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First published online 30 May 2007
doi: 10.1242/dev.007088
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MRC Centre for Developmental Neurobiology and Randall Division for Cell and Molecular Biophysics, New Hunt's House, Guy's Campus, King's College London, London SE1 1UL, UK.
* Author for correspondence (e-mail: simon.hughes{at}kcl.ac.uk)
Accepted 23 April 2007
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SUMMARY |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Mef2c, Mef2d, Myosin, Muscle, Zebrafish, Myofibril, Somite, tnnc, Myogenin, Hoover, prdm1, eng2a, acta1, actc1, smyhc1, myhz1, tpma, mybpc1, hsp90a
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INTRODUCTION |
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SUMMARY
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Xu et al.,
2000). Consistent with this, one model of myofibril assembly
suggests that myofibrils are initiated on actin stress fibres by formation of
z bodies, aggregates containing thin filament proteins found later in mature z
lines, which align and anchor the thin filaments
(van der Ven et al., 1999;
Wang et al., 2005). Myosin and
other thick filament proteins would then integrate into the thin filament
structure, perhaps mediated by the giant molecular ruler titin
(Lange et al., 2006). What
triggers expression of myosin and other thick filament proteins is
unknown.
High throughput studies are revealing the complex temporal succession of
gene expression during and following myoblast terminal differentiation in
culture (Penn et al., 2004;
Tapscott, 2005). As terminal
differentiation leads on to myofibrilogenesis, distinct combinations of
transcription factors are expressed
(Tapscott, 2005). Among such
transcription factors, members of the myocyte enhancer factor 2 (Mef2) and
serum response factor (SRF) families of MADS domain-containing proteins are
expressed in muscle from jellyfish to humans and upregulated during muscle
terminal differentiation (Black and Olson,
1998; Spring et al.,
2002). In culture, Mef2 can collaborate with MyoD-family proteins
to enhance myogenic conversion of non-muscle cells
(Molkentin et al., 1995). In
vivo, both Mef2 and SRF proteins can regulate many heart and skeletal muscle
genes (Balza and Misra, 2006;
Black and Olson, 1998;
Niu et al., 2005). SRF
proteins can drive C. elegans myogenesis, are required for murine
myocardiogenesis and also regulate cytoskeletal components in non-muscle cells
(Fukushige et al., 2006;
Niu et al., 2005;
Posern and Treisman, 2006).
Thus, these MADS proteins appear to regulate the specialised muscle
cytoskeleton. Yet the precise functions of Mef2 proteins during myoblast
differentiation in vivo remain unclear.
In invertebrates, the requirement for Mef2 genes is highly variable,
suggesting evolutionary flexibility as animal phyla diverged
(Dichoso et al., 2000;
Lilly et al., 1995). In
vertebrates, Mef2c is required for cardiac morphogenesis and right
ventricle formation and Mef2a mutants suffer from structural defects
in cardiac muscle (Lin et al.,
1997; Naya et al.,
2002). Mutations in MEF2A in humans are also associated
with cardiovascular disease (Bhagavatula et
al., 2004; Gonzalez et al.,
2006). However, understanding of how vertebrate Mef2 proteins
function to regulate skeletal muscle development in vivo is lacking. Mef2
function in vertebrate skeletal myogenesis is unclear because several Mef2
genes are expressed in the early myotome, and because mice lacking
Mef2c die early in development from cardiovascular defects
(Lin et al., 1997). As fish
embryos can develop for several days without a functioning heart because
oxygen is delivered by diffusion, we set out to study the in vivo function of
the Mef2 gene family in zebrafish muscle.
In zebrafish, several populations of skeletal muscle precursors arise and
undergo terminal differentiation to make contractile muscle within hours of
formation of the mesoderm (Stickney et
al., 2000). As in mice, Mef2 genes in zebrafish are expressed in
both cardiac and skeletal muscle precursors and in neural tissue
(Ticho et al., 1996)
(www.zfin.org).
Here we show that mef2d mRNA and protein are expressed in muscle
precursors, whereas mef2c appears after muscle terminal
differentiation. Mutant or antisense-mediated knockdown reveals that Mef2s are
required to upregulate several genes encoding the major components of the
thick filament, but not those of thin filaments. These findings reveal a
molecular mechanism controlling myofibril assembly.
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MATERIALS AND METHODS |
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MATERIALS AND METHODS
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DISCUSSION
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),
smob641 (Barresi et
al., 2000) and transgenic Tg(acta1:GFP)
(Higashijima et al., 1997)
D. rerio lines were maintained on King's wild-type background, and
staging and husbandry were as described
(Westerfield, 1995).
