1 UMR144, CNRS Institut Curie, 26, rue d'Ulm, 75248 Paris Cedex 05,
France
2 U 368, INSERM Ecole Normale Supérieure, 46, rue d'Ulm 75230
Paris Cedex 05, France
3 Max Planck Institute of Biochemistry, Department of Molecular Medicine,
Martinsried, 82152, Germany
Author for correspondence (e-mail:
sylvie.dufour{at}curie.fr)
Accepted 30 April 2004
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SUMMARY |
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Key words: Integrin, Peripheral nervous system, Neural crest cells, Conditional knockout
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Introduction |
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Constitutive knockout of the ß1-integrin gene leads to the loss of
twelve members of the integrin family and leads to death of the embryo during
the peri-implantation period due to inner cell mass failure
(Fässler and Meyer, 1995;
Stephens et al., 1995
). This
constitutive ß1-knockout model demonstrates the essential role of
ß1-integrins during early embryogenesis, but is of no use for
investigating integrin functions during later stages of development or in
differentiated cells, such as NCC derivatives. By contrast, neither the
invalidation of individual
-integrin subunits (for reviews, see
Bouvard et al., 2001
;
Hynes, 2002
) nor the
production of knockout chimaeric mice
(Fässler and Meyer, 1995
)
has made it possible to delineate precisely the roles of specific integrins
during NCC development, with the exception of
5ß1-integrins, which
have been implicated in cranial NCC survival
(Goh et al., 1997
), and
5ß1- and
4ß1-integrins, which have been implicated in
the proliferation or survival of glial cell precursors
(Haack and Hynes, 2001
).
The Cre-LoxP conditional gene disruption system has been used to
investigate the effect of integrins on NCC ontogeny in more detail.
P0-Cre mice crossed with mice carrying a floxed ß1-integrin
allele generated animals with the conditional mutation targeted specifically
to Schwann cells (SC), which are derived from NCC
(Feltri et al., 2002). This
conditional knockout revealed the role played by ß1-integrin receptors in
postnatal PNS development. The loss of ß1-integrins impedes interactions
between SC and axons, causing dysmyelinating neuropathy
(Feltri et al., 2002
).
We recently developed the Ht-PA-Cre transgenic mouse line, in which Cre
recombinase activity is targeted specifically to NCC at the start of migration
(Pietri et al., 2003). We
report here the effects of specific inactivation of the ß1-integrin gene
in migratory neural crest cells. The crossing of Ht-PA-Cre mice with ß1
floxed mice generated animals in which the NCC produce no ß1-integrin but
do produce ß-galactosidase under the control of the endogenous
ß1-integrin promoter (Potocnik et
al., 2000
). The mutation targets the glial and sensory neuronal
compartments of the PNS as well as the other NCC derivatives.
We show here that ß1-integrin receptors control PNS development at both early and late stages. The absence of ß1-integrins delayed the migration of SC along nerves and resulted in abnormal axo-glial segregation, especially for sensory axons. Substantial changes were observed in the pattern of subcutaneous and muscular innervation. In addition, the arborisation and fasciculation of the innervating fibres and their targeting to the distal tissues were abnormal, and neuromuscular synaptogenesis appeared to be blocked in a state of immaturity. Finally, the mutation caused the death of the animals by the age of three weeks, owing to multiple defects affecting various NCC derivatives.
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Materials and methods |
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Reagents and antibodies
The partially purified mouse monoclonal antibody (clone 2H3) against the
160 kDa neurofilament (NF-160) protein was obtained from Developmental Studies
Hybridoma Bank. Mouse monoclonal antibodies against the nuclear factor Hu-D
(clone 16A11) and against the major myelin protein P0 were gifts
from J. A. Weston (Marusich et al.,
1994) and J. J. Archelos
(Archelos et al., 1993
),
respectively. The rabbit polyclonal antibody against laminins was obtained
from Sigma. The rabbit polyclonal antibodies against tenascin and S100, and
the mouse monoclonal antibody against fibronectin were obtained from Chemicon.
