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First published online January 13, 2009
doi: 10.1242/10.1242/dev.028019
Randall Division for Cell and Molecular Biophysics and MRC Centre for Developmental Neurobiology, New Hunt's House, Guy's Campus, King's College London, SE1 1UL, UK.
Author for correspondence
(simon.hughes{at}kcl.ac.uk)
Accepted 17 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Muscle, Zebrafish, Myosin, Slow, Fibre, Fast, Head, Fin, mrf4 (myf6), myod, myf5, Myogenin, Hedgehog, prdm1, pax3, meox1, hsp90, mef2d
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Human diseases and murine genetic manipulations that affect specific muscle
groups suggest that such differences in muscle form are underlain by distinct
populations of myogenic cells (Sambasivan
and Tajbakhsh, 2007). How muscle diversified at a molecular
evolutionary level is unclear, but changes in the utilisation and function of
myogenic regulatory factors of the myod family (MRFs) are possible
contributors. Analysis of murine MRF function has revealed that Myf5,
Myod and Mrf4 (Myf6) are not, individually, essential
for viability (Kassar-Duchossoy et al.,
2004; Kaul et al.,
2000; Rudnicki et al.,
1992; Zhang et al.,
1995). Yet mice lacking function of all three of these genes fail
to make skeletal muscle (Kassar-Duchossoy
et al., 2004; Rudnicki et al.,
1993). Lack of one MRF usually delays myogenesis until another MRF
is expressed, although there is debate about the cell autonomy of compensation
(Braun and Arnold, 1996;
Braun et al., 1994;
Gensch et al., 2008;
Haldar et al., 2008;
Kassar-Duchossoy et al., 2004;
Tajbakhsh et al., 1997). Owing
to the extensive growth of secondary muscle fibres, it has been difficult to
determine whether specific differentiated muscle cells are lacking in single
MRF mutant adults. An exception is the lack of tail epaxial muscle in
Myf5-null alleles
(Kassar-Duchossoy et al.,
2004), which emphasises one overriding theme to emerge from murine
MRF knockout studies of somite myogenesis: that Myf5 function is
initially required for myogenin (Myog) and Myod expression
and myogenesis in the epaxial somite domain
(Kablar et al., 1997;
Kassar-Duchossoy et al., 2004;
Tajbakhsh et al., 1997).
Perturbation of the Myf5/Mrf4 locus also prevents early hypaxial
Myod expression, possibly owing to loss of Mrf4 function
(Kassar-Duchossoy et al.,
2004; Tajbakhsh et al.,
1997). