1 Friedrich Miescher Institute for Biomedical Research, Maulbeerstr. 66, 4058
Basel, Switzerland
2 Departamento de Biologia Celular, Universidad de Valencia, 46100 Burjassot,
Spain
3 Department of Physiology, University of Basel, 4051 Switzerland
* Present address: The Scripps Research Institute, 10550 North Torrey Pines
Road, ICND 222, La Jolla, CA 92037, USA
Author for correspondence (e-mail:
umueller{at}scripps.edu)
Accepted 3 February 2003
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SUMMARY |
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Key words: Erbb2, Nrg1, Muscle spindle, Synapse, Neuromuscular junction, Mouse
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INTRODUCTION |
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During formation of the NMJ, synaptic proteins such as acetylcholine
receptors (AChRs), rapsyn, Musk, Nrg1 and its Erbb receptors become clustered
in the postsynaptic muscle membrane. Furthermore, the expression of genes that
encode postsynaptic proteins becomes restricted to subsynaptic nuclei
(Burden, 1998;
Sanes and Lichtman, 1999
).
Clustering of proteins and synaptic gene expression are dependent on agrin
that is released from motor nerve terminals and activates the Musk receptor
tyrosine kinase in muscle. Accordingly, in mice that lack agrin or Musk, the
formation of stable synaptic AChR clusters and synaptic gene expression are
defective (DeChiara et al.,
1996
; Gautam et al.,
1996
). Agrin/Musk is not only necessary, but also sufficient for
postsynaptic differentiation. Ectopic expression of agrin or of constitutively
active Musk in muscle induces the formation of a postsynaptic apparatus and
the upregulation of genes normally restricted to subsynaptic nuclei
(Cohen et al., 1997
;
Jones et al., 1997
;
Jones et al., 1999
;
Meier et al., 1997
;
Moore et al., 2001
).
Nrg1 and its Erbb receptors have also been implicated in the regulation of
gene expression in subsynaptic nuclei. Several Nrg1 isoforms are generated
from the Nrg1 gene by alternative splicing. Isoforms containing an
EGF domain induce AChR gene expression in cultured myotubes in vitro
(Buonanno and Fischbach, 2001;
Schaeffer et al., 2001
). Mice
heterozygous for a targeted mutation ablating an Ig-domain encoding exon of
the Nrg1 gene have decreased numbers of synaptic AChRs, suggesting
that Ig-domain containing Nrg1 isoforms are important in vivo
(Sandrock et al., 1997
). The
cellular source of Nrg1 that regulates synaptic gene expression is at present
unclear. Mice that lack Nrg1 specifically in motoneurons still develop NMJs
(Schaeffer et al., 2001
;
Yang et al., 2001
), suggesting
that Nrg1 may be provided by another cellular source, e.g. by muscle fibers.
Consistent with this interpretation, Nrg1/Erbb receptors are clustered at
postsynaptic sites induced by ectopic expression of agrin in muscle fibers in
the absence of nerve terminals (Jones et
al., 1996
; Jones et al.,
1999
; Meier et al.,
1998
; Moore et al.,
2001
). Interestingly, expression of synapse specific genes in
ectopically induced postsynaptic membranes is decreased when the function of
Erbb2 or Erbb4 is blocked in muscle (Jones
et al., 1996
; Jones et al.,
1999
; Meier et al.,
1998
; Moore et al.,
2001
). Nrg1/Erbb are also concentrated at endogenous NMJs, but
fail to cluster in mice that lack agrin or Musk
(DeChiara et al., 1996
;
Gautam et al., 1996
). Taken
together, these data suggest that muscle derived Nrg1 acts downstream of
nerve-derived agrin.
Genetic evidence implicating Erbb receptors in NMJ formation is still
missing. Synapse formation and synaptic gene expression are initiated in mice
that carry null mutations in the genes encoding Erbb2 and
Erbb3, but the synaptic band is broadened
(Gassmann et al., 1995;
Lee et al., 1995
;
Meyer and Birchmeier, 1995
;
Riethmacher et al., 1997
). It
is at present unclear whether the changes in the synaptic band arise due to
defects in muscle fibers or in Schwann cells that are also affected in the
mutants (Lin et al., 2000
;
Morris et al., 1999
;
Riethmacher et al., 1997
;
Woldeyesus et al., 1999
;
Wolpowitz et al., 2000
).
Furthermore, Erbb4 that is expressed in muscle
(Zhu et al., 1995
) may
compensate for the loss of Erbb2/b3, and account for the activation of
synapse-specific gene expression.
Little is known about the signaling mechanisms that lead to muscle spindle
differentiation. Muscle spindles consist of multiple fibers such as nuclear
bag1 and bag2 fibers and multiple nuclear chain fibers.
Nuclear bag2 fibers differentiate first, followed by the formation
of nuclear bag1 fibers and multiple nuclear chain fibers. The
timing of ingrowths of nerve fibers relative to the time when spindle
development is initiated suggests that muscle fibers are recruited to develop
into spindle-forming intrafusal fibers by signals derived from the innervating
sensory Ia afferent neurons (Maier,
1997; Walro and Kucera,
1999
). Mice with a targeted mutation in the genes for neurotrophin
3 (Ntf3) and its TrkC (Ntrk3 Mouse Genome
Informatics) receptor lack Ia afferents and muscle spindles, providing genetic
evidence that contact between sensory neurons and developing muscle fibers is
required for muscle spindle development
(Ernfors et al., 1994
;
Farinas et al., 1994
;
Klein et al., 1994
;
Tessarollo et al., 1994
).
