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First published online November 21, 2006
doi: 10.1242/10.1242/dev.02696
1 Developmental Biology Program, Sloan-Kettering Institute, New York, NY 10021,
USA.
2 Howard Hughes Medical Institute, University of Colorado Health Sciences
Center, Department of Pediatrics, Section of Developmental Biology, Aurora, CO
80045, USA.
* Authors for correspondence (e-mail: s-weatherbee{at}ski.mskcc.org; Lee.Niswander{at}uchsc.edu)
Accepted 5 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Lrp4, Neuromuscular junction, Limb development, Lung hypoplasia, Oligosyndactyly, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Schneider and Nimpf, 2003). In
addition, several members of the LRP family are also known to have specific
roles in the development and function of the mammalian nervous system, as
receptors for both known and unknown ligands
(May and Herz, 2003). For
example, mutant mice that lack the Wnt co-receptor Lrp6 have defects in the
development of the thalamus and radial glia
(Zhou et al., 2004) while Lrp2
(also known as Megalin) is required for normal forebrain development, perhaps
through regulation of Bmp4 clearance
(Spoelgen et al., 2005;
Willnow et al., 1996). Vldlr
and Lrp8 (also known as apoER2) are receptors for reelin and are required for
normal layering of cortical neurons
(D'Arcangelo et al., 1999;
Hiesberger et al., 1999;
Trommsdorff et al., 1999).
Lrp1 is localized to postsynaptic specializations in the central nervous
system, where it associates with NMDA-type glutamate receptors
(Bu et al., 1994;
Ishiguro et al., 1995;
May et al., 2004;
Moestrup et al., 1992). Taken
together, the results of these studies indicate that Lrps have distinct roles
in neural development and synaptic connectivity.
In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in
the mouse, we identified two mutants that had striking and similar defects in
limb development, as well as an intriguing set of other developmental defects.
Both mutations proved to be alleles of Lrp4 [also known as
Megf7 (Nakayama et al.,
1998)]. Three other alleles of Lrp4 have been described
that produce similar defects in limb development; however, those mutants are
viable and no other developmental defects have been described
(Johnson et al., 2005;
Simon-Chazottes et al., 2006).
Additionally, they appear to be hypomorphic alleles, whereas the ENU-induced
alleles are null. The phenotypes of the null alleles reveal that Lrp4 is
required for viability and for normal development of the lung, kidney and
ectodermal organs. One of the striking phenotypes caused by a complete lack of
Lrp4 is paralysis at birth, due to an early block in the development of a
specialized synapse, the neuromuscular junction (NMJ).
During the formation of the NMJ, acetylcholine receptors (AChRs) are
clustered in the central region of muscle before the nerve arrives at the site
of the prospective synapse (Flanagan-Steet
et al., 2005; Panzer et al.,
2005; Panzer et al.,
2006; Lin et al.,
2001; Pun et al.,
2002; Yang et al.,
2001; Yang et al.,
2000). Signals from the motor axon are required to refine and
maintain this pre-pattern so that AChRs are stably restricted to synaptic
sites (Lin et al., 2005;
Misgeld et al., 2005). Agrin,
a large glycoprotein that is expressed by motoneurons and deposited into the
synaptic basal lamina, is a crucial nerve-derived signal
(Denzer et al., 1997;
Ruegg and Bixby, 1998;
Rupp et al., 1991). Agrin
regulates synaptic differentiation by activating MuSK (Musk - Mouse Genome
Informatics) (Hopf and Hoch,
1998), a receptor tyrosine kinase that is expressed selectively by
skeletal muscle and is concentrated at synaptic sites
(Bowen et al., 1998;
Glass et al., 1996;
Jennings et al., 1993;
Liyanage et al., 2002;
Trinidad et al., 2000;
Valenzuela et al., 1995). Mice
lacking either Agrin or MuSK fail to form neuromuscular synapses, cannot move
or breathe and die at birth (DeChiara et
al., 1996; Gautam et al.,
1999; Gautam et al.,
1996; Gautam et al.,
1995).
Previous studies have shown that MuSK plays a key role in all aspects of
postsynaptic differentiation, including the early nerveindependent
pre-patterning of AChRs and the subsequent stabilization of AChRs at synaptic
sites (Lin et al., 2001;
Yang et al., 2001;
Yang et al., 2000). However,
the mechanisms that lead to MuSK activation, both in the Agrin-independent
step during prepatterning and in the Agrin-dependent stage after contact with
motor axons, are poorly understood.
