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First published online 29 March 2006
doi: 10.1242/dev.02346
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1 UMR144, CNRS-Institut Curie, 26, rue d'Ulm, 75248 Paris cedex 05,
France.
2 Max Planck Institute of Biochemistry, Department of Molecular Medicine,
Martinsried, Germany.
Author for correspondence (e-mail:
sylvie.dufour{at}curie.fr)
Accepted 3 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Enteric nervous system, ß1 integrins, Neural crest, Migration, Conditional knockout, Hirschsprung's disease, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Newgreen and
Young, 2002b). In mice, as in humans, the ENS is composed of
interconnected ganglia organised in two concentric plexuses in the gut wall:
the myenteric and the submucosal plexuses. Each ganglion contains glial cells
and several subtypes of neurons. In mice, the ENS arises from vagal and sacral
neural crest cells (NCCs). Vagal NCCs invade the foregut at E9.5, then
progress through the gut mesenchyme in a rostrocaudal direction and reach the
rectum at E14 (Young et al.,
1998). Sacral NCCs give rise to a fraction of neurons and glial
cells in the distal intestine by colonising the hindgut from the pelvic plexus
in a caudorostral wave (Kapur,
2000; Druckenbrod and Epstein,
2005).
During their progression in the gut wall and the later development of the
ENS, the enteric NCCs (ENCCs, including vagal and sacral NCCs within the gut)
interact with their environment, which is composed of mesenchymal cells and
extracellular matrix (ECM) components, like fibronectin, laminin, tenascin and
chondroitin sulfate proteoglycan (Newgreen
and Hartley, 1995;
Simon-Assmann et al., 1995;
Rauch and Schafer, 2003). ENS
precursors express various receptors for ECM, such as 
4
(Bixby et al., 2002;
Kruger et al., 2002), 6
and ß1 integrins (Iwashita et al.,
2003) (current study), and the 110 kDa laminin receptor
(Chalazonitis et al., 1997).
Integrins comprise a large family of 24 ß heterodimers that can
activate signalling pathways that control cell proliferation, survival,
migration or differentiation (Hynes,
2002). The ß1 integrins represent the largest subfamily, as
the ß1 chain can associate with 12 different subunits.
Although naturally occurring and targeted mutations in rodents have
identified several genes implicated in ENS formation (reviewed by
Newgreen and Young, 2002a;
Newgreen and Young, 2002b),
little is known about the role of ECM components and their receptors in the
process. In mice, the homozygous disruption of the ß1 integrin gene
results in an early death of the embryo at E5.5
(Fassler and Meyer, 1995;
Stephens et al., 1995). The
analysis of chimaeric embryos did not provide any information about the roles
of ß1 integrins during ENS development
(Fassler and Meyer, 1995). In
order to circumvent the early lethality caused by the knockout of the ß1
integrin gene, we restricted its invalidation to the migrating NCCs, through
the use of the Ht-PA-Cre mouse line. We have previously shown that this line
drives the Cre-mediated recombination specifically in NCCs as they emerge from
the neural tube. This allows a precocious targeting of virtually all the NCC
derivatives, including the ENS (Pietri et
al., 2003; Pietri et al.,
2004). We show that the loss of the ß1 integrins in ENCCs
causes an incomplete colonisation of the gut and an abnormal organisation of
the enteric ganglia network. This phenotype is mostly due to a migration
defect, linked to an increased aggregation of ß1-null ENCCs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Samples processing and immunostaining
Immunostaining was performed as described by Pietri et al.
(Pietri et al., 2004) for
whole tissues and paraffin sections, and by Delannet et al.
(Delannet et al., 1994) for
frozen sections. The primary antibodies we used are listed in Table S1 in the
supplementary material.
The ß-galactosidase (ß-gal) activity was detected by X-Gal
staining on whole tissues as described in Dufour et al.
(Dufour et al., 1994). The
ß-gal activity of the protein extracts was measured using the
ß-galactosidase Enzyme Assay System (Promega).
For 5-bromo-2'-deoxyuridine (BrdU) incorporation, mice were injected intraperitoneally with 100 µg of BrdU/g of body weight 2 hours prior to sacrifice.
Organotypic cultures
For graft experiments, segments of control or mutant distal midgut were
grafted onto segments of wild-type distal hindgut. The explants were placed on
the filter of a Millicell chamber (Millipore) and cultured in growth
factor-reduced Matrigel (Becton Dickinson), in DMEM/F12 medium (Invitrogen)
supplemented with 5% horse serum (Babco). In orienting the explants, special
care was taken to respect the rostrocaudal direction of ENCC migration. Only
explants without a gap between the two grafted segments were further analysed
(five independent experiments, control: n=11, mutant: n=10).