Interestingly, NT3 regulates the survival of proprioceptive sensory neurons
prior to innervation of muscle spindles, but is also required for the survival
of Ia afferent neurons once they reach their target
(ElShamy and Ernfors, 1996
;
ElShamy et al., 1998
;
Farinas et al., 1996
;
Kucera et al., 1995
;
Oakley et al., 1995
;
Oakley et al., 1997
;
Ockel et al., 1996
;
Ringstedt et al., 1997
;
Wright et al., 1997
). NT3 is
expressed in spindles, and maintenance of NT3 expression is dependent on nerve
contact (Chen et al., 2002
;
Copray and Brouwer, 1994
).
These data suggest the existence of a regulatory loop, where yet to be defined
neuronal signals induce muscle spindle formation and NT3 expression, while NT3
serves to maintain Ia afferent neurons.
To investigate the function of Erbb2 receptors in muscle, we have generated a mouse line carrying a floxed allele of the Erbb2 gene, and a second mouse line expressing Cre in developing skeletal muscle. Using these mice, we have inactivated Erbb2 expression in muscle. We demonstrate that Erbb2 is not essential for the development of NMJs, although the Erbb2-deficient synapses contain reduced numbers of AChRs and are less efficient. Unexpectedly, the mutant mice also have proprioceptive defects. Consistent with this finding, we demonstrate that Erbb2 is essential for muscle spindle development.
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MATERIALS AND METHODS |
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Analysis of Cre-mediated recombination
DNA was isolated and analyzed by PCR or Southern blot as described
(Graus-Porta et al., 2001;
Muller et al., 1997
). The
sequence of the PCR primers for the Erbb2 allele
are as follows: 5'-CTCCCAAGTCTGGGCTCTTTCTC-3',
5'GCGTGTTTTGCCTGTGTGTATGTC-3' and
5'-CCTTGGGAAAAGCGCCTCCCCTAC-3'. Primers that amplify Cre were used
to genotype HSA-Cre mice: 5'GACATGTTCAGGGATCGCCAGGCG-3'
and 5'-GACGGAAATCCATCGCTCGACCAG-3'. Primers to identify the
recombined Erbb2flox allele were as follows:
5'-CTGTTGCAAACAAATGCCTGC-3' and
5'-CAGAATGGCTAAATCTGGGATC-3'.
PCR conditions
Erbb2 allele: 5 minutes at 95°C followed by
35 amplification cycles (30 seconds at 95°C; 30 seconds at 62°C; 30
seconds at 72°C) and 10 minutes final extension at 72°C.
Cre transgene: 10 minutes at 94°C followed by 30 amplification cycles (1 minute at 94°C; 1 minute at 65°C; 1 minute at 72°C) and 10 minutes extension at 72°C.
Recombined Erbb2flox allele: as for Cre, but 35 amplification cycles.
PCR reaction products were 520 bp (Erbb2+), 330 bp (Erbb2), 170 bp (recombined Erbb2flox) and 600 bp (Cre). For Southern blot analysis of Cre transgenic animals, the full-length cDNA encoding Cre was used.
Histology and electron microscopy
Tissues were dissected, fixed overnight at 4°C in 4% PFA in PBS without
Mg2+ and Ca2+ (PBS), and incubated
overnight at 4°C in 30% sucrose in PBS prior to freezing
in OTC. Alternatively, fresh tissue was embedded in OTC. Cryosections were
stained with Hematoxylin and Eosin as described
(Graus-Porta et al., 2001;
Muller et al., 1997
).
lacZ staining was performed as described
(Farinas et al., 1996
), either
on whole-mount embryos or on 20-30 µm cryosections of isolated soleus
muscle, after fixation in 2% PFA in PBS. For electron
microscopy, embryos were collected at different developmental stages and
immersion fixed in 4% PFA/2%glutaraldehyde in PBS at 4°C
overnight. Hindlimbs were osmicated in 2% osmium tetroxide for 2 hours,
stained with 2% uranyl acetate in 70% ethanol en bloc, dehydrated and embedded
in araldite (Durcupan, Fluka). Complete serial of transverse 2 µm sections
through the thigh were stained with 1% Toluidine Blue for light microscopic
inspection. Those sections containing nerve bundles of recognizable muscle
spindles were sectioned into 70 nm sections for EM analysis after lead citrate
staining.
