific relationship with one another. Also,
this may not be limited to enteric motor nerves but may also apply to
possible extrinsic afferent fibers that form close anatomic
relationships with ICC (7). How this targeting is achieved
and maintained is an important question for enteric neurobiology in the future.
 |
ACKNOWLEDGEMENTS |
Work on this manuscript was supported by National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-57236 to S. Ward.
K. Sanders and S. Ward have also been supported by a Program Project
Grant (DK-41315) for much of their work on ICC.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: S. M. Ward, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (E-mail:
Sean{at}physio.unr.edu).
 |
REFERENCES |
1.
Bernax, F,
De Sepulveda P,
Kress C,
Elbaz C,
Delouis C,
and
Panthier JJ.
Spatial and temporal patterns of c-kit-expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos.
Development
122:
3023-3033,
1996[Abstract/Free Full Text].
2.
Burns, AJ,
Lomax AEJ,
Torihashi S,
Sanders KM,
and
Ward SM.
Interstitial cells of Cajal mediate inhibitory neurotransmission in the stomach.
Proc Natl Acad Sci USA
93:
12008-12013,
1996[Abstract/Free Full Text].
3.
Cajal, SR.
Histologie du système nerveux de l'homme et des vertébrés. Paris: Maloine, 1911, vol. 2, p. 891-942.
4.
Daniel, EE,
and
Berezin I.
Interstitial cells of Cajal: are they major players in control of gastrointestinal motility?
J Gastrointest Motil
4:
1-24,
1992.
5.
Epperson, A,
Hatton WJ,
Callaghan B,
Walker R,
Sanders KM,
Ward SM,
and
Horowitz B.
Molecular components expressed in cultured and freshly isolated interstitial cells of Cajal.
Am J Physiol Cell Physiol
279:
C529-C539,
2000[Abstract/Free Full Text].
6.
Faussone-Pellegrini, MS,
Cortesini C,
and
Romagnoli P.
Sull'ultrastruttura della tunica muscolare della prozione cardiale dell'esofago e dello stomaco umano con particolare riferimento alle cosiddette cellule inerstiziali di Cajal.
Arch Ital Anat Embriol
82:
157-177,
1977[Medline].
7.
Fox, EA,
Phillips RJ,
Martinson FA,
Baronowsky EA,
and
Powley TL.
Vagal afferent innervation of smooth muscle in the stomach and duodenum of the mouse: morphology and topography.
J Comp Neurol
428:
558-576,
2000[ISI][Medline].
8.
Huizinga, JD,
Thuneberg L,
Kluppel M,
Malysz J,
Mikkelsen HB,
and
Bernstein A.
W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity.
Nature
373:
347-349,
1995[ISI][Medline].
9.
Imaizumi, M,
and
Hama K.
An electromicroscopic study on the interstitial cells of the gizzard in the love bird (Uronloncha domestica).
Z Zellforsch Mikrosk Anat
97:
351-357,
1969[ISI][Medline].
10.
Komuro, T.
Three-dimensional observation of the fibroblast-like cells associated with the rat myenteric plexus, with special reference to the interstitial cells of Cajal.
Cell Tissue Res
255:
343-351,
1989[ISI][Medline].
11.
Kluppel, M,
Huizinga JD,
Malysz J,
and
Bernstein A.
Developmental origin and Kit-dependent development of the interstitial cells of cajal in the mammalian small intestine.
Dev Dyn
211:
60-71,
1998[ISI][Medline].
12.
Lecoin, L,
Gabella G,
and
Le Douarin N.
Origin of the c-kit-positive interstitial cells in the avian bowel.
Development
122:
725-733,
1996[Abstract/Free Full Text].
13.
Liu, LW,
Thuneberg L,
and
Huizinga JD.
Development of pacemaker activity and interstitial cells of Cajal in the neonatal mouse small intestine.
Dev Dyn
213:
271-282,
1998[ISI][Medline].
14.
Maeda, H,
Yamagata A,
Nishikawa S,
Yoshinaga K,
Kobayashi S,
Nishi K,
and
Nishikawa S.
Requirement of c-kit for development of intestinal pacemaker system.
Development
116:
369-375,
1992[Abstract/Free Full Text].
15.