The quantification of the migration was achieved by measuring the distance
between the most caudal ß-gal+ cell in the wild-type hindgut
and the boundary separating the two grafted segments.
For cultures on a 2D substrate, rings (500 µm thick) of distal midgut
were plated on a mixture of ECM Gel (Sigma) at 150 µg/ml and fibronectin
(Sigma) at 10 µg/ml, and cultured in DMEM/F12 medium supplemented with 3%
horse serum (4 independent experiments, control: n=17, mutant:
n=20). For the dissociation experiments, mutant explants were
incubated at 37°C either in 0.001% trypsin, 1 mM EDTA in HCMF (low
trypsin-EDTA), or in 0.01% trypsin in HMF (trypsin-Ca2+)
(Nakagawa and Takeichi,
1995).
For cultures in collagen gel, segments of proximal midguts were placed in 1.5 mg/ml collagen type I gel (Sigma) on the filter of a Millicell chamber, and cultured in DMEM/F12 medium supplemented with Insulin-Transferrin-Selenium (Gibco), either in the presence or absence of GDNF at 10 ng/ml (Promega) (three independent experiments; control; n=9; mutant, n=7).
Semi quantitative RT-PCR and western blotting
For RT-PCR, total RNA was isolated from E13.5 guts with RNAeasy Protect
Mini kit (Qiagen). RNA (1.5 µg) was reverse transcribed with Mo-MuLV
reverse transcriptase (200 U, Promega) primed with random hexamers (1 µg,
Roche). cDNA was subjected to PCR using murine gene-specific primers listed in
Table 2 in the supplementary material. Expression levels of intercellular
adhesion molecules were determined relative to ß-gal expression.
For western blotting, guts were homogenised for 45 minutes on ice in Triton
X-100 1%, sodium desoxycholate 10%, SDS 0.1% in PBS with Ca2+ and
Mg2+, complemented with protease inhibitor cocktail (Roche). The
extracts were then processed as described by Dufour et al.
(Dufour et al., 1999).
Expression levels were normalised with respect to the ß-gal activity of
the extracts.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) mice
to target the ß1 gene disruption in the ENS precursors. Two genotypes
were obtained: Ht-PA-Cre; ß1+/ß1fl mice, used
as controls, and Ht-PA-Cre; ß1-/ß1fl mice,
referred to as mutants, which show several defects of the peripheral nervous
system and die around 3 weeks after birth
(Pietri et al., 2004). In both
control and mutant mice, ß-gal gene expression is controlled by the
ß1 integrin promoter after the Cre-mediated recombination at the ß1
floxed locus. This allowed us to follow the targeted cells during embryonic
and postnatal development.
In embryonic control guts, the Cre-mediated recombination targeted a
population of cells that colonised the gut in a rostrocaudal wave from stage
E9.5 to E14 (see Fig. S1 in the supplementary material), with similar
migration timetable and radial location as the vagal NCCs
(Kapur et al., 1992;
Young et al., 1998;
Young et al., 2004). No
ß-gal+ cells were seen in the hindgut before these cells
arrived at E14. The pelvic plexus was labelled similarly to that found in the
guts of Ht-PA-Cre; R26R mice (Pietri et
al., 2003), suggesting that sacral NCCs contributing to the
hindgut ENS are also targeted (not shown). The ß-gal+ cells
expressed two markers for vagal NCCs, Phox2b and p75NTR
(Young et al., 1998) (not
shown), and colocalised with the neuronal markers HuD and NF160
(Fig. 5) and the glial marker
B-FABP (not shown) at different stages of development. Taken together, these
results show that the Ht-PA-targeted enteric cells correspond to the enteric
vagal and sacral NCCs (referred to as ENCCs) and to their neuronal and glial
derivatives.
ENCCs aggregated into myenteric ganglia between E12.5 and E16.5 (compare
Fig. S1E-H with Fig. S1C,D in the supplementary material). At E16.5, a few
isolated ß-gal+ cells were found in the submucosal zone of the
small intestine, which are likely to belong to the future submucosal plexus
(Fig. S1F). However, in the colon, no labelled cells were observed in this
zone (Fig. S1H). This confirms that the submucosal plexus appears earlier in
the small intestine than in the colon, as described before
(McKeown et al., 2001). In
E16.5 small intestines, ß-gal+ cells were observed, the nuclei
of which appeared to be passing through the circular muscle layer. It is
likely that they were migrating from myenteric ganglia towards the submucosal
zone (Fig. S1I).
