Physiology and pathophysiology of the interstitial cell of Cajal: from bench to bedside. I. Functional development and plasticity of interstitial cells of Cajal networks

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.     

Effects of the gap junction blocker glycyrrhetinic acid on gastrointestinal smooth muscle cells

In the tunica muscularis of the gastrointestinal (GI) tract, gap junctions form low-resistance pathways between pacemaker cells known as interstitial cells of Cajal (ICCs) and between ICC and smooth muscle cells. Coupling via these junctions facilitates electrical slow-wave propagation and responses of smooth muscle to enteric motor nerves. Glycyrrhetinic acid (GA) has been shown to uncouple gap junctions, but previous studies have shown apparent nonspecific effects of GA in a variety of tissues. We tested the effects of GA using isometric force measurements, intracellular microelectrode recordings, the patch-clamp technique, and the spread of Lucifer yellow within cultured ICC networks. In murine small intestinal muscles, beta-GA (10 muM) decreased phasic contractions and depolarized resting membrane potential. Preincubation of GA inhibited the spread of Lucifer yellow, increased input resistance, and decreased cell capacitance in ICC networks, suggesting that GA uncoupled ICCs. In patch-clamp experiments of isolated jejunal myocytes, GA significantly decreased L-type Ca(2+) current in a dose-dependent manner without affecting the voltage dependence of this current. The IC(50) for Ca(2+) currents was 1.9 muM, which is lower than the concentrations used to block gap junctions. GA also significantly increased large-conductance Ca(2+)-activated K(+) currents but decreased net delayed rectifier K(+) currents, including 4-aminopyridine and tetraethylammonium-resistant currents. In conclusion, the reduction of phasic contractile activity of GI muscles by GA is likely a consequence of its inhibitory effects on gap junctions and voltage-dependent Ca(2+) currents. Membrane depolarization may be a consequence of uncoupling effects of GA on gap junctions between ICCs and smooth muscles and inhibition of K(+) conductances in smooth muscle cells.     

Immunoelectron-microscopic study of Kit-expressing cells in the jejunum of wildtype and Ws/Ws rats

Interstitial cells of Cajal (ICC) are responsible for generating electrical slow waves in the gastrointestinal (GI) tract. Slow waves regulate the frequency of contractions of the tunica muscularis, and therefore ICC are critical for normal motility in the small intestine. ICC express Kit, the gene product of c-kit, a protooncogene that encodes a receptor tyrosine kinase. Physiological evidence demonstrating that ICC are pacemakers has come from experiments on W-mutant mice which have few Kit-positive cells at the level of the myenteric plexus (IC-MY) and also lack electrical slow waves. In the past identification of ICC required the use of electron microscopy, however the discovery that ICC express Kit has facilitated studies of the distribution of ICC in several species. Immunoelectron microscopy to relate ultrastructure to Kit expression has only been performed in a limited number of studies of mice. We examined the ultrastructure of Kit-expressing cells in the rat using immunoelectron microscopy and an anti-Kit antibody. We compared the presence and appearance of Kit-expressing ICC in wildtype and Ws/Ws rats, which carry a mutation in the white spotting locus and have a phenotype similar to W/Wv mutant mice. Kit-expressing cells could be detected in the myenteric plexus (MY) and deep muscular plexus (DMP) regions of the small intestine of wildtype animals. In Ws/Ws rats, Kit-expressing cells were not observed in the region of MY, but were observed in the DMP. The density of Kit-positive cells in the DMP of Ws/Ws rats was similar to those in wildtype rats. Electron microscopy showed that Kit-expressing cells at the level of the MY of the rat had similar ultrastructural features as IC-MY in wildtype mice. IC-DMP in the rat of both wildtype and Ws/Ws mutants were similar in structure to IC-DMP of the mouse. We conclude that wildtype rats have IC-MY and IC-DMP in the tunica muscularis of the jejunum. ICC express Kit-like immunoreactivity (Kit-LI) in the rat as in the mouse. IC-MY are absent in the small intestine of Ws/Ws rats, and this corresponds to the lack of Kit-labeling in this region. Ws/Ws rats, however, possess IC-DMP with normal ultrastructural features and Kit-LI. The absence of IC-MY of Ws/Ws rats is likely to account for the abnormal contractile activity of the GI tract observed in these mutants. The present study suggests that Ws/Ws rats could provide an interesting model to investigate the physiological significance of pacemaker activity because they manifest a defect in IC-MY.     

Enteric motor neurons form synaptic-like junctions with interstitial cells of Cajal in the canine gastric antrum

Morphological studies have shown synaptic-like structures between enteric nerve terminals and interstitial cells of Cajal (ICC) in mouse and guinea pig gastrointestinal tracts. Functional studies of mice lacking certain classes of ICC have also suggested that ICC mediate enteric motor neurotransmission. We have performed morphological experiments to determine the relationship between enteric nerves and ICC in the canine gastric antrum with the hypothesis that conservation of morphological features may indicate similar functional roles for ICC in mice and thicker-walled gastrointestinal organs of larger mammals. Four classes of ICC were identified based on anatomical location within the tunica muscularis. ICC in the myenteric plexus region (IC-MY) formed a network of cells that were interconnected to each other and to smooth muscle cells by gap junctions. Intramuscular interstitial cells (IC-IM) were found in muscle bundles of the circular and longitudinal layers. ICC were located along septa (IC-SEP) that separated the circular muscle into bundles and were also located along the submucosal surface of the circular muscle layer (IC-SM). Immunohistochemistry revealed close physical associations between excitatory and inhibitory nerve fibers and ICC. These contacts were synaptic-like with pre- and postjunctional electron-dense regions. Synaptic-like contacts between enteric neurons and smooth muscle cells were never observed. Innervated ICC formed gap junctions with neighboring smooth muscle cells. These data show that ICC in the canine stomach are innervated by enteric neurons and express similar structural features to innervated ICC in the murine GI tract. This morphology implies similar functional roles for ICC in this species.     

Primary cilia in gastric Gastrointestinal Stromal Tumours (GISTs): an ultrastructural study

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal (non‐epithelial) neoplasms of the human gastrointestinal (GI) tract. They are thought to derive from interstitial cells of Cajal (ICCs) or an ICC progenitor based on immunophenotypical and ultrastructural similarities. Because ICCs show primary cilium, our hypothesis is based on the possibility that some of these neoplastic cells could also present it. To determine this, an exhaustive ultrastructural study has been developed on four gastric GISTs. Previous studies had demonstrated considerable variability in tumour cells with two dominating phenotypes, spindly and epithelioid. In addition to these two types, we have found another cell type reminiscent of adult ICCs with a voluminous nucleus surrounded by narrow perinuclear cytoplasm with long slender cytoplasmic processes. We have also noted the presence of small undifferentiated cells. In this study, we report for the first time the presence of primary cilia (PCs) in spindle and epithelioid tumour cells, an ultrastructural feature we consider of special interest that has hitherto been ignored in the literature dealing with the ultrastructure of GISTs. We also point out the frequent occurrence of multivesicular bodies (MVBs). The ultrastructural findings described in gastric GISTs in this study appear to be relevant considering the critical roles played by PCs and MVBs recently demonstrated in tumourigenic processes.     

Interstitial cells in the primate gastrointestinal tract

Kit immunohistochemistry and confocal reconstructions have provided detailed 3-dimensional images of ICC networks throughout the gastrointestinal (GI) tract. Morphological criteria have been used to establish that different classes of ICC exist within the GI tract and physiological studies have shown that these classes have distinct physiological roles in GI motility. Structural studies have focused predominately on rodent models and less information is available on whether similar classes of ICC exist within the GI tracts of humans or non-human primates. Using Kit immunohistochemistry and confocal imaging, we examined the 3-dimensional structure of ICC throughout the GI tract of cynomolgus monkeys. Whole or flat mounts and cryostat sections were used to examine ICC networks in the lower esophageal sphincter (LES), stomach, small intestine and colon. Anti-histamine antibodies were used to distinguish ICC from mast cells in the lamina propria. Kit labeling identified complex networks of ICC populations throughout the non-human primate GI tract that have structural characteristics similar to that described for ICC populations in rodent models. ICC-MY formed anastomosing networks in the myenteric plexus region. ICC-IM were interposed between smooth muscle cells in the stomach and colon and were concentrated within the deep muscular plexus (ICC-DMP) of the intestine. ICC-SEP were found in septal regions of the antrum that separated circular muscle bundles. Spindle-shaped histamine⁺ mast cells were found in the lamina propria throughout the GI tract. Since similar sub-populations of ICC exist within the GI tract of primates and rodents and the use of rodents to study the functional roles of different classes of ICC is warranted.     

Distribution of the intermediate filament nestin in the muscularis propria of the human gastrointestinal tract

The intermediate filament nestin is expressed in neural stem cells, neuroectodermal tumors and various adult tissues. In the gastrointestinal (GI) tract, nestin has been reported in glial cells. Recently, nestin has been reported in interstitial cells of Cajal (ICC) and in gastrointestinal stromal tumors, thought to derive from ICC. Here we investigated nestin immunoreactivity (-ir) in the normal human GI tract, with emphasis on Kit-ir ICC. Two different antibodies specific for human nestin and multicolor high-resolution confocal microscopy were used on material from our human GI tissue collection. The staining pattern of both nestin antibodies was similar. In labeled cells, nestin-ir appeared filamentous. Most intramuscular ICC in antrum and all myenteric ICC (ICC-MP) in small intestine were nestin-ir, while nestin-ir was not detected in deep muscular plexus ICC. In the colon, some - but not all - ICC-MP and most ICC in the circular musculature were nestin-ir while nestin-ir was not detected in ICC in the longitudinal musculature and in the submuscular plexus. In addition, many Kit-negative cells were nestin-ir in all regions. Neurons and smooth muscle cells were consistently nestin negative, while most S100-ir glial cells were nestin-ir. In addition, nestin-ir was also present in some CD34-ir fibroblast-like cells, in endothelium and in other cell types in the mucosa and serosa. In conclusion, nestin-ir is abundantly present in the normal human GI tract. Among a number of cell types, several, but not all, subpopulations of Kit-ir ICC were nestin-ir. The functional significance of nestin in the GI tract remains obscure.     

Biological and clinical review of stromal tumors in the gastrointestinal tract

Submucosal tumors of the gastrointestinal tract (GI tract) mainly consist of gastrointestinal mesenchymal tumors (GIMTs) that are distributed in the GI tract from the esophagus through the rectum. GIMTs include myogenic tumors, neurogenic tumors and gastrointestinal stromal tumors (GISTs). The term "GIST" is now preferentially used for the tumors that express CD34 and KIT. GIMTs are composed of spindle or epithelioid cells, and 20% to 30% show malignant behavior, including peritoneal dissemination and hematogenous metastasis. KIT expression and mutations in the c-kit gene are found only in GISTs, but not in myogenic or neurogenic tumors. Mutation in the c-kit gene is associated with aggressive features and poor prognosis, and malignant GISTs frequently have mutations in the c-kit gene. The clinicopathological features of GISTs with or without c-kit mutations are markedly different. Therefore, GIMTs may be divided into four major categories based on histochemical and genetic data: myogenic tumors; neurogenic tumors; GISTs with c-kit mutation; and GISTs without c-kit mutation. The origin of GISTs is not fully understood. However, phenotypical resemblance to the interstitial cells of Cajal (ICCs) and gain-of-function mutations in the c-kit gene may suggest origin from ICCs and/or multipotential mesenchymal cells that differentiate into ICCs.     