Immunohistochemistry
Immunohistochemistry with antibodies to Erbb2 (Santa Cruz, sc-284) on
histological sections of soleus muscle or with primary muscle cells in culture
was carried out as described (Trinidad et
al., 2000). For staining with antibodies to
-dystroglycan
and utrophin (antibodies kindly provided by M. Ruegg) 30 µm cryosections of
freshly embedded soleus muscle were fixed for 10 minutes with 2% PFA in
PBS, washed three times for 5 minutes with
PBS and permeabilized for 10 minutes with 1% Triton X-100 in
PBS. Sections were incubated in 100 mM glycine in
PBS for 10 minutes at room temperature, and in 3% normal
goat serum (NGS), 0.3% Triton X-100 in PBS for 1 hour at
room temperature. Sections were incubated overnight at 4oC with
primary antibody in 3% NGS, 0.3% Triton X-100 in PBS. For
immunofluorescence detection, sections were washed in PBS
and incubated for 1 hour at room temperature with FITC-, or TRITC-labeled
secondary antibodies (Jackson ImmunoResearch), or FITC- or TRITC-labeled
-bungarotoxin (Molecular Probes). Sections were washed in
PBS and mounted in Mowiol (Calbiochem).
In experiments using an antibody against parvalbumin (Swant), detection was performed with peroxidase-coupled secondary antibodies and the Vectastain ABC kit (Vector Laboratories). Embryonic muscles were dissected, fixed in 4% PFA for 2 hours at 4°C and incubated overnight at 4°C in 30% sucrose in PBS. Tissue was embedded in OTC and sections were cut at various thickness. Prior to staining, sections were fixed again in 4% PFA for 10 minutes at room temperature, washed three times in PBS and endogenous peroxidase was inactivated in 10% methanol, 3% H2O2 in PBS for 30 minutes. Blocking was performed in 0.4% Triton X-100, 10% NGS, 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. Primary antibody was diluted in blocking solution and incubated on sections at 4°C overnight. Samples were further processed following the instructions in the Vectastain ABC kit. Digital images were collected on a Deltavision microscope (Applied Precision) and processed by deconvolution.
Primary muscle cell cultures
Hindlimb muscles from P2 animals were dissected, minced and incubated at
37°C for 15 minutes in HBSS-CMF (Gibco-BRL) containing collagenase type IV
(100 U/ml, Sigma) and dispase type II (2.4 U/ml, Roche Molecular Diagnostics).
The solution was triturated to obtain a single cell suspension, and cell
clumps were allowed to settle. The supernatant was collected and supplemented
with 1/10 of the volume with fetal calf serum (FCS). The solution was filtered
through a cell strainer (Becton Dickinson), cells were harvested by
centrifugation at room temperature and resuspended in DMEM supplemented with
25% 199 medium (Gibco-BRL), 10% FCS and antibiotic-antimycotic (Gibco-BRL).
Cells were plated onto plastic dishes coated with 1% gelatine (Sigma) and
cultured in a humidified atmosphere at 37°C/5% CO2. To induce
differentiation into myotubes, medium was changed to DMEM supplemented with
25% 199 medium, 2% horse serum, antibiotic-antimycotic and 10 µg/ml insulin
(Sigma) when myoblast cultures had reached subconfluency.
Postsynaptic differentiation was induced by adding a C-terminal fragment of
the neuronal isoform of chick agrin (200 pM-2 nM)
(Ruegg, 1996) to myotubes in
culture. Myotubes were analyzed 24 hours after induction by
immunohistochemistry as described above.
Electrophysiology
Muscle fibers of the diaphragm were voltage clamped to 70 mV with a
conventional two-electrode clamp system at endplates identified visually, and
miniature endplate currents (mepcs) were recorded at room temperature in
Tyrode solution. Amplitudes and decay phases of 20-50 mepcs per synapse were
analyzed individually, assuming a single exponential decay. Amplitudes and
decay time constants (reflecting mean burst durations of the AChR channels)
from 20-50 mepcs per synapse were averaged. Only synapses with mepcs with rise
times of less than 700 µs were included in the analysis.
Quantitative immunofluorescence measurements
In order to measure AChR density in mutant versus wild-type endplates,
confocal image stacks were recorded with calibrated photomultiplier settings.
Images were truncated at a lower intensity threshold of 10 because preliminary
analysis showed that all voxels displaying intensity above 10 were associated
with endplates. The remaining -bungarotoxin Texas Red-positive voxels
were processed as follows: the number of voxels at each intensity was
multiplied by this intensity and the products were summed. This sum was
divided by the total number of voxels remaining after truncation, thus
yielding an estimate of average voxel intensity.
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RESULTS |
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Inactivation of Erbb2 in skeletal muscle
We next crossed the HSA-Cre transgene on an
Erbb2+/ background. These mice were crossed with
Erbb2flox/flox mice. Offspring that inherited
one Erbb2 allele, one Erbb2flox
allele and the HSA-Cre transgene were born with the expected
Mendelian frequency and were viable. DNA analysis confirmed that Cre induced
efficient recombination of the Erbb2flox allele in muscle
(data not shown). To confirm that Erbb2 protein was absent from muscle, we
carried out immunohistochemistry with antibodies against Erbb2. In wild-type
mice, Erbb2 immunoreactivity was concentrated at NMJs that were visualized by
co-staining with -bungarotoxin, to label AChR clusters
(Fig. 2A). In the mutants, AChR
clusters still formed, but Erbb2 was not expressed at the synapse
(Fig. 2B). Low levels of
immunoreactivity for Erbb2 were still detectable adjacent to synaptic sites,
probably reflecting Erbb2 expression in terminal Schwann cells that surround
the synapses, but where the HSA promoter used to drive Cre expression was not
active.
|
In summary, we conclude that we had effectively inactivated Erbb2 expression in developing muscle fibers. Furthermore, the data demonstrate that agrin can induce AChR clusters in myotubes in vitro in the absence of Erbb2 expression.
