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RESEARCH ARTICLE 3597
Development 136, 3597-3606 (2009) doi:10.1242/dev.035857
Talin 1 and 2 are required for myoblast fusion, sarcomere
assembly and the maintenance of myotendinous junctions
Francesco J. Conti1, Sue J. Monkley2, Malcolm R. Wood1, David R. Critchley2 and Ulrich Müller1,*
Talin 1 and 2 connect integrins to the actin cytoskeleton and regulate the affinity of integrins for ligands. In skeletal muscle, talin 1
regulates the stability of myotendinous junctions (MTJs), but the function of talin 2 in skeletal muscle is not known. Here we show
that MTJ integrity is affected in talin 2-deficient mice. Concomitant ablation of talin 1 and 2 leads to defects in myoblast fusion and
sarcomere assembly, resembling defects in muscle lacking 1 integrins. Talin 1/2-deficient myoblasts express functionally active 1
integrins, suggesting that defects in muscle development are not primarily caused by defects in ligand binding, but rather by
disruptions of the interaction of integrins with the cytoskeleton. Consistent with this finding, assembly of integrin adhesion
complexes is perturbed in the remaining muscle fibers of talin 1/2-deficient mice. We conclude that talin 1 and 2 are crucial for
skeletal muscle development, where they regulate myoblast fusion, sarcomere assembly and the maintenance of MTJs.
INTRODUCTION
Skeletal muscle development and function are dependent on 1
integrins, a family of cell surface receptors that are formed by
heterodimerization of the 1 subunit with different  subunits
(Hynes, 1992). In skeletal muscle, 1 integrins localize to
costameres and myotendinous junctions (MTJs), where they
establish a link between the cytoskeleton and the extracellular
matrix (ECM) (Mayer, 2003). These connections are important
for transmitting mechanical forces and for maintaining skeletal
muscle fibers. Accordingly, defects in integrin function lead to
muscle fiber degeneration: mutations in the gene encoding the 7
integrin subunit cause congenital myopathy in humans (Hayashi
et al., 1998), and genetic ablation of either the 5 or 7 integrin
subunits causes muscular dystrophy in mice (Mayer et al., 1997;
Taverna et al., 1998). Integrins appear to be particularly important
at MTJs. Inactivation of the 7 integrin subunit leads to
detachment of MTJs from the ECM (Miosge et al., 1999),
whereas inactivation of integrin-linked kinase (ILK) and talin 1
lead to detachment of integrin adhesion complexes from the
muscle fiber cytoskeleton (Wang et al., 2008; Conti et al., 2008).
Integrins also have important functions during skeletal muscle
development, as ablation of the murine 1 integrin subunit gene,
which leads to loss of all 1 integrins, causes defects in
myoblast fusion and sarcomere assembly (Schwander et al.,
2003). The mechanisms by which integrins carry out their
function in skeletal muscle are still incompletely understood.
Integrins assemble signaling complexes at the plasma membrane,
which contain proteins that bind to the integrin cytoplasmic domains
or are recruited indirectly (Geiger et al., 2001; Liu et al., 2000).
Several lines of evidence suggest that talin 1 is central for integrin
signaling. Talin 1 interacts with the cytoplasmic domain of 1
integrins (as well as several other  subunits) and with focal
adhesion components such as focal adhesion kinase (FAK) and
vinculin (Nayal et al., 2004). Talin 1 also binds to F-actin,
establishing a link between 1 integrins and the cytoskeleton (Nayal
1
The Scripps Research Institute, Department of Cell Biology and Institute of
Childhood and Neglected Diseases, La Jolla, CA 92037, USA. 2University of Leicester,
Department of Biochemistry, Leicester LE1 9HN, UK.
*Author for correspondence ([email protected])
Accepted 1 September 2009
et al., 2004). The assembly of focal adhesions is regulated by
mechanical force, which controls the recruitment of vinculin into
focal adhesions (Balaban et al., 2001; Choquet et al., 1997; Galbraith
et al., 2002; Riveline et al., 2001). Talin 1 is required for the forcedependent recruitment of vinculin and strengthens the interactions
between integrins and the cytoskeleton (Giannone et al., 2003).
Binding of talin 1 to the integrin cytoplasmic domain also enhances
the strength of integrin adhesion to ligands (inside-out activation)
(Nieswandt et al., 2007; Petrich et al., 2007; Tadokoro et al., 2003).
However, it is less clear whether inside-out activation is essential for
interactions with insoluble ligands; integrins can be activated
directly by binding to insoluble ligands (outside-in activation) (Du
et al., 1991), and talin ablation in Drosophila causes detachment of
myofibers from integrins without loss of adhesive contact with the
ECM (Brown et al., 2002).
Previously, we have shown that inactivation of the talin 1 gene
(Tln1) in skeletal muscle leads to a progressive myopathy, caused by
mechanical failure of MTJs (Conti et al., 2008). The phenotype
resembles the defect observed in mice with a mutation in the gene
encoding the integrin 7 subunit (Itga7) (Mayer et al., 1997; Miosge
et al., 1999), suggesting that talin 1 mediates integrin 71 functions
at MTJs. The Tln1-deficient mice did not show the defects in
myoblast fusion and sarcomere assembly that have been observed
in integrin 1-deficient mice (Schwander et al., 2003). Because
vertebrates contain two genes encoding two talins (talin 1 and 2)
(McCann and Craig, 1997; McCann and Craig, 1999; Monkley et
al., 2001), and because talin 1 and 2 have redundant functions in
integrin-mediated attachment of fibroblasts (Zhang et al., 2008), we
argued that talin 2 might compensate for loss of talin 1 in skeletal
muscle. Talin 2 is expressed at higher levels in skeletal muscle than
talin 1 (Conti et al., 2008; Monkley et al., 2001; Senetar and
McCann, 2005), and talin 2 expression is upregulated during
myotube formation (Senetar et al., 2007). Therefore, to determine
the function of talin 2 in skeletal muscle, we generated Tln2deficient mice (referred to as Tln2-KO), and mice lacking both talin
1 and 2 in skeletal muscle (referred to as Tln1/2-dKO). We show
here that ablation not only of the talin 1 gene but also of the talin 2
gene leads to defects in the maintenance of MTJs, and we provide
evidence that talin 1 and 2 mediate 1 integrin functions in myoblast
fusion and sarcomere assembly.
