Molecular Neurobiology Program, Skirball Institute, NYU Medical School,
NY 10016, USA
* Present address: Brain Research Institute, University of Vienna, Spitalgasse
4, A-1090 Vienna
Author for correspondence (e-mail:
ruth.herbst{at}univie.ac.at)
Accepted 30 August 2002
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SUMMARY |
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Key words: Agrin, Acetylcholine receptor, Synapse-specific transcription, Receptor tyrosine kinase, Neuromuscular synapse, Mouse
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INTRODUCTION |
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Domains in Musk that are important for Musk signaling have been defined by
introducing wild-type or mutant forms of Musk into Musk mutant muscle
cell lines (Herbst and Burden,
2000; Zhou et al.,
1999
). These studies have revealed an essential role for one of
the two juxtamembrane tyrosine residues in Musk (Y553), as mutation of this
tyrosine abrogates the ability of agrin to induce clustering or tyrosine
phosphorylation of AChRs in cultured myotubes. This tyrosine appears to have a
dual function in Musk signaling, as this tyrosine is required both to activate
Musk kinase activity fully and to recruit a signaling component(s) that
functions downstream from Musk (Herbst and
Burden, 2000
). Evidence for such a dual role is based upon
analysis of Musk/TrkA chimeric receptors. Agrin stimulates tyrosine
phosphorylation of a chimeric receptor composed of the extracellular and
transmembrane regions of Musk, and the intracellular region of TrkA (Nrtk1
Mouse Genome Informatics), but phosphorylation of this chimeric
receptor fails to induce AChR clustering or tyrosine phosphorylation in muscle
cell lines. The same Musk/TrkA chimera, but including a substitution of
thirteen amino acids from the juxtamembrane region of Musk, including Y553,
with the comparable region in TrkA is similarly tyrosine phosphorylated by
agrin stimulation, but tyrosine residue phosphorylation of this chimera leads
to clustering and tyrosine phosphorylation of AChRs in cultured myotubes
(Herbst and Burden, 2000
).
These experiments indicate that the juxtamembrane region of Musk, even in the
context of a different kinase domain, is sufficient to activate a signaling
pathway leading to the clustering and tyrosine phosphorylation of AChRs in
muscle cell lines. Nonetheless, as cultured muscle cells are not amenable to
study all aspects of synaptic differentiation, these experiments could not
address whether this juxtamembrane region of Musk is sufficient to confer
additional aspects of Musk signaling, including clustering of additional
postsynaptic proteins, synapse-specific transcription, presynaptic
differentiation and synapse formation. Moreover, requirements for synaptic
proteins may differ in cell culture and in vivo; for example, rapsyn is
required to cluster Musk in cultured cells but not at synapses in vivo
(Gillespie et al., 1996
;
Moscoso et al., 1995
), and the
ectodomain of Musk is required to cluster AChRs in cultured cells but not in
vivo (Apel et al., 1997
;
Sander et al., 2001
). Thus, in
vivo studies are required to delineate the signaling mechanisms that lead to
the complex biological response initiated by Musk at synapses.
In addition to Y553 in the juxtamembrane region of Musk, agrin stimulates
the phosphorylation of five tyrosine residues in the kinase domain of Musk
(Watty et al., 2000). In other
RTKs, recruitment of different adaptor proteins to distinct phosphotyrosine
docking sites leads to activation of disparate signaling pathways, which are
often coupled to different biological responses
(Madhani, 2001
;
Pawson and Scott, 1997
). For
example, in TrkA, the receptor for nerve growth factor, phosphorylation of a
juxtamembrane tyrosine leads to Ras and PI3-kinase activation, which are
important for cell survival and neurite outgrowth
(Greene and Kaplan, 1995
;
Huang and Reichardt, 2001
),
whereas phosphorylation of a tyrosine in the C-terminal region of TrkA leads
to PLC
activation, which is crucial for NGF-dependent Na+
channel and VRI channel regulation (Choi et
al., 2001
; Chuang et al.,
2001
). Moreover, in TrkB, phosphorylation of a single
juxtamembrane tyrosine is required for nearly all of NT4-dependent signaling
whereas phosphorylation of other tyrosine residues are required to mediate
BDNF-dependent signaling in vivo
(Minichiello et al., 1998
).