Here we show that the phenotypes of the two ENU-induced alleles of Lrp4 define an essential role for Lrp4 in the initial establishment of the NMJ. Lrp4 is required for clustering of AChRs at the synapse, at both early and late stages of synapse formation. Expression of the Lrp4 gene, like other genes that encode proteins enriched at the synapse, is upregulated in the postsynaptic endplate region, and Lrp4 is required for this specialized transcription. Lrp4 is required for proper localization of MuSK, and Lrp4 and Musk mutants appear to have identical phenotypes at the neuromuscular junction. These studies indicate that Lrp4 acts at the first known step of assembly of the neuromuscular junction.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kasarskis et al., 1998). The
mitaine and mitten mutants (renamed
Lrp4mte and Lrp4mitt after
identification of the gene) were identified in two independent screens.
Recombination mapping utilized simple-sequence length polymorphism markers to
test for association between phenotypes and regions of the C57BL/6 genome
(Kasarskis et al., 1998). Both
mutations mapped on Chromosome 2 between markers D2Mit14 (49.6 cM) and
D2Mit221 (50.3 cM).
Myotube cultures
Myoblasts and fibroblasts were dissociated from forelimb and hind limb
muscles of embryonic day (E) 17.5 embryos and plated on collagen-coated tissue
culture plates in Ham's F10 Medium containing 20% fetal bovine serum, 2.5
ng/ml basic fibroblast growth factor (recombinant human, Promega) and
antibiotics. Cells were selectively passaged twice over 8 days to enrich for
myoblasts. Cultures were then switched to Matrigel (BD Biosciences)-coated
tissue culture plates in Dulbecco's modified Eagle Medium containing 5% horse
serum and antibiotics for 6 to 7 days to promote fusion into myotubes.
Cultures were then treated overnight with 20 ng/ml of C-terminal Agrin
(recombinant rat, R&D systems). The next day (18 hours later), these
cultures were incubated with 
-bungarotoxin, rinsed in PBS, fixed in 2%
paraformaldehyde (PFA) and mounted for immunofluorescence.
Expression analysis
In situ analysis was performed on whole-mount specimens, paraffin sections
or cryosections of embryos fixed in 4% PFA at 4°C for 16-24 hours using
published techniques (Holmes and
Niswander, 2001). An antisense riboprobe (nucleotides 4481-5752)
from mouse Lrp4 cDNA was utilized for transcript detection.
Immunofluorescence was performed using standard methods
(Eggenschwiler and Anderson,
2000). Antibodies used were: anti-synaptophysin (polyclonal;
Zymed), anti-utrophin (monoclonal; Novacastra), anti-MuSK (polyclonal;
Affinity BioReagents). The monoclonal antibodies SV2 (developed by Kathleen
Buckley) and 2H3 (neurofilament, developed by Thomas Jessell and Jane Dodd)
were obtained from the Developmental Studies Hybridoma Bank developed under
the auspices of the NICHD and maintained by The University of Iowa, Department
of Biological Sciences, Iowa City, IA 52242. Alexafluor594-conjugated
-bungarotoxin was obtained from Molecular Probes. For detection of
ß-galactosidase activity, embryos were stained with X-Gal
(5-bromo-4-chloro-3-indolyl ß-D-galactoside) as described
(Hogan and Lacy, 1994).
Antibodies used on lung sections (see Fig. S2 in the supplementary
material) were: phospho-histone H3 (polyclonal, Upstate), ProSurfactant
Protein C (polyclonal, Chemicon), PECAM-1 (polyclonal, Pharmingen), Clara Cell
Protein 26 kDa (polyclonal, Chemicon) and acetylated -tubulin
(monoclonal, Zymed). The monoclonal antibody 8.1.1 (T1-) developed by
Andrew Farr was obtained from the Developmental Studies Hybridoma Bank. TUNEL
assay was performed as per manufacturer's recommendations (Roche).
Skeletal preparations
Alcian Blue/Alizarin Red staining was performed using standard methods
(Hogan and Lacy, 1994).