In some cases, we used a rabbit polyclonal antibody against S100 produced by
Dako. The mouse monoclonal antibody (clone SY38) against synaptophysin was
obtained from Progen. The rabbit polyclonal antibody against cleaved caspase 3
(Asp175) was obtained from Cell Signaling. The rabbit polyclonal antibody
against Ki67 was obtained from NovoCastra and the goat polyclonal antibody
against parvalbumin was purchased from Swant. The
-bungarotoxin
(
-BTX) biotin-conjugated compound extracted from Bungarus
multicinctus venom was purchased from Molecular Probes. Secondary
antibodies conjugated with Alexa 488, cyanin 3 or horseradish peroxidase (HRP)
were purchased from Molecular Probes, Jackson Laboratories and Amersham
Pharmacia Biotech, respectively.
Histology, immunohistochemistry and microscopy
For the detection of ß-galactosidase activity, embryos or organs were
dissected in cold phosphate-buffered saline (PBS), pH 7.6, supplemented with
5% foetal calf serum (FCS), were fixed in toto in freshly prepared ice-cold 1%
formaldehyde/0.2% glutaraldehyde/0.02% Nonidet-P40 for 2-4 hours and processed
as described elsewhere (Dufour et al.,
1994).
Whole-mount immunostaining was performed on embryos fixed in methanol Carnoy fixative or 4% PFA. Samples were incubated overnight in blocking solution composed of 0.3% Triton X100, 0.5x Blocking Reagent (Roche Applied Science), and 10% foetal calf serum in PBS and then for 2 days each with primary and secondary Alexa 488- or HRP-conjugated antibodies. Samples were thoroughly washed between incubations. HRP activity was detected with the VECTOR® peroxidase substrate kit, according to the manufacturer's instructions (Vector Laboratories), followed by benzyl-benzoate solution (BABB) treatment for the detection of deep stained structures.
For histological and immunohistochemical analysis of sections, fixed samples were dehydrated and embedded in paraplast Plus. They were then cut into serial sections of 7-10 µm and the wax removed. Some whole-mount stained embryos were serially sectioned at a thickness of 200 µm on a vibratome. Sections were incubated for 2 hours in blocking solution consisting of 10% FCS/0.1% Triton X100/0.5x Blocking Reagent (Roche Applied Science) in PBS. The sections were then incubated overnight at 4°C with primary antibodies in blocking solution, rinsed several times in PBS and incubated with secondary antibodies for 2 hours at room temperature in the dark.
Cell proliferation was assessed by counting more than 2500 DRG cells in every second section of three different embryos of each genotype and analysing staining for the Ki67 marker. Results are expressed as the percentage of cells that were Ki67-positive. Statistical significance was determined with the ANOVA unpaired t-test.
Acetylcholine receptors (AChR) were detected with BTX. The
abdominal, diaphragm, soleus and gastrocnemius muscles of animals at times P1
and P21 were dissected in cold PBS, pH 7.6, and were then incubated for 2-3
hours at 37°C in
BTX in minimum essential culture medium. The
samples were rinsed three times, for 1 hour each, in cold PBS, fixed in 4% PFA
for thirty minutes and processed as for whole-mount samples used for
immunostaining.
Whole-mount stained samples and histological sections were photographed under a Leica MZ8 stereomicroscope (Leica Microsystems SA) equipped with a JVC 3CCD colour camera. Confocal images were obtained for muscle preparations on a Leica TCS4D confocal microscope based on a DM microscope interfaced with an Ar/Kr laser. Stacks of images were mounted with Metamorph 5.0, and normalised to the same width on the z-axis.
Semi-thin sections and electron microscopy
Postnatal sciatic nerves were isolated from control and mutant animals
perfused with 0.5% glutaraldehyde in phosphate buffer (PB, pH 7.4), fixed in
0.5% glutaraldehyde for 1 hour at 4°C and washed in PB.
ß-Galactosidase activity was detected histochemically by incubating
samples in PB supplemented with 5 mM potassium ferricyanide, 5 mM potassium
ferrocyanide, 2 mM magnesium chloride and 1 mg/ml Bluo-gal
(5-bromo-3-indolyl-ß-D-galactoside, Sigma) as a substrate for 12 hours at
30°C. The following day, samples were post-fixed by incubation in 1.6%
glutaraldehyde in PB for 1 hour at 4°C followed by 1 hour in 1%
OsO4 (Sigma) in PB. They were then washed in PB, dehydrated in
ethanol (30%, 50%, 70%), stained by incubation with 1% uranyl acetate in 70%
ethanol for 1 hour, dehydrated in ethanol (80%, 90%, 100%), infiltrated and
embedded in Durcupan (Fluka). Ultrathin sections were cut and stained with
uranyl acetate and lead citrate. The sections were observed with a Tecnai 12
(FEI, Phillips) electron microscope.