Similarly, in Met, the receptor for hepatocyte growth factor, a binding site
for Grb2 is essential for late steps in myogenesis
(Maina et al., 1996
), a
binding-site for PI3-kinase is essential for placental development, hepatocyte
survival and myoblast migration, and a Src binding-site is essential for motor
axon outgrowth (Maina et al.,
2001
). To determine whether clustering of postsynaptic proteins,
synapse-specific transcription and presynaptic differentiation depend upon
multiple, distinct docking sites in Musk, we produced mice that expressed the
Musk/TrkA chimera, containing thirteen amino acids from the juxtamembrane
region of Musk and the kinase domain of TrkA, and we crossed this transgene
into Musk mutant mice. We found that expression of this chimeric
receptor could restore all aspects of postsynaptic and presynaptic
differentiation that were defective in Musk mutant mice. Moreover, we
found that accumulation of Musk protein at synaptic sites was not dependent
upon synaptic localization of Musk mRNA. These results indicate that
the juxtamembrane region of Musk, including a single phosphotyrosine docking
site, even in the context of a different kinase domain, is sufficient to
activate the multiple pathways that lead to presynaptic and postsynaptic
differentiation in vivo.
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MATERIALS AND METHODS |
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Mouse genotyping
Tail DNA of F1 mice was digested with EcoRI and
analyzed by Southern blotting using probes from the rat Musk
(nucleotides 2080-2708) and rat TrkA (nucleotides 2191-2572) kinase
domains (Meakin et al., 1992).
Subsequent generations of mice were genotyped by PCR using primers that are
specific for sequences in the extracellular region of the mouse Musk
cDNA (5'-GAAGCAACCTTTCCTTCCTGAG-3' and
5'-ATTTTCCCTGAGAGCATTGTCC-3') using the following conditions: one
cycle of 94°C for 2 minutes, 40 cycles of 94°C for 30 seconds,
58°C for 30 seconds and 72°C for 40 seconds followed by 1 cycle of
72°C for 2 minutes. The wild-type and mutant Musk alleles
(DeChiara et al., 1996
) were
detected by PCR, using primers that are specific for sequences in the
Musk kinase domain and the neo gene
(5'-ATGCCGCCCGAGTCTATGTTCTAC-3',
5'-TTCTCCTGGCAAACAATCAACTGG-3' and
5'-CATAGCCTGAAGAACGAGATCAGCAGC-3'), using the following
conditions: one cycle of 94°C for 2 minutes, 40 cycles of 94°C for 30
seconds, 56°C for 30 seconds and 72°C for 40 seconds followed by one
cycle of 72°C for 2 minutes. We examined three MCK-Musk lines and
two MCK-MMT lines; analyzed mice were heterozygous for
MCK-Musk or MCK-MMT. We noted attrition in large litters up
to the time of weaning, indicating that Musk-/-;
MCK-MMT and, in particular, Musk-/-;
MCK-Musk pups competed poorly with their wild-type littermates for
nourishment. Musk-/-; MCK-MMT and
Musk-/-; MCK-Musk mice are smaller than their
littermates during this period, but attain the same weight as their
littermates by one month after birth. We assessed presynaptic and postsynaptic
differentiation in embryos and in 3-week postnatal mice, when neuromuscular
synapses are fully mature. Musk-/-; MCK-Musk and
Musk-/-; MCK-MMT adult mice appeared to have
normal motility as they performed identically to wild-type mice on a Rotarod
at a constant speed (32 rpm). Rescued adult mice show no signs of a shortened
longevity, as they have been maintained for an excess of 1 year.
Immunohistochemistry
Diaphragm muscles were dissected from embryos or three week old mice, fixed
for 90 minutes in 1% formaldehyde, rinsed in PBS and incubated with 0.1 M
glycine in PBS for 15 minutes. After dissection of the overlying connective
tissue, the muscles were permeabilized in 0.5% Triton X-100 in PBS (PBST) for
5 minutes, incubated overnight at 4°C with rabbit polyclonal antibodies
against neurofilament (1:500, Chemicon) and synaptophysin (1:5, Zymed) in 4%
goat serum/2% BSA/PBST, washed three times for 20 minutes in PBST, incubated
overnight at 4°C with fluorescein-conjugated goat anti-rabbit IgG (1:200,
Jackson Immunoresearch) and Texas Red-conjugated -BGT (Molecular
Probes). The muscles were washed twice for 20 minutes in PBST, twice for 20
minutes in PBS, postfixed in 1% formaldehyde for 10 minutes, rinsed in PBS,
flat mounted in Vectashield (Vector Labs) and viewed with optics selective for
either fluorescein or Texas Red.
