A discovery that the protooncogene encoding the receptor tyrosine kinase, c-kit, is allelic with the Dominant white spotting (W) locus establishes that c-kit plays a functional role in the development of three cell lineages, melanocyte, germ cell, and hematopoietic cell which are defective in W mutant mice. Recent analyses of c-kit expression in various tissues of mouse, however, have demonstrated that c-kit is expressed in more diverse tissues which are phenotypically normal in W mutant mice. Thus, whether or not c-kit expressed outside the three known cell lineages plays a functional role is one of the important questions needing answering in order to fully elucidate the role of c-kit in the development of the mouse. Here, we report that some of the cells in smooth muscle layers of developing intestine express c-kit. Blockade of its function for a few days postnatally by an antagonistic anti-c-kit monoclonal antibody (mAb) results in a severe anomaly of gut movement, which in BALB/c mice produces a lethal paralytic ileus. Physiological analysis indicates that the mechanisms required for the autonomic pacing of contraction in an isolated gut segment are defective in the anti-c-kit mAb-treated mice, W/Wvmice and even W/+ mice. These findings suggest that c-kit plays a crucial role in the development of a component of the pacemaker system that is required for the generation of autonomic gut motility.

c-kit is a proto-oncogene encoding a receptor tyrosine kinase in PDGF/CSF-1 receptor family (Yarden et al., 1987; Qui et al., 1988). The findings that the dominant white spot - ting (W) locus is allelic with c-kit (Chabot et al., 1988; Geissler et al., 1988) and subsequent molecular analyses of the c-kit gene in a number of W mutants have facilitated an understanding of in vivo function of c-kit (Nocka et al., 1990; Reith et al., 1990; Tan et al., 1990; Hayashi et al., 1991). Previous studies have demonstrated that develop-mental failure of three cell lineages; hematopoietic cells, melanocyte and germ cells, is the characteristics of W mutant mice (for review see Russell, 1979; Silvers, 1979). Consistent with this, recent in situ analyses of c-kit expression showed that the stem cells of these three cell lineages express c-kit (Orr-Urtreger et al., 1990; Manova et al., 1990; Keshet et al., 1991; Nishikawa et al., 1991; Motro et al., 1991; Yoshinaga et al., 1991; Ogawa et al., 1991; Papayannopoulou et al., 1991). More interestingly, the ligand for c-kit (Copeland et al., 1990; Huang et al., 1990; Zsebo et al., 1990) has been shown to be expressed in the cells closely adjacent to these c-kit+stem cells (Matsui et al., 1990; Keshet et al., 1991; Motro et al., 1991), suggesting that the ligand is presented to the c-kit receptor on these cells.

In contrast to the consistency between function and expression of c-kit in these three cell lineages, recent in situ analyses have identified highly contiguous expression pat-terns of c-kit and its ligand in more diverse tissues, while no obvious phenotypic changes have been detected in W mutant mice (Keshet et al., 1991; Motro et al., 1991).

There could be a number of reasons for the disparity between the expression and function of c-kit in a given tissue. First, c-kit, although expressed, is not functioning. Second, the defect is too subtle to be detected by a super-ficial inspection. Third, another signalling pathway might compensate for the function of c-kit required for the normal developmental process. Finally, some c-kit-dependent processes in postnatal life might have remained undetected because complete disruption of the c-kit gene is lethal. In any case except the first, the complementary expression pat-terns of c-kit and its ligand in quite diverse tissues suggest that further experiments, if properly designed, could reveal additional c-kit-dependent process. Recently, we have shown that an in vivo injection of monoclonal antibody ACK2, which antagonizes c-kit function, provides a useful method to reveal ongoing c-kit-dependent processes (Nishikawa et al., 1991; Ogawa et al., 1991; Yoshinaga et al., 1991). Previously, we had only employed this method on the developmental process of three known cell lineages, but we have now attempted to use the same method to reveal the existence of other c-kit-dependent processes which have remained undescribed. In this report we focused on neonatal development, because it is easy to manipulate by ACK2 injection but difficult to analyze by phenotype analyses of the mutant mice as a complete loss of c-kit function is lethal. We report here that a fraction of cells in intestinal smooth muscle layers expresses c-kit and its postnatal development is suppressed by the injection of ACK2 into neonates, resulting in a severe disorder of gut motility, which in BALB/c mice causes a lethal paralytic ileus. Thus, this report not only identifies a novel c-kit-dependent process in mouse development but also provides a clear insight into the cellular component regulating autonomic gut movement.

Animals

Pregnant C57BL/6, BALB/c, DBA/2, C3H, CBA mice were purchased from Japan SLC Inc. (Shizuoka, Japan). WBB6F1-W/Wv mice were obtained by mating WB-W/ + females with B6-Wv/ + males.

Preparation of antibodies

Preparation of ACK2 and ACK4, rat anti-murine-c-kit mAbs was described previously (Nishikawa et al., 1991; Ogawa et al., 1991). ACK2, ACK4 and M1/70.15.11.5 (Springer et al., 1979) mAbs were purified from the ascites fluid by ammonium sulfate precipitation followed by DE52 ion-exchange column chromatograpy. 200 μg mAb suspended in 50 μl PBS was injected intraperitoneally each time. Smooth muscle cell specific mAb (HM 19/2; Voller et al., 1987) was purchased from Boehringer-Mannheim GmbH.

Assay for motility of isolated gut segments

Segments of the small intestine (5 mm in length) at the ileo-cecal region were excised from the animals and placed vertically in a 10 ml water-jacket bath. The distal end of the excised intestine was fixed to the bottom of the bath and the other end was fixed for tension measurement. The tissues were bathed in a Tyrode’s solution of the following composition (mM); NaCl, 137; KCl, 4.0; MgCl2, 0.25; CaCl2, 3.0; glucose, 5.5; Hepes, 10. This solution was aerated with 95% O2 and 5% CO2 and maintained at 35±0.5°C. The tissues were then allowed to equilibrate for at least 30 minutes, during which time the tension was adjusted to maintain a 20–50 mg stable resting tension throughout the experiment. Isometric tension along the longitudinal axis generated by the intestine was recorded with a force displacement transducer and a ink-writing recorder.

Histology

Osmic acid-zinc iodide staining for neuronal components was per-formed as described previously (Maillet, 1963; Kobayashi, 1990). Briefly, whole-mount preparations containing the myenteric plexus layer, deep muscular plexus layer, submucous layer with lamina muscularis mucosae and periglandular plexus layers were made under the dissecting microscope using watch-maker’s for-ceps. They were mounted on chrome alum glass slides, stained by osmic acid-zinc iodide solution, dehydrated, and embedded in Enthelan.

For immunohistochemistry, the ileum was embedded in Tissue Tek (Miles) and quick frozen at −80°C. Sections were cut at 6 μm along the long axis by a Microm cryostat. The sections were fixed for 10 minutes with cold acetone, washed in phosphate-buffered saline at pH7.4 and incubated with either ACK2 or the rat anti-smooth muscle mAb. Antibody binding to tissue sections was detected by a streptavidin-biotin-peroxidase technique, using Histostain-SP kit (Zymed Labs.). Specificity of antibody binding was checked by comparing the samples with controls that had been treated with non-immune rabbit serum or anti-Mac1 anti-body.

Paralytic ileus induced by anti-c-kit mAb, ACK2

In order to investigate whether acute blockade of c-kit func-tion by ACK2 injection can induce notable morphological abnormalities, we injected varying doses of ACK2 into neonates. To take into account genetic variations among strains of mice, we carried out the same experiment on C57BL/6, C3H/He, CBA, DBA/2, and BALB/c mice.

Confirming our previous reports, ACK2 injection into neonates blocks the proliferation of melanoblasts, sper-matogonia, and hematopoietic precursors irrespective of mouse strains (data not shown). In contrast, with an ACK2 dose which allows pup survival (< 500 μg/pup), no gross abnormality has been produced in any tissues other than melanocytes, germ cells and hematopoietic cells of C57BL/6, C3H/He, CBA, or DBA mice (Fig. 1B, and unpublished observation). In BALB/c mice, however, injec-tion of more than 100 μg ACK2 on alternate days from day 0 to 8 post partum (pp.) induced severe waning of the pups, anatomical examination of which showed that the proximal portion of small intestine was distended without an obstructing lesion, and all contents in the alimentary tract were stained by bile (Fig. 1D). On the other hand, ACK2 injection from day 4 to 10 pp. even into BALB/c mice could not induce gross abnormality (Fig. 1E). Because neither a class matched control mAb, M1/70 nor non-antagonistic anti-c-kit mAb, ACK4 could induce such an morphological abnormality (Figs. 1A,C, and unpublished observation), it is likely that this dysfunction of the intestine of BALB/c mice is an outcome of the specific blockade of c-kit function.

