Normal peristaltic and segmental motor activity requires

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1 GASTROENTEROLOGY 2001;120: Development of Interstitial Cells of Cajal in a Full-Term Infant Without an Enteric Nervous System JAN D. HUIZINGA,*, IRENE BEREZIN,*, KANISHKA SIRCAR, BRYAN HEWLETT, GRAEME DONNELLY,*, PREMYSL BERCIK,*, CATHERINE ROSS, TALAL ALGOUFI, PETER FITZGERALD,, TARA DER,*, ROBERT H. RIDDELL, STEPHEN M. COLLINS,*, and KEVAN JACOBSON, Departments of *Medicine, Pathology and Molecular Medicine, Pediatrics, and Surgery and Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario, Canada The relationship between the development of the enteric nervous system and interstitial cells of Cajal (ICC) in the human small intestine was investigated in a full-term infant who presented with intestinal pseudo-obstruction. Immunohistochemistry revealed absence of enteric nerves and ganglia but abundant c-kit immunoreactivity associated with Auerbach s plexus (ICC-AP). However, c-kit immunoreactivity associated with the deep muscular plexus (ICC-DMP) and intermuscular ICC was absent. Electron microscopy showed ICC-AP with a normal ultrastructure; ICC-DMP were seen but were severely injured, suggesting degeneration. In vitro recording of intestinal muscle showed slow wave activity as well as response to cholinergic stimulation. Fluoroscopic examination of the small bowel showed a variety of motor patterns, including rhythmic, propagating contractions. In conclusion, total absence of enteric nerves was associated with absence of normal ICC-DMP. However, a normal musculature, including a network of ICC-AP, allowed for generation of rhythmic, propagating contractile activity, suggesting the presence of functional motor activity. Normal peristaltic and segmental motor activity requires cooperation between the musculature, enteric nerves, and interstitial cells of Cajal (ICC). 1 The absence of ICC associated with Auerbach s plexus (ICC- AP) impairs normal distention-induced peristalsis in the small intestine. 2 ICC-AP initiate the intestinal pacemaker activity, the so-called slow waves that determine frequency and propagation characteristics of intestinal motor activity. 3,4 Functional roles of intermuscular ICC and ICC associated with the deep muscular plexus (ICC- DMP) are less well understood, but they are involved in enteric innervation. 5 All ICC have an intimate morphologic and functional relationship with enteric nerves that could indicate a mutual dependence during development. Information from some animal models exists, 6,7 but no data are available on human ICC development as it relates to neural development. There are only a few reports on the role of ICC in the pathophysiology of gut motor function in infants, but delayed maturation of ICC, with normal development of enteric nerves, has been shown to be associated with a failure to develop normal peristaltic motor activity. 8 Hence, delayed maturation of ICC may underlie the etiology of transient neonatal pseudo-obstruction. The first objective of this case study was to provide knowledge about the developmental relationships between ICC and enteric nerves. A second aim was to determine whether the musculature was capable of generating functional motor patterns in the presence of pacemaker cells without the contribution of enteric nerves. Case Report A full-term male was delivered vaginally to a 29-yearold nulliparous woman after an uncomplicated pregnancy. The antenatal history, family history, and antenatal ultrasound examination were unremarkable. The delivery was uneventful, the infant s Apgar scores were 9 at 5 minutes and 10 at 10 minutes, and physical examination did not show anything remarkable. His birth weight was 3155 g. By 18 hours postpartum, the infant appeared jaundiced. Blood tests revealed an unconjugated hyperbilirubinemia that was likely a result of ABO incompatibility between mother (O ) and son (B ). At 36 hours postpartum, he developed bilious vomiting, he had not yet passed meconium, and a plain radiograph of the abdomen showed dilated loops of small bowel. A lower gastrointestinal single-contrast study (Omnipaque 240; Nycomed Imaging, Oslo, Norway) performed 48 hours postpartum showed a small-caliber colon with multiple Abbreviations used in this paper: AP, Auerbach s plexus; DJ, duodenojejunal; DMP, deep muscular plexus; ICC, interstitial cells of Cajal; ICC-AP, ICC associated with AP; ICC-DMP, ICC associated with DMP; PGP 9.5, protein gene product by the American Gastroenterological Association /01/$35.00 doi: /gast