DEVELOPMENT
KEY WORDS: Integrin, Talin, Muscular dystrophy, Dystrophin, Mice
3598 RESEARCH ARTICLE
MATERIALS AND METHODS
Generation of mice
Tln2-KO mice were generated by ablating the first coding exon of Tln2 (see
Results). Mice were genotyped by PCR using the following primers: (a) 5⬘CAAACTGAATGAAGGCCCAACAG-3⬘; (b) 5⬘-TCTCCACTTACTCCTTGCCC-3⬘; (c) 5⬘-GCCGAGGCTACATGGAGTCAGTAT-3⬘. Tln1/2dKO mice and control mice were obtained by crossing Tln1flox/flox;Tln2+/–
mice with Tln1flox/+;Tln2+/–;HSA-CRE+/– mice. Tln1flox/flox and HSA-CRE
mice have been described previously (Conti et al., 2008; Leu et al., 2003).
Histology, western blotting, electron microscopy and CK levels
Muscle sections were stained with hematoxylin and eosin (H&E). To
determine the number of fibers with central nuclei, random areas across the
muscle were photographed and nuclei quantified. The number of fields
analyzed depended on the size of the muscle type (soleus, n3;
gastrocnemius n9; tibialis anterioris, n6). Three mice per genotype and
time-point were analyzed. The mean ±s.d. was determined and a Student’s
t-test performed. Immunohistochemistry, analysis of cell proliferation and
western blotting were carried out as described (Conti et al., 2008; Schwander
et al., 2003) using the following antibodies: (1) monoclonal against vinculin
(Sigma), MHCf (Sigma), ILK (Li et al., 1999), tenascin C (Sigma), MHCs
(Leica), CD9 (BD Pharmingen), sarcomeric -actinin (Sigma), BrdU
(Pharmingen); (2) rabbit polyclonal against 7 integrin (kindly provided by
U. Meyer, University of East Anglia, Norwich, UK), v integrin (Chemicon),
1 integrin (Schwander et al., 2003), collagen type IV (Chemicon), laminin
2 (Chemicon) and CX43 (Abcam). Polyclonal antibodies specific for talin
1 and 2 have been described previously (Conti et al., 2008), and correspond
to residues 1830-1850 (talin 1) and 940-957 (talin 2). Electron microscopy
and Evans blue dye (EBD) uptake assays were performed as described
previously (Conti et al., 2008). Measurements of creatine kinase (CK) levels
were performed by Antech Diagnostics (Irvine, CA, USA).
Primary cultures of fetal myoblasts
Primary cultures of fetal myoblasts were prepared from hindlimb muscle
from E17.5 embryos as described previously (Schwander et al., 2003). Cells
were plated onto coverslips coated with 0.1% gelatin (Sigma) and grown in
medium consisting of 65% DMEM (Gibco), 25% Media 199 (Gibco) and
10% fetal bovine serum (Gibco). Differentiation was induced by transferring
the cultures to medium consisting of 70% DMEM, 28% Media 199, 2%
horse serum (Gibco) and 0.1 mg/ml insulin (Sigma). After 3 days, cells were
fixed, permeabilized with 0.5% TX-100 and stained with antibodies to actinin and MHCf and then with a secondary anti-mouse Alexa 488
antibody. Myonuclei were stained with DAPI (Sigma). The fusion index was
determined as the ratio of myonuclei in cells with three or more nuclei to the
total number of nuclei. To measure adhesion, 6⫻104 cells were plated on
poly-D-lysine, collagen type IV, laminin or fibronectin. After 90 minutes,
cells were washed, fixed and myoblasts immunostained with antibodies to
7 integrin or -actinin. Nuclei were stained with DAPI. The number of
cells adhering to each substrate was normalized to the number of cells
adhering to poly-D-lysine. The mean ±s.d. was determined and a Student’s
t-test performed. Cells were photographed using an Olympus AX70
microscope and counted using Metamorph.
Development 136 (21)
cassette flanked by LoxP sites (Fig. 1A). A third LoxP site was
inserted between exons 2 and 3. Chimeric mice were generated that
transmitted the targeted Tln2 allele through the germline. Crossing
of these mice with a CRE deleter mouse (Schwenk et al., 1995)
generated three recombination events leading to mouse lines with
different Tln2 alleles: (1) mice lacking the neomycin cassette (rec);
(2) mice lacking exon 2 (data not shown); and (3) mice lacking exon
2 and the neomycin cassette (Tln2–; Fig. 1A). Mice homozygous for
the Tln2– allele were identified by genotyping using the PCR
primers indicated in Fig. 1A, which amplify a 569-base-pair band
specific for Tln2-KO mice (Fig. 1B). Tln2-KO mice were viable and
fertile and did not differ in general appearance from wild-type mice
(Fig. 2A,B). To confirm that expression of talin 2 was ablated, we
analyzed protein expression by western blot using antibodies
specific for talin 2 (Conti et al., 2008). Two bands corresponding to
intact and cleaved talin 2 were detected in muscle extracts from 1month-old wild-type but not Tln2-KO mice (Fig. 1C). We also
immunostained sections of gastrocnemius muscle in 1-month-old
mice. As reported previously (Conti et al., 2008), talin 2 was
concentrated at MTJs in wild-type mice, but not in Tln2-KO muscle
fibers (Fig. 1D). We conclude that talin 2 expression was effectively
ablated in muscle from Tln2-KO mice.
Tln2-KO mice develop a myopathy with centrally
nucleated fibers
We next analyzed histological sections from Tln2-KO mice for
skeletal muscle defects. In several mouse models of muscular
dystrophy, muscle fibers undergo cycles of degeneration and
regeneration. While the nuclei of healthy muscle fibers are located
close to the sarcolemma, regenerating fibers display centrally
located nuclei, providing a useful readout for muscle defects
Myoblasts were analyzed by fluorescence-activated cell sorting (FACS)
based on 7 integrin subunit expression as described previously (BlancoBose et al., 2001). Cells were harvested and resuspended in PBS containing
3% BSA. To detect 7 expression, cells (106/sample) were incubated with
phycoeritrin (PE)-conjugated antibodies to 7 (MBL). To detect expression
of active 1 integrins, cells were then incubated with antibody 9EG7 (BD
Biosciences), followed by a FITC-conjugated secondary antibody. Cells
were analyzed in a LSR II 2 flow cytometer (BD Biosciences).
RESULTS
Generation of Tln2-KO mice
To analyze talin 2 function during skeletal muscle development we
inactivated Tln2 in mice. A gene-targeting vector was generated that
included, 5⬘ of the first Tln2 coding exon, a neomycin (PGK-Neo)
Fig. 1. Generation of Tln2-KO mice. (A)Schematic representation of
the targeting strategy. (B)PCR result from genotyping using the primers
in (A) with DNA from 1-month-old wild-type (WT) and Tln2-KO mice
(T2-KO). 325-bp and 569-bp bands indicative of wild-type and Tln2alleles, respectively, were observed. (C)Protein extracts from 1-month
old gastrocnemius muscle were analyzed by western blot. Talin 2
expression was ablated in Tln2-KO mice. Membranes were probed with
-tubulin as a loading control. (D)Longitudinal sections of 1-month-old
gastrocnemius muscle were stained with antibodies against talin 2. Talin
2 was undetectable in Tln2-KO muscle. Scale bar: 50mm.
DEVELOPMENT
Flow cytometry
Talin 1 and 2 in skeletal muscle development
RESEARCH ARTICLE 3599
(Pierson et al., 2005). At 1 month of age, skeletal muscles from Tln2KO mice and controls were largely indistinguishable, but a slight
increase in the number of centrally nucleated fibers was noticeable
in the mutants (Fig. 2C,D,I). By 7 months of age, the number of
centrally located nuclei was drastically increased in Tln2-KO mice.
Similar observations were made in gastrocnemius, soleus and tibialis
muscles (Fig. 2E-I; data not shown). As observed in other mouse
models for muscular dystrophy (Straub et al., 1997), the severity of
the phenotype showed differences between distinct muscles. In
Tln2-KO mice, a significantly higher percentage of centrally located
nuclei was observed in the soleus when compared with the
gastrocnemius and tibialis muscles (gastrocnemius: wild-type
1.27±0.93%, Tln2-KO9.55±0.31%; soleus: wild-type0.81±
0.16%, Tln2-KO35.03±2.26%; tibialis: wild-type0.11±0.09%;
Tln2-KO 9.27±1.34%; in all instances n3, values are mean ±s.d.).
The higher proportion of centrally nucleated fibers in the soleus
muscle suggests that deficiency of talin 2 might predominantly affect
slow (type I) fibers. To determine whether this was the case, muscle
fibers were co-immunostained with DAPI and with antibodies to
slow and fast myosin heavy chain isoforms (Fig. 2L-N, data not
shown). Centrally located nuclei were found in both type I and type
II fibers. While central nuclei were found more frequently in slow
fibers, once the relative proportion of fast versus slow fibers in the
soleus muscle was taken into account, no significant difference was
observed (Fig. 2R). Co-immunostaining for MHCf and talin 2
showed that talin 2 was expressed at the MTJs of both fast and slow
fibers (Fig. 2O-Q). Finally, analysis by western blot showed that,
although talin 2 is expressed at higher levels in muscle than talin 1
(Conti et al., 2008; Senetar and McCann, 2005), levels of talin 1 and
talin 2 did not differ between different muscles (Fig. 2S,T). We
therefore conclude that deficiency of talin 2 equally affects slow and
fast fiber types. The higher number of centrally nucleated fibers in
soleus muscle could be explained by it being a postural muscle
(Vandervoort and McComas, 1983), experiencing more stress than
the gastrocnemius and tibialis muscles. Interestingly, muscles in
Tln1-KO mice, including the soleus, do not show centrally nucleated
myofibers (Conti et al., 2008), which could reflect the fact that talin
2 levels in muscle are higher than talin 1 levels.
No evidence for sarcolemmal damage in Tln2-KO
mice
Centrally nucleated myofibers are frequently found in dystrophic
muscle fibers that also show defects in the stability of the
sarcolemma, leading to efflux of proteins from muscle fibers. For
DEVELOPMENT
Fig. 2. Tln2-KO mice develop a myopathy with
centrally nucleated fibers. (A,B)Normal appearance of
Tln2-KO mice. (C-H)Sections of gastrocnemius muscle
were stained with H&E. Centrally nucleated fibers were
evident in gastrocnemius (C-F) and soleus muscles (G,H) of
mice that were 1 (C,D) and 7 (E-H) months old.
(I)Quantification of centrally nucleated fibers (CNF) in
gastrocnemius and soleus muscles. The number of affected
fibers increased with the age of Tln2-KO mice (P0.006,
gastrocnemius; *, P0.0007, soleus). (J)Serum creatine
kinase (CK) levels were normal in 5-month-old Tln2-KO
mice (n4-7 per genotype). (K)EBD was injected into 5month-old mice. No dye incorporation was noted in
muscles of Tln2-KO mice. Occasionally, dye incorporation
was observed irrespective of genotype (control), validating
the experimental set up (n4-7 per genotype). (L-N,R) Coimmunostaining of Tln2-KO muscle for MHCf (green) and
DAPI (blue) revealed that fast and slow fibers were
affected; laminin staining (red) highlights muscle fiber
contours. (O-Q)Co-immunostaining for MHCf (red) and
talin 2 (green) revealed that talin 2 was expressed at the
MTJs of fast and slow fibers. Yellow and white arrows point
to fast and slow fibers, respectively. (R)Quantification of
the distribution of central nuclei (CNF) in slow and fast
fibers (*, P0.02). (S,T) Expression of talin 1 and talin 2 was
evaluated by western blot. Equivalent expression levels
were observed in soleus and gastrocnemius muscles. Scale
bars: 100mm.