The ß1-null ENCCs fail to colonise the entire gut
In conditional mutants, ß1 integrins were barely detectable at the
cell surface of mutant ENCCs when they start to invade the foregut, at E9.5
(Fig. 1A-D), and completely
absent from E11.5 (Fig. 1E-H).
Thus, our genetic system permits the efficient removal of the ß1
integrins from the beginning of the gut colonisation by ENCCs. The ß1
integrin loss did not have a strong influence on the expression of other
ß integrins, as ß3 and ß5 integrins were similarly expressed in
mutant and control ENCCs (Fig.
1I-P).
In all the mutant animals (number of X-Gal-stained samples: mutant,
n=37; control, n=48), the ENCCs showed a delay in the gut
colonisation, detected from E11.5. At this stage, the control ENCCs migratory
front was located at the base of the caecum, although the mutant front was
still located in the distal midgut (not shown). At E12.5, chains of control
ENCCs had invaded the hindgut, whereas mutant ENCCs had entered the proximal
caecum (Fig. 2A,B). At E16.5,
control ENCCs had reached the rectum, whereas the ß1-null ENCC migratory
front was located in the middle of the hindgut
(Fig. 2C,D). Thus, the
difference in the distance travelled by control and mutant ENCCs increased
with time. This defect did not appear to be just a delay, as the ß1-null
ENCCs did not subsequently invade the caudal hindgut, leading to an
aganglionosis of the descending colon after birth that resembles the human
Hirschsprung's disease (HSCR) (Passarge,
2002) (Fig. 2E,F).
The majority of the new-born mutants had a distended ascending colon and
caecum (megacolon) (Fig. 2G,H).
In addition, as it has been documented in HSCR, bundles of extrinsic neurons
innervated the aganglionic segments (Fig.
2F). These bundles were ß-gal+ and expressed
tyrosine hydroxylase (not shown), revealing their sympathetic neuronal origin.
We did not detect any anomaly in the radial location of ENCCs during their
progression in the gut, or any delay in the formation of the submucosal plexus
in the mutant (not shown). Thus, in contrast to the migration across the
longitudinal axis, the loss of ß1 integrins does not affect the radial
distribution of ENCCs in the gut.
The ß1-null ENCCs show increased aggregation properties
In addition to their inability to completely colonise the intestinal tract,
the mutant ENCCs gave rise to an abnormal ganglia network. At E12.5, mutant
ENCCs began to form clusters of cells in the distal midgut and the caecum
(Fig. 2A,B). At E16.5 and
thereafter, this defect was seen in the different parts of the mutant guts,
with ENCCs forming abnormal aggregates surrounded by ENCC-free spaces
(Fig. 2K,M,O, compare with
J,L,N). As a consequence, the organisation of the enteric network
was severely altered at P14 (Fig.
2P,Q).
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The defects of the mutant ENS are not due to a decreased number of migrating ENCCs
The incomplete gut colonisation by mutant ENCCs could be explained by a
decrease in their number during migration, owing to survival and/or
proliferation defects. However, apoptotic neurons were not detected either in
control or mutant guts at E13.5 (Fig.
4A,B) and other stages (E10.5 and E16.5, not shown). To compare
the proliferation state of control and mutant ENCCs, we analysed their
expression of the PCNA antigen (Fig.
4C,D) and their BrdU incorporation
(Fig. 4F,G) at E12.5. No
significant difference was found in the percentage of proliferative ENCCs
between mutant and control with these two methods
(Fig. 4E,H). Moreover, the
ß-gal activity measured in protein extracts prepared from E14.5 guts was
equivalent between mutants (15.13±1.79 units/g protein) and controls
(16.34±1.56 units/g protein), supporting the fact that the number of
ENCCs is not significantly reduced in mutants. Taken together, these results
strongly suggest that the partial gut colonisation by mutant ENCCs is due to a
migration and/or a differentiation defect, and not to survival or
proliferation alteration decreasing their number.
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;
Young et al., 2004). We
therefore examined the neuronal differentiation of ENCCs in relation to the
gut colonisation, by labelling the neuron cell bodies and the neuronal
processes on E12.5 X-Gal stained guts (Fig.