Reduced synaptic transmission at Erbb2-deficient NMJs
We next analyzed by immunohistochemistry and electrophysiology whether the
structure and function of NMJs was affected in vivo in the absence of Erbb2.
Staining with -bungarotoxin revealed that AChR clusters had formed in
the mutants, and that additional postsynaptic proteins, such as b-dystroglycan
and utrophin were appropriately localized to synaptic sites. In addition, no
defects were apparent in the characteristic pretzel-shape of the NMJs
(Fig. 3A-C).
|
The reduced efficiency of NMJs in Erbb2-deficient mice could be due to
reduced AChR levels at synaptic sites. We therefore measured AChR levels by
quantitative immunofluorescence. Clusters of AChR at NMJs in wild-type and
mutant mice were stained with Texas Red-labeled -bungarotoxin, and
serial optical sections were taken and quantified. Integration of fluorescent
intensities revealed a decrease by about 20% in AChR numbers at endplates of
mutant compared with wild-type muscles
(Fig. 3F; 27 wild-type and 27
mutant endplates, from five wild-type and five mutant diaphragms), consistent
with the electrophysiological data. Taken together, the data show that Erbb2
is not essential for NMJ formation. However, in the absence of Erbb2, the
synapses are less efficient and contain reduced numbers of AChRs.
Muscle spindle development
Mice that lacked Erbb2 in muscle showed abnormal hindlimb extension
reflexes and ataxia (Fig. 4A,
panels 1-3). When positioned on a slippery surface, the mutant mice were
unable to position their limbs properly
(Fig. 4A, panel 4). They also
showed spastic movements (data not shown). This behavior is consistent with
proprioceptive defects, suggesting that muscle spindle development or function
may be defective in the mutants.
|
To test whether Erbb2 was expressed in muscle spindles, we stained
transverse sections through skeletal muscle with antibodies to Erbb2.
Unfortunately, Erbb2 expression was difficult to detect in early developing
muscle spindles, presumably because it was expressed at low levels and
distributed diffusely (data not shown). However, we could detect Erbb2 protein
in mature muscle spindles, where it became concentrated at synaptic sites
where -motoneurons innervate muscle spindles
(Fig. 4C). These data suggest,
that Erbb2 is already expressed in early developing muscle spindles, and
becomes clustered upon synapse formation.
We next analyzed the embryonic development and differentiation of muscle
spindles in wild-type and Erbb2-deficient muscles using electron microscopy
(EM) to determine whether Erbb2 signaling in muscle was required for muscle
spindle induction, differentiation and/or maintenance
(Fig. 5). In wild-type mice,
muscle spindle morphogenesis in hindlimb muscle is initiated around
E15.5-16.5, when sensory Ia afferents reaching the muscle first contact
myotubes. At this developmental time, most nascent spindles are formed by a
single myotube that is approached by one or more sensory afferents arising
from a neighboring nerve bundle (Kucera
and Walro, 1995). EM examination of E16.5 muscles revealed Ia
afferent nerve fiber-myotube contact in both wild-type and mutant muscles,
suggesting that initial contact is not dependent of Erbb2 expression in
myotubes (Fig. 5A,B). These
contacts between sensory Ia afferent axons and muscle fibers can readily be
distinguished from putative contacts between motoneurons and muscle fibers
because they lack a basal lamina between the axon terminal and the muscle
fiber (Landon, 1972
). Overall,
mutant muscle did not appear to have fewer sensory terminalsmyotube contacts.
The contacted myotubes represented early nuclear bag2 intrafusal
fibers and those present in the mutant muscle appeared less differentiated
than in wild types, with smaller size and less developed packages of
myofibrils (data not shown). At E17, most spindles in wild-type muscles
contained an extra myotube (nuclear bag1) in addition to the
initial nuclear bag2 fiber (Fig.
5C). Normal morphogenesis of wild-type muscle spindles was also
characterized by the lateral expansion of the axon-associated Schwann cells
around the intrafusal bag fibers and by an increase in the myotube surface
covered by the sensory axon terminals (Fig.
5C). By contrast, mutant spindles at E17.5 had the characteristic
appearance of spindles at E16.5 (Fig.
5D). The initial contacts between sensory afferents and the
primary myotube were still evident, but morphological differentiation was
halted. Morphological analysis of serial sections indicated that in
Erbb2-deficient muscle the initially contacted myotube did not become a
nuclear bag fiber and that a second myotube was never recruited. Moreover,
Schwann cells in the mutants extended laterally following the axon terminals
but they failed to surround the muscle fiber that was contacted by the nerve
ending (Fig. 5D). At E18.5,
wild-type muscle spindles continue to develop normally but spindles in the
Erbb2-deficient muscles still resembled the initial stages of muscle spindle
formation (Fig. 5E,F). Despite
the fact that normal morphological differentiation of muscle spindles was
abrogate, sensory innervation became more extensive and numerous around mutant
myotubes (Fig. 5F). At birth,
when muscle spindles in wild-type muscle can be easily identified by their
characteristic surrounding capsule and are composed of a few muscle bag fibers
innervated in much of their surface (Fig.