2 562 HUIZINGA ET AL. GASTROENTEROLOGY Vol. 120, No. 2 filling defects consistent with meconium plugging. Subsequent plain films of the abdomen revealed retained contrast in the colon. Rectal biopsies 72 hours postpartum showed aganglionosis. Normal sweat chloride concentrations ruled out cystic fibrosis. Day 6 postpartum, the baby underwent a laparotomy to determine the extent of the aganglionosis. Intraoperatively, some dilatation of the proximal small bowel was observed; however, contractile activity was seen in this segment of bowel. The colon and ileum appeared small and featureless. On frozen section, ganglion cells were absent from the appendix and from 10 seromuscular biopsy specimens taken from the ileum and jejunum. A biopsy specimen taken at approximately 7 cm from the duodenojejunal (DJ) flexure was initially thought to contain ganglion cells. On day 15 postpartum, a further laparotomy was performed for G-tube insertion, jejunostomy, and placement of ileostomy with mucous fistula. A significant amount of meconium was milked from the ileum, and the bowel was flushed with saline. At 50 cm from the DJ flexure, a jejunostomy was fashioned on the left side of the abdomen, and the mucous fistula was created on the right side of the abdomen. Approximately 5 cm of small bowel 50 cm distal to the DJ flexure was removed for histologic and functional examination, and a seromuscular biopsy specimen from the anterior wall of the stomach was obtained for histologic examination. Total parenteral nutrition (TPN) was commenced on day 2 postpartum, and continuous G-tube feeds with pregestemil were instituted after the second surgery. The baby s enteral feeding tolerance was limited by ostomy fluid and sodium losses and later by recurrent intestinal bleeds associated with portal hypertension. He was only able to tolerate pregestimil (0.67 kcal/ml) at an infusion rate of 4 ml/h for 8 hours twice a day with a rest period of 4 hours between infusions. The baby s ostomy losses averaged 137 ml/d and his G-tube losses 85 ml/d. His fluid intake averaged 160 to 170 ml kg 1 day 1 and his total caloric intake 113 kcal kg 1 day 1. The child remained on full TPN awaiting a multivisceral transplant, but unfortunately died on day 119 postpartum from hepatic complications associated with TPN. Materials and Methods Routine 5- m H&E staining was performed on multiple tissues from rectum to stomach (taken at days 3, 6, and 15) and examined for the presence of ganglion cells. Immunohistochemistry was performed for the presence of c-kit as a marker for ICC, protein gene product 9.5 (PGP 9.5), and acetylcholinesterase as markers for nerves. No nerves and ganglion cells were found from stomach to colon, but this report will focus solely on the intestine. At day 15, a 5-cm segment of small bowel was taken 50 cm distal to the DJ flexure for electron microscopy, 9 immunohistochemistry, 10 and electrophysiologic studies at the time of placing an ostomy. Electrophysiologic studies were performed on a segment of the tissue resected on day 15 postpartum. This was pinned flat in a dissecting dish. The mucosa was removed from the external muscle layers, and studies were done on 5 mm 10 mm sections. 2 Fluoroscopy images were recorded for a 10-minute period after applying barium contrast intragastrically via the gastrostomy tube. Video images were digitized at a frequency of 10 images per second. As described previously, changes in optical density dependent on the contractile status of the intestine were evaluated, and spatiotemporal maps were created. 11 Frequency and velocity of contraction, as well as coordination between segments, were examined. Results Morphology Innervation of the intestine. Immunohistochemical detection of the neural marker PGP 9.5 showed an absence of ganglia and enteric nerves at the site of Auerbach s plexus (AP), within the circular and longitudinal muscle layers, and in the deep muscular plexus (DMP) area (Figure 1). Immunohistochemistry using antibodies against the Schwann cell marker S100 and acetylcholinesterase confirmed the absence of nerve structures (not shown). Sporadic PGP positivity was seen identifying extrinsic nerves associated with submucosal blood vessels and within the AP region (Figure 1B). Electron microscopy of the jejunum showed absence of enteric nerve fibers, neuronal cell bodies, and associated Schwann cells in the AP region and within both muscle layers. There was a sparse presence of extrinsic nerve fibers in the AP region (Figure 2B) and adjoining longitudinal muscle, as well as extrinsic nerve fibers associated with blood vessels in the submucosa, distinguished from enteric nerves by the presence of a complete or partial perineurial sheath (Figure 2B). Extrinsic nerve fascicles were composed of bundles of up to 20 small axons ( m in diameter). ICC-AP. The ICC were by far the most prominent cells in the area between the circular and longitudinal muscle layers (Figure 3A and B), their features similar to the previously published descriptions of ICC-AP in normal human adult tissue. 12 The ICC-AP processes formed close-apposition contacts with each other and nearby smooth muscle cells (Figure 3A), but no gap junctions were found. No close contacts were found between ICC-AP and extrinsic nerves. ICC-AP and groups of overlapping processes extended into the septa of the circular muscle layer. Fibroblasts were present uniformly throughout the entire muscularis externa and in the submucosa. The AP region harbored many small blood vessels, associated pericytes, collagen fibers, macrophages, and occasional mast cells and lymphocytes. The musculature. The organization of the circular and longitudinal muscle layers appeared normal on

3 February 2001 ICC IN ABSENCE OF ENTERIC NERVES 563 Figure 1. Immunohistochemistry revealed absence of PGP reactivity, indicating absence of enteric nerves and ganglia but abundant presence of ICC in the AP region (arrows). (A) H&E staining of small intestinal musculature, 50 cm distal to the pylorus, including part of the longitudinal muscle (top), circular muscle, and part of the submucosa. (B) Adjacent section stained with PGP. Note the absence of staining in the AP region but scattered staining of small extrinsic nerves (arrowhead) and staining associated with blood vessels in the submucosa. (C ) Immunohistochemistry on the adjacent section with c-kit antibodies revealing presence of ICC in AP, but absence of staining within the muscle layers and in the DMP. Note positive mast cells in the submucosa. (D) c-kit immunohistochemistry of another section of proximal intestine revealing a normal presence of ICC in the AP with some presence in the firs part of the septa. light (Figure 1A) and electron microscopy. The circular muscle layer was comprised of a thin inner layer and a thick outer layer and was divided by a narrow zone of connective tissue. There were no prominent degenerative changes in the circular muscle cells that would indicate cell injury (Figure 2A and C). Only a few circular muscle cells displayed an abnormal abundance of rough endoplasmic reticulum, ribosomes, and Golgi complexes in their perinuclear cytoplasm, indicating the possibility of a rudimentary myofibroblast transformation. The smooth muscle cells formed many close apposition contacts with each other. Muscle cells of the outer circular layer were also interconnected by numerous gap junctions (Figure 2A). There was no apparent increase of collagen or extracellular space between muscle cells of the circular muscle layer nor evidence of hypertrophy. ICC-DMP. In the inner third part of the circular muscle layer, where nerves and ICC of the DMP are usually found in normal adult human tissue, 13,14 no normal ICC were detected. Between the inner and outer subdivision of the circular muscle layers, in the zone of connective tissue where ICC-DMP are normally found, ramified cells were encountered in various stages of injury (Figure 2C). They were characterized by partial or complete destruction of their organelle contents (Figure 2C), had long thin processes, and frequently were found in extended close contacts with adjacent circular muscle cells. Some of these ramified cells were characterized by the presence of plasma membrane caveolae and well-developed Golgi complexes (Figure 2C). Because of their location, frequent contacts with smooth muscle cells, presence of caveolae, and their branched cellular profiles, these damaged cells were identified as ICC-DMP. Immune cells. Macrophages were the predominant immune cell found regularly in both muscle layers in close contact with smooth muscle cells, at the level of AP and in the submucosa. Neutrophils were seen only in the lumens of blood vessels. Mast cells

4 564 HUIZINGA ET AL. GASTROENTEROLOGY Vol. 120, No. 2 Figure 2. Ultrastructure of smooth muscle, extrinsic nerves, ICC-DMP, and immune cells. (A) Circular muscle cells are normal interconnected by large gap junctions (arrows). (B) A small extrinsic nerve bundle (N) composed of several axons (A) enclosed by Schwann cell cytoplasm (SwC) can be seen in the AP region. Note the complete perineurial covering (P) with perineurial caveolae (arrow) and Schwann cell basal lamina (arrowheads). Original magnificatio 11,330. (C ) Damaged ICC-DMP (ICC*) from the inner region of the circular muscle. It is identifie as ICC-DMP by location, presence of plasma membrane caveolae (arrows), perinuclear well-developed Golgi complexes (G), and thin cellular processes. Its cell body displays electron-lucent