Download Requirement of c-kit for development of intestinal pacemaker system

Development 116, 369-375 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
369
Requirement of c-kit for development of intestinal pacemaker system
HITOMI MAEDA 1, AKIHIRO YAMAGATA2, SATOMI NISHIKAWA1, KAZUYA YOSHINAGA3, SHIGERU
KOBAYASHI4, KATSUHIDE NISHI2 and SHIN-ICHI NISHIKAWA 1*
1Institute
of Molecular Embryology and Genetics, 2Department of Pharmacology, and 3Department of Anatomy, Kumamoto University School of
Medicine, 2-2-1 Honjo, Kumamoto 860, Japan
4Department of Anatomy, Nagoya University, School of Medicine, Nagoya, Japan
*Author
for correspondence
Summary
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/Wv mice 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.
Introduction
gesting 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 patterns 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 superficial 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 patterns 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
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 developmental 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), sug-
Key words: c-kit, Steel factor, gut development, intestinal
pacemaker.
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H. Maeda and others
(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.
Materials and methods
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 intraperi toneally 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±0.5oC. 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 performed 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 forceps. 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 −80oC. 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 phosphatebuffered 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 antibody.
Results
Paralytic ileus induced by anti-c-kit mAb, ACK2
In order to investigate whether acute blockade of c-kit function 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, spermatogonia, 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, injection 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.
Disturbance of the gut motility induced by neonatal ACK2injection
Because it was suspected upon superficial inspection that
the ileus was functional, we isolated segments from various 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 without external stimulation. However, the ileal segment of the
ACK2-treated BALB/c mice showed almost no phasic contraction except transient contractions which occurred randomly. Such a disturbance of the motility of isolated gut
c-kit function in intestinal pacemaker
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).
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 acetylcholineor 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 synchronously. These physiological features suggest that the paralytic ileus may be due to loss of mechanisms that generate
normal motility patterns of isolated gut segments. To determine whether this physiological abnormality of the intestine 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/Wv mice. Despite the absence of
notable morphological abnormalities, the intestine of the
12-day-old W/Wv mice 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 motility, but the abnormalities were not as severe as in the
371
ACK2-treated BALB/c mice or the W/Wv mice (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/Wv mice 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 fertile, 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/Wv mice.
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 gametogenesis 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 disorder 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 ckit-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 disorder, 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 ACK2injected 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 mus-
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H. Maeda and others
cular 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
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 ACK2injected 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(ACK2treated) 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 ACK2treated 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.
c-kit function in intestinal pacemaker
373
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 iodideosmium (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) .
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.
Discussion
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 ACK2treated 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 pacemaker 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 ultrastructural characteristics of ICCs are as abundant as c-kit+ cells
in the circular layers of the gastroduodenal junction. However, due to the technical difficulty in using our mAbs for
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H. Maeda and others
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.
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 ckit+. 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 development 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 defeca-
c-kit function in intestinal pacemaker
tion, 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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(Accepted 6 July 1992)