Frozen sections (10 µm) from fixed, adult leg muscles were stained with
antibodies as described previously
(DeChiara et al., 1996;
Zhu et al., 1995
). The
following primary antibodies were used: affinity-purified rabbit anti-rapsyn
(1:500) (Herbst and Burden,
2000
), mouse anti-utrophin C terminus (1:20, Vector Labs), rabbit
anti-Na+ channel (1:1000, Upstate), rabbit anti-ErbB4 (antibody #
616) (Zhu et al., 1995
),
rabbit anti-synaptophysin (1:20, Zymed), mouse anti-SV2 (1:10)
(Buckley and Kelly, 1985
) and
rabbit anti-Musk (#83033, 1:4000 and #24908, 1:50)
(Watty et al., 2000
). We were
unable to detect fluorescence from the Musk-GFP transgene.
Whole-mount in situ hybridization
Intercostal muscles were fixed in 4% formaldehyde, dehydrated in methanol,
digested with Proteinase K, probed with a digoxigenin-labeled riboprobe
transcribed from an AChR (DeChiara
et al., 1996
), AChR
subunit
(Simon et al., 1992
) or a rat
Musk cDNA (nucleotides 1-1663) and processed as described elsewhere
(Wilkinson, 1992
). A low level
of uniform staining was observed with control, sense probes for the AChR
and
subunits.
Immunoprecipitation and western blotting
Tissues were homogenized and lysed as described previously
(Bruning et al., 1998). Tissues
were homogenized in lysis buffer [50 mM HEPES (pH 7.4), 50 mM sodium
pyrophosphate, 0.1 M sodium fluoride, 10 mM EDTA, 10 mM sodium orthovanadate,
10 µg/ml aprotinin, 10 µg/ml leupeptin and 2 mM PMSF]. After addition of
an equal volume of lysis buffer containing 2% Triton X-100, the lysates were
incubated at 4°C for 30 minutes and pre-cleared by centrifugation (20
minutes at 100,000 g in a TLA 100.3 rotor in a TL-100
ultracentrifuge) (Beckman). Proteins were immunoprecipitated and analyzed by
western blotting as described previously
(Herbst and Burden, 2000
).
Quantitation of AChR density and synaptic area
The density of AChRs at synapses in diaphragm muscles from 3-week-old mice
was quantitated from data captured with a Zeiss 510 confocal microscope using
a 3D software program provided by the manufacturer. In each experiment,
diaphragms from wild-type and mutant mice were stained together. While viewing
a wild-type muscle, the gain of the amplifier was adjusted to a subsaturating
level, and this setting was maintained while viewing mutant muscles. We
examined at least three mice from each genotype, and images from at least ten
synapses in each muscle were included in the analysis.
Quantitation of axon growth
Diaphragm muscles were stained with antibodies to NF and synaptophysin.
Images were captured on a CCD camera (Princeton Instruments), attached to a
Zeiss Axioskop and analyzed using Metamorph. We measured the total number of
pixels in two thresholded areas of the left hemi-diaphragm muscle, each
adjacent to the main intramuscular nerve. The value in the first area, which
is immediately adjacent to the main intramuscular nerve, provides an estimate
of the number of axons that branched from the main nerve, while the value in
the second area, which is adjacent to the main nerve but extends further
toward the edge of the muscle, provides an estimate of the extent of axon
growth away from the main intramuscular nerve. The value in each area of
wild-type muscle was assigned as 100%, and the values in mutant muscles were
expressed relative to wild type. By late embryonic stages, many motor axons in
Musk mutant mice have reached the edge of the muscle; owing to the
irregular shape of the muscle, however, we measured axon outgrowth in an area
that did not extend to the edge of the muscle. Consequently, our procedure
underestimates the increase in motor axon outgrowth in Musk mutant
mice. Images from at least three diaphragm muscles from each genotype, at
three different stages (E14.5, E16.5 and E18.5), were included in the
analysis. We found that the number of axons that branch from the main
intramuscular nerve was similar in wild-type and mutant embryos.