cytoplasm, the result of depletion of most cytoplasmic filament (circle). No depletion of filament is seen in the muscle cells (CM). Some mitochondria are swollen or have burst (*), probably as a result of lack of oxygen. Original magnificatio 21,920. (D) Small lymphocyte (Ly) within the longitudinal muscle layer (LM). Original magnificatio 11,330. were frequently observed in the submucosa and inside the circular muscle layer. Lymphocytes were occasionally seen between the 2 main muscle layers and between smooth muscle cells adjoining the AP region (Figure 2D). Electrophysiology Slow wave activity occurred at cycles/ min (Figure 4) with an amplitude of mv arising from a resting membrane potential of 61.2 mv. Duration of the slow wave was seconds. Slow wave activity responded to cholinergic stimulation (carbachol, 10 6 mol/l) by increasing amplitude ( mv) and duration ( seconds). The frequency of the slow wave was reduced to cycles/min. The resting membrane potential was also affected by carbachol, decreasing to 46.9 mv. In Vivo Motor Activity At day 51 postpartum, a significantly delayed gastric emptying was observed with an emptying halftime of 124 minutes (upper limit of normal, 90 minutes). A small bowel meal radiography was performed at day 55 postpartum with barium 100 gravity injected via

5 February 2001 ICC IN ABSENCE OF ENTERIC NERVES 565 the gastrostomy tube. The duodenum showed both segmental contractile activity and propagating contractions in both the oral and aboral direction. A few centimeters distal to the DJ flexure, the small bowel was somewhat distended and showed uncoordinated muscular contractions. Image analysis confirmed the occurrence of propagating contractions, mainly in the aboral direction, which appeared at a frequency of cycles/min at a propagation velocity of 1.4 cm/s (Figure 5). However, under the conditions of the radiology examination, the predominant pattern was irregular contractile activity with segments of intestine appearing to contract independent of each other. This uncoordinated activity was seen in both distended and contracted regions. Figure 3. Normal gut pacemaker cells. (A) A cross section through the AP region shows circular (CM) and longitudinal muscle cells (LM), ICC-AP cell body (ICC), and a group of overlapping ICC-AP processes (ICC*). Note close contact between ICC-AP process and longitudinal muscle projection (arrow). Arrowheads indicate close apposition contacts between overlapping ICC processes. Original magnificatio 15,800. (B) High magnificatio of ICC-AP cell body (ICC) from A shows main ultrastructural features of human ICC-AP: presence of caveolae (arrows), well-developed Golgi complexes (G), abundant intermediate filament (circles), and scattered cisternae of rough endoplasmic reticulum (rer). Nu, nucleus. Original magnificatio 31,350. Discussion The presence of normal ICC-AP was observed in the small intestine of an infant, 15 days postpartum, in which the enteric nervous system had not developed. This is the first electron microscopy description of ICC-AP in a newborn, but the ICC had most features in common with previously published descriptions of ICC-AP in normal adult human tissue. 12,15 The ICC-AP processes formed many close apposition contacts with each other, indicating the existence of an ICC network within the AP region. Close contacts were not found between the sporadic extrinsic nerves and ICC-AP. The normal distribution of ICC-AP and their normal close apposition contacts with smooth muscle cells appeared to be sufficient for the generation of regular rhythmic slow wave activity that responded to cholinergic stimulation. Carbachol (10 6 mol/l) depolarized the muscle cells and increased the slow wave duration. The presence of regular slow wave activity suggested that rhythmic contractile activity was possible, as well as slow wave driven peristalsis. Fluoroscopic observations revealed propagating rhythmic peristaltic contractions at a dominant frequency of approximately 12 waves/min. This was probably close to normal because in 9 preterm infants weeks postconception, the maximum frequency of rhythmic motor activity in vivo was 11.5 cycles/min. 16 In the present study, during a 5-minute recording session, propagating contractions occurred over short distances, in both oral and aboral directions, as Figure 4. Slow wave activity recorded from the proximal small intestine with microelectrodes recording intracellular electrical activity from single cells within tissue. (A) Slow wave activity recorded from the circular muscle. (B) Same cell, but after cholinergic stimulation with 10 6 mol/l carbachol. Note depolarization and changes in slow wave frequency and duration.