5G), it was still possible to observe isolated muscle fibers
completely surrounded by afferent fibers in Erbb2-deficient muscle
(Fig. 5H). Some of the nerve
terminals in the mutants appeared altered, with a swollen morphology. Mature
muscle spindles were never observed in the Erbb2 conditional mutants. Taken
together, the data suggest that lack of Erbb2 signaling in muscle fibers is
not essential for the initial contact between sensory Ia afferent neurons and
myotubes but is necessary for the subsequent development of the structure.
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DISCUSSION |
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Erbb receptors in NMJ formation
The function of Erbb receptors in NMJ formation has been studied
previously. Skeletal muscle fibers express Erbb2, Erbb3 and Erbb4 receptors.
NMJ formation and gene expression in subsynaptic nuclei was observed in mice
that lack Erbb2 or Erbb3 (Gassmann et al.,
1995; Lee et al.,
1995
; Meyer and Birchmeier,
1995
; Riethmacher et al.,
1997
). However, the previously described genetically modified mice
died at birth, preventing a more detailed analysis of Erbb2/3 receptor
function in NMJ development. We now show that mice genetically modified to
lack Erbb2 specifically in muscle survive into adulthood and develop NMJs, but
the synapses are less efficient and contain reduced numbers of AChRs. As the
Erbb receptor ligand Nrg1 has been implicated in mediating its effect on
muscle fibers by regulating gene expression in subsynaptic nuclei
(Burden, 1998
;
Sanes and Lichtman, 1999
), our
data are consistent with a model where Nrg1 activates via Erbb2 gene
expression in subsynaptic nuclei. In the absence of Erbb2, Nrg1 signaling may
be less effective, leading to decreased gene expression and reduced levels of
synaptic proteins.
Muscle fibers express not only Erbb2, but also Erbb3 and Erbb4
(Zhu et al., 1995). It is
therefore possible that Erbb3/Erbb4 may have partially compensated for a loss
of Erbb2. Unfortunately, mice that carry a null mutation in the Erbb4
gene die during embryogenesis (Gassmann et
al., 1995
), preventing an analysis of its function in NMJ
formation by conventional genetic approaches. However, our recent data
implicate Erbb4 in NMJ formation. When postsynaptic differentiation is induced
in Erbb2-deficient muscle fibers by ectopic expression of agrin, clustering of
proteins in the postsynaptic membrane is less efficient. This defect can be
rescued by overexpressing Erbb4 (Moore et
al., 2001
), suggesting that both Erbb2 and Erbb4 are sufficient to
induce gene expression in subsynaptic nuclei. As we observed in vivo decreased
levels of AChRs in muscle that only lacked Erbb2, the data also suggest that
in the more physiological setting where Erbb2 and Erbb4 are expressed at lower
levels than achieved by ectopic overexpression, both receptors may be
essential for maximal expression of AChR subunit genes. To test for a
partially redundant function of Erbb2 and Erbb4 in muscle fibers, it will be
important to inactivate both receptors simultaneously in muscle fibers.
Erbb2 functions in muscle spindle development
Our data show that Erbb2 expression in skeletal muscle fibers is essential
for muscle spindle development. Previous studies have provided strong evidence
that muscle spindle development is initiated by a signal from sensory Ia
afferent neurons. We show here that contact of Ia afferents with myotubes is
initiated in the absence of Erbb2 expression in myotubes, but muscle spindle
formation does not progress and the rudimentary spindles degenerate. This
suggests that Erbb2 is not required for the establishment of the initial
contact between sensory neurons and myotubes, but for the subsequent
differentiation of muscle spindles. Interestingly, during development of
sympathetic neurons, NT3 expression is activated by Nrg1
(Verdi et al., 1996).
Likewise, muscle spindles express NT3
(Copray and Brouwer, 1994
) and
muscle spindles are absent in NT3-deficient mice
(Farinas et al., 1994
). This
raises the possibility that NT3 is more generally a downstream effector of
Nrg1/Erbb signaling, and that it is an essential component by which Erbb2
regulates muscle spindle development. In this model, activation of Erbb2 in
muscle fibers may regulate expression of NT3. As NT3 promotes survival of Ia
afferent neurons (ElShamy and Ernfors,
1996
; ElShamy et al.,
1998
; Farinas et al.,
1996
; Oakley et al.,
1995
; Oakley et al.,
1997
; Ockel et al.,
1996
; Ringstedt et al.,
1997
; Wright et al.,
1997
), and because ectopic expression of Ntf3 induces muscle
spindle development (Wright et al.,
1997
), Nrg1/Erbb signaling may be essential to maintain NT3
expression in muscle spindles during their development, which in turn may
affect sensory Ia afferent neurons. As the zinc-finger transcription factor
Egr3 appears to regulate spindle specific NT3 expression
(Chen et al., 2002
), and is
essential for muscle spindle maintenance
(Tourtellotte and Milbrandt,
1998
), it is possible that Erbb2 may act at least in part upstream
of Egr3 thereby regulating NT3 expression.