5). In both types of guts, the neuronal differentiation front,
represented by the most caudal HuD+ cell body, was located just
rostral to the migratory front. Thus, the leading ß-gal+ cells
were not HuD+, suggesting that they were in an undifferentiated
state, in controls as well as in mutants
(Fig. 5E,F). The proportion
between neurons and differentiating neurons
(ß-gal+HuD+) and undifferentiated ENCCs
(ß-gal+HuD-) was similar in control and mutant
guts, near the migratory front and elsewhere (not shown). ENCCs at the
migratory front were closely associated with NF160+ neuronal
processes (Fig. 5E,F),
suggesting that the progression of ENCCs in the gut is linked to neuronal
processes elongation. Rostral to the front, in the distal midgut, control
neurons and non-neuronal ENCCs formed a scattered network occupying all the
available space (Fig. 5C). By
contrast, ß1-null neurons and other ENCCs were strongly associated with
fasciculated neuronal processes, forming thick bundles surrounded by ENCC-free
areas (Fig. 5D). Moreover, the
position of the glial differentiation front analysed with the B-FABP marker,
was similar in both types of guts at E12.5 (not shown). Taken together, these
results suggest that the timing of the neuronal and glial differentiation is
not affected in mutants. Moreover, analysing the neuronal differentiation
revealed an abnormal association between mutant ENCCs and neuronal processes.
This may reflect their incapacity to explore and occupy the gut environment
properly. We then examined the acquisition of different neuronal subtypes (VIP, NPY, SP, NOS and CGRP) by control and mutant neurons. At P7, all these markers were expressed in both control and mutant enteric ganglia (not shown), showing that ß1-null neurons retain the capacity to differentiate in various neuronal subtypes. However, at P7 and P4, the VIP staining revealed an innervation defect of the villi in the mutant small intestines, probably resulting from a neuronal process degeneration (see Fig. S3 in the supplementary material).
The ß1-null ENCCs show a migration defect
Our results concerning ENCC apoptosis, proliferation and differentiation
strongly suggest a migration defect. In order to compare the migratory
behaviours of control and mutant ENCCs in a 3D tissue environment, we
performed graft experiments at E12.5. Segments of distal midgut, already
colonised by ENCCs, were grafted onto segments of wild-type distal hindgut,
devoid of ENCCs (Fig. 6A).
After 3 days in culture, the explants were stained with X-Gal to label the
ENCCs that had entered the wild-type hindguts. Both control and mutant ENCCs
were able to enter the wild-type hindguts, but the distance travelled by the
ß1-null ENCCs was significantly reduced
(Fig. 6B-E). On average, mutant
ENCCs covered 66.5% of the control ENCC distance, showing that ß1-null
ENCCs lost part, but not all, of their migratory abilities, as suggested by
the in vivo mutant phenotype. Moreover, these data indicate that the distal
hindgut environment is not repellant to ß1-null ENCCs.
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To further characterise this migration defect, we cultured segments of
E13.5 proximal midguts in 3D collagen gels, in the presence or absence of
GDNF, a known chemoattractant for ENCCs. In the absence of GDNF, no cell was
found outside the explants, in both control and mutant cultures
(Fig. 8A,B,E,F). As previously
described (Natarajan et al.,
2002; Iwashita et al.,
2003), in the presence of GDNF, a large number of control neurons
(both cell bodies and processes) and glial cells emigrated out of the explant
(Fig. 8C,D). By contrast, in
the mutant, neuronal processes had penetrated into the collagen gel, but not
neuron cell bodies or glial cells (Fig.
8G,H). These results confirm our in vivo and in vitro data about
the alteration of the ß1-null ENCC migratory abilities. In addition, they
support the idea that neurite outgrowth of enteric neurons is ß1 integrin
independent, suggesting that ENCC migration and neurite extension in response
to GDNF are regulated by distinct mechanisms.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Neuron survival depends on ß1-mediated cell-matrix
contacts in vitro (Rozzo et al.,
1997; Bonfoco et al.,
2000; Gary et al.,
2003). In addition, 4ß1 and 5ß1 integrin
are required for in vitro peripheral glial cell survival and proliferation,
respectively (Haack and Hynes,
2001). We have shown that the loss of the ß1 integrins in
ENCCs does not affect their survival and proliferation during the gut
colonisation. Moreover, because our data suggest that the number of ENCCs is
not decreased in mutants, a reduction in the pool of vagal NCCs before their
entry in the gut is unlikely to contribute to the phenotype. Indeed, the
complex environment in vivo probably counteracts the apoptotic effect due to
the loss of ß1 integrins in vitro. ß1-Null ENCCs may receive signals
through growth factor receptors, other ECM receptors and/or intercellular
adhesion molecules that are sufficient for their survival and proliferation.