Telocytes express PDGFRα in the human gastrointestinal tract

Telocytes (TC), a cell population located in the connective tissue of many organs of humans and laboratory mammals, are characterized by a small cell body and extremely long and thin processes. Different TC subpopulations share unique ultrastructural features, but express different markers. In the gastrointestinal (GI) tract, cells with features of TC were seen to be CD34‐positive/c‐kit‐negative and several roles have been proposed for them. Other interstitial cell types with regulatory roles described in the gut are the c‐kit‐positive/CD34‐negative/platelet‐derived growth factor receptor α (PDGFRα)‐negative interstitial cells of Cajal (ICC) and the PDGFRα‐positive/c‐kit‐negative fibroblast‐like cells (FLC). As TC display the same features and locations of the PDGFRα‐positive cells, we investigated whether TC and PDGFRα‐positive cells could be the same cell type. PDGFRα/CD34, PDGFRα/c‐kit and CD34/c‐kit double immunolabelling was performed in full‐thickness specimens from human oesophagus, stomach and small and large intestines. All TC in the mucosa, submucosa and muscle coat were PDGFRα/CD34‐positive. TC formed a three‐dimensional network in the submucosa and in the interstitium between muscle layers, and an almost continuous layer at the submucosal borders of muscularis mucosae and circular muscle layer. Moreover, TC encircled muscle bundles, nerve structures, blood vessels, funds of gastric glands and intestinal crypts. Some TC were located within the muscle bundles, displaying the same location of ICC and running intermingled with them. ICC were c‐kit‐positive and CD34/PDGFRα‐negative. In conclusion, in the human GI tract the TC are PDGFRα‐positive and, therefore, might correspond to the FLC. We also hypothesize that in human gut, there are different TC subpopulations probably playing region‐specific roles.     

Roles of Interleukin-9 in the Growth and Cholecystokinin-Induced Intracellular Calcium Signaling of Cultured Interstitial Cells of Cajal

Interstitial cells of Cajal (ICC) are pacemaker cells in the gastrointestinal (GI) tract and loss of ICC is associated with many GI motility disorders. Previous studies have shown that ICC have the capacity to regenerate or restore, and several growth factors are critical to their growth, maintenance or regeneration. The present study aimed to investigate the roles of interleukin-9 (IL-9) in the growth, maintenance and pacemaker functions of cultured ICC. Here, we report that IL-9 promotes proliferation of ICC, and culturing ICC with IL-9 enhances cholecystokinin-8-induced Ca²⁺ transients, which is probably caused by facilitating maintenance of ICC functions under culture condition. We also show co-localizations of cholecystokinin-1 receptor and IL-9 receptor with c-kit by double-immunohistochemical labeling. In conclusion, IL-9 can promote ICC growth and help maintain ICC functions; IL-9 probably performs its functions via IL-9 receptors on ICC.     

The "fish-specific" Hox cluster duplication is coincident with the origin of teleosts

The Hox gene complement of zebrafish, medaka, and fugu differs from that of other gnathostome vertebrates. These fishes have seven to eight Hox clusters compared to the four Hox clusters described in sarcopterygians and shark. The clusters in different teleost lineages are orthologous, implying that a "fish-specific" Hox cluster duplication has occurred in the stem lineage leading to the most recent common ancestor of zebrafish and fugu. The timing of this event, however, is unknown. To address this question, we sequenced four Hox genes from taxa representing basal actinopterygian and teleost lineages and compared them to known sequences from shark, coelacanth, zebrafish, and other teleosts. The resulting gene genealogies suggest that the fish-specific Hox cluster duplication occurred coincident with the origin of crown group teleosts. In addition, we obtained evidence for an independent Hox cluster duplication in the sturgeon lineage (Acipenseriformes). Finally, results from HoxA11 suggest that duplicated Hox genes have experienced diversifying selection immediately after the duplication event. Taken together, these results support the notion that the duplicated Hox genes of teleosts were causally relevant to adaptive evolution during the initial teleost radiation.     

Interstitial cell network volume is reduced in the terminal bowel of ageing mice

Ageing is associated with impaired neuromuscular function of the terminal gastrointestinal (GI) tract, which can result in chronic constipation, faecal impaction and incontinence. Interstitial cells of cajal (ICC) play an important role in regulation of intestinal smooth muscle contraction. However, changes in ICC volume with age in the terminal GI tract (the anal canal including the anal sphincter region and rectum) have not been studied. Here, the distribution, morphology and network volume of ICC in the terminal GI tract of 3‐ to 4‐month‐old and 26‐ to 28‐month‐old C57BL/6 mice were investigated. ICC were identified by immunofluorescence labelling of wholemount preparations with an antibody against c‐Kit. ICC network volume was measured by software‐based 3D volume rendering of confocal Z stacks. A significant reduction in ICC network volume per unit volume of muscle was measured in aged animals. No age‐associated change in ICC morphology was detected. The thickness of the circular muscle layer of the anal sphincter region and rectum increased with age, while that in the distal colon decreased. These results suggest that ageing is associated with a reduction in the network volume of ICC in the terminal GI tract, which may influence the normal function of these regions.     