It has recently been reported that Erbb2 is required for survival of muscle
spindles and myoblasts, and that regeneration of muscle fibers is impaired in
the absence of Erbb2 (Andrechek et al.,
2002). We cannot substantiate these findings. We show that muscle
fiber development and maintenance is not impaired in the absence of Erbb2 in
vivo. Primary myoblasts isolated from the genetically modified mice described
here are not impaired in their ability to survive and replicate in vitro, and
they fused to form muscle fibers. In addition, the regenerative capacity of
Erbb2-deficient muscle is unaltered (H.R.B., M.L., and U.M., unpublished).
Likewise, although our data do not exclude a function for Erbb2 in muscle
spindle maintenance, they clearly show an earlier function in spindle
development. In the previous study it was demonstrated that muscle spindles
were absent in postnatal Erbb2-deficient muscle
(Andrechek et al., 2002
). As a
likely explanation, the previously described defects were not caused by a
defect in muscle spindle maintenance, but rather in development.
Alternatively, differences in genetic background could have contributed to the
different results.
Muscle spindle development and central projections of Ia afferent
neurons
It has remained unclear whether spindle development is required for the
development and maintenance of the monosynaptic reflex circuit. The data
presented here provide evidence that large parts of the monosynaptic reflex
circuit form in the absence of normal muscle spindle development. Sensory Ia
afferent neurons not only develop their peripheral projections into muscle.
They also develop central projections towards the ventral spinal cord to the
motoneuron pools. These central projections are maintained even in early
postnatal animals when no signs of muscle spindle development can be detected.
However, although our studies demonstrate that sensory Ia afferent neurons
develop normal projections, it will be important to establish whether they
also develop functional synaptic connections. The availability of the
genetically modified mice described here will allow testing this
hypothesis.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Aigner, L., Arber, S., Kapfhammer, J. P., Laux, T., Schneider, C., Botteri, F., Brenner, H. R. and Caroni, P. (1995). Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice. Cell 83,269 -278.[Medline]
Andrechek, E. R., Hardy, W. R., Girgis-Gabardo, A. A., Perry, R.
L., Butler, R., Graham, F. L., Kahn, R. C., Rudnicki, M. A. and Muller,
W. J. (2002). ErbB2 is required for muscle spindle and
myoblast cell survival. Mol. Cell Biol.
22,4714
-4722.
Brennan, K. J. and Hardeman, E. C. (1993).
Quantitative analysis of the human alpha-skeletal actin gene in transgenic
mice. J. Biol. Chem.
268,719
-725.
Brown, A. G. (1981). In Organization in the Spinal Cord, pp. 154-214.New York: Springer.
Buonanno, A. and Fischbach, G. D. (2001). Neuregulin and ErbB receptor signaling pathways in the nervous system. Curr. Opin. Neurobiol. 11,287 -296.[CrossRef][Medline]
Burden, S. J. (1998). The formation of
neuromuscular synapses. Genes Dev.
12,133
-148.
Carr, P. A., Yamamoto, T., Karmy, G., Baimbridge, K. G. and Nagy, J. I. (1989). Analysis of parvalbumin and calbindin D28k-immunoreactive neurons in dorsal root ganglia of rat in relation to their cytochrome oxidase and carbonic anhydrase content. Neurosci. 33,363 -371.[CrossRef][Medline]
Chen, H. H., Tourtellotte, W. G. and Frank, E.
(2002). Muscle spindlederived neurotrophin 3 regulates synaptic
connectivity between muscle sensory and motor neurons. J.
Neurosci. 22,3512
-3519.
Cohen, I., Rimer, M., Lomo, T. and McMahan, U. J. (1997). Agrin-induced postsynaptic-like apparatus in skeletal muscle fibers in vivo. Mol. Cell. Neurosci. 9, 237-253.[CrossRef][Medline]
Copray, J. C. and Brouwer, N. (1994). Selective expression of neurotrophin-3 messenger RNA in muscle spindles of the rat. Neuroscience 63,1125 -1135.[CrossRef][Medline]
DeChiara, T. M., Bowen, D. C., Valenzuela, D. M., Simmons, M. V., Poueymirou, W. T., Thomas, S., Kinetz, E., Compton, D. L., Rojas, E., Park, J. S. et al. (1996). The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo. Cell 85,501 -512.[Medline]
ElShamy, W. M. and Ernfors, P. (1996). A local action of neurotrophin-3 prevents the death of proliferating sensory neuron precursor cells. Neuron 16,963 -972.[Medline]
ElShamy, W. M., Fridvall, L. K. and Ernfors, P. (1998). Growth arrest failure, G1 restriction point override, and S phase death of sensory precursor cells in the absence of neurotrophin-3. Neuron 21,1003 -1015.[Medline]
Ernfors, P., Lee, K. F., Kucera, J. and Jaenisch, R. (1994). Lack of neurotrophin-3 leads to deficiencies in the peripheral nervous system and loss of limb proprioceptive afferents. Cell 77,503 -512.[Medline]
Farinas, I., Jones, K. R., Backus, C., Wang, X. Y. and Reichardt, L. F. (1994). Severe sensory and sympathetic deficits in mice lacking neurotrophin-3. Nature 369,658 -661.[CrossRef][Medline]
Farinas, I., Yoshida, C. K., Backus, C. and Reichardt, L. F. (1996). Lack of neurotrophin-3 results in death of spinal sensory neurons and premature differentiation of their precursors. Neuron 17,1065 -1078.[Medline]
Gassmann, M., Casagranda, F., Orioli, D., Simon, H., Lai, C., Klein, R. and Lemke, G. (1995). Aberrant neural and cardiac development in mice lacking the ErbB4 neuregulin receptor. Nature 378,390 -394.[CrossRef][Medline]
Gautam, M., Noakes, P. G., Moscoso, L., Rupp, F., Scheller, R. H., Merlie, J. P. and Sanes, J. R. (1996). Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell 85,525 -535.[Medline]
Graus-Porta, D., Blaess, S., Senften, M., Littlewood-Evans, A., Damsky, C., Huang, Z., Orban, P., Klein, R., Schittny, J. C. and Muller, U. (2001). Beta1-class integrins regulate the development of laminae and folia in the cerebral and cerebellar cortex. Neuron 31,367 -379.[Medline]
Gu, H., Zou, Y. R. and Rajewsky, K. (1993). Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73,1155 -1164.[Medline]
Jones, G., Herczeg, A., Ruegg, M. A., Lichtsteiner, M., Kroger,
S. and Brenner, H. R. (1996). Substrate-bound agrin
induces expression of acetylcholine receptor epsilon-subunit gene in cultured
mammalian muscle cells. Proc. Natl. Acad. Sci. USA
93,5985
-5990.