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Results |
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PNS development is altered in mutant embryos
At E10.5, the condensing DRG displayed no phenotypic alteration in mutants.
These structures, which displayed ß-galactosidase activity, were similar
in size and structure to those of control embryos
(Fig. 3A,B, respectively). Hu-D
was distributed similarly in the DRG of mutant and control mice
(Fig. 3C,D, respectively). By
contrast, 70% of the E10.5 mutant embryos (n=17, from four different
litters) displayed changes in the patterning of the cranial nerves
(Fig. 3F) with respect to
control embryos (n=15; Fig.
3E). Fifty-nine percent of the mutants exhibited a decrease in the
number of vagus nerve (X) roots (Fig.
3F, white arrowhead), 24% had a fusion of nerves IX and X
(Fig. 3F, black arrowhead) and
12% had an absence of nerve IX, whereas the placode-derived ganglion IX was
present (not shown). The phenotypically altered mutants often displayed two of
these defects (see Fig.
3F).
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At E12.5-E13.5, the trunk nervous system displayed abnormalities, with the partial disappearance of ß-galactosidase staining at the sites of muscular and subcutaneous innervation of the thoracic-abdominal body wall and limbs in the mutant embryos (Fig. 4B,D). Four different litters were analyzed and all the mutants exhibited this phenotype. On 200 µm transverse sections, ß-galactosidase-positive structures were not observed in the distal part of the developing nervous system in mutant embryos (white arrows, Fig. 4D), in contrast to what was observed in controls (Fig. 4A,C).
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By contrast, on E16.5, SC were found at the apex of the nerves in the mutant embryos, as in controls (not shown). The transitory absence of SCP along axons may be due to a decrease in SCP proliferation or to an increase in the apoptosis of these cells. Because it is difficult to quantify the glial cells number along the spinal nerves and because SCP are mainly originating from the DRG, we analyzed the proliferation and apoptosis rate within that structure. No significant difference was observed in the percentage of proliferative DRG cells on E12.5 and E16.5 for control (30.69±0.88% and 14.07±3.34%) and mutant (33.17±4.04% and 19.78±1.89%) embryos, as shown by detection of the Ki67 marker. In addition, very few DRG cells expressed the activated cleaved caspase 3 apoptosis marker and the numbers of such cells were similar in both mutant and control embryos at these stages (not shown). These results could not account for the lack of SCP on E13.5 in the distal part of the developing PNS. We therefore conclude that this phenotype is probably related to a delayed migration of these cells along the axons.
The subcutaneous nerve network at various sites, such as the area around the eyes, the lateral body wall and in the limbs, displayed similar morphological defects in all of the mutant embryos analyzed (n=13). At E13.5 and E14.5, the nerves in the mutants were always thinner and displayed a different pattern of arborisation from controls, as shown by whole-mount NF-160 immunostaining viewed from the ventral (Fig. 5A,C) and lateral (Fig. 5B-D and 5G-H) sides of the embryos and at the hindlimb level (Fig. 5E,F). In addition, the subcutaneous emergence sites of the sensory nerves always differed between the mutants and the controls (not shown). By contrast, at these stages, the extent of axonal growth was not significantly affected, even at the most distal part of the limbs in the mutants.
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ß1-Integrin gene deletion leads to major changes in radial sorting in the sciatic nerves
Morphological, ultrastructural and immunohistochemical analyses of sciatic
nerves revealed a defect in SC differentiation, modified axon segregation
(Fig. 7), and changes in ECM
composition in the mutant animals (Fig.
8). Extensive ß-galactosidase labelling of the mutant and
control sciatic nerves revealed the activity of the ß-galactosidase in
the axons (in contrast to whole-mount embryonic staining) and therefore the
identification of the sensory axons (ß-galactosidase +) and the motor
axons (ß-galactosidase ) within the nerve. At P21, the sciatic
nerve appeared to have a smaller diameter in the mutants
(Fig. 7C) than in the controls
(Fig. 7A). This difference was
already apparent at stage P1, but was less marked (not shown). The sciatic
nerves of P21 control animals were composed of myelinated sensory axons
(Fig. 7B, red arrow) and motor
axons (Fig. 7B, black arrow) of
medium to large diameter, and small clusters of small-diameter unmyelinated
sensory axons (Fig. 7B, red
arrowhead). The mutant nerves were composed of myelinated motor axons
(Fig. 7D, black arrow) and
large clusters of unsorted axons of small to large diameter
(Fig. 7E) mostly of sensory
origin (as revealed by their light blue staining, ß-galactosidase +;
Fig. 7D, black arrowheads).
Only a few myelinated sensory axons were observed in the mutant nerves
compared with the control nerves (red arrows in
Fig. 7D and 7B, respectively).
However, owing to the significant importance of the clusters of unsorted
axons, we could not exclude that a small number of motor axons are present
within these clusters and remain consequently unsorted.
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In addition to the axo-glial segregation defect, loose basal lamina could be detected surrounding the myelinated axons as well as the clusters of unsorted axons at P21 (Fig. 7E). This suggests strong linkage alterations of these structures to the ECM. However, the axons within the clusters appeared not to contain basal lamina. Schwann cells are known to secrete various ECM proteins of which integrins represent the major receptors. We therefore assessed the expression level of major components of the ECM such as fibronectin, laminin, tenascin and collagen IV, in control and mutant sciatic nerves at P1 and P21 (Fig. 8I-R). At P1 and P21, laminin levels were lower in the sciatic nerves of mutants (Fig. 8N,O) than in controls (Fig. 8I,J). Fibronectin levels increased in controls between P1 and P21 (Fig. 8K,L), but decreased in mutants over this period, with fibronectin levels at P21 being lower in mutants than in controls, despite having been higher in the mutants on P1 (Fig. 8P,Q). Tenascin was not detected in the sciatic nerves of the two types of animals at P1. However, it was detected at P21, in larger amounts in the sciatic nerves of mutant animals than in those of controls (Fig. 8M,R). Collagen IV levels (not shown) were similar in the sciatic nerves of mutants and controls at both stages. Interestingly, at P21, some ECM-free spaces were detected in the mutant, reinforcing the idea that unsorted axons were not surrounded by a basal lamina as observed in Fig. 7E. In the mutants, the general organisation of the ECM was strongly disrupted at P1 and P21. This disruption was correlated with the delayed maturation of SC and changes in axo-glial segregation.
ß1-Integrin loss in SC leads to neuromuscular innervation defects
The muscular activity is regulated by neuromuscular junction (NMJ)
activity, which in turn requires coordinated interaction between SC, axons and
muscle fibres (Burden, 1998;
Sanes and Lichtman, 1999
). We
analysed the development of NMJs for various muscles obtained from P1 and P21
control animals and from mutant animals at P1 and from those at P21 that have
developed motor and posture defects. Synaptophysin (synapto) and
-bungarotoxin (
-BTX) were used to detect the pre- (axonal
terminal end) and post- (clusters of acetylcholine receptor, AChR) synaptic
compartments of the synapse. The mutants differed from the controls in having
a lower level of intramuscular innervation and in the formation of NMJs in
abdominal muscles (Fig. 9) and
the diaphragm, soleus and gastrocnemius muscles (not shown). At P1, the
control abdominal muscle displayed a high density of AChR clusters
co-localised with synaptophysin (Fig.
9A, inset), indicating the apposition of the pre- and
post-synaptic compartments, as previously described
(Lin et al., 2001
). At P21,
fewer AChR clusters (Fig. 9C,
insert) were detected, demonstrating that the known postnatal regulation of
receptor number had occurred. By contrast, at P1, the muscles of mutants
displayed high densities of AChR clusters, but most of these receptors were
not connected to the post-synaptic apparatus
(Fig. 9B, inset). At P21, the
density of AChR clusters remained high in the mutant muscles, indicating that
no postnatal regulation of receptor number had occurred. Some of these
receptors were connected to nerve terminals
(Fig. 9D, inset). The synapses
in the P21 mutants appeared as ring-like structures
(Fig. 9D, inset), resembling
immature NMJs (Sanes and Lichtman,
1999
).
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Discussion |
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ß1-Integrins transiently control embryonic development of the cranial nerves
The conditional deletion of the ß1-integrin gene began in the cephalic
region of the embryos at stage E8 and followed a rostral to caudal progression
in line with the generation of NCC. On E10, in most mutants, significant
changes were observed in the epibranchial ganglia and nerves, of NCC origin,
with altered vagus nerve roots and also fusions of the IXth and Xth nerves.
Interestingly, defects of this type are not observed in the trigeminal
ganglia, despite the NCC origin of the glial and neuronal compartments
(Pietri et al., 2003). These
defects, observed during a period in which the loss of ß1-integrins from
the cell surface is incomplete, illustrate differences between the
epibranchial ganglia and nerves and the trigeminal ganglia to integrin levels
during development.
Indeed ß1-integrins were not lost immediately after recombination of
the locus and ceased to be detectable at the cell surface only after 3 days of
detectable ß-galactosidase activity. This time lag to the loss of
ß1-integrins in the targeted structures limits the functional analysis of
ß1-integrin requirements during the early stages of NCC migration. In
particular, no defects are observed in the branchial arches or in the
colonisation of the heart in our mutants. A previous study showed that
4-integrin-null embryos die during early development and display
abnormalities in heart development (Yang
et al., 1995
). In mouse and avian embryos, perturbation
experiments with competitors have shown that
4-integrin controls
cranial NCC migration (Kil et al.,
1996
; Kil et al.,
1998
). This suggests that, in our mutants, sufficient amounts of
ß1-integrins remain at the cell surface of the cranial NCC at these
stages to support the correct development of these morphological processes.
Consistent with this hypothesis, the elimination of ß1-integrins earlier
in development, when the NCC are still resident in the neural tube, results in
the early death of the embryo (E12.5) (T.P., unpublished). In this case, the
embryos die from haemorrhaging, accompanied by defects in NCC cardiac
derivatives and in neural tube closure. Thus, various early morphological
processes may occur if ß1-integrins are expressed at the cell surface at
low levels, but the complete loss of ß1-integrins strongly inhibits these
processes. An increase in the apoptosis of cephalic NCC has been described in
5-integrin-null mice (Goh et al.,
1997
). This phenomenon has not been observed in other knockout
mouse models (reviewed by De Arcangelis
and Georges-Labouesse, 2000
;
Sheppard, 2000
). In our
conditional mutants, the rate of apoptosis in the branchial arches was similar
in mutants and controls (T.P., unpublished). This suggests that the increase
in the rate of apoptosis observed in
5-null cranial NCC may be
triggered by the combined effect of the loss of
5ß1-integrin and a
possibly defective ECM microenvironment in constitutive
5-integrin
knockout embryos.
The phenotypic effects on the cranial nerves of conditional mutants seem to be transient because on the following day, the cranial nerves and ganglia appeared similar to those of the controls. At birth, mutant and control embryos displayed no obvious differences in sensorial capacities, with both groups responding to external noise and light stimuli. This suggests the existence of a compensatory mechanism, operating after E10.5, establishing a new path to guide the altered nerve to its normal destination, leading to normal sensorial behaviour later on. Our results indicate that ß1-integrins are not required for the further development and maintenance of craniofacial structures.
ß1-Integrins control embryonic development of the PNS
In vitro studies have clearly implicated ß1-integrins in the control
of neurite outgrowth for peripheral neurons
(Tomaselli et al., 1993;
Ivins et al., 2000
;
Vogelezang et al., 2001
). In
our conditional ß1-integrin knockout embryos, the loss of
ß1-integrins in sensory neurons does not affect their axonal growth. The
general trajectory of the proximal portion of these nerves appeared to be
minimally affected in the mutant animals, with the nerve endings reaching
distal targets. However, the organisation of the network is visibly affected
because, from E12, the distal region of the nerves of the body wall appeared
less branched and fasciculated. In addition, the pathfinding of the distal
region of the spinal nerves was perturbed, particularly when they reach the
subcutaneous level. The spinal nerves contain a mixture of motor and sensory
axons along most of their length, and the specific axon bundles segregate from
one another only once they are in close proximity to their targets. Therefore,
the motor axons, which are not targeted for the conditional deletion of the
ß1-integrin gene in our mutant animals, in the mixed nerves may help to
compensate for or to mask the potential ß1-integrin-dependent guidance
defect of the neighbouring sensory axons.