In situ mRNA hybridisation and immunohistochemistry
In situ mRNA hybridisation and immunohistochemistry were performed as
described (Hammond et al.,
2007). Fluorescein- or digoxigenin-tagged probes used were
mef2c and mef2d (Ticho
et al., 1996), myod and myogenin
(Weinberg et al., 1996),
smyhc1 (Bryson-Richardson et al.,
2005), myhz1, mylz2, mybpc1, tnnc, tpma
(Xu et al., 2000), acta1,
actc1, hsp90a (I.M.A.G.E. clones 6997034, 7284336 and 7259827,
respectively). Anti-Mef2 was raised in rabbit against a C-terminal peptide of
human MEF2 (c-21, Santa Cruz; used at 1:200). Anti-Mef2c is a rabbit
polyclonal made against aa 140-238 of human MEF2C
(McDermott et al., 1993)
(81.8% identity to aa 139-237 of zebrafish Mef2c; 1:1000), A4.1025 [recognises
all myosin heavy chain (MyHC) proteins
(Dan-Goor et al., 1990); 1:5],
F59 and S58 [anti-slow MyHC, DSHB (Devoto
et al., 1996); 1:5], EB165 (anti-fast MyHC, DSHB; 1:1),
anti-
-actinin (Sigma; 1:500), anti-tropomyosin (CH1, Sigma; 1:1000),
anti-titin T12 (a gift from D. Furst, University of Bonn, Germany; 1:1),
anti-cardiac actin (Ac1-20.4.2, Progen), anti-MyBP-C (rabbit polyclonal, gift
from M. Gautel, King's College London, UK), anti-Pax3/7 (DP312)
(Hammond et al., 2007). Slow
MyHC was detected with S58 in dual staining with EB165 and with F59 elsewhere.
Secondary reagents were Alexa (Invitrogen) or peroxidase (Vector) conjugates.
Embryos were dissected, flatmounted in glycerol or Citifluor (Agar) and images
recorded on a Zeiss Axiophot with Axiocam using Openlab software, or on a
Zeiss LSM510. Except where stated otherwise, confocal images are short stacks
of one side of the embryo at around somites 10-13.
Embryo manipulation
Antisense morpholino oligonucleotides (MOs) (Gene-Tools, 0.5-8 ng per
embryo, see Fig. 1D for
sequences) and plasmid DNA were injected into 1-to 2-cell stage embryos.
myogenin:GFP plasmid DNA (Du et
al., 2003) was kindly provided by S. J. Du (University of Maryland
Biotechnology Center, Baltimore, MD). Cell transplantations from donor embryos
injected at the 1-to 2-cell stage with 
1% FITC-dextran (Invitrogen) were
made at sphere stage into age-matched hosts. Rescue experiments were conducted
by co-injecting MOs with hs-mef2d-IRES-GFP into 1-to 2-cell stage
embryos. hs-mef2d-IRES-GFP was made by cloning the full-length coding
sequence of mef2d, generated by PCR using the 5' primer
5'-TCTAGATCTAGAATGGGACGAAAGAAAATTCAGATTCAGC-3' with a 5 nt
mismatch to the mef2d/c and mef2c/d MOs and the 3'
primer 5'-GTCGACTTATGTGACCCAGGTGTCCA-3', shuttling through
pGEM-Teasy (Promega) into the XbaI and SalI sites of
hsp70-4-MCS-IRES-mGFP6 plasmid (gift of S. Gerety and D. Wilkinson,
NIMR, London, UK). DNA sequence was verified. Embryos were heat shocked at
39°C for 1 hour, recovered for 1 hour at 28.5°C and then fixed for
immunofluorescence. For quantitative data supporting all experiments, see
Table S1 in the supplementary material.