example, this phenotype is observed in patients affected by
Duchenne muscular dystrophy and in the mdx mouse (Dmd –
Mouse Genome Informatics), which bear mutations in the
dystrophin gene (Carpenter and Karpati, 1979; Schmalbruch, 1975;
Straub et al., 1997; Weller et al., 1990). By contrast, mutations in
the Itga7 and Tln1 genes cause muscle defects with little or no
evidence of membrane damage, and the former but not the latter
have centrally nucleated myofibers (Conti et al., 2008; Hayashi et
al., 1998; Rooney et al., 2006). To further evaluate whether talin 2, talin 1- and integrin 7-deficient muscle fibers shared other
phenotypic features, we analyzed sarcolemmal damage by
measuring creatine kinase (CK) levels in the plasma of 5-monthold mice. Tln2-KO mice presented no evidence for membrane
damage (Fig. 2J). We also injected EBD in the tail vein.
Occasionally, EBD-positive fibers (which by histology appeared
damaged; data not shown) were detected irrespective of the
genotype, validating the experimental set up (‘control’ in Fig. 2K);
however, no obvious EBD accumulation was noted in Tln2-KO
mice (Fig. 2K). Finally, while immune-cell infiltration and fibrosis
are observed in mice with mutations that affect the dystrophin
complex (Stedman et al., 1991), these histopathological
abnormalities were not present in muscle from Tln2-KO mice (data
not shown).
We conclude that Tln2-KO and 7 integrin mutant mice show
signs of skeletal muscle fiber degeneration that differ from the
phenotype associated with mutations affecting the dystrophin
complex, providing evidence that these protein complexes regulate
muscle fiber maintenance in different ways.
Development 136 (21)
Talin 2 is not essential for the assembly of the
muscle fiber cytoskeleton
Talin is essential for focal adhesion assembly and turnover (Franco
et al., 2004; Priddle et al., 1998). However, sarcomere and costamere
assembly were maintained when Tln1 was ablated in skeletal muscle
(Conti et al., 2008). As talin 2 is expressed in muscle at higher levels
than talin 1 (Conti et al., 2008; Senetar and McCann, 2005), we
determined whether the muscle fiber cytoskeleton was abnormal in
Tln2-KO mice. Sarcomere integrity was evaluated by electron
microscopy in 3-month-old mice. In wild-type muscle, the structure
of the sarcomere was well maintained, with well-defined Z- and Mbands (Fig. 3A). In Tln2-KO muscle, necrotic material was observed
within myofibers (Fig. 3B,C). Although the M-band (asterisk in Fig.
3B) was not always evident in all areas of mutant muscle fibers (Fig.
3C), the Z-line was present and the overall striation pattern was
maintained. By contrast, major defects were observed at MTJs. In
wild-type muscle, actin filaments reached the sarcolemma at the end
of muscle fibers (Fig. 3D,G). In Tln2-KOs, actin filaments detached
from the MTJs, and necrotic and membranous material localized in
the gap left by retracting myofilaments (Fig. 3E,H,I). The
perturbations at MTJs of Tln2-KO mice resemble the defects in mice
lacking Tln1 in skeletal muscle but were considerably more severe
and prominent at an earlier age (3 instead of 6 months) (Conti et al.,
2008). Unlike in Tln1-KO mice, lateral detachment of the
cytoskeleton from the sarcolemma was also occasionally noted in
Tln2-KO mice (Fig. 3F). These data are consistent with talin 2 being
the major talin isoform in skeletal muscle (Conti et al., 2008; Senetar
and McCann, 2005).
Fig. 3. Talin 2 is required for MTJ integrity but not
for sarcomere organization. (A-C)EM micrographs of
gastrocnemius (A,B) and soleus (C) isolated from
3-month-old mice. Disorganization was evident to
varying degrees in Tln2-KO muscle fibers, which
accumulated necrotic material. Muscle fibers appeared
contracted, but the Z-line and A-band were evident
(white arrows and asterisks, respectively). (D-I)Electron
micrographs of MTJ from soleus (D,E) and gastrocnemius
(G-I) of 3-month-old mice. In wild-type mice,
myofilaments reached the end of muscle fibers (D,G,
arrowhead). In Tln2-KO mice, myofilaments were
detached from the MTJ, and necrotic material
accumulated in the gaps (E,H,I, asterisks). Lateral
detachment of the cytoskeleton from the sarcolemma
was occasionally noted (F, arrowheads). (J-Q)Longitudinal
sections of gastrocnemius muscle were immunostained
with antibodies to 7 integrin (J,K), ILK (L,M), vinculin
(Vn) (N,O) and talin 1 (Tln1) (P,Q). All proteins were
localized at MTJs, but talin 1 staining was increased in
the mutants (arrow in Q). Scale bars: 2mm in A-C; 5mm
in D-I; 100mm in J-Q.
DEVELOPMENT
3600 RESEARCH ARTICLE
Talin 1 and 2 in skeletal muscle development
RESEARCH ARTICLE 3601
Assembly of integrin complexes and increased
recruitment of talin 1 to the MTJ of Tln2-KO mice
To determine whether assembly of integrin complexes was
compromised in Tln2-KO mice, we evaluated the distribution of
integrins and their effectors. In control muscle, 7 integrin and
vinculin were localized at costameres and MTJs, whereas ILK was
only found at MTJs. The distribution of these proteins was normal
in Tln2-KO mice, although it appeared more diffuse, possibly
reflecting MTJ disorganization (Fig. 3J-O). Importantly, talin 1
expression was undetectable or very low in wild-type mice (Fig. 3P)
but was readily detectable at the MTJs of Tln2-KO mice (Fig. 3Q,
arrow). Talin 1 accumulation at MTJs was likely to be caused by
redistribution of talin 1 protein as western blot analysis showed that
total talin 1 levels in muscle remained unchanged (see Fig. S1A in
the supplementary material). We conclude that talin 2 is not essential
for the assembly of integrin complexes in skeletal muscle fibers, and
that talin 1 is likely to compensate for a lack of talin 2. However,
defects at MTJs are observed in muscle lacking either talin 1 (Conti
et al., 2008) or talin 2 (this study), suggesting that the two talin
isoforms cannot completely compensate for each other or that a
reduction in the total amount of talin (1 and 2) protein caused the
defects at MTJs (see Discussion).
Fig. 4. Tln1/2-dKO mice die at birth with skeletal muscle defects.
(A)Tln1/2-dKO embryos (dKO) had a contracted posture compared
with that of wild-type (WT) embryos. (B,C)Immunostaining of sections
from E18.5 wild-type and Tln1/2-dKO muscle showed that talin 2 was
effectively ablated from costameres and MTJs (white arrows).
(D-K)Sections showing intracostal muscle in E18.5 wild-type (D,F) and
Tln1/2-dKO embryos (E,G,H-K) stained with H&E. Myofibers from
Tln1/2-dKO mice had abnormal morphology and variation in fiber size
(black arrows in G-K). Scale bars: 50mm in B,C; 100mm in D-E; 50mm
in F-K.
number of proliferating cells per unit area (mm2) muscle tissue was
observed between control and Tln1/2-dKO embryos. Next, we
immunostained E16.5-E17.5 embryos with antibodies to desmin
(see Fig. S2A-D in the supplementary material, red), -actinin (see
Fig. S2G,H in the supplementary material) and MHCf (see Fig. S2I,J
in the supplementary material). These markers were normally
expressed in Tln1/2-dKO embryos, indicating that the differentiation
of myoblasts was not affected in the mutants. We therefore
hypothesized that, similar to mice lacking 1 integrins (Schwander
et al., 2003), defects in skeletal muscle development in Tln1/2-dKO
mice might be a consequence of perturbations in myoblast fusion
and sarcomere assembly.
Defects in sarcomere organization in Tln1/2-dKO
mice
Although no alterations in sarcomere organization were noted in
Tln1-KO or Tln2-KO mice (Fig. 3) (Conti et al., 2008), simultaneous
inactivation of Tln1 and Tln2 caused severe defects. In control
DEVELOPMENT
Severe defects in skeletal muscle development in
Tln1/2-dKO mice
We have previously shown that mice lacking 1 integrins in skeletal
muscle (refereed to as Itgb1-KOs) have a considerably more severe
phenotype than mice lacking either talin 1 or 2 (Conti et al., 2008;
Schwander et al., 2003). This, together with the observed increased
localization of talin 1 at the MTJ in Tln2-KO mice (Fig. 3P,Q),
prompted us to test whether the functions of talin 1 and 2 might
overlap. We took advantage of our previous observation that a
Tln1flox allele can be effectively inactivated in developing skeletal
muscle by an HSA-CRE transgene (Conti et al., 2008). Using these
mice and the Tln2-KO mice described here, we generated Tln1/2dKO mice, which lack both talin 1 and talin 2 in skeletal muscle (see
Materials and methods). Similar to Itgb1-KO mice (Schwander et
al., 2003), Tln1/2-dKO mice had a contracted posture (Fig. 4A) and
died shortly after birth. Immunohistochemical analysis confirmed
that expression of talin 2 was effectively ablated in muscle from
E17.5 Tln1/2-dKO embryos (Fig. 4B,C). While expression of talin
1 in skeletal muscle was too low to be detected at this stage even in
wild-type mice, the severity of the phenotype of the Tln1/2-dKO
mice compared with single mutants suggests that Tln1 was
effectively inactivated. In addition, PCR analysis of forelimb
muscles confirmed recombination of the Tln1flox/flox allele (see Fig.
S1C in the supplementary material).
Histological analysis of embryos at embryonic day 17.5 (E17.5)
revealed severe defects in muscle development throughout the body.
Although muscles could still be detected, they had an abnormal
morphology similar to the phenotype of Itgb1-KO mice (Schwander
et al., 2003). In wild-type embryos, muscle fibers were well
developed and possessed a uniform size (Fig. 4D,F). In Tln1/2-dKO
mice, a general disorganization of the muscles was evident, with
striking variations in fiber size (Fig. 4G,H,I,K).
We next determined whether skeletal muscle defects were
accompanied by changes in cell proliferation or differentiation. To
evaluate proliferation, we carried out BrdU labeling experiments.
Proliferating cells were located in several muscle groups, including
intercostals and semispinalis muscles, and were identified by coimmunostaining for BrdU and desmin (see Fig. S2A,B,E in the
supplementary material; data not shown). No difference in the
Fig. 5. Defective sarcomere assembly in Tln1/2-dKO muscle.
(A-F)Sections from E18.5 embryos were immunostained with
antibodies against vinculin (Vn) (A,B), laminin (Lm) (C,D) and tenascin C
(Tn C) (E,F). Expression levels of vinculin were reduced in Tln1/2-dKO
embryos. Laminin localized around myofibers but appeared
disorganized in mutants (arrow in D). Tenascin C was exclusively
localized at the MTJ and in periosteum in controls (E) but was expressed
in Tln1/2-dKOs in extrajunctional areas as well (arrow in F).
(G-I)Electron micrographs of intercostal muscles from wild-type (G) and
Tln1/2-dKO embryos (H,I). The cytoskeletal structure appeared
immature and disorganized in Tln1/2-dKO embryos. Disorganized
filamentous material accumulated throughout the myofiber, and
Z-bands appeared to be incompletely assembled (arrows in H,I)
compared with those of controls (arrow in G). Scale bars: 25mm in A-D;
50mm in G,H; 2mm in I.
embryos, vinculin was evenly localized at the sarcolemma (Fig. 5A).