In particular, both control and mutant ENCCs were found to express the ß5
integrin chain, and small amounts of ß3 integrin, at E11.5 (not shown)
and E13.5 (Fig. 1I-P).
Moreover, N-cadherin, which is expressed by ß1-null ENCCs, is known to
activate pro-survival signalling pathways
(Li et al., 2001;
Tran et al., 2002).
|
The spatial restriction of the aganglionosis to the descending colon is not due to the late requirement of ß1 integrins at the time of distal hindgut invasion, because (1) our graft experiments suggest that the distal hindgut environment is permissive for ß1-null ENCC migration, and (2) the delay of the mutant ENCCs is observed from E11.5, well before the hindgut colonisation. This delay increases with time. This could be due to a progressive loss of the ß1 integrin subunit at the ENCC surface. However, it is unlikely, because the ß1 integrin loss is mostly complete from the beginning of the gut invasion (E9.5). Although ß1 integrins are stably expressed at the cell surface with a slow turnover, the high vagal NCC proliferation rate is likely to dilute the ß1 subunit before their entry in the foregut, explaining its rapid loss.
|
). It is possible that the ß1-null ENCC population is
less capable of forming these `advanced cells' in the caecum, then making a
longer pause in this region. After the caecum invasion, the ß1-null ENCC
are misplaced in regard to the hindgut environment. It has been shown that if
they are late, ENCCs entering the hindgut have to migrate in a growing and
differentiating environment (Newgreen and
Hartley, 1995; Newgreen et
al., 1996), which may prevent them from reaching the rectum. This
might be the case in the conditional ß1 mutants.
The migration defect of ß1-null ENCCs could be linked to an abnormal
deposition of ECM by mutant ENCCs, but no differences were detected in the
expression of various ECM components (not shown). This migration defect could
also be linked to a premature neuronal differentiation. However, our analysis
of the ENCC differentiation does not support this hypothesis. Moreover, we
examined the expression of tyrosine hydroxylase (TH), which is present in a
subpopulation of differentiating neurons between E10.5 and E12.5
(Young et al., 1999). No
differences were observed between mutant and control in the number of
TH+ cells, or in the location of the most caudal TH+
cell, at E11.5 and E12.5 (not shown). Taken together, our data suggest that
the timing of neuronal and glial differentiation is unchanged in mutants. This
conclusion is supported by the fact that no significant reduction of ENCC
proliferation can be detected in mutants.
ß1 Integrins are required for the formation and the maintenance of the gut neuronal network
In addition to the incomplete gut colonisation, a severe alteration of the
network rostral to the migratory front is observed in mutants. Previous
studies have suggested that in the gut, leading ENCCs form a scaffold on which
more rostral ENCCs migrate and neuronal processes elongate
(Young et al., 2002;
Young et al., 2004). The
association between leading cells and neuronal processes at the control and
mutant migratory fronts supports the idea of a link between ENCC progression
and the elongation of neuronal processes. As the ß1-null leading cells do
not migrate correctly, they might be unable to form a correct scaffold
necessary for the following ENCCs to organise the ganglia network. In
addition, it has been observed that early in ENS development, small regions
remained empty behind the migratory front and were subsequently filled in by a
secondary migration of ENCCs (Young et
al., 2004). So, in addition to the migration defect at the front,
the empty spaces we observe from E12.5 in mutant guts reveal the inability of
ß1-null ENCCs to fill these cell-free regions by a second wave of
migration. Indeed, ß1-null ENCCs appear to aggregate and associate with
neuronal processes instead of occupying all the available space as in
controls. In the mutant, ENCC-free spaces were also devoid of neuronal
processes (Fig. 5D). However,
the ß1-null neurite outgrowth appeared to be normal, in vivo as well as
in vitro. This suggests that migration and neurite outgrowth are controlled by
different mechanisms in the ENS. The absence of neuronal processes in the
ENCC-free areas might be due to the incapacity of ß1-null ENCC to fill
these regions and thus to leave guidance cues for the neurite outgrowth of
surrounding neurons. Thus, our data support the idea that the pre-patterning
of the gut environment by ENCCs, or cues deposited by ENCCs, is necessary for
neurite extension (Young et al.,
2002).