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RESULTS |
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),
mef2d expression was seen to follow that of myod in skeletal
muscle precursors and was maintained in differentiated fibres
(Fig. 1A,D), but was
undetectable in the developing heart (data not shown). In skeletal muscle,
mef2d mRNA accumulated in parallel with actin mRNA and
before expression of genes encoding myosin heavy chain (MyHC) proteins
(Fig. 1A). By contrast,
mef2c mRNA is first detected in skeletal muscle fibres just as they
undergo differentiation and appears early in heart primordial cells
(Fig. 1A,D)
(Ticho et al., 1996). Both
mef2d and mef2c mRNAs persist until after 24 hpf
(Fig. 1A). Using a general
anti-Mef2 antibody, Mef2 proteins were detected in differentiated
cardiomyocytes and their precursor cell nuclei in heart primordium and in
skeletal myoblasts and differentiated fibres in the somites
(Fig. 1B,D and see Fig. S1 in
the supplementary material). A specific anti-Mef2c antibody detected nuclear
Mef2c protein in heart and differentiated skeletal muscle, but not in skeletal
myoblasts (Fig. 1B,D and see
Fig. S2 in the supplementary material). Anti-Mef2c does not react with tissues
expressing mef2d in the absence of mef2c mRNA, and is
therefore Mef2c-specific (Fig.
1D). Thus, accumulation of Mef2 proteins matches their mRNAs.
Several morpholino/mutant combinations deplete Mef2 proteins
We designed four antisense morpholino oligonucleotides (MOs) to block
translation of the mRNAs encoding Mef2d and Mef2c
(Fig. 1C). We also searched for
mutants affecting Mef2 and noticed a loss of Mef2c-specific immunoreactivity
and mRNA in skeletal muscle of fish carrying the hoover
(hoo) mutation (see Fig. S2C,D in the supplementary material). Like
hoo mutants, mice lacking Mef2c in neural crest lineages show jaw
defects (Verzi et al., 2007).
Using the general anti-Mef2 and specific anti-Mef2c antibodies, we showed that
each MO knocks down the predicted target(s) (summarised in
Fig. 1D, for data see Figs S1,
S2 in the supplementary material). Importantly: (1) the mef2d MO
abolishes Mef2 immunoreactivity from regions where mef2d but not
mef2c is expressed; (2) the mef2c MO ablates Mef2c protein
from muscle; (3) injection of the mef2d/c MO or mef2c/d MO
alone, which are each predicted to knockdown both Mef2c and Mef2d, or of
mef2c+mef2d MOs, eliminates all Mef2 immunoreactivity from
muscle, as does injection of the mef2d MO into the hoo
mutant (see Fig. S1F in the supplementary material). Thus, we have three
independent ways to eliminate Mef2 proteins from skeletal muscle that all gave
similar results; we hereafter refer to these treated fish as `mef2
morphants'.
Mef2 is required for myofibrilogenesis and muscle function
What is the effect of Mef2 loss? mef2 morphants lacked somitic
Mef2 protein and had a severe skeletal muscle defect
(Fig. 2 and see Figs S1-S3 in
the supplementary material). Embryos were ventrally curved with little or no
motility at any stage (Fig. 2A)
and a dramatic loss of MyHC accumulation
(Fig. 2B-D). These
manipulations did not have common effects on the heart, which in most cases
appeared wild-type at 24 hpf (data not shown). Neither knockdown of Mef2c or
Mef2d alone, nor hoo mutation, gave any obvious phenotype during the
segmentation period (see Fig. S2C and Fig. S3 in the supplementary material).
Thus, loss of Mef2c and Mef2d proteins in skeletal muscle causes a severe
defect in muscle structure and function.
mef2 morphant muscle failed to mature after terminal differentiation. In fast muscle cells of mef2 morphants, almost no MyHC was detected with anti-MyHC antibodies (Fig. 2B,C arrowheads). Nevertheless, injecting mef2d/c MO into embryos of the muscle actin reporter line Tg(acta1:GFP), which marks all terminally differentiated muscle, confirmed that both slow and fast fibres differentiated, elongated and migrated normally in mef2 morphants (Fig. 2D). Co-injecting mef2d/c MO and myogenin:GFP plasmid DNA, which expresses chimaerically but specifically in fast muscle precursors, showed that these cells undergo fusion into multinucleate fibres with three to four nuclei (Fig. 2E). Despite the presence of terminally differentiated and fused fast fibres, fast MyHC was absent from fast fibres at both protein and mRNA levels (Fig. 2C,F, arrowheads). Thus, in the absence of Mef2 proteins, fast fibre development is halted after terminal differentiation but prior to myosin expression and myofibrilogenesis.
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). mef2 morphants initiated normal terminal
differentiation of slow muscle and expressed smyhc1 and
myhz1 at the 15-somite stage (Fig.
2F and Fig. 3A,B).
Moreover, mef2 morphant slow fibres migrated to the lateral myotome
surface as normal (Fig. 2B-D).