Fig. 1.

Gross morphological abnormality induced by the injection of an anti-c-kit mAb into BALB/c neonates. 200 μg ACK2, a mAb that blocks the function of murine c-kit was injected peritoneally into C57BL/6 (B) or BALB/c neonates (D,E) by either regimen 1; day 0,2,4,6 and 8 pp. (B,D), or by regimen 2; day 4, 6, 8 and 10 pp. (E). Control C57BL/6 (A) or BALB/c (C) mice were injected with a class matched (IgG2b) mAb, M1/70.15.11.5 (anti-Mac1) by the regimen 1. Note that ACK2 injection into C57BL/6 neonates results in a complete coat-color dilution, suggesting that ACK2 is effective (B). Only in the BALB/c mice treated with ACK2 by regimen 1, was a severe distension of the small intestine observed (D).

Fig. 1.

Gross morphological abnormality induced by the injection of an anti-c-kit mAb into BALB/c neonates. 200 μg ACK2, a mAb that blocks the function of murine c-kit was injected peritoneally into C57BL/6 (B) or BALB/c neonates (D,E) by either regimen 1; day 0,2,4,6 and 8 pp. (B,D), or by regimen 2; day 4, 6, 8 and 10 pp. (E). Control C57BL/6 (A) or BALB/c (C) mice were injected with a class matched (IgG2b) mAb, M1/70.15.11.5 (anti-Mac1) by the regimen 1. Note that ACK2 injection into C57BL/6 neonates results in a complete coat-color dilution, suggesting that ACK2 is effective (B). Only in the BALB/c mice treated with ACK2 by regimen 1, was a severe distension of the small intestine observed (D).

Disturbance of the gut motility induced by neonatal ACK2-injection

Because it was suspected upon superficial inspection that the ileus was functional, we isolated segments from vari-ous parts of the intestine, and examined the motility of the intestine in vitro at day 12 pp. Fig. 2 illustrates traces of the movement of isolated ileum. Ileal segments excised from normal mice displayed phasic contractions which occurred at the regular rate of 20 to 30 cycles/minute with-out external stimulation. However, the ileal segment of the ACK2-treated BALB/c mice showed almost no phasic contraction except transient contractions which occurred ran-domly. Such a disturbance of the motility of isolated gut was observed at every level of the small intestine from the ACK2-treated mice, although it was most conspicuous in the ileum. As shown in Fig. 2B, presence of acetylcholine- or bradykinin-induced contraction suggests that the smooth muscle is functioning in the intestine of the ACK2-treated mice. In normal intestine the pattern of phasic contractions was not disturbed by these agonists, but tone was slightly increased. In contrast, the intestines of the ACK2-treated mice showed a rather simple contraction-relaxation pattern suggesting that smooth muscle cells behaved synchro-nously. These physiological features suggest that the para-lytic ileus may be due to loss of mechanisms that generate normal motility patterns of isolated gut segments. To deter-mine whether this physiological abnormality of the intes-tine is specific to BALB/c mice, we examined the motility of morphologically normal ileal segments from the C57BL/6 mice, which had been injected with ACK2, and from an untreated W/Wvmice. Despite the absence of notable morphological abnormalities, the intestine of the 12-day-old W/Wvmice also displayed irregular patterns of movement (Fig. 1A) and hyperreactivity to acetylcholine and bradykinin (data not shown). The intestines of the ACK2-treated C57BL/6 mice showed disturbance in motil-ity, but the abnormalities were not as severe as in the

Fig. 2.

Representative isometric tension recordings of the isolated mouse intestine. Tension recordings of the distal end of ileum were carried out at day 12 pp. (A) Patterns of intestine from an untreated BALB/c mouse, a BALB/c mouse ACK2-injected by the regimen 1 (see the legend of Fig. 1), a C57BL/6 ACK2-injected by the regimen 1, an untreated W/Wv mouse, and a BALB/c mouse ACK2-injected by the regimen 2. (B) Effects of bradikinin (BK) and acetycholine (ACh) on isolated segments of the intestine of a BALB/c mouse treated with ACK2 by the regimen 1(ACK2-treated) or untreated (control). BK (0.1 pg/ml in a total concentration in the bathing solution) applied at the time indicated by an arrow in a ACK2-treated mouse induced a prominent contraction followed by a relaxation. ACh (0.01, 0.1, 1 μg/ml) applied in a cumulative fashion at the point indicated by arrows increased the tone of the intestine in a step-wise manner in a ACK2-treated mouse, but not in the control mouse. (C) Patterns of intestine from an untreated W/+ mouse and its +/+ littermate. W/+ and +/+ were discriminated by the presence of white spot. (Silvers, 1979) Erythrocyte counts and hematocrit values measured at the time of death were 6.9 million and 41% for the W/+ mouse and 6.8 million and 41% for the +/+ mouse.

