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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MORPHOLOGICAL IDENTIFICATION OF...
DEVELOPMENT OF ICC WITHIN...
SUMMARY AND CONCLUSIONS
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MORPHOLOGICAL IDENTIFICATION OF...
DEVELOPMENT OF ICC WITHIN...
SUMMARY AND CONCLUSIONS
REFERENCES

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).


    MORPHOLOGICAL IDENTIFICATION OF ICC
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MORPHOLOGICAL IDENTIFICATION OF...
DEVELOPMENT OF ICC WITHIN...
SUMMARY AND CONCLUSIONS
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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.

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.


    DEVELOPMENT OF ICC WITHIN THE GI TRACT
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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 gamma -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.

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 beta -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 beta -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.

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).

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 Cgamma , 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
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MORPHOLOGICAL IDENTIFICATION OF...
DEVELOPMENT OF ICC WITHIN...
SUMMARY AND CONCLUSIONS
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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
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INTRODUCTION
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SUMMARY AND CONCLUSIONS
REFERENCES

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Am J Physiol Gastrointest Liver Physiol 281(3):G602-G611
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