In addition to the ENS patterning across the longitudinal axis of the gut,
ENCCs have to elaborate a radial network, with the submucosal plexus formation
and the innervation of the different gut layers. In E16.5 control small
intestines, we saw cells belonging to the myenteric plexus migrating towards
the submucosal zone. This is consistent with the hypothesis of a radial
migration of myenteric ENCCs towards the epithelium, giving rise to the
submucosal plexus (Burns and Le Douarin,
2001; McKeown et al.,
2001). As no anomaly in the submucosal plexus formation was
detected in mutants, it seems that ß1 integrins are not required for this
radial ENCC migration. However, ß1 integrins are necessary for the
innervation of the small intestine epithelial villi after birth. Nevertheless,
the neuronal processes innervated the full length of the villi at earlier
stages (see Fig. S3E-H in the supplementary material), suggesting that this
alteration is not due to a neurite outgrowth defect during development but
rather to a degeneration of neuronal processes.
|
|
) and ß3 and ß5 integrins, or intercellular adhesion
molecules in their migration through gut tissues. The importance of
intercellular adhesion in ENCC progression is supported by the various
repertoire of intercellular adhesion proteins we found to be expressed by
ENCCs. In addition, a recent time-lapse microscopy study of the migration
revealed the importance of intercellular contacts between migrating and
post-migrating ENCCs (Young et al.,
2004). In the case of ENCCs, intercellular adhesion and migration
must not be antagonist processes. Their chain migration may depend on a
delicate balance between cell-matrix and intercellular adhesion. The increased
aggregation of mutant ENCCs correlated to their impaired adhesion to ECM
suggest that this balance is perturbed in ß1-null ENCCs. Several studies
have described crosstalk between ß1 integrins and intercellular adhesion
systems, such as cadherins, in vitro
(Monier-Gavelle and Duband,
1997; Yano et al.,
2004; Schaller,
2004) and in vivo (Marsden and
De Simone, 2003). However, we have found no difference in the
pattern and level of expression of the intercellular adhesion proteins between
control and mutant ENCCs. Nevertheless, dissociation experiments of mutant
aggregates suggested that cadherins are implicated in their formation. Further
analysis is required to elucidate the mechanisms by which cadherins enhance
intercellular interactions in ß1-null ENCCs, such as mobilisation of a
cytoplasmic pool or activation of cadherins at the cell surface.
As it is known that strong cell-cell contacts can inhibit cell migration
(Monier-Gavelle and Duband,
1997; Dufour et al.,
1999), the abnormal aggregation of ß1-null ENCCs could
participate to their migration defect. Moreover, it has been suggested that a
high ENCC density promotes their migration
(Young et al., 2004). The
diminution of the population pressure caused by the mutant ENCC aggregation
could also be an additive mechanism by which they do not reach the caudal
hindgut.
ß1 Integrins and HSCR
HSCR is the most common human congenital defect affecting the ENS (1 in
over 5000 births). It consists of a distal aganglionosis of variable length,
leading to a potentially fatal intestinal obstruction
(Passarge, 2002). The
mutations responsible for half of the cases have not been identified yet, and
to date, no mutation of the ß1 integrin gene has been reported in
individuals with HSCR. As the ß1 integrin is ubiquitously expressed,
complete loss-of-function mutations of the gene must be lethal at early stages
of human development, as in the mouse. Two signalling systems, mediated by the
receptor tyrosine kinase Ret and the G-protein-coupled receptor Ednrb, have
been shown to play crucial roles in ENS development
(Hearn et al., 1998;
Barlow et al., 2003;
Kruger et al., 2003). Integrin
signalling could interact with the signalling pathway of these receptors. In
particular, the endothelin pathway is known to interact with cell matrix and
intercellular adhesion systems (Koyama et
al., 2003; Bagnato et al.,
2004). Moreover, mutations in genes coding for endothelin 3 or its
receptor Ednrb give rise to defects which are similar to the ß1 mutant
phenotype (Kapur et al., 1992;
Wu et al., 1999). Taken
together, these observations suggest a possible interaction between ß1
integrin and endothelin 3 signalling pathways in ENCCs.
Our study demonstrates that the removal of ß1 integrins in ENCCs leads to a partial colonisation of the intestinal tract by ENCCs and a severe alteration of the ENS network organisation. These defects are mostly due to a defective migration linked to an increased cell aggregation. Our study also reveals a role for ß1 integrins in the late maintenance of villi innervation. In conclusion, ß1 integrins are crucial at various key steps of the ENS development.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/9/1725/DC1
* Present address: Institute of Neuroscience, University of Oregon, Eugene,
OR 97403-1254, USA 
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2 test, P>0.05). Scale
bar: 100 µm in A,B; 20 µm in C,D,F,G.