Antibodies
Antibodies (#83033) to Musk were produced by immunizing rabbits with a
conjugate between KLH and an extracellular sequence (SCGALQVKMKPKITRPPINV) in
Musk (Research Genetics). Antibodies to rapsyn were produced and purified as
described previously (Herbst and Burden,
2000). The monoclonal antibody (M2) to the FLAG epitope was
purchased from Sigma. Polyclonal antibodies to TrkA (C14) were purchased from
Santa Cruz. The monoclonal antibody to phosphotyrosine (mAB 4G10) was
purchased from Upstate. The monoclonal antibody to GFP was purchased from
Clontech.
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RESULTS |
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We produced and analyzed two MCK-MMT transgenic lines, as well as
three MCK-Musk control transgenic lines. These mice express the
transgenes in a muscle-specific manner, as assessed by western blotting
(Fig. 1), and the protein
encoded by each transgene is tyrosine phosphorylated
(Fig. 1). MCK-MMT and
MCK-Musk transgenic mice were crossed to Musk+/-
mice, and progeny were crossed to Musk+/- mice to generate
mice that lack endogenous Musk but express either MCK-MMT or
MCK-Musk. Unlike Musk mutant mice, which lack neuromuscular
synapses and die at birth (DeChiara et al.,
1996), Musk mutant mice expressing either
MCK-MMT or MCK-Musk move, breathe and survive after birth
(Table 1).
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We stained wholemounts of diaphragm muscle from E18.5 mice with a mixture
of antibodies to neurofilament and synaptophysin, to label axons and
terminals, respectively, and with Texas Red -Bungarotoxin (TR
-BGT) to label acetyl choline receptors (AChRs). We found that nerve
terminals differentiate and that AChRs cluster in the rescued newborn mice
(Fig. 2A). Moreover, synapses
are maintained and continue to mature postnatally
(Fig. 2B). These experiments
demonstrate that the juxtamembrane region of Musk, even in the context of a
kinase domain from a different tyrosine kinase, is sufficient to activate a
signaling cascade that leads to clustering of AChRs in vivo. Furthermore, as
MMT expression, selectively in muscle, restores presynaptic differentiation,
these results indicate that the juxtamembrane region of Musk has a central
role in producing and/or organizing retrograde signals required for
presynaptic differentiation and synapse formation.
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The density of AChRs at synaptic sites in rescued mice
We measured the density of AChRs at synaptic sites
(Fig. 2B) and found that the
density of synaptic AChRs is modestly lower in Musk-/-;
MCK-Musk mice than in wild-type mice (line #23, 79±3.2%,
n=3; line #54, 77%, n=1). These results indicate that the
MCK regulatory region confers sufficient Musk expression to restore
AChR clustering at synapses but that the level and/or pattern of
MCK-Musk expression differs from wild-type Musk expression,
resulting in a 20% reduction in AChR density at synapses. In
Musk-/-; MCK-MMT mice, AChRs are likewise clustered at
synapses, but at a reduced density (line #5, 66±3.4%, n=3;
line #29, 48±0.8%, n=3). The lower density of synaptic AChRs
in rescued mice expressing MMT may be due to a modestly (twofold) lower
expression of the MMT transgene (see supplementary data at
http://dev.biologists.org/supplemental/)
or to sequences in Musk that are absent from MMT. Nonetheless, the MMT chimera
also restores AChR clustering at synapses.
MMT chimera contains sequences that are sufficient for clustering
postsynaptic proteins
Agrin/Musk signaling is necessary and sufficient to cluster several
muscle-derived proteins in addition to AChRs
(DeChiara et al., 1996;
Gautam et al., 1996
;
Meier et al., 1997
;
Rimer et al., 1998
). We
therefore examined whether the MMT chimera contains sequences sufficient for
clustering these proteins at synaptic sites. We stained frozen sections of
muscle from three week old mice with antibodies to: (1) rapsyn, an
intracellular, peripheral membrane protein associated with AChRs; (2)
utrophin, a component of the dystrophin-associated glycoprotein complex; (3)
ErbB4, a receptor for neuregulin; and (4) Na+ channels
(Sanes and Lichtman, 1999
).