Briefly, embryos were eviscerated and skinned, followed by dehydration in
ethanol. After processing for Alcian Blue and Alizarin Red staining, embryos
were cleared through a graded series of potassium hydroxide and glycerol.
Quantitative real-time RT-PCR
The quality of RNA was ensured before processing by analyzing 100 ng of
each sample using the RNA 6000 NanoAssay and a Bioanalyzer 2100 (Agilent). One
microgram of total RNA was reverse-transcribed using the iScript cDNA
Synthesis Kit (Bio-Rad) at 52°C for 1 hour. Twenty nanograms of resultant
cDNA was used in a quantitative real-time RT-PCR reaction using an iCycler
(Bio-Rad) and pre-designed TaqMan Gene expression Assays for MuSK
(Mm00448006_m1) and HPRT (Mm00446966_m1). Amplification was carried out for 40
cycles (95°C for 15 seconds, 60°C for 1 minute). Data from the linear
phase of amplification were used to calculate MuSK and HPRT levels based on
the standard curve method. MuSK levels were normalized to HPRT and triplicates
values were averaged.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We used positional cloning to identify the locus disrupted by the
mte and mitt mutations. The mte/mitt mutations were
mapped by meiotic recombination to a 0.7 cM interval on Chromosome 2 (see
Materials and methods). Sequencing of candidate genes in the interval showed
that both mutant chromosomes had lesions in the Lrp4 gene. The
extracellular domain of Lrp4 contains four YWTD ß-propeller domains, five
EGF-like domains and seven LDLR type A (LA; ligand binding) domains
(Fig. 1A)
(Springer, 1998). The
mitt allele had two mutations: an early stop codon C-terminal to the
LA domains, which should delete most of the protein, and a second splice site
mutation that would introduce a second premature stop
(Fig. 1A). The mte
allele was associated with a missense mutation at a conserved position
(Asp1436Gly) in the most C-terminal YWTD domain; mutations at the same
position in human LDLR (Day et al.,
1997; Ward et al.,
1995; Webb et al.,
1996) appear to disrupt protein folding and stability
(Jeon et al., 2001). Based on
the nature of these sequence changes and the identical phenotypes of
mte and mitt homozygous embryos, it is likely that both of
these mutations caused a complete loss of Lrp4 function.
Distal limb development depends upon Lrp4
The limb phenotype of Lrp4mte and
Lrp4mitt homozygotes included a reduction in digit number,
shortening of the autopod elements, ectopic phalanges or digits dorsal to the
normal plane of the hand, and fusions of the central digits
(Fig. 1B-E). The earliest
defect in limb patterning of Lrp4 mutants was an expansion of the
apical ectodermal ridge (AER) along the dorsoventral axis of the limb bud.
This was detected as an expanded domain of Fgf8
(Fig. 1F,G) and Msx2
(Fig. 1H,I) expression in the
limb ectoderm. Wnt7a, a marker of dorsal limb ectoderm that normally
extends to the dorsal boundary of the AER, was found only in proximal ectoderm
(Fig. 1J,K), in a domain
complementary to that of the dorsally expanded AER. A targeted allele and two
insertional mutations in Lrp4 produce skeletal defects similar to
those seen in Lrp4mte and Lrp4mitt
mutants (Johnson et al., 2005;
Simon-Chazottes et al., 2006),
and the gene expression changes we observed
(Fig. 1; see Fig. S1 in the
supplementary material) are consistent with the phenotype reported for the
targeted Lrp4 allele (Johnson et
al., 2005).
Expression and requirement for Lrp4 in a variety of tissues
Lrp4 was expressed in limb ectoderm at early stages (E9.5)
(Fig. 2A) and later was
restricted to the AER (E11.5) (Fig.
2B) (Johnson et al.,
2005). We found that Lrp4 was also expressed in a variety
of other tissues, and many of those tissues developed abnormally in the
Lrp4mte and Lrp4mitt mutants, although
these defects have not been described for the targeted and insertional
alleles. For example, Lrp4 was expressed throughout kidney
development: it was expressed in the epithelial tips of the ureteric buds
during formation of the ureteric bud (E11.5),
(Fig. 2C); by E16.5, expression
was restricted to the tips of the growing ureter in the nephrogenic zone
(Fig. 2D). The majority of
Lrp4mte and Lrp4mitt mutants exhibited
unilateral or bilateral kidney agenesis (66%, 59/89). The targeted allele of
Lrp4 displayed variable craniofacial defects
(Johnson et al., 2005), and we
noted Lrp4 expression in tooth primordia
(Fig. 2G), as well as in a
variety of other ectodermal organs such as whisker vibrissae
(Fig. 2E), hair follicles
(Fig. 2F) and mammary buds
(Fig. 2H); each of these
tissues developed abnormally in mutant embryos (not shown).