Jones, G., Meier, T., Lichtsteiner, M., Witzemann, V., Sakmann,
B. and Brenner, H. R. (1997). Induction by agrin of
ectopic and functional postsynaptic-like membrane in innervated muscle.
Proc. Natl. Acad. Sci. USA
94,2654
-2659.
Jones, G., Moore, C., Hashemolhosseini, S. and Brenner, H.
R. (1999). Constitutively active MuSK is clustered in the
absence of agrin and induces ectopic postsynaptic-like membranes in skeletal
muscle fibers. J. Neurosci.
19,3376
-3383.
Klein, R., Silos-Santiago, I., Smeyne, R. J., Lira, S. A., Brambilla, R., Bryant, S., Zhang, L., Snider, W. D. and Barbacid, M. (1994). Disruption of the neurotrophin-3 receptor gene trkC eliminates la muscle afferents and results in abnormal movements. Nature 368,249 -251.[CrossRef][Medline]
Kucera, J., Fan, G., Jaenisch, R., Linnarsson, S. and Ernfors, P. (1995). Dependence of developing group Ia afferents on neurotrophin-3. J. Comp. Neurol. 363,307 -320.[Medline]
Kucera, J. and Walro, J. M. (1995). Origin of intrafusal fibers from a subset of primary myotubes in the rat. Anat. Embryol. 192,149 -158.[Medline]
Landon, D. N. (1972). The fine structure of developing muscle spindles in the rat. J. Anat. 111,512 -513.
Lee, K. F., Simon, H., Chen, H., Bates, B., Hung, M. C. and Hauser, C. (1995). Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378,394 -398.[CrossRef][Medline]
Lin, W., Sanchez, H. B., Deerinck, T., Morris, J. K., Ellisman,
M. and Lee, K. F. (2000). Aberrant development of
motor axons and neuromuscular synapses in erbB2-deficient mice.
Proc. Natl. Acad. Sci. USA
97,1299
-1304.
Maier, A. (1997). Development and regeneration of muscle spindles in mammals and birds. Int. J. Dev. Biol. 41,1 -17.[Medline]
Mao, X., Fujiwara, Y. and Orkin, S. H. (1999).
Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions
in mice. Proc. Natl. Acad. Sci. USA
96,5037
-5042.
Meier, T., Hauser, D. M., Chiquet, M., Landmann, L., Ruegg, M.
A. and Brenner, H. R. (1997). Neural agrin induces
ectopic postsynaptic specializations in innervated muscle fibers.
J. Neurosci. 17,6534
-6544.
Meier, T., Masciulli, F., Moore, C., Schoumacher, F.,
Eppenberger, U., Denzer, A. J., Jones, G. and Brenner, H. R.
(1998). Agrin can mediate acetylcholine receptor gene expression
in muscle by aggregation of muscle-derived neuregulins. J. Cell
Biol. 141,715
-726.
Meyer, D. and Birchmeier, C. (1995). Multiple essential functions of neuregulin in development. Nature 378,386 -390.[CrossRef][Medline]
Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfessel, C. and Sakmann, B. (1986). Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature 321,406 -411.[Medline]
Moore, C., Leu, M., Muller, U. and Brenner, H. R.
(2001). Induction of multiple signaling loops by MuSK during
neuromuscular synapse formation. Proc. Natl. Acad. Sci.
USA 98,14655
-14660.
Morris, J. K., Lin, W., Hauser, C., Marchuk, Y., Getman, D. and Lee, K. F. (1999). Rescue of the cardiac defect in ErbB2 mutant mice reveals essential roles of ErbB2 in peripheral nervous system development. Neuron 23,273 -283.[Medline]
Müller, U., Wang, D., Denda, S., Meneses, J. J., Pedersen, R. A. and Reichardt, L. F. (1997). Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell 88,603 -613.[CrossRef][Medline]
Muscat, G. E. and Kedes, L. (1987). Multiple 5'-flanking regions of the human alpha-skeletal actin gene synergistically modulate muscle-specific expression. Mol. Cell. Biol. 7,4089 -4099.[Medline]
Oakley, R. A., Garner, A. S., Large, T. H. and Frank, E.