Fig. 3 shows that rapsyn,
ErbB4, utrophin and Na+ channels are each concentrated at synapses
in Musk-/-; MCK-MMT mice. These results indicate that the
MMT chimera contains sequences that are sufficient to restore the clustering
of most, if not all postsynaptic proteins.
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MMT chimera induces extrasynaptic AChR clusters
In innervated muscle fibers from wild-type and Musk-/-;
MCK-Musk mice, AChR clusters are restricted to synaptic sites. In
Musk-/-; MCK-MMT mice, however, AChR clusters are readily
evident at ectopic sites in addition to neuromuscular synapses
(Fig. 4). Because these ectopic
AChR clusters are found on most muscle fibers and are often located
immediately adjacent to synaptic sites, marked by terminal arbors and synaptic
AChR clusters, these ectopic AChR clusters are not associated with muscle
denervation. Moreover, other synaptic proteins, including rapsyn, ErbB4,
utrophin and Na+ channels, are each co-clustered with ectopic AChR
clusters in Musk-/-; MCK-MMT mice
(Fig. 4).
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Synapse-specific transcription is activated by MMT
Agrin/Musk signaling is necessary to activate synapse-specific
transcription, leading to an enrichment of AChR mRNA in the central,
synapse-rich region of muscle (DeChiara et
al., 1996; Gautam et al.,
1996
). We therefore used in-situ hybridization to determine
whether AChR genes are expressed selectively in synaptic nuclei of
Musk-/-; MCK-MMT mice.
Fig. 5 shows that AChR
and
subunit mRNAs are each enriched in the central region of
intercostal muscle from Musk-/-; MCK-MMT newborn mice,
demonstrating that the Musk juxtamembrane region has a key role in activating
a pathway leading to synapse-specific gene expression.
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Musk and MMT proteins are concentrated at synaptic sites despite
uniform Musk and MMT RNA expression
In wild-type mice, Musk RNA, like AChR mRNA, is expressed
preferentially at synaptic sites
(Valenzuela et al., 1995).
Because the MCK gene is expressed in nuclei throughout the myofiber
(Tang et al., 1994
), the
MCK regulatory region, as expected, confers Musk and
MMT mRNA expression uniformly throughout the muscle
(Fig. 6). We examined whether
Musk protein, encoded by the transgene, is expressed uniformly in the muscle
or concentrated at synaptic sites in Musk-/-; MCK-Musk
mice. We stained sections of muscle from Musk-/-; MCK-Musk
mice with antibodies to Musk and found that Musk is clustered at synaptic
sites (Fig. 6). Likewise, we
found that MMT is clustered at synaptic sites in Musk-/-;
MCK-MMT mice (Fig. 6).
Thus, Musk protein can be clustered at synaptic sites even if Musk
mRNA is expressed uniformly in muscle.
|
Motor axons extend until Musk is expressed, yet postsynaptic
differentiation is limited to a discrete site on the muscle fiber
Motor axons enter developing skeletal muscle at E12.5 and form a main
intramuscular nerve in the middle of the muscle
(Sanes and Lichtman, 1999). In
wild-type mice, motor axons branch and terminate adjacent to the main
intramuscular nerve, resulting in a narrow, distinct endplate zone in the
middle of the muscle, marked by presynaptic nerve terminals and a high
concentration of AChRs (Fig.
7). In Musk mutant mice, motor axons branch from the main
intramuscular nerve, but these axons fail to terminate and instead wander
across the muscle surface without forming specialized nerve terminals
(DeChiara et al., 1996
).
|
Based on the expression of transgenes containing the MCK enhancer and
promoter, the endogenous MCK gene is activated in skeletal muscle at
E15.5 (S. Hauschka, personal communication), 1 day after motor axons
first enter the muscle. Expression of the MCK gene increases modestly
(
20-fold) between E13.5 and birth, and dramatically after birth
(>300-fold between birth and P21) (S. Hauschka, personal communication). We
therefore reasoned that axon growth would be exuberant in
Musk-/-; MCK-Musk mice, as in Musk mutant mice,
prior to the onset of Musk expression. Fig.