Fig. 2.

Representative isometric tension recordings of the isolated mouse intestine. Tension recordings of the distal end of ileum were carried out at day 12 pp. (A) Patterns of intestine from an untreated BALB/c mouse, a BALB/c mouse ACK2-injected by the regimen 1 (see the legend of Fig. 1), a C57BL/6 ACK2-injected by the regimen 1, an untreated W/Wv mouse, and a BALB/c mouse ACK2-injected by the regimen 2. (B) Effects of bradikinin (BK) and acetycholine (ACh) on isolated segments of the intestine of a BALB/c mouse treated with ACK2 by the regimen 1(ACK2-treated) or untreated (control). BK (0.1 pg/ml in a total concentration in the bathing solution) applied at the time indicated by an arrow in a ACK2-treated mouse induced a prominent contraction followed by a relaxation. ACh (0.01, 0.1, 1 μg/ml) applied in a cumulative fashion at the point indicated by arrows increased the tone of the intestine in a step-wise manner in a ACK2-treated mouse, but not in the control mouse. (C) Patterns of intestine from an untreated W/+ mouse and its +/+ littermate. W/+ and +/+ were discriminated by the presence of white spot. (Silvers, 1979) Erythrocyte counts and hematocrit values measured at the time of death were 6.9 million and 41% for the W/+ mouse and 6.8 million and 41% for the +/+ mouse.

ACK2-treated BALB/c mice or the W/Wvmice (Fig. 2A). The gut segments from the ACK2-treated C57BL/6 mice were also hyperreactive to these two agonists (data not shown). These findings, particularly that the morphologically normal intestine from the untreated W/Wvmice shows the same physiological disorder in the intestinal motility as the intestine from ACK2-treated BALB/c mice indicate that this disorder is a consequence of c-kit disfunction rather than a nonspecific effect of mAbs. Interestingly, the gut segments from W/+ mice, which are non-anemic and fer-tile, also showed abnormality in the autonomic gut motility (Fig. 2C). This finding indicates that c-kit is directly involved in the development of the autonomic gut movement rather than that the disorder of the gut motility is secondary to the anemia in ACK2-treated and W/Wvmice. Moreover, like the coat color formation, the development of the gut motility appears to be more sensitive to a simple reduction of c-kit-expression than hematopoiesis and game-togenesis which are almost normal in the W/+ mice (Geissler et al., 1981).

In order to specify the critical phase of c-kit-dependency during gut development, we delayed the initial injection of ACK2. Starting the ACK2 injection regimen on day 2 pp. induced the same physiological disorder (data not shown), but if the initial injection was delayed until day 4, the dis-order did not develop and the normal phasic contractions were observed (Fig. 2A). Thus, the physiological disorder of gut movement is a result of developmental failure of c-kit-dependent process during an early neonatal stage rather than a result of direct involvement of c-kit in the generation or regulation of motility. Our previous result that ACK2 is cytostatic rather than cytotoxic suggests that this developmental failure is induced by the blockade of c-kit binding to its ligand. Consistent with this, a non-antagonistic anti-c-kit mAb, ACK4 had no ability to induce the ileus (data not shown).