Orr-Urtreger, A,
Avivi A,
Zimmer Y,
Givol D,
Yarden Y,
and
Lonai P.
Developmental expression of c-kit, a proto-oncogene encoded by the W locus.
Development
109:
911-923,
1990[Abstract].
16.
Sanders, KM.
A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.
Gastroenterology
111:
492-515,
1996[ISI][Medline].
17.
Sanders, KM,
Torihashi S,
Ordog T,
Koh SD,
and
Ward SM.
Development and plasticity of interstitial cells of Cajal.
Neurogastroenterol Motil
11:
311-338,
1999[ISI][Medline].
18.
Taxi, J.
Contribution á l'étude des connexions des neurones moteurs du systeme nerveux sutonome.
Ann Sci Nat Zool Biol Anim
7:
413-674,
1965.
19.
Thuneberg, L.
Interstitial cells of Cajal: intestinal pacemaker cells.
Adv Anat Embryol Cell Biol
71:
1-130,
1982[ISI][Medline].
20.
Torihashi, S,
Ward SM,
Nishikawa SI,
Nishi K,
Kobayashi S,
and
Sanders KM.
c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract.
Cell Tissue Res
280:
97-111,
1995[ISI][Medline].
21.
Torihashi, S,
Ward SM,
and
Sanders KM.
Development of c-Kit-positive cells and the onset of electrical rhythmicity in murine small intestine.
Gastroenterology
112:
144-155,
1997[ISI][Medline].
22.
Torihashi, S,
Yoshida H,
Nishikawa SI,
Kunisada T,
and
Sanders KM.
Enteric neurons express steel factor-lacZ transgene in the murine gastrointestinal tract.
Brain Res
738:
323-328,
1996[ISI][Medline].
23.
Ward, SM,
Beckett EAH,
Wang XY,
Baker F,
Khoyi M,
and
Sanders KM.
Interstitial cells of Cajal mediate cholinergic neurotransmission from enteric motor neurons.
J Neurosci
20:
1393-1403,
2000[Abstract/Free Full Text].
24.
Ward, SM,
Brennan MF,
Jackson M,
and
Sanders KM.
Role of PI3-Kinase in the development of interstitial cells of Cajal and pacemaking in murine gastrointestinal smooth muscle.
J Physiol (Lond)
516:
835-846,
1999[Abstract/Free Full Text].
25.
Ward, SM,
Burns AJ,
Torihashi S,
Harney SC,
and
Sanders KM.
Impaired development of interstitial cells and intestinal electrical rhythmicity in steel mutants.
Am J Physiol Cell Physiol
269:
C1577-C1585,
1995[Abstract/Free Full Text].
26.
Ward, SM,
Burns AJ,
Torihashi S,
and
Sanders KM.
Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
J Physiol (Lond)
480:
91-97,
1994[Abstract].
27.
Ward, SM,
Harney SC,
Bayguinov JR,
McLaren GJ,
and
Sanders KM.
Development of electrical rhythmicity in the murine gastrointestinal tract is specifically encoded in the tunica muscularis.
J Physiol (Lond)
505:
241-258,
1997[Abstract].
28.
Ward, SM,
Morris G,
Reese L,
Wang XY,
and
Sanders KM.
Interstitial cells of Cajal mediate enteric inhibitory neurotransmission in the lower esophageal and pyloric sphincters.
Gastroenterology
115:
314-329,
1998[ISI][Medline].
29.
Ward, SM,
Ordog T,
Bayguinov JR,
Horowitz B,
Epperson A,
Shen L,
Westphal H,
and
Sanders KM.
Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves.
Gastroenterology
117:
584-594,
1999[ISI][Medline].
30.
Wu, JJ,
Rothman TP,
and
Gershon MD.
Development of the interstitial cell of Cajal: origin, kit dependence and neuronal and nonneuronal sources of kit ligand.
J Neurosci Res
59:
384-401,
2000[ISI][Medline].
31.
Young, HM,
Ciampoli D,
Southwell BR,
and
Newgreen DF.
Origin of interstitial cells of Cajal in the mouse intestine.
Dev Biol
180:
97-107,
1996[ISI][Medline].
Am J Physiol Gastrointest Liver Physiol 281(3):G602-G611
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