Thereafter, however, fibre maturation and myofibrilogenesis failed. Fast MyHC
persisted with slow MyHC in slow fibres
(Fig. 2C,F) and the normal
downregulation of myogenin mRNA failed to occur in mef2
morphants (Fig. 2H). At 24 hpf,
the mononucleate slow fibres in control embryos had thick, regularly striated
myofibril bundles (Fig. 2D). By
contrast, mef2 morphant slow fibres had aggregates of MyHC at their
ends, connected by a thin myofibril with striped MyHC bands, similar to
younger fibres in unmanipulated embryos (compare 24 hpf morphants in
Fig. 2C,D and
Fig. 3D with control at 22
somites in Fig. 3C). Normal
maturation of slow fibres involves downregulation of prdm1
(Baxendale et al., 2004) and
expression of eng2a; both events failed in mef2 morphants
(Fig. 3F,G). In summary, slow
fibres in mef2 morphants fail to mature beyond the initial terminal
differentiation step and have a severe defect in myofibrilogenesis.
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Mef2 acts cell-autonomously to rescue myofibrilogenesis
To prove that Mef2 acts cell-autonomously within muscle fibres to control
myofibrilogenesis, we transplanted fluorescein-labelled cells from
mef2d/c MO donor embryos into a wild-type host and found that the
donor-derived slow cells did not contain Mef2 and failed to assemble mature
myofibrils (Fig. 4A).
Conversely, when wild-type donor cells were implanted into a mef2d/c
morphant host, single donor slow fibres contained nuclear Mef2 and had a
significantly more mature structure than the surrounding host fibres
(Fig. 4B). We conclude that
Mef2 proteins act cell-autonomously within the mononucleate slow fibres to
promote myofibril assembly.
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To prove that morphant defects are due to loss of Mef2 function and to confirm the cell-autonomous requirement for Mef2d for myofibril assembly, mef2 morphants were rescued by overexpression of a mef2d cDNA engineered to lack the MO target sequence. We injected hs-mef2d-IRES-GFP plasmid DNA together with the mef2d/c MO into 1-to 2-cell stage embryos and applied heat shock at 22 hpf. Both within somites and elsewhere, GFP-labelled cells contained immunoreactive Mef2d protein, whereas surrounding cells lacking GFP did not contain Mef2 (Fig. 4C). All GFP-expressing slow fibres had significantly better-assembled myofibrils as compared with adjacent slow fibres lacking GFP (Fig. 4C). Thus, Mef2d expression at a late stage is sufficient to rescue slow fibre maturation.
Mef2 is required for expression of major components of thick filaments
We next examined expression of genes encoding major components of the
sarcomere. In mef2 morphants, mRNAs encoding certain thick filament
proteins were downregulated, whereas mRNAs encoding thin filament proteins
were, if anything, upregulated (Fig.
5 and see Fig. S4A,C in the supplementary material). hoo
mutants treated with the mef2d MO showed similar changes (see Fig.
S4B in the supplementary material). Fast fibres failed to express myosin genes
myhz1 and mylz2 (Fig.
2B,C, Fig. 5A-C).
Interestingly, the fast myosin light chain (MyLC) gene mylz2 has
several Mef2 elements in its proximal promoter that are essential for
activation in vitro (Xu et al.,
1999). As described above, slow fibres commence normal expression
of both myosin genes smyhc1 and myhz1
(Bryson-Richardson et al.,
2005; Xu et al.,
2000), but remain immature, failing to downregulate
myhz1. Expression of the slow fibre-specific thick filament gene,
mybpc1, was greatly reduced (Fig.
5D). Thus, with the exception of the MyHC genes expressed in
nascent slow fibres, all genes examined encoding thick filament-associated
proteins were downregulated in mef2 morphants. By contrast, probes to
thin filament genes encoding skeletal and cardiac actin, acta1 or
actc1, and Tg(acta1:GFP) and troponin C
(tnnc), showed no change in mRNA level in mef2 morphant
somites (Fig. 2D,
Fig. 5E,G and data not shown),
whereas -tropomyosin (tpma) and hsp90a, a
gene implicated in myosin folding (Barral
et al., 2002), were upregulated
(Fig. 5F,H). We conclude that
Mef2c and Mef2d redundantly drive expression of genes involved in thick
filament assembly in zebrafish skeletal muscle.
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). Although
MyHC accumulation in fast fibres failed completely, some MyHC clusters and
single myofibrils did form in slow fibres
(Fig. 6A,F). In mef2
morphants, actin was present in the cytoplasm of both fast and slow fibres,
but was only located in a regular sarcomeric array in the residual thin
myofibrils of slow fibres (Fig.