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Lrp4 is required for formation of the NMJ
The phenotypes of several mutants have pointed to the importance of muscle
structure and activity in the proper morphogenesis of the lung
(Inanlou et al., 2005). In the
absence of skeletal and/or diaphragm muscles, embryos lack fetal breathing
movements, which causes reduced lung size, reduced sacculation and lung
patterning defects (Ackerman et al.,
2005; Inanlou and Kablar,
2003; Inanlou and Kablar,
2005a; Inanlou and Kablar,
2005b). Similarly, muscles cannot contract in Musk
mutants due to the absence of NMJs, and these embryos display pulmonary
hypoplasia (DeChiara et al.,
1996). The late lung hypoplasia phenotype observed in the
Lrp4 mutants (Fig. 3D)
did not appear to be the result of absence of normal muscles, as the
intercostal and diaphragm muscles appeared macroscopically normal (data not
shown). However, the failure of Lrp4 mutant mice to move at birth
suggested that the mutants might have a general defect in neuromuscular
function. Although Lrp4 was not expressed in motoneurons
(Fig. 2J), Lrp4
transcripts were detected in the diaphragm and other muscles at E12.5, before
neuromuscular synapse formation (Fig.
2I and data not shown), which would be consistent with a
postsynaptic requirement for Lrp4.
To assess whether normal synaptogenesis took place at the NMJ of
Lrp4 mutants, we examined the organization of AChRs in mutant
muscles. In normal muscles, each muscle fiber bears a single AChR cluster
apposed by a nerve terminal (Fig.
3I-K). By contrast, no AChR clusters could be detected by staining
with labeled -bungarotoxin in either Lrp4mitt or
Lrp4mte mutant diaphragms
(Fig. 3L-N) or intercostal
muscles (Fig. 3E,F) at E18.5.
The absence of AChR clusters in these muscles is sufficient to account for the
failure of the mutants to breathe, and therefore can explain the abnormal
morphology of the mutant lungs. No AChR clustering was detected in limb
muscles of Lrp4 mutants (Fig.
3G,H), and the lack of embryonic and perinatal movement suggests
that Lrp4 is required globally for NMJ organization.
To confirm that Lrp4 was acting in the postsynaptic cell, we derived
myotubes from E18.5 wild-type and Lrp4mitt or
Lrp4mte mutant forelimb and hind limb muscles. In
wild-type cultured myotubes, spontaneous clustering of AChRs occurs in the
absence of motor axons (Fig.
4M) and treatment with Agrin increases the number and frequency of
AChR clusters (Fig. 4O)
(Campanelli et al., 1991).
However, in Lrp4 mutant myotubes, clusters of AChRs were not observed forming
spontaneously or following stimulation with Agrin
(Fig. 4N). These results
confirm that Lrp4 is required in the postsynaptic cell for the clustering of
AChRs.
|
). We
therefore examined AChR clusters in wild-type and Lrp4 mutant mice at
E13.5 and 14.5. In wild-type animals, AChR preclustering in the endplate
region was visible in E13.5 diaphragms, as motor axons were exiting the main
body of the phrenic nerve (Fig.
4B). However, Lrp4 mutants lack this early AChR clustering before
innervation (Fig. 4E). At
E14.5, as at E18.5, AChR clusters were prominent in wild type
(Fig. 4H), but could not be
detected in Lrp4 mutant muscle
(Fig. 4K). This early NMJ
defect is similar to that observed in Musk mutants
(Lin et al., 2001). Also
similar to Musk mutants, the motor axons in Lrp4 mutants did
not stop adjacent to the main intramuscular nerve, but instead grew
excessively across the muscle (Fig.
4L, Fig. 3N). These
phenotypes suggest that Lrp4 is essential for AChR clustering and
acts at the early, Agrin-independent, MuSK-dependent step in this process.