Role of Interstitial Cells of Cajal and their relationship with the enteric nervous system.

The term 'Interstitial cells of Cajal' (ICC) designates several groups of mesenchymal cells present along the gastro-intestinal tract (GI), in close association with smooth muscle cells and elements of the enteric nervous system (ENS). For years, transmission electron microscopy (TEM) has been the only reliable tool to study ICC. Whilst TEM remains the golden standard for identification of ICC, the observation that the tyrosine kinase receptor c-kit plays a crucial role in their development recently resulted in numerous immunohistochemical studies and also led to a better characterization of their roles. ICC form extensive networks of electrically coupled cells and certain groups of ICC are currently regarded as the source of the spontaneous slow waves of the gut musculature (pacemaker cells). Other ICC appear to be involved in the transduction of the relaxation of smooth muscle triggered by nitric oxide. Abnormal distribution of ICC has been reported in several human diseases and abnormal functioning of ICC might actually be involved in many disorders of GI transit. This review addresses (1) the morphology and relationships of ICC along the GI tract in man and mouse, mainly based on data from immunohistochemistry and confocal microscopy, (2) the emerging role of ICC in the pathophysiology of human diseases, like infantile hypertrophic pyloric stenosis (a common disorder with a dysfunction of the pyloric sphincter), Hirschsprung's disease (aganglion-osis coli) and intestinal pseudo-obstruction, (3) developmental issues, (4) recent reports suggesting a possible link between ICC and gastrointestinal stromal tumors.     

Development of the cerebellum and cerebellar neural circuits

The cerebellum, a structure derived from the dorsal part of the most anterior hindbrain, is important for integrating sensory perception and motor control. While the structure and development of the cerebellum have been analyzed most extensively in mammals,recent studies have shown that the anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost) species, including zebrafish. In the mammalian and teleost cerebellum,Purkinje and granule cells serve, respectively, as the major GABAergic and glutamatergic neurons. Purkinje cells originate in the ventricular zone (VZ), and receive inputs from climbing fibers. Granule cells originate in the upper rhombic lip (URL) and receive inputs from mossy fibers. Thus, the teleost cerebellum shares many features with the cerebellum of other vertebrates, and isa good model system for studying cerebellar function and development. The teleost cerebellum also has features that are specific to teleosts or have not been elucidated in mammals, including eurydendroid cells and adult neurogenesis. Furthermore, the neural circuitry in part of the optic tectum and the dorsal hindbrain closely resembles the circuitry of the teleost cerebellum; hence,these are called cerebellum-like structures. Here we describe the anatomy and development of cerebellar neurons and their circuitry, and discuss the possible roles of the cerebellum and cerebellum-like structures in behavior and higher cognitive functions. We also consider the potential use of genetics and novel techniques for studying the cerebellum in zebrafish.     

Loss of interstitial cells of Cajal after pulsating electromagnetic field (PEMF) in gastrointestinal tract of the rats.