6A). Actin, along with other thin filament components,
-tropomyosin and -actinin, showed diffuse cytoplasmic staining,
often in puncta, concentrated at fibre ends and near the plasma membrane. In
slow fibres, actin extended well beyond regions of residual MyHC accumulation
(Fig. 6A). In morphant embryos,
titin arrays and ordered I-Z-I structure were only observed where
MyHC-containing sarcomeres formed (Fig.
6B-D,F). Most of the rudimentary sarcomeric structure that formed
in mef2-morphant slow fibres was concentrated near the somite border.
Cytoplasmic puncta and the level of thick filament components, such as MyHC
and slow myosin-binding protein C, were greatly reduced as compared with thin
filament proteins (Figs2,
3;
Fig. 6A,E). Overall, there is a
more severe disruption of thick filament components, and aggregates of thin
filament proteins form, often at the cell periphery. |
DISCUSSION |
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DISCUSSION
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;
Wang et al., 2005;
Xu et al., 2000). Moreover,
isoforms of thin and thick filament proteins are substituted at different
times during fibre maturation (Schiaffino
and Reggiani, 1996). How are the genes independently regulated?
Actin expression rapidly succeeds myod expression in zebrafish, which
we suggest constitutes a first phase of myofibrilogenesis. Our data show that
Mef2s drive thick filament gene expression in nascent fast fibres, indicating
that Mef2 activity directs a second phase of myofibrilogenesis. It is striking
that the Mef2-related protein Srf regulates the actin cytoskeleton in both
muscle and non-muscle cells (Niu et al.,
2005; Posern and Treisman,
2006; Treisman,
1987). Srf is highly expressed as zebrafish muscle differentiates
(Vogel and Gerster, 1999)
(www.zfin.org),
raising the possibility that it drives thin filament genes during the first
phase. The widespread low-level expression of Srf and Mef2 proteins in
non-muscle tissue at later stages of mammalian development might indicate
roles for each, beyond muscle, in regulation of the actin and myosin
cytoskeletons, respectively.
Our findings show that Mef2 proteins are required for the maturation and
assembly of sarcomeric structure in nascent muscle fibres and provide in vivo
evidence consistent with one suggested mechanism of myofibril assembly.
Myofibrilogenesis is initiated by formation of pre-myofibrils, in which
z-bodies decorate actin stress fibres near the cell periphery
(Wang et al., 2005). The
actin-, -actinin-, titin z-line region- and
-tropomyosin-containing puncta observed in mef2 morphants
might be z-bodies. Thus, in the absence of the second phase of
myofibrilogenesis, zebrafish muscle appears to be stuck in a pre-myofibril
state. Mef2 is required to permit these z-body-like structures to assemble
efficiently into large z-lines. In fast fibres, the absence of thick filament
components might account for the failure of sarcomere assembly, as occurs when
MyLC is lacking in heart (Rottbauer et
al., 2006). The ability of slow fibres, which provide a
mononucleate fibre scaffold for the myotome, to initiate myofibrilogenesis in
mef2 morphants is consistent with their early myosin expression and
weak early motility and is reminiscent of the lack of muscle defects in the
mononucleate muscles of C. elegans embryos lacking MEF-2
(Dichoso et al., 2000).
However, mef2 morphant slow myofibrils are immature: they retain fast
MyHC but lack MyBP-C and fail to grow, similar to the phenotype of mice
lacking titin's M-line region (Weinert et
al., 2006). Strikingly, although fast fibres are devoid of
myofibrils, all slow fibres appear to have a single thin myofibril spanning
their length. This suggests that myofibril initiation and growth are two
separate processes, at least in slow fibres. In slow fibres, the role of Mef2
appears to be to permit growth of these initial myofibrils. Therefore, the
data implicate Mef2 in a second transcriptional phase occurring in both fast
and slow nascent fibres.