By contrast, vinculin distribution was strongly reduced and patchy
in the disorganized and short muscle fibers of Tln1/2-dKO mice
(Fig. 5B). ECM proteins such as collagen type IV (data not shown)
and laminin were deposited around Tln1/2-deficient muscle fibers
but showed signs of disorganization and discontinuity (Fig. 5C,D).
Tenascin C expression is associated with processes of degeneration
and regeneration in dystrophic muscle (Settles et al., 1996; Taverna
et al., 1998). In control muscle fibers, tenascin C was confined at the
periosteum and the tendon (Fig. 5E). In Tln1/2-dKO muscle fibers,
tenascin C was expressed in areas distal from the MTJs (Fig. 5F).
Severe defects in sarcomere organization were noted at the
ultrastructural level. In control embryos, myofilaments and the
striation of sarcomeres were well defined (Fig. 5G). In Tln1/2-dKO
muscle, the organization of myofilaments was disrupted, and the
assembly of Z-bands appeared rudimentary (Fig. 5H,I). Amorphous
filamentous material was abundant between myofilaments. We
conclude that the skeletal muscle fiber cytoskeleton is severely
disrupted in Tln1/2-dKO muscle.
Defects in the assembly of integrin adhesion
complexes in Tln1/2-dKO mice
Immunohistochemical evaluation of the expression and
localization of components of integrin adhesion complexes in
skeletal muscle of E17.5 embryos revealed severe defects in
Tln1/2-dKO mice. In wild-type embryos, 7-, v- and 1-integrins
were clustered at MTJs (Fig. 6A,C; data not shown), whereas none
Development 136 (21)
of these proteins was localized at MTJs in Tln1/2-dKO mice (Fig.
6B,D; data not shown). Likewise, the localization of vinculin and
ILK was compromised (Fig. 6E-H, see also Fig. 5A,B), indicating
that talin 1 and 2 are essential for the assembly and clustering of
integrin adhesion complexes at MTJs. Consistent with these data,
MTJs were rarely detected in Tln1/2-dKO mice by ultrastructural
analysis. When present, they appeared abnormal: myofilaments
and the electron-dense plaque at the muscle terminus were absent
(arrows in Fig. 6I,J). Notably, these defects at MTJs differ from
those in muscle from Itgb1-KO mice, where MTJs develop
normally (Schwander et al., 2003). A possible explanation for this
difference is the presence of the integrin v subunit at MTJs
(Hirsch et al., 1994). Unlike the integrin 7 subunit, v
heterodimerizes with several  integrin subunits in addition to 1
(Hynes, 1992). The expression and localization of v were not
affected in muscle from Itgb1-KO mice, but v was no longer
present at MTJs from Tln1/2-dKO mice (Fig. 6C,D), suggesting
that the v subunit with a heterodimeric partner other than 1 is
sufficient to direct the assembly of MTJs. Taken together, our data
indicate that, in Tln1/2-dKO mice, the formation of integrin
adhesion complexes in skeletal muscle was affected, leading to
defects in the formation of the MTJs and the assembly of the
muscle fiber cytoskeleton.
Impaired fusion of Tln1/2-dKO myoblasts
Myoblast fusion depends on the alignment of the membranes of
myoblasts, the formation of prefusion complexes characterized by
electron-dense plaques, and the subsequent breakdown of the
plasma membrane. In wild-type mice, it is difficult to capture
myoblasts containing the electron-dense plaques because prefusion
complexes are rapidly resolved (Schwander et al., 2003). By
contrast, we could readily observe in developing skeletal muscle
from Tln1/2-dKO embryos unfused myoblasts containing the
electron-dense plaques (Fig. 7A-C), suggesting that fusion was
perturbed.
To evaluate directly whether fusion was affected, we established
primary cultures of fetal myoblasts from the hindlimbs of E17.5
embryos and analyzed myotube formation in vitro. After 3 days in
culture, myoblasts isolated from wild-type embryos formed
numerous long myotubes (Fig. 7D,F). By contrast, Tln1/2-dKO
myoblasts attached to the underlying fibroblast layer but failed to
fuse (Fig. 7E,F). Myotubes formed occasionally, but they were short
and assembled a rudimentary cytoskeleton: -actinin and MHCf
were recruited in a striated pattern in wild-type myotubes, but their
localization was altered in double mutants (Fig. 7G-J).
The defects in sarcomere assembly and myoblast fusion in
Tln1/2-dKO mice were similar to those observed in Itgb1-KO mice
(Schwander et al., 2003). In other cells types, such as fibroblasts and
platelets, talin mediates the assembly of integrin complexes and
integrin activation (Nieswandt et al., 2007; Petrich et al., 2007;
Tadokoro et al., 2003). Defects in either of these processes (or both)
could affect myoblast fusion. We therefore evaluated 1 integrin
expression and activation in primary cultures of fetal myoblasts from
Tln1/2-dKO mice by FACS analysis using the 9EG7 antibody, which
specifically recognizes an exposed epitope when 1 integrins are in
an active conformation (Bazzoni et al., 1995). To distinguish
myoblasts from contaminating fibroblasts, we used an antibody to
7-integrin, which is specifically expressed by myoblasts (BlancoBose et al., 2001) (Fig. 7K). 7-integrin expression levels were
normal in myoblasts from double-mutant mice (Fig. 7L,N),
excluding that defects in muscle fiber development were caused by
a reduction of the amount of integrin expressed at the cell surface of
DEVELOPMENT
3602 RESEARCH ARTICLE
Talin 1 and 2 in skeletal muscle development
RESEARCH ARTICLE 3603
myoblasts. To our surprise, labeling by the 9EG7 antibody of Tln1/2dKO myoblasts was also comparable to that of controls (Fig. 7L,M;
myoblasts are represented by the gated population), indicting that
integrin activation was not affected. To confirm these findings
further, we seeded myoblasts on collagen type IV, laminin and
fibronectin. No significant difference in adhesion was noted on any
of the tested substrates (Fig. 7O). We conclude that defects in
development of Tln1/2-dKO muscle are not likely to be caused by
an inability of 1 integrins to interact with ligands.