In addition to defects in nerve branching and fasciculation, we observed
that the distal region of the nerves was not covered by migrating SCP, as
shown by that lack of ß-galactosidase-expressing cells along the nerve
fibres at E12-13.5, in the mutants. This lack of SCP could give account into
the developmental defect of the innervation network in mutants. Consistent
with this hypothesis, similar embryonic PNS alterations in nerve branching and
pathfinding have been described in the ErbB2, ErbB3, neuregulin 1 and Sox10
mutants (Erickson et al.,
1997; Woldeyesus et al.,
1999
; Morris et al.,
1999
; Britsch et al.,
2001
; Riethmacher et al.,
1997
), which display alterations in the development of NCC
derivatives and lack SC. For example, in ErbB2 mutants in which the heart
development defect has been rescued by the myocardial-dependent expression of
ErbB2, the cutaneous sensory nerves display normal overall trajectories but
are free of SC precursors, poorly fasciculated and disorganised
(Woldeyesus et al., 1999
;
Morris et al., 1999
). Taken
together, these and our results strongly suggest that migratory SCP are
required for correct organisation of the innervation network and nerve
morphology.
The 4ß1- and
5ß1-integrins have been implicated in
control of the survival and proliferation rates of SC obtained from explants
of embryos lacking
4- and
5-integrins
(Haack and Hynes, 2001
). The
SCP migrating along nerves are originated from the DRG. However, we detected
no difference in the proliferation or apoptosis rates of the ß1-null NCC
derivatives in the DRG of our mutants at E12.5 or E16.5. In addition, the lack
of SCP along nerves was transient because, by E16.5, as for the controls, SCs
were detected in the distal region of the spinal nerves in mutants. Although
we cannot exclude the possibility that a modification of proliferative or
apoptosis rate may occur on SCP located along the nerves, the loss of
ß1-integrins (which is effectively complete by this stage) seems to
reduce the rate of migration of the SC along nerves. This is consistent with
previous observations showing that SC lacking ß1-integrins survive and
proliferate normally in vivo (Feltri et
al., 2002
). As spatiotemporal regulation of the integrin
repertoire occurs during SC ontogeny
(Bronner-Fraser et al., 1992
;
Stewart et al., 1997
;
Milner et al., 1997
;
Previtali et al., 2001
) other
integrins, such as
6ß4,
Vß3 and
Vß8, may
have compensated for the possible migratory defect of SC induced by the loss
of ß1-integrins, by E16.5 in the mutants.
ß1-Integrins are required for radial sorting of the sensory axons in sciatic nerves
In the course of development of peripheral nerves, cytoplasmic processes of
promyelinating SC penetrate into the axon bundles to progressively achieve a
one to one ratio between SC and axons. This phenomenon, starting just before
birth, is named radial sorting and precedes the myelination process
(Mirsky et al., 2002). In the
mutant, the radial sorting of motor axons (not targeted by the mutation)
occurs and the subsequent myelination of motor axon-associated SC is not
affected. By contrast, there was an almost complete absence of myelinated
sensory axons (distinguishable by ß-galactosidase activity). Sensory
axons remain in large dense clusters of unsorted axons, indicating that a
defect in axo-glial segregation occurs in the mutant nerves.