|
; Nakagawa et al.,
2005; Sandmann et al.,
2006; Tapscott,
2005; van Oort et al.,
2006; Xu et al.,
2006; Haberland et al.,
2007)? In cultured cells, Mef2d seems to act together with
MyoD-family transcription factors to promote a late step in terminal
differentiation of myoblasts (Penn et al.,
2004; Tapscott,
2005). In zebrafish, as in mice, the Myod family drives
differentiation of the earliest muscle fibres
(Hammond et al., 2007) and
these cells express Mef2d but not Mef2c. Nevertheless, the earliest fish
muscle precursors undergo apparently normal terminal differentiation, actin
expression, elongation and fusion into myotubes in the absence of detectable
Mef2 protein. This is surprising when one considers the potent ability of Mef2
to cooperate with Myod to drive myogenic conversion of 10T1/2 cells
(Molkentin et al., 1995). Our
data indicate a need to reexamine myogenic conversion assays performed using
expression of thick filament components as a readout: perhaps, in vivo, Mef2
is not normally required for cell-cycle exit and initiation of muscle gene
expression indicative of terminal differentiation. Despite the lack of
immunologically detectable Mef2 protein, morpholino knockdowns might not
represent a null condition. However, we did not observe under-representation
of knockdown cells in muscle in chimaeras. We conclude that normal levels of
Mef2c and Mef2d are not required for muscle fibre terminal
differentiation.
Could the mef2a gene, which is expressed in fast muscle after
differentiation (Hammond et al.,
2007; Ticho et al.,
1996; Wang et al.,
2006)
(www.zfin.org),
compensate for loss of other Mef2s? Our mef2 morphants lack Mef2
immunoreactivity, even though the anti-Mef2 serum was raised against human
MEF2A and detects zebrafish Mef2a protein in the brain (Y.H. and S.M.H.,
unpublished). So Mef2a is unlikely to provide significant compensation. Our
data show that Mef2c or Mef2d is required for thick filament mRNA and protein
accumulation in fast fibres. Interestingly, body curvature is seen after
mef2a knockdown in older embryos
(Wang et al., 2006). We
suggest that Mef2a contributes to myofibrilogenesis at later stages.
The failure of fibre maturation and growth in mef2 morphants fits
well with the suggested roles of Mef2 in a late differentiation step and in
the adult fibre response to electrical activity and physical load
(Jordan et al., 2005;
Nakagawa et al., 2005;
Penn et al., 2004;
Wu et al., 2001). In cultured
myotubes, Mef2 binds to many genes, and functional targets are likely to be
diverse (Black and Olson, 1998;
Nakagawa et al., 2005). Our
data indicate that the defects in sarcomere assembly result from a requirement
for Mef2 in the activation of various thick filament genes. However, Mef2
might not act directly on all thick filament genes, although many contain Mef2
sites and E boxes, through which Mef2 can act
(Beylkin et al., 2006;
Molkentin et al., 1995). Mef2
might also act elsewhere, for example on genes required for muscle fibre
attachment, which also appears defective (Y.H. and S.M.H., unpublished). In
Drosophila, Mef2 binds to and is required for correct quantitative
expression of hundreds of genes (Sandmann
et al., 2006), but we see no contradiction to our finding that
Mef2 is not essential for terminal differentiation in fish. One explanation,
given that Drosophila and C. elegans Mef2 differ greatly in
their importance for myogenesis (Dichoso
et al., 2000), is that the role of Mef2 has evolved after
divergence of vertebrates and invertebrates. Alternatively, the first
essential role of Drosophila Mef2 in somatic muscle might be similar
to that in fish: Mef2-null flies still form nascent myofibres, but
fail to express much myosin or form proper attachments
(Lilly et al., 1995;
Bour et al., 1995;
Ranganayakulu et al., 1995;
Prokop et al., 1996;
Gunthorpe et al., 1999).
Indeed, muscle defects in adult flies lacking Mef2 arise in the absence of
changes in myofibrillar actin expression
(Baker et al., 2005). Our
analysis is restricted to the earliest stages of skeletal muscle formation,
but reveals the importance of Mef2d and Mef2c in turning the nascent myotube
into a mature fibre.
The finding of a new regulatory step in embryonic myofibrilogenesis prompts
further analysis of the role of Mef2 in fetal and adult animals. Mef2 has been
implicated in regulating size, metabolism and type of adult muscle cells
(Kolodziejczyk et al., 1999;
Liu and Olson, 2002;
Wu et al., 2000). Our work
raises the possibility that myofibrilogenic thick filament protein turnover
might control muscle strength and character in the adult. Interestingly, the
defects we observe in nascent fibres are reminiscent of those in some forms of
human acute quadriplegic myopathy, in which thick filament gene expression can
be specifically depleted leading to catastrophic paralysis in a significant
fraction of critical care patients
(Larsson et al., 2000).
Although Mef2 target genes are likely to have diversified as complex muscle
evolved, it will be interesting to determine which aspects of Mef2 function in
nascent fibres persist in the adult.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/13/2511/DC1
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ACKNOWLEDGMENTS |
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DISCUSSION
REFERENCES |
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