(1995). Muscle sensory neurons require neurotrophin-3 from
peripheral tissues during the period of normal cell death.
Development 121,1341
-1350.
Oakley, R. A., Lefcort, F. B., Clary, D. O., Reichardt, L. F.,
Prevette, D., Oppenheim, R. W. and Frank, E. (1997).
Neurotrophin-3 promotes the differentiation of muscle spindle afferents in the
absence of peripheral targets. J. Neurosci.
17,4262
-4274.
Ockel, M., Lewin, G. R. and Barde, Y. A.
(1996). In vivo effects of neurotrophin-3 during sensory
neurogenesis. Development
122,301
-307.
Riethmacher, D., Sonnenberg-Riethmacher, E., Brinkmann, V., Yamaai, T., Lewin, G. R. and Birchmeier, C. (1997). Severe neuropathies in mice with targeted mutations in the ErbB3 receptor. Nature 389,725 -730.[CrossRef][Medline]
Ringstedt, T., Kucera, J., Lendahl, U., Ernfors, P. and Ibanez,
C. F. (1997). Limb proprioceptive deficits without neuronal
loss in transgenic mice overexpressing neurotrophin-3 in the developing
nervous system. Development
124,2603
-2613.
Ruegg, M. A. (1996). Agrin, laminin beta 2 (s-laminin) and ARIA: their role in neuromuscular development. Curr. Opin. Neurobiol. 6, 97-103.[CrossRef][Medline]
Sandrock, A. W., Jr, Dryer, S. E., Rosen, K. M., Gozani, S. N.,
Kramer, R., Theill, L. E. and Fischbach, G. D. (1997).
Maintenance of acetylcholine receptor number by neuregulins at the
neuromuscular junction in vivo. Science
276,599
-603.
Sanes, J. R. and Lichtman, J. W. (1999). Development of the vertebrate neuromuscular junction. Annu. Rev. Neurosci. 22,389 -442.[CrossRef][Medline]
Schaeffer, L., de Kerchove d'Exaerde, A. and Changeux, J. P. (2001). Targeting transcription to the neuromuscular synapse. Neuron 31,15 -22.[Medline]
Tessarollo, L., Vogel, K. S., Palko, M. E., Reid, S. W. and
Parada, L. F. (1994). Targeted mutation in the neurotrophin-3
gene results in loss of muscle sensory neurons. Proc. Natl. Acad.
Sci. USA 91,11844
-11848.
Tinsley, J. M., Potter, A. C., Phelps, S. R., Fisher, R., Trickett, J. I. and Davies, K. E. (1996). Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature 384,349 -353.[CrossRef][Medline]
Tourtellotte, W. G. and Milbrandt, J. (1998). Sensory ataxia and muscle spindle agenesis in mice lacking the transcription factor Egr3. Nat. Genet. 20, 87-91.[CrossRef][Medline]
Trinidad, J. C., Fischbach, G. D. and Cohen, J. B.
(2000). The Agrin/MuSK signaling pathway is spatially segregated
from the neuregulin/ErbB receptor signaling pathway at the neuromuscular
junction. J. Neurosci.
20,8762
-8770.
Verdi, J. M., Groves, A. K., Farinas, I., Jones, K., Marchionni, M. A., Reichardt, L. F. and Anderson, D. J. (1996). A reciprocal cell-cell interaction mediated by NT-3 and neuregulins controls the early survival and development of sympathetic neuroblasts. Neuron 16,515 -527.[Medline]
Walro, J. M. and Kucera, J. (1999). Why adult mammalian intrafusal and extrafusal fibers contain different myosin heavy-chain isoforms. Trends Neurosci. 22,180 -184.[CrossRef][Medline]
Woldeyesus, M. T., Britsch, S., Riethmacher, D., Xu, L.,
Sonnenberg- Riethmacher, E., Abou-Rebyeh, F., Harvey, R., Caroni, P.
and Birchmeier, C. (1999). Peripheral nervous system
defects in erbB2 mutants following genetic rescue of heart development.
Genes Dev. 13,2538
-2548.
Wolpowitz, D., Mason, T. B., Dietrich, P., Mendelsohn, M., Talmage, D. A. and Role, L. W. (2000). Cysteine-rich domain isoforms of the neuregulin-1 gene are required for maintenance of peripheral synapses. Neuron 25,79 -91.[Medline]
Wright, D. E., Zhou, L., Kucera, J. and Snider, W. D. (1997). Introduction of a neurotrophin-3 transgene into muscle selectively rescues proprioceptive neurons in mice lacking endogenous neurotrophin-3. Neuron 19,503 -517.[Medline]
Yang, X., Arber, S., William, C., Li, L., Tanabe, Y., Jessell, T. M., Birchmeier, C. and Burden, S. J. (2001). Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation. Neuron 30,399 -410.[CrossRef][Medline]
Zhu, X., Lai, C., Thomas, S. and Burden, S. J. (1995). Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses. EMBO J. 14,5842 -5848.[Abstract]