Alterations in the radial sorting and myelination process have been
described in P0-Cre;ß1/ß1fl
mutants (Feltri et al., 2002)
where postnatal nerves were composed of numerous clusters of unsorted axons
and of few myelinated fibres. In the
P0-Cre;ß1/ß1fl mutants, the
conditional disruption of the ß1-integrin gene is restricted to the glial
lineage; the SCP are targeted between E13.5 and E14.5 and the loss of the
integrin subunit on these cells is achieved by E17.5, whereas the neuronal
components of the sciatic nerve are not targeted. Therefore, in this study the
phenotype appears to be SC autonomous. The authors attributed the partial loss
of myelinated axons in the adult sciatic nerve as the result of a random
escape process because of compensation by others receptors. In the
Ht-PA-Cre;ß1/ß1fl mutants, the
conditional mutation is made on SC and in sensory neurons early during
development, and we can visualize the targeted cells by their
ß-galactosidase activity. The defects obtained on radial sorting of the
Ht-PA-Cre;ß1/ß1fl mutant sciatic nerves
are in agreement with a crucial role of ß1-integrins in SC to achieve the
axo-glial segregation. We are able to discriminate the sensory axons from the
motor axons within the sciatic nerves. This has allowed us to show a much more
prominent defect of radial sorting on the sensory compartment than on the
motor compartment. This observation suggests that the ß1-integrins in
neurons could be also required for this process. In addition, the absence of
myelinated sensory axons indicates that the myelination process requires the
ß1-integrin functions both in SC and neurons. However, in the
P0-Cre;ß1/ß1fl mutants
(Feltri et al., 2002
) the
discrimination between the sensory and motor axons could not be made and it
was not possible to determine whether the radial sorting defects affected only
one or both types of neuronal fibres within the sciatic nerve. Therefore, it
is possible that in the
P0-Cre;ß1/ß1fl mutants, as
for the Ht-PA-Cre;ß1/ß1fl mutants, the
radial sorting defect is restricted to the sensory compartment. In this case,
additional hypotheses can explain the differential response of sensory and
motor compartment of the sciatic nerves to the inactivation of the
ß1-integrin gene: (1) owing to their distinct embryonic origin, sensory
and motor neurons may express different sets of cell adhesion molecules
involved in the radial sorting process such that the sensory axon sorting
mechanism required ß1-integrins on glia; and (2) SC could be specialized
in two distinct types, each ensheathing only motor axons or only sensory
axons, and with distinct requirement for ß1-integrin function. To
definitively conclude on the specific role of ß1-integrins in neurons and
SC during the development of the PNS, it would be interesting to compare the
radial sorting process of sensory and motor axons in animals in which the
invalidation of ß1-integrin gene is restricted to SC, to motor or sensory
neurons, or to both SC and neuronal compartments.
The delay in the myelination of SC in the
Ht-PA-Cre;ß1/ß1fl sciatic nerves is
consistent with results of two other studies that have implicated
ß1-integrins in the control of the myelination process. One of these
studies shows that ß1-integrin blocking antibodies prevent SC co-cultured
with DRG neurons to myelinate the sensory axons
(Fernandez-Valle et al.,
1994). Another study shows that glial cells that have been
genetically modified to express dominant negative forms of the
ß1-integrins, were unable to remyelinate after their transplantation in
the dorsal funiculus (Relvas et al.,
2001
). In addition, several lines of evidence demonstrate that
laminins and their receptors are involved in PNS development and that SC
require contacts both with the basal lamina and with axons for their survival
and maturation during development (reviewed by
Jessen and Mirsky, 1999
;
Previtali et al., 2001
;
Previtali et al., 2003
;
Mirsky et al., 2002
). These
interactions facilitate the activation of the signalling pathways mediated by
various molecules including the focal adhesion kinase and paxillin
(Chen et al., 2000
) (reviewed
by Previtali et al., 2001
),
further implicating ß1-integrins in the initiation of myelination. These
observations are consistent with our observation that myelination was delayed
in mutant sciatic nerves. Together with changes to the ECM in the sciatic
nerves, the general delay in myelination in mutants strongly suggests that the
loss of ß1-integrins alters the interaction of the SC with the basal
lamina, as previously suggested (Feltri et
al., 2002
), and with axons. In addition, the loss of
ß1-integrins in targeted cells appears also to affect the ECM and basal
lamina organisation. The presence of a basal lamina surrounding unsorted axons
in the Ht-PA-Cre;ß1/ß1fl sciatic nerves
is consistent with a retraction of SC cytoplasmic processes and previous
reports for several types of peripheral demyelinating neuropathies (reviewed
by Dyck et al., 1992
). This
similarity is corroborated by quantitative modifications of the ECM, in
particular tenascin deposition in the sciatic nerves on P21, and indicates
that demyelination may occur in the mutants. The absence of basal lamina
surrounding the unsorted axons within the clusters in the mutant sciatic
nerves at P21, strongly suggests that the SC did not individually ensheath
those axons in earlier stages of the maturation of the nerves. Therefore, our
data strongly indicate that both processes complete absence and
retraction of SC cytoplasmic process may occur concomitantly in the
mutant sciatic nerve.