7 shows that motor axon growth is similarly exuberant in
Musk-/-, Musk-/-; MCK-Musk and
Musk-/-; MCK-MMT mice at E14.5 and E16.5. In addition,
postsynaptic clustering of AChRs is absent at E14.5 and only weakly detectable
at E16.5 in Musk-/-; MCK-Musk and Musk-/-;
MCK-MMT mice. Between E16.5 and E18.5 in Musk-/-;
MCK-Musk mice, the rate of axon growth reverts to normal and postsynaptic
differentiation commences (Fig.
7). In Musk-/-; MCK-MMT mice, the
rate of axon outgrowth also reverts between E16.5 and E18.5, but incompletely,
like AChR clustering. Owing to the late expression of the MCK gene,
motor axons extend well beyond their normal termination zone prior to forming
neuromuscular synapses, resulting in a substantially wider end-plate zone,
which persists in rescued adult mice (Fig.
8).
|
Because motor axons in Musk-/-; MCK-Musk mice grow over a substantial region of muscle prior to E16.5, we reasoned that postsynaptic differentiation, initiated by neurally deposited agrin, might occur over an unusually large region of each muscle fiber once Musk expression began. We found, however, that the size of AChR clusters was normal (102±4%, n=3) in muscle from Musk-/-; MCK-Musk newborn mice (Fig. 2). These results raise the possibility that agrin is preferentially available, or active, at or near the growth cone of the motor axon, and that the resulting bias in Musk activation consolidates Musk and AChR clustering to a discrete patch on the muscle fiber surface (see Discussion).
Terminal arbors are immature in Musk-/-;
MCK-MMT mice
Neuromuscular synapses undergo several structural and functional
transitions during the first few weeks after birth, as the number of
presynaptic inputs at individual synapses is reduced to one, and the single,
remaining nerve terminal arbor becomes more complex
(Sanes and Lichtman, 1999). To
determine whether synaptic sites become singly innervated and nerve terminal
arbors become more complex in Musk mutant mice expressing MMT, we
stained wholemounts of diaphragm muscle from 3-week-old mice with antibodies
to neurofilament/synaptophysin and with TR-
-BGT. In
Musk-/-; MCK-Musk and
Musk-/-; MCK-MMT mice, as in wild-type mice,
synaptic sites are singly innervated (Fig.
2B, Fig. 9). Thus,
synapse elimination appears normal. In Musk-/-;
MCK-Musk mice, motor axon terminals arborize and invariably form a
complex, branched endplate (Fig.
2B, Fig. 9). In
muscle from Musk-/-; MCK-MMT mice, however,
terminal branching is less extensive, resulting in less complex terminal
arbors (Fig. 2B,
Fig. 9). Some (
10%)
terminal arbors are remarkably simplified, as terminal branches are fragmented
into individual boutons (Fig.
9). These results indicate that MMT can restore presynaptic
differentiation, although the extent of terminal arborization is incomplete.
Because terminal arbor differentiation and AChR clustering are restored to a
similar degree in Musk-/-; MCK-MMT mice, Musk
activity in the postsynaptic cell appears to be limiting for presynaptic
differentiation.
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DISCUSSION |
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The precise role for Musk in synapse-specific expression is poorly
understood. Analysis of rapsyn mutant mice indicates that Musk may activate a
signaling pathway that directly stimulates synapse-specific transcription
(Gautam et al., 1995).
Expression of ectopic agrin or activated Musk in adult myofibers, however,
stimulates AChR transcription in an ErbB-dependent manner, suggesting that
synapse-specific expression may require the Musk-dependent recruitment of a
Nrg-1/ErbB signaling complex to synaptic sites
(Jones et al., 1999
;
Meier et al., 1997
;
Moore et al., 2001
;
Rimer et al., 1998
). Our
experiments indicate that the Musk juxtamembrane region contains sequences
that are crucial for synapse-specific transcription, but they do not shed
light on whether these sequences act in a manner that is independent or
dependent on Nrg-1/ErbB signaling. Thus, the Musk juxtamembrane region could
recruit a signaling complex that directly regulates transcription in synaptic
nuclei, or the Musk juxtamembrane region could regulate synapse-specific
transcription indirectly by regulating the distribution of a Nrg-1/ErbB
signaling complex, analogous to the action of Musk on AChR protein. In either
case, our experiments indicate that the Musk juxtamembrane region, rather than
other potential phosphotyrosine docking sites in the Musk kinase domain, has a
crucial role in synapse-specific transcription.