Lrp4 is required for synaptic protein localization at the NMJ
AChR clustering depends on the cytoplasmic, peripheral membrane protein
Rapsn (also known as rapsyn), which localizes to NMJs in response to MuSK
(DeChiara et al., 1996) and
binds directly to AChRs (Bartoli et al.,
2001; Burden et al.,
1983). To examine whether Lrp4 might act with Rapsn, we tested
whether Rapsn was localized correctly in Lrp4 mutant muscles. We
marked synaptic sites with antibodies to synaptophysin or SV2, which are
components of presynaptic vesicles (Gautam
et al., 1995). Rapsn was concentrated at postsynaptic regions in
wild-type muscles, but was not localized in Lrp4 mutants
(Fig. 5A',D').
Utrophin, a component of the muscle cytoskeleton, is specifically localized
with AChRs at the crests of postjunctional folds
(Blake et al., 1994;
Blake et al., 1996) and depends
upon a scaffold of proteins, including MuSK and Rapsn, for its localization.
In Lrp4 mutant muscle, utrophin was not detectable at the synapse
(Fig. 5B',E'). Thus
Lrp4 is required for proper localization of Rapsn at the synapse and because
Rapsn fails to localize correctly, neither AChRs nor utrophin
(Gautam et al., 1995) localize
to the NMJ of Lrp4 mutants.
Failure of postsynaptic specialization in Lrp4 mutants
The subset of muscle cell nuclei located near synaptic sites is specialized
to express high levels of proteins enriched at the postsynaptic membrane
(Sanes et al., 1991).
Synapse-specific transcription is disrupted in Musk, but not in
Rapsn, mutants (Gautam et al.,
1999); thus MuSK has a second activity in addition to its role in
localization of Rapsn and Rapsn-dependent proteins. Transcripts encoding the
AChR and 
subunits and MuSK were concentrated in the central
region of diaphragm muscles in E14.5 controls
(Fig. 6A-C); however, these
transcripts were more broadly expressed in Lrp4mte and
Lrp4mitt mutants (Fig.
6E-G and data not shown), indicating that their expression had not
been restricted to synaptic nuclei. By E18.5, the Musk, AChR
and AChR genes were expressed only at very low levels in
Lrp4 mutant muscles and were not localized to the endplate region
(Fig. 6M-O).
Moreover, Lrp4 mRNA itself was highly enriched in the synaptic region of diaphragm and intercostal muscles from wild-type animals (Fig. 6D,L and data not shown), as seen for proteins that are enriched at the synapse. Lrp4 RNA was not properly localized in Lrp4mte and Lrp4mitt mutant muscles (Fig. 6H,P and data not shown), although it was detectable via quantitative RT-PCR (data not shown). Thus Lrp4 is required for both aspects of MuSK activity, assembly of the AChR scaffold and specialization of postsynaptic nuclei.
The similarity of the Musk and Lrp4 phenotypes suggested that Lrp4 and MuSK regulate a common step in formation of the NMJ. We found that MuSK was not concentrated at synapses in Lrp4 mutant muscles (Fig. 5C',F') even though MuSK transcripts were present at similar levels in wild type and mutants before postsynaptic specialization based on quantitative real-time RT-PCR (see Fig. S3 in the supplementary material). Thus, Lrp4 is required for the first known step of postsynaptic assembly, the accumulation of MuSK at the synapse.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
and targeted (Johnson et al.,
2005) alleles of the gene, which are homozygous viable and
fertile. One of the ENU-induced alleles has an early stop codon that would
remove over 80% of the protein, and the second ENU-induced allele carries a
missense mutation at the same position as an allele of the LDL receptor that
probably destabilizes the protein (Jeon et
al., 2001). These two ENU-induced mutations are therefore likely
to be null alleles. The Lrp4mdig and
Lrp4dan alleles appear to be hypomorphic, as some
wild-type transcript is present in each mutant
(Simon-Chazottes et al.,
2006). The targeted allele of Lrp4 removes the
transmembrane and cytoplasmic domains of Lrp4, but could express an intact
extracellular domain. Approximately 35% of homozygous targeted mutants die
between P0 and P5, although the cause of lethality was not determined
(Johnson et al., 2005). Based
on the phenotypes of the Lrp4mte and
Lrp4mitt mutant alleles, the incompletely penetrant
lethality of the targeted mutants could be due to renal agenesis or defects in
postsynaptic specialization. In principle, differences in genetic background
could account for the phenotypic differences between the ENU-induced and
targeted alleles, as the Lrp4mte and
Lrp4mitt alleles were analyzed on mixed C3H-C57Bl/6 and
congenic C3H backgrounds, while the targeted allele was studied on a mixed
129SvEv-C57Bl/6 background. In either case (different allele strength or
different genetic background), we conclude that reduced levels of Lrp4 cause a
common limb phenotype, and complete loss of Lrp4 function causes additional
phenotypes that include defects in the formation of multiple embryonic
tissues, perinatal lethality and failure to organize the NMJ.