ß1-Integrins are required in the terminal SC for the targeting of nerves and maturation of the neuromuscular junctions
The defects in intramuscular innervation observed in several muscles at
birth and following postnatal stages in the mutant animals suggest that SC
with ß1-integrins are required for axonal target selection. It was
recently suggested that glial cells may be involved in this process in
Drosophila (Poeck et al.,
2001), but no similar mechanism has yet been described in mammals.
By contrast to control animals, in which axons penetrated the muscle to
innervate specific fascicles, most of the nerve network in the mutants
remained along the surface of each muscle layer and failed to penetrate. On
stages P1 and P21 in the mutant animals, SC covered the entire length of the
motor nerves from base to tip and were found at the nerve endings. This
suggests that ß1-integrins are required for the interactions of SC with
axons or basal lamina and to facilitate the entry of axons into the muscle
layers and the targeting of AChR clusters. The perturbation of intramuscular
innervation was more apparent on P21 than on P1 in the mutant animals,
indicating the lack of a compensatory mechanism. At the postsynaptic level, on
P1, no difference was observed in the number and structure of AChR clusters in
the mutants, indicating that this failure for the axons to meet the targets
was not due to the absence of targets. The mismatch between nerve endings and
AChR clusters (pre- and post-synaptic compartments, respectively) was striking
on both P1 and P21, confirming the independence of the formation of AChR
clusters from the presynaptic apparatus.
The mutants responded to tactile stimulation at P1, which implies that there were some functioning NMJs at that stage. However this ability to respond deteriorated thereafter, suggesting that SC and sensory innervation were involved in this process.
Even in the presence of the three components of the synapse (axons,
terminal SC and postsynaptic apparatus), NMJ observed at P21 in the mutant
resemble to an immature NMJ as they have a ring-like structure. This type of
structures is normally observed in the normal course of NJM development from
P7 to P10 (Burden, 1998;
Sanes and Lichtman, 1999
). In
addition, a lack of regulation of the number AChR clusters in the mutant
occurred, at least until P21. In wild-type rodents, this mechanism occurred
within the first 2 weeks after birth. Therefore, NMJ maturation in the mutants
appears to be blocked at an early stage of their postnatal development.
Several studies examining muscle re-innervations have suggested that terminal
SC are involved in the maintenance and remodelling of the synapse
(Son and Thompson, 1995
). Our
results point out the ß1-integrins as major receptors for the proper
activity of the terminal SC in the course of the NMJ development.
In normal NMJ development (reviewed by
Sanes and Lichtman, 1999) or
after nerve regeneration (Rich and
Lichtman, 1989
), a significant number of synapses are eliminated
during organisation of the nerve-muscle connection. Synapse elimination
involves the withdrawal of the axons and SC processes. This process was
observed in the control animals, in which the number of AChR clusters and
nerve terminals in the abdominal muscles decreased from P1 to P21. By
contrast, in the mutants, the number of AChR clusters increased from P1 to
P21. This phenomenon resembles the responses of muscles to denervation, by
increasing the number of AChR clusters
(Blondet et al., 1989
). The
increase in the number of AChR clusters in mutants coincides with progressive
muscle fibre atrophy, reflecting the absence of stimulatory innervation. This
suggests that the failure of many axons to establish functional NMJs leads to
muscle to produce more AChR clusters and that the process of synapse
elimination is probably not initiated. The specific alteration of the
elaboration and maturation of the NMJs in our mutant animals shows a SC
autonomic effect for the muscular innervation and reveals the major
involvement of the ß1-integrin specifically in the terminal SC
functions.
We conclude from our results that ß1-integrins are important for correct interactions between glial cells and axons, SC migration, axon morphology and the organisation of spinal nerves into networks en route to their peripheral targets. This study also highlights the functions of ß1-integrins for both the neuronal and glial lineages of the PNS and at several steps in the differentiation of peripheral nerves, such as radial sorting and myelination, as well as the muscular synaptogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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