6 566 HUIZINGA ET AL. GASTROENTEROLOGY Vol. 120, No. 2 Figure 5. Spatiotemporal map of a motor pattern demonstrating regular propagating contractions. Time is represented on the y-axis, and the position along the intestine on the x-axis. Light bands in the map represent contracted regions. The contractions originate in the proximal part and regularly propagate in the distal direction at a frequency of 12 cycles/min. The most proximal part of the map represents a distended region of the intestine that did not exhibit contractile activity during this time. well as irregular contractile activity. From the necessarily short observation period, with little control over the extent of distention by the barium infusion, it was not possible to conclude that the motor patterns were normal. The only conclusion that can be made is that the musculature was capable of peristaltic contractile activity. The absence of the enteric nervous system will be associated with intestinal dysmotility, which may limit the use of enteral feeding. Our data indicate that, through preservation of the ICC-AP, some organized motor activity was preserved in the complete absence of the enteric nervous system. In this patient, this finding led to the introduction of enteral feeding. Unfortunately, the infant s feeding tolerance was limited by his impaired gastric emptying, his high ostomy fluid and sodium losses, and the recurrent intestinal bleeds associated with portal hypertension. Thus, despite showing some organized small bowel motor activity both in vivo and in vitro, the child failed to tolerate adequate enteral feeds and therefore developed TPN-induced hepatocellular complications, which ultimately led to death. Whether appropriate motor patterns develop will depend on whether or not the musculature is properly stimulated. Without enteric nerves, it is obvious that many motor patterns dependent on, or usually modified by, enteric nerves will not develop. Enteral feeding may have to be complemented by pharmacologic stimulation of the musculature. Based on c-kit immunohistochemistry, an ICC-AP network is present at weeks of gestation 17 and at birth. 18 Our case report shows that in humans, the development of ICC-AP occurs independently from the enteric nervous system. This is consistent with data from animal models. 7 In the normal adult human small intestine DMP, ICC-DMP have a very close and distinct association with nerve structures. 13 Identification using c-kit immunohistochemistry is uncertain because of weak staining. 18,19 Nevertheless, immunoreactivity to c-kit antibodies was seen in the DMP at 27 weeks postconception 18 and 42 weeks. 8 In the mouse, ICC-DMP are fully developed at birth, whereas ICC-AP mature a few days after birth. 20 We found no c-kit positive cells within the muscle layers or at the level of the DMP. Electron microscopy did not find a normal array of ICC-DMP; however, isolated cells resembling ICC-DMP were seen throughout the DMP region. All these cells were found in various states of injury suggestive of degeneration. This tissue was studied 15 days postpartum, and it appears that the ICC-DMP developed, but were unable to survive without enteric innervation. This is consistent with the hypothesis that a close functional relationship exists between ICC-DMP and nerves; studies in the mouse small intestine suggest that ICC-DMP may supply nitric oxide to the smooth muscle cells after activation by enteric inhibitory nerves. 5 Interestingly, in WW v mice that lack a fully functional Kit receptor, ICC-AP develop through the gestation period but do not further develop after birth, 21 confirming that factors supporting development can be different at various stages of development. There appears to be a difference between the development of ICC-DMP in humans and mice. In glial cell line derived neurotrophic factor knockout mice that lack enteric nerves, ICC-AP developed normally. 22 It was mentioned that ICC-DMP also developed normally, but only a few ICC-DMP were found by immunohistochemistry. Confirmation of the normality of the ICC-DMP in these tissues by electron microscopy may be required. The general organization of both muscle layers was similar to adult small intestine. 14 The presence of many macrophages and the infiltration of lymphocytes, as well as the sporadic appearance of smooth muscle cells undergoing myofibroblast transformation, suggest the possibility of an onset of inflammation. In conclusion, in the absence of the entire enteric nervous system, ICC-AP develop normally but ICC- DMP do not survive. The normal musculature, including the ICC-AP pacemaker system, generates slow wave ac-