c-kit+cells in intestine

To determine the histological basis of this physiological dis-order, we next examined the organization of the cells in the intestine of ACK2-treated mice. Despite extensive histological analysis using various histochemical methods for neuronal components and electron microscopy, we failed to detect any significant defects of myenteric ganglion cells or neural network (Fig. 3 and unpublished observations). More correctly, we failed to detect any histological abnormalities in the cellular organization of the intestine of ACK2-injected animals (Fig. 3). Thus, from a series of attempts to identify a lost cellular component, we could only conclude that the pathology of the intestine of ACK2-injected mice is different from that of human Hirschprung’s disease or of mutant mice with megacolon in which developmental failure of myenteric ganglions is the primary cause of the functional disorders (Lane, 1966; Lane and Liu, 1984). Since the cause of this paralytic ileus is the injection of ACK2 at few days pp., we attempted to determine the types of cells that express c-kit. As shown in Fig. 4, most c-kit+ cells in the intestine are elongated and exist within the smooth muscle layers. Although rarely, we also see mast cells which are c-kit+, but they are not elongated and localize in the submucosal region. In regions with thick muscular layers such as the gastroduodenal junction, they are dispersed both in longitudinal and circular muscle layers, whereas in the ileum they are located at the border of two layers. This localization of c-kit+ cells is clearly different from the distribution of myenteric ganglion cells. In fact, most myenteric ganglia are demarcated clearly by c-kit+cells (Fig. 4C, D). These histological observations suggest that the intestinal c-kit+cells are not neurons and confirm a previous report of Orr-Urtreger et al. (1990) showing that c-kit mRNA is localized within the muscle layers in the intestine. ACK2 injection into neonates reduced the number of c-kit+ cells in the muscular layers at every levels of the intestine (Fig. 4E, F), and this is the only histological defect we have been able to identify.

Fig. 3.

Absence of abnormalities in the cellular organization of the ileum from ACK2 treated BALB/c mice. Ileum from untreated or ACK2 injected (by the regimen 1) BALB/c mice was isolated at day 12 pp. (A, B) Hematoxylin-eosin staining. (C, D) Neural components in the muscular layers visualized by Champy-Maillet zinc iodide-osmium (ZIO) staining. (E, F) Immunohistochemical staining by smooth muscle cell specific mAb. Most cells in the muscular layers are stained by this mAb except myenteric neurons. Notable histological difference could not be detected between untreated (A, C, E) and ACK2-injected BALB/c mice (B, D, F).

Fig. 3.

Absence of abnormalities in the cellular organization of the ileum from ACK2 treated BALB/c mice. Ileum from untreated or ACK2 injected (by the regimen 1) BALB/c mice was isolated at day 12 pp. (A, B) Hematoxylin-eosin staining. (C, D) Neural components in the muscular layers visualized by Champy-Maillet zinc iodide-osmium (ZIO) staining. (E, F) Immunohistochemical staining by smooth muscle cell specific mAb. Most cells in the muscular layers are stained by this mAb except myenteric neurons. Notable histological difference could not be detected between untreated (A, C, E) and ACK2-injected BALB/c mice (B, D, F).

Fig. 4.

c-kit+ cells in the muscular layers of small intestines from untreated or ACK2-injected BALB/c mice. Cryostat sections were prepared from either ileum (A, C, E) or gastroduodenal junctions (B, D, F) of newborn (A,B) or 12-day-old normal BALB/c mice (C, D). Sections were also prepared from a 12-day-old BALB/c mouse which was treated with ACK2 by the regimen 1 (E, F). (A, B) A few c-kit+ cells are already present in the ileum (indicated by arrows) and the pylorus. (C, D) The number of c-kit+ cells increases during neonatal development. While their elongated shape is clearly seen in the longitudinal muscular layer in these sections, transverse sections of the same parts showed that c-kit+ cells in the circular muscular layers are also elongated (data not shown). Myenteric ganglia (GC) are often demarcated by c-kit+cells. (E, F) The number of c-kit+ cells is reduced dramatically by ACK2-injection.

Fig. 4.

c-kit+ cells in the muscular layers of small intestines from untreated or ACK2-injected BALB/c mice. Cryostat sections were prepared from either ileum (A, C, E) or gastroduodenal junctions (B, D, F) of newborn (A,B) or 12-day-old normal BALB/c mice (C, D). Sections were also prepared from a 12-day-old BALB/c mouse which was treated with ACK2 by the regimen 1 (E, F). (A, B) A few c-kit+ cells are already present in the ileum (indicated by arrows) and the pylorus. (C, D) The number of c-kit+ cells increases during neonatal development. While their elongated shape is clearly seen in the longitudinal muscular layer in these sections, transverse sections of the same parts showed that c-kit+ cells in the circular muscular layers are also elongated (data not shown). Myenteric ganglia (GC) are often demarcated by c-kit+cells. (E, F) The number of c-kit+ cells is reduced dramatically by ACK2-injection.