To investigate the mechanisms that might cause the fusion
defect in Tln1/2-dKO myoblasts further, we analyzed the
expression of connexin 43 (CX43; also known as Gja1) and the
tetraspanin CD9, which have been shown to regulate cell fusion.
CX43 is upregulated in myoblasts preceding fusion (Araya et al.,
2005; Gorbe et al., 2007) and was expressed in control and mutant
myoblasts (Fig. 7P,Q). Although the localization of CD9 was
affected in 1 integrin mutant myoblasts (Schwander et al., 2003),
it was normally expressed in myoblasts isolated from Tln1/2-dKO
mice (Fig. 7R,S). It is interesting to note that CD9 and 7 integrin
were detected, although not exclusively, at the contact surface
between talin1/2-deficient myoblasts (Fig. 7P-S, arrows),
indicating that their recruitment was not dependent on talin1/2.
Talin 1/2 are therefore likely required at a subsequent step in
myoblast fusion, potentially by linking integrins to the actin
cytoskeleton. Unfortunately, analysis of the organization of the Factin cytoskeleton of myoblasts using phalloidin was not
informative because of the small size and rounded morphology of
these cells (data not shown).
DISCUSSION
We show here that talin 1 and 2 are essential for skeletal muscle
development and function. Tln2-KO mice are viable and fertile but
develop a myopathy with centrally located nuclei that is associated
with defects in the maintenance of MTJs. When talin 1 and 2 are
inactivated simultaneously, severe defects in myoblast fusion and
sarcomere assembly are observed that are not present in the single
mutants. The defects in skeletal muscle development in Tln1/2-dKO
mice closely resemble the phenotype of muscle lacking 1 integrins.
As talin1/2-deficient myoblasts expressed functionally active 1
integrins, defects in muscle development are likely not primarily
caused by lack of an ability of 1 integrins to bind to ECM ligands
but by the disruption of their interaction with the cytoskeleton.
Consistent with this finding, recruitment of integrin effectors is
perturbed in the remaining small muscle fibers of talin1/2-deficient
mice.
Previous studies in invertebrates have shown that talin is required
for the attachment of skeletal muscle fibers (Brown et al., 2002;
Cram et al., 2003). The findings presented here and in our previous
report (Conti et al., 2008) extend these findings and show that talin
has an evolutionarily conserved function in skeletal muscle
attachment. Consistent with the higher expression levels of talin 2
in skeletal muscle compared with those of talin 1 (Conti et al., 2008;
Senetar and McCann, 2005), Tln2-KO mice developed a more
severe myopathy than talin 1 mutants, which is characterized by
centrally nucleated myofibers and prominent MTJ defects.
Nevertheless, MTJ defects were also present in muscle lacking talin
1 (Conti et al., 2008). This result could be explained by two
mechanisms. First, talin protein levels might be important. Talin 1
was redistributed to MTJs in Tln2-KO mice, but overall talin 1 levels
were not changed. Therefore, a reduction of total talin levels due to
loss of talin 2 might have caused MTJ instability. Alternatively, talin
1 and 2 at MTJs might not be entirely interchangeable. Although
talin 1 shares 74% identity with talin 2, differences in the remaining
amino acids could possibly affect protein function. This model is
consistent with previous findings. Although myoblasts express
integrin 1A, muscle fibers express the 1D isoform, which binds
with higher affinity to F-actin (Belkin et al., 1997; Belkin et al.,
1996; van der Flier et al., 1997). The I/LWEQ module of talin 2
binds with higher affinity to muscle -actin than the corresponding
module in talin 1, which in turn binds with higher affinity to nonmuscle -actin (Senetar et al., 2004). These data delineate a model
whereby the expression of 1D-integrin and talin 2 might be
important to confer a strong mechanical link between integrins and
the cytoskeleton. As ILK-deficient mice also show defects at MTJs
(Wang et al., 2008), it appears that several integrin effectors
cooperate in this process. Although speculative, talin 1 might be
more important for dynamic connections in other cells such as
fibroblasts and platelets.
Our studies also provide evidence that talin 1 and 2 cooperate to
regulate muscle fiber development. Tln1/2dKO mice show defects
in myoblast fusion and sarcomere assembly similar to 1 integrindeficient mice (Schwander et al., 2003), suggesting that talin1/2 are
DEVELOPMENT
Fig. 6. Compromised assembly of integrin
complexes in Tln1/2-dKO mice. (A-H)Sections from
E18.5 embryos were immunostained with antibodies
against 7 and v-integrins and to vinculin (Vn) and ILK.
The localization of integrins and their effectors at MTJs
was disrupted in Tln1/2-dKO muscle. Dotted lines
highlight the location of the MTJ in Tln1/2-dKO muscle.
(I,J)Electron micrographs of the MTJ of intercostal
muscles from E18.5 wild-type (I, arrows) and Tln1/2dKO (J, arrows) embryos. Muscle fibers close to MTJs in
Tln1/2-dKO were disorganized (asterisk in J). Scale bars:
50mm in A-H; 2mm in I,J.
3604 RESEARCH ARTICLE
Development 136 (21)
required for integrin functions in muscle. Previous studies have
shown that talin1/2 regulate both integrin activation and their
linkage to the cytoskeleton. Although defects in either of these
processes could lead to the skeletal muscle defects in Tln1/2dKO
mice, our data suggest that defects in cytoskeletal linkage are the
cause for the phenotype in the mutant mice. Consistent with this
model, 1 integrin expression and activation were not affected in
primary cultures of fetal myoblasts from Tln1/2-dKO mice. Similar
to our findings, it has previously been shown that talin-mediated
integrin activation is also not essential for the initial adhesion of
fibroblasts to ECM substrates (Zhang et al., 2008). Although
integrin activation by talin is essential in other cell types such as
platelets (Petrich et al., 2007; Nieswandt et al., 2007), it might be
less important in myoblasts. Kindlin-3 synergizes with talin in
regulating integrin activation in platelets (Moser et al., 2009;
Svensson et al., 2009), and other proteins, such as ILK, regulate this
process (Honda et al., 2009). Therefore, the ligand binding activity
of integrins might be regulated by different mechanisms in a celltype-specific manner.