The diverse phenotypes caused by loss of Lrp4 have no obvious common
molecular basis. Musk mutants do not show limb defects, and it has
been suggested that Lrp4 limb phenotypes are the result of excessive
canonical Wnt signaling (Johnson et al.,
2005), which is supported by our in vivo analysis of a Wnt
reporter in Lrp4mte and Lrp4mitt
mutants (see Fig. S1A,G in the supplementary material), although the mechanism
by which Lrp4 could antagonize Wnt signaling has not been defined. By
contrast, the kidney hypoplasia and agenesis phenotype is more akin to Wnt
loss of function (Carroll et al.,
2005; Majumdar et al.,
2003; Pinson et al.,
2000; Stark et al.,
1994) than to excess Wnt signaling. Thus it appears that Lrp4
affects different signaling pathways in specific tissues.
Defects in NMJ formation, maintenance or function can lead to human
disorders such as muscular dystrophy and myasthenia syndromes
(Engel et al., 2003;
Ervasti and Campbell, 1993;
Hughes et al., 2005).
Myasthenia syndromes are characterized by muscle weakness; severe cases can
affect breathing and thus are life threatening. Several congenital myasthenia
syndromes have been mapped to mutations in proteins localized in the
presynaptic and postsynaptic regions of the NMJ. Myasthenia gravis is a group
of acquired autoimmune disorders wherein some patients produce antibodies
against AChR subunits, while others test positive for antibodies that
recognize MuSK or Rapsn. Targeting of NMJ components by the host immune system
results in a reduction in the number of AChR clusters (up to an 80% decrease)
in voluntary muscles. In some cases of Myasthenia gravis, the autoantigen has
not been identified, and the genetic basis of a subset of congenital
myasthenia syndromes has not been elucidated. It will be important to test
whether any of these individuals have mutations in the Lrp4 gene or generate
antibodies against the Lrp4 protein.
The requirement of Lrp4 for NMJ formation is reminiscent of the role of
Lrp1 at central nervous system synapses. Lrp1 associates with postsynaptic
proteins, including neurotransmitter receptors, and is required for normal
synaptic function (reviewed by May et al.,
2005). The biochemical activities of other members of the Lrp
family suggest two possible mechanisms for the function of Lrp4 at the
synapse. Like Lrp5 and Lrp6, Lrp4 might act as a co-receptor that, together
with MuSK, binds a ligand that activates MuSK activity. No ligand has been
identified that activates MuSK kinase activity early in the formation of the
synapse, and it is possible that Lrp4 helps bind that ligand. Alternatively,
because the cytoplasmic domain of Lrp4, unlike Lrp5 and Lrp6, contains
clustering motifs (NPxY, di-leucine YxxL), similar to those in Ldlr, Lrp1 and
Lrp2 (He et al., 2004), Lrp4
might facilitate its own clustering or clustering of another protein such as
MuSK. Before our work, the only proteins previously known to be required to
initiate postsynaptic differentiation was MuSK and a recently described
binding partner of MuSK, Dok7 (Okada et
al., 2006). The discovery that Lrp4 is a crucial component for the
localization of MuSK provides a new entry point to define the early events
that lead to synaptic specialization. The findings that Lrp4 has been isolated
from synaptic fractions of rat brain extracts and that Lrp4 interacts with
postsynaptic scaffold proteins in neurons
(Tian et al., 2006) suggests
that Lrp4 may play a broad role in synapse formation.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/24/4993/DC1
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ACKNOWLEDGMENTS |
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