In the present study we have identified a novel c-kit-dependent process in mouse postnatal development. In the smooth muscle cell layers of mouse intestine, c-kit+ cells are present in the embryo (Orr-Urtreger et al., 1990), and their numbers increase for several days postnatal. A mAb, ACK2, which antagonizes c-kit function, blocks this developmental process and the resulting intestine without c-kit+ cells displays no regular phasic contractions which are regularly detected in vitro in an isolated segment of normal intestine. What then is the c-kit-dependent process during postnatal development of intestine? The physiological disorder detected in this study is the disturbance of the phasic contractions of isolated gut segment, which is highly autonomic and is not disturbed by the application of tetradotoxin which disconnects smooth muscle cells from the neuronal control.

Thus, the most attractive possibility is that c-kit is required for the development of a pacemaker system for the gut motility. The fact that the number of c-kit+ cells is severely reduced in the paralytic gut segments from the ACK2-treated neonates accords best with a notion that these c-kit+ cells are actually a component of the intestinal pacemaker. One distinct cell type which has been suspected as a pace-maker component of gut is a fibroblast-like cell called interstitial cells of Cajal (ICC) (Thuneberg, 1982; Faussone-Pellegrini, 1984; Kobayashi, 1990; Langton et al., 1990). ICCs have been claimed to localize in two specific regions, the border of the longitudinal and circular muscle layers and the submucosal edge of the circular layers. Interestingly, the localization and developmental process of c-kit+ cells in the murine intestine is quite similar to those of murine ICCs studied by electron microscopy (Faussone-Pellegrini, 1984). For example, c-kit+ cells are most frequently found around myenteric ganglia (Fig. 4C, D) where electron dense fibroblast-like cells are populated most abundantly (data not shown). Furthermore, the cells which fulfil the ultrastruc-tural characteristics of ICCs are as abundant as c-kit+ cells in the circular layers of the gastroduodenal junction. How-ever, due to the technical difficulty in using our mAbs for immunoelectronmicroscopy as well as the absence of specific markers for ICCs, whether c-kit+ cells are identical to ICCs or a special form of smooth muscle cells is yet to be determined. Because a considerable number of ICCs still remain in the intestine from ACK2-treated mice (our unpublished observation), we think either that c-kit+ cells are different from ICCs or that only a part of ICCs are c-kit+. Whichever is the case, from these results on mice treated with ACK2 and on W/Wv mice, c-kit, and presumably its ligand, appears to be critical for normal develop-ment of the intestinal pacemaker system. In fact, recent papers have demonstrated that both c-kit and its ligand are expressed in the alimentary tract of mouse (Orr-Urtreger et al., 1990; Matsui et al., 1990; Keshet et al., 1991; Motro et al., 1991) indicating that this signal transduction machinery is available throughout the developmental process of the intestine. Moreover, some physiological disorders of alimentary tract such as high frequency of gastric ulcer (Shimada, 1980) and unregulated secretion of bile (Yokoyama et al., 1982) have been noted as characteristic phenotypes of W mutant mice. More recently, a paper on a human piebaldism described that chronic constipation (Giebel et al., 1991) is one of characteristic symptoms. Thus, it would be plausible that all these diverse symptoms are outcomes of the disorder of the pacing system of the intestinal motility, although the exact pathophysiology of each symptom is yet to be investigated. Nevertheless, to elucidate the pathogenesis of these physiological disorders associated with c-kit dysfunction, it would be important to characterize the physiological properties of c-kit+ cells in each organs at the single cell level. Thus, besides using ACK2 to identify a pacemaker component of intestine and manipulate its development as described in this paper, this mAb can provide a method for separating the c-kit+cells from intestine. Patch-clump analysis of ion channels expressed in the intestinal c-kit+cells is currently in progress in our laboratories.

Finally, it is of note that even with such a severe disorder of the autonomic gut movement, most mouse strains including W/Wv and W/+ mice manage to maintain defecation, and thereby escape from such a lethal paralytic ileus as observed in BALB/c mice. This suggests that other regulators of gut movement, presumably an autonomic nervous system, may compensate for the defect. While the molecular basis underlying this genetic variation is totally obscure, this would be the major reason why such an intriguing phenotype related to the c-kit molecule has not been recognized very well.

We thank Drs. M.Naito, and K.Takahashi for helpful discussion. We are grateful to Dr. K. M. Sanders for a critical review of the manuscript. This work was supported by the grants from The Ministry of Education, Science and Culture (Monbusho) and from The Institute for Physical Chemistry (RIKEN).

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