In wild-type mice, Musk mRNA and protein are concentrated at
neuromuscular synapses (Valenzuela et al.,
1995). In Musk mutant mice carrying MCK-Musk,
Musk mRNA is expressed throughout the muscle, yet Musk protein is
concentrated at synaptic sites. These experiments demonstrate that
accumulation of Musk protein at synaptic sites is not dependent upon synaptic
localization of Musk mRNA. Thus, it may be more important that muscle
fibers express an adequate level of mRNAs encoding synaptic proteins, such as
Musk and AChR, rather than restricting these mRNAs to synaptic sites.
Moreover, in Musk mutant mice carrying MCK-MMT, MMT mRNA is
likewise expressed throughout the muscle, yet the chimeric protein is
concentrated at synaptic sites. These results suggest that activation of the
chimeric protein by agrin is sufficient to recruit additional chimeric protein
from non-synaptic regions, via a positive feedback mechanism
(Jones et al., 1999
). Our
findings, both from in vitro and in vivo experiments, therefore, underscore
the crucial role of the Musk juxtamembrane domain in clustering Musk as well
as AChRs. The mechanisms by which activated Musk recruits Musk are not
understood, but this positive feedback loop may be important for achieving an
adequate level of Musk expression at the synapse, sufficient to cluster more
than ten million AChR molecules per synapse. Nonetheless, it seems likely that
this positive feedback mechanism is restrained, as Musk activation and
clustering would otherwise proceed beyond the synaptic site.
We showed previously that the MMT chimera is less responsive than Musk to
agrin, as agrin stimulates half the number of AChR clusters in MMT-expressing
myotubes than in Musk-expressing myotubes
(Herbst and Burden, 2000). We
found that the density of synaptic AChRs is lower in Musk mutant mice
rescued with MMT than in Musk mutant mice rescued with Musk. The
lower density of synaptic AChRs could be due to the reduced responsiveness of
MMT to agrin, poorer (approx. half) expression of the MMT transgene, or both.
Because MMT is less responsive to agrin in vitro, we favor the idea that
sequences in the intracellular domain of Musk, not present in the MMT chimera,
contribute to clustering of AChRs at synapses as well. Consistent with this
idea, Y576, in the N-terminal lobe of the kinase domain, is phosphorylated in
activated Musk and contributes to AChR clustering in cultured muscle cells
(Herbst and Burden, 2000
).
Alternatively, maximal AChR clustering may depend upon sequences in Musk, not
represented in the MMT chimera, that bind proteins independently of tyrosine
phosphorylation (Strochlic et al.,
2001
).
We showed previously that MMT-expressing myotubes have more
agrin-independent AChR clusters than Musk-expressing myotubes
(Herbst and Burden, 2000).
Consistent with these results, we found ectopic AChR clusters in innervated
myofibers from Musk mutant mice rescued with MMT but not in
Musk mutant mice rescued with Musk. These data support the idea that
sequences in Musk restrain Musk activation in vivo, and that the absence of
this auto-inhibitory sequence in the MMT chimera results in adventitious Musk
activity (Till et al.,
2002
).
During the first few weeks after birth, the structure and function of the
neuromuscular synapse is modified (Sanes
and Lichtman, 1999). At birth, multiple motor axons terminate at a
single, elliptical synaptic site on each myofiber. During the next few weeks,
all but one of these motor axons is withdrawn, leading to innervation of the
single synaptic site by a single motor axon. In addition, the shape of the
synaptic site becomes more complex, as the presynaptic terminal grows and
branches, and the shape of synaptic AChR clusters, ovoid at birth, becomes
correspondingly complex. Although the elimination of polyneuronal innervation
appears to occur normally in Musk mutant mice expressing
MCK-MMT, presynaptic terminal arbors are often less complex and lack
extensive terminal branching. Indeed, some terminal arbors are so simplified
that the synaptic site is composed of only a few boutons. These aberrations in
terminal branching could arise from a failure to branch adequately when
synapses first form or from remodeling and simplification, rather than growth
and elaboration of branches later in development
(Balice-Gordon and Lichtman,
1990
). In either case, presynaptic differentiation, like
postsynaptic differentiation, is incomplete in Musk mutant mice
expressing MCK-MMT, and these results reinforce the idea that
sequences other than the critical juxtamembrane region of Musk, though not
essential for synapse formation, contribute to postsynaptic differentiation
and presynaptic terminal arborization. Moreover, although prior studies of
rapsyn and Musk mutant mice demonstrated that presynaptic
differentiation is linked to postsynaptic differentiation
(DeChiara et al., 1996
;
Gautam et al., 1995
), the
experiments described demonstrate that the extent of presynaptic and
postsynaptic differentiation are matched, indicating that Musk activity in the
postsynaptic cell is limiting for presynaptic differentiation. Despite the
simplified structure of synapses in Musk-/-; MCK-MMT mice,
these mice are viable, fertile and behave normally in simple behavioral
paradigms (see Materials and Methods).