7 February 2001 ICC IN ABSENCE OF ENTERIC NERVES 567 tivity, and propagating, slow wave driven peristaltic movements do occur in the small bowel. Whether this ability to generate functional motor patterns could be sufficient to support enteral feeding to some extent requires further study. References 1. Huizinga JD, Thuneberg L, Vanderwinden JM, Rumessen JJ. Interstitial cells of Cajal as pharmacological targets for gastrointestinal motility disorders [review]. Trends Pharmacol Sci 1997;18: Der-Silaphet T, Malysz J, Arsenault AL, Hagel S, Huizinga JD. Interstitial cells of Cajal direct normal propulsive contractile activity in the small intestine. Gastroenterology 1998;114: Thomsen L, Robinson TL, Lee JCF, Farraway L, Hughes MJG, Andrews DW, Huizinga JD. Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 1998;4: Koh SD, Sanders KM, Ward SM. Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine. J Physiol (Lond) 1998;513: Ward SM, Morris G, Reese L, Wang XY, Sanders KM. Interstitial cells of Cajal mediate enteric inhibitory neurotransmission in the lower esophageal and pyloric sphincters. Gastroenterology 1998; 115: Faussone-Pellegrini MS, Matini P, Stach W. Differentiation of enteric plexuses and interstitial cells of Cajal in the rat gut during pre- and postnatal life. Acta Anat (Basel) 1996;155: Young HM, Ciampoli D, Southwell BR, Newgreen DF. Origin of interstitial cells of Cajal in the mouse intestine. Dev Biol 1996; 96: Kenny SE, Vanderwinden JM, Rintala RJ, Connell MG, Lloyd DA, Vanderhaegen JJ, De Laet MH. Delayed maturation of the interstitial cells of Cajal: a new diagnosis for transient neonatal pseudoobstruction. Report of two cases. J Pediatr Surg 1998; 33: Liu LWC, Farraway L, Berezin I, Huizinga JD. Interstitial cells of Cajal: mediators of communication between longitudinal and circular muscle cells of canine colon. Cell Tissue Res 1998;294: Sircar K, Hewlett BR, Huizinga JD, Chorneyko K, Berezin I, Riddell RH. Interstitial cells of Cajal as precursors of gastrointestinal stromal tumors. Am J Surg Pathol 1999;23: Bercik P, Bouley L, Dutoit P, Blum AL, Kucera P. Quantitative analysis of intestinal motor patterns: spatiotemporal organization of nonneural pacemaker sites in the rat ileum. Gastroenterology 2000;119: Rumessen JJ, Thuneberg L. Interstitial cells of Cajal in human small intestine. Ultrastructural identificatio and organization between the main smooth muscle layers. Gastroenterology 1991; 100: Rumessen JJ, Mikkelsen HB, Thuneberg L. Ultrastructure of interstitial cells of Cajal associated with deep muscular plexus of human small intestine. Gastroenterology 1992;102: Faussone Pellegrini MS, Cortesini C. Some ultrastructural features of the muscular coat of human small intestine. Acta Anat (Basel) 1983;115: Faussone-Pellegrini MS. Histogenesis, structure and relationships of interstitial cells of Cajal (ICC): from morphology to functional interpretation. [Review]. Eur J Morphol 1992;30: Bisset WM, Watt J, Rivers RP, Milla PJ. Postprandial motor response of the small intestine to enteral feeds in preterm infants. Arch Dis Child 1989;64: Wester T, Eriksson L, Olsson Y, Olsen L. Interstitial cells of Cajal in the human fetal small bowel as shown by c-kit immunohistochemistry. Gut 1999;44: Torihashi S, Horisawa M, Watanabe Y. c-kit immunoreactive interstitial cells in the human gastrointestinal tract. J Auton Nerv Syst 1999;75: Romert P, Mikkelsen HB. c-kit immunoreactive interstitial cells of Cajal in the human small and large intestine. Histochem Cell Biol 1998;109: Liu LWC, Thuneberg L, Huizinga JD. Development of pacemaker activity and interstitial cells of Cajal in the neonatal mouse small intestine. Dev Dyn 1998;213: Klüppel M, Huizinga JD, Malysz J, Bernstein A. Developmental origin and Kit-dependent development of the interstitial cells of Cajal in the mammalian small intestine. Dev Dyn 1998;211: Ward SM, Ordog T, Bayguinov JR, Horowitz B, Epperson A, Shen L, Westphal H, Sanders KM. Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves. Gastroenterology 1999;117: Received February 15, Accepted September 6, Address requests for reprints to: Jan D. Huizinga, Ph.D., McMaster University, HSC-3N5C, 1200 Main Street West, Hamilton L8N 3Z5, Canada. huizinga@mcmaster.ca; fax: (905)