Exposure to the magnetic field has remarkably increased lately due to fast urbanization and widely available magnetic field in diagnosis and treatment. However, biological effects of the magnetic field are not well recognized. The myoelectric activity recorded from the gastrointestinal and urinary systems is generated by specialized electrically active cells called interstitial cells of Cajal (ICCs). Thus it seems rational that ICC have significant vulnerability to physical factors like an electromagnetic field. The aim of this study was to evaluate the influence of pulsating electromagnetic field (PEMF) (frequency 10 kHz, 30ms, 300 muT burst, with frequency 1Hz) on ICCs density in the rat gastrointestinal tract. Rats were divided into two groups (n=32). The first group was exposed to PEMF continuously for 1, 2, 3, and 4 weeks (n = 16), and the second group (n=16) served as a control. Tissue samples of the rat stomach, duodenum and proximal colon were fixed and paraffin embedded. The tangential sections of 5 microm thickness were stained immunohistochemically with anti-c-Kit (sc-168) antibody and visualized finally by DAB as chromogen (brown end product). C-Kit positive branched ICC-like cells were detected under the light microscope, distinguished from the c-kit-negative non-branched smooth muscle cells and from the c-kit positive but non-branched mast cells and quantitatively analyzed by MultiScan computer program. Apoptosis detection was performed with rabbit anti-Bax polyclonal antibody (Calbiochem, Germany) and LSAB 2 visualization system. The surface of c-Kit immunopositive cells decreased after exposure to PEMF in each part of the gastrointestinal tract. Reduced density of ICCs was related to exposure time. The most sensitive to PEMF were ICCs in the fundus of the stomach and in the duodenum, less sensitive were ICCs in the colon and pacemaker areas of the stomach. No marked changes in ICC density in the pyloric part of the stomach were observed. We demonstrate that the PEMF induced apoptosis dependent decrease in ICC expression.     

Immunohistochemical and ultrastructural characteristics of interstitial cells of Cajal in the rabbit duodenum. Presence of a single cilium

Santiago Ramón y Cajal discovered a new type of cell related to the myenteric plexus and also to the smooth muscle cells of the circular muscle layer of the intestine. Based on their morphology, relationships and staining characteristics, he considered these cells as primitive neurons. One century later, despite major improvements in cell biology, the interstitial cells of Cajal (ICCs) are still controversial for many researchers. The aim of study was to perform an immunohistochemical and ultrastructural characterization of the ICCs in the rabbit duodenum. We have found interstitial cells that are positive for c-Kit, CD34 and nestin and are also positive for Ki67 protein, tightly associated with somatic cell proliferation. By means of electron microscopy, we describe ICCs around enteric ganglia. They present triangular or spindle forms and a very voluminous nucleus with scarce perinuclear chromatin surrounded by a thin perinuclear cytoplasm that expands with long cytoplasmic processes. ICC processes penetrate among the smooth muscle cells and couple with the processes of other ICCs located in the connective tissue of the circular muscle layer and establish a three-dimensional network. Intercellular contacts by means of gap-like junctions are frequent. ICCs also establish gap-like junctions with smooth muscle cells. We also observe a population of interstitial cells of stellate morphology in the connective tissue that sur-rounds the muscle bundles in the circular muscle layer, usually close to nervous trunks. These cells establish different types of contacts with the muscle cells around them. In addition, the presence of a single cilium showing a structure 9 + 0 in an ICC is demonstrated for the first time. In conclusion, we report positive staining c-Kit, CD34, nestin and Ki 67. ICCs fulfilled the usual transmission electron microscopy (TEM) criteria. A new ultrastructural characteristic of at least some ICCs is demonstrated: the presence of a single cilium. Some populations of ICCs in the rabbit duodenum present certain immunohistochemical and ultrastructural characteristics that often are present in progenitor cells.     

Ultrastructural observations of the tunica muscularis in the small intestine of Xenopus laevis, with special reference to the interstitial cells of Cajal

The distribution and ultrastructure of the interstitial cells of Cajal (ICC) has been examined in the small intestine of the frog Xenopus laevis, as the physiological significance of these cells remains obscure in amphibians and other lower vertebrates. The present study has revealed the existence of a special type of interstitial cell in the tunica muscularis of the small intestine of Xenopus; this cell is characterized by the presence of numerous caveolae, many small mitochondria, and the formation of intercellular connections with the same type of cell. Since these ultrastructural features are shared with mammalian ICC, the cells in the small intestine of Xenopus probably correspond to ICC. These cells also form close contacts with neighboring smooth muscle cells and with nerve varicosities containing accumulations of synaptic vesicles. These cellular networks are likely to be involved in the transmission of nerve impulses to muscle cells, as has been suggested for mammalian tissues. However, true gap junctions have not been detected; they occur neither between the same type of cells nor between the putative ICC and smooth muscle cells. The widespread distribution of ICC or equivalent cells in different groups of vertebrates, together with the conservation of their ultrastructural features, suggests that they differentiated early in vertebrate evolution to play key regulatory roles in gastrointestinal movement.