Musk mutant mice carrying a MCK-Musk transgene first
express Musk when motor axons have already extended well beyond their normal
termination zone. Thus, it seemed possible that agrin, which is deposited by
motor axons growing exuberantly along the muscle, might initiate postsynaptic
differentiation over an unusually extensive area of the muscle once Musk
expression begins. The shape and size of AChR clusters, however, are similar
in wild-type mice and in Musk mutant mice that express
MCK-Musk. The mechanisms that regulate release and retention of agrin
from motor axons are poorly understood
(Cohen et al., 1994;
Ma et al., 2000
), but our
results are consistent with the idea that agrin accumulates, or is more
active, at or near the growth cone and that an ensuing bias in Musk activation
leads to focal clustering of Musk and AChRs.
The timing of MCK-Musk expression correlates well with the cessation of motor axon growth and the onset of nerve terminal differentiation in Musk-/-; MCK-Musk mice. Prior to transgene expression, motor axon growth proceeds well beyond the normal synaptic zone in the muscle. Motor axon growth halts and the differentiation of nerve terminals begins in Musk-/-; MCK-Musk and Musk-/-; MCK-MMT mice, only after Musk, or MMT, is expressed from the MCK regulatory region. These results confirm prior studies of Musk and agrin mutant mice and provide direct evidence that Musk regulates the organization, or synthesis of a stop signal(s) for axon growth and nerve terminal differentiation.
The C terminus of Musk contains a binding-site for PDZ domain-containing
proteins. Clustering of AChRs, at least in cultured muscle cells, however, is
not dependent upon this sequence, as agrin stimulates AChR clustering in
myotubes expressing a C-terminal Musk mutant
(Zhou et al., 1999). As the
MCK-Musk transgene, which is studied here, is a fusion between
Musk and GFP, this gene fusion encodes a protein that lacks
a C-terminal binding site for PDZ domains. Because this transgene fully
rescues the presynaptic and postsynaptic defects of Musk mutant mice,
our experiments indicate that recruitment of PDZ domain-containing proteins to
the C-terminus of Musk is not required for Musk to stimulate presynaptic and
postsynaptic differentiation in vivo.
In chick and Xenopus, Musk expression is not restricted to
skeletal muscle. In chick embryos, Musk is expressed transiently in
the brain and liver (Ip et al.,
2000), and in Xenopus embryos, Musk is expressed widely
in the CNS (Fu et al., 1999
).
These findings raised the possibility that low and/or transient Musk
expression in motoneurons of mice may have escaped attention and that a loss
of motorneuron-derived Musk could be responsible for the presynaptic deficits
in Musk mutant mice. We find that muscle-specific expression of Musk
is sufficient to restore presynaptic as well as postsynaptic differentiation
in Musk mutant mice. Thus, these data strongly support the idea that
activation of Musk in skeletal muscle is required to initiate a signaling
pathway that leads to production and/or clustering of a retrograde signal for
presynaptic differentiation.
The steps that follow Musk activation and that lead to neuromuscular
synapse formation are poorly understood
(Mittaud et al., 2001;
Mohamed et al., 2001
;
Smith et al., 2001
;
Weston et al., 2000
). Our
results indicate that phosphorylation of the Musk juxtamembrane region
initiates a signaling pathway that regulates nerve terminal differentiation,
synapse-specific transcription and clustering of postsynaptic proteins. Thus,
the Musk juxtamembrane region, including a single phosphotyrosine docking
site, controls multiple pathways leading to presynaptic and postsynaptic
differentiation in vivo.
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ACKNOWLEDGMENTS |
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Footnotes |
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