Instead, our findings suggest that defects in Tln1/2-deficient
skeletal muscle fibers are caused by defects in the interaction of
integrins with the cytoskeleton. In support of this model, we
observed in the mutants detachment of the muscle fiber
cytoskeleton from MTJs and defects in the linkage of integrin
adhesion complexes to costameres. The fact that integrins in talin
1/2-deficient myoblasts effectively bound to ligands suggests that
steps subsequent to adhesion also led to defects in myoblast
fusion. Fusion defects in mice lacking 1 integrins are
accompanied by reduced recruitment of the tetraspanin CD9 to
the site of fusion (Schwander et al., 2003). However, CD9 was
still recruited normally in talin1/2-deficient myoblasts, suggesting
that talin1/2 act at a subsequent step. Interestingly, several
proteins that regulate integrin function and actin dynamics are
implicated in myoblast fusion. For example, the guaninenucleotide exchange factors (GRFs) Dock180 and Brag2/GEP100
are required for the fusion of myoblasts and macrophages (Laurin
et al., 2008; Pajcini et al., 2008); genetic ablation of Dock180 in
mice leads to impaired myoblast fusion (Laurin et al., 2008);
DEVELOPMENT
Fig. 7. Defective fusion but normal integrin activation in myoblasts from Tln1/2-dKO mice. (A-C)Electron micrographs of muscle from
E18.5 Tln1/2-dKO embryos revealed myoblasts at intermediate stages of fusion. Plasma membranes were aligned (arrows in A,B) and electrondense adhesion plaques were evident (arrows in C). (D-J)Cell fusion was evaluated in primary cultures of fetal myoblasts. (D,E)Cultures were
immunostained with antibodies against -actinin to label myotubes and myoblasts. In cultures from Tln1/2-dKO mice, myoblast fusion was
impaired; only a few short, dysmorphic myotubes were detected. (F)The fusion index was determined (number of nuclei in myoblasts/total number
of nuclei) (n3 mice per genotype) (*P0.035). (G-J)Immunostaining with antibodies against -actinin (G,H) and MHCf (I,J) revealed that the
cytoskeleton in myofibers from Tln1/2-dKO remained immature. Arrowheads in G,I refer to costameres. (K-O)Analysis of integrin expression and
activation by FACS. (K)Cell surface expression of 7 integrin was used to distinguish myoblasts from fibroblasts (bracket indicates 7-integrinpositive population). (L)Representative dot plots of FACS sorted myoblasts from wild-type and Tln1/2-dKO mice analyzed for 7 integrin expression
and presence of the 9EG7 epitope (detecting activated 1-integrins). Gated area represents myoblasts. (M)Histogram representing frequency of the
9EG7 epitope on myoblasts. No significant difference was observed between myoblasts from wild-type and Tln1/2-dKO mice. (N)Surface expression
levels of 7-integrin were normal in Tln1/2-dKOs (n3 controls, 5 double mutants). (O)Adhesion to collagen type IV (Coll IV), laminin (Lm) and
fibronectin (Fn) was evaluated in primary cultures of fetal myoblasts. No adhesion defects of Tln1/2-dKO myoblasts were observed (n2 per
genotype). The mean ± s.d. are indicated. (P-S)Myoblasts from E17.5 embryos were transferred to differentiation medium and stained for 7
integrin (red), CX43 (P,Q) and CD9 (R,S). The expression levels of CX43 and CD9 were normal in Tln1/2-dKO cells. 7 integrin, CD9 and occasionally
CX43, were localized at the interface of fusing myoblasts (arrows). Scale bars: 5mm in A; 500 nm in B; 250 nm in C; 20mm in P-S.
ablation of filamin C leads to defects in myogenesis (Dalkilic et
al., 2006); furthermore, ablation of FAK affects cell fusion and
myofiber regeneration (Quach et al., 2009). This raises the
interesting possibility that integrins, and their effectors such as
talin1/2, FAK and filamin C, might cooperate to regulate actin
reorganization during myoblast fusion.
Centrally nucleated skeletal muscle fibers, as observed in Tln2KO mice, are also present in several myopathies (Pierson et al.,
2005). In patients and mice with mutations that affect the
dystrophin complex, centrally nucleated fibers are an indication
of fiber degeneration and regeneration that is caused by plasma
membrane breakdown (Straub et al., 1997). By contrast, centrally
nucleated fibers in Tln2-KO mice accumulated without noticeable
plasma membrane breakdown. This resembles the situation in
mice and humans with mutations in the gene encoding the integrin
7 subunit (Hayashi et al., 1998; Mayer et al., 1997). The findings
suggest that mechanisms associated with defects in the dystrophin
and integrin adhesion complexes differ. Interestingly, mutations
in genes encoding proteins that are indirectly involved in actin
reorganization or membrane trafficking, such as myotubularin 1
(Mtm1), dynamin 2 (Dnm2) and g-actin, also lead to centrally
nucleated skeletal muscle fibers without plasma membrane
breakdown (Bitoun et al., 2005; Buj-Bello et al., 2002;
Sonnemann et al., 2006). The Tln2-KO mice presented here
provide a useful model for studying the molecular mechanisms
that lead to fiber degeneration in the absence of plasma membrane
damage. Our data also suggest that it will be important to
sequence the TLN1 and TLN2 genes in patients affected with
genetically uncharacterized congenital myopathies.
Acknowledgements
We thank Heather Elledge for technical assistance and members of the
laboratory for comments on the manuscript. This work was funded by NIH
grants NS046456 and MH078833 (U.M.). Deposited in PMC for release after
12 months.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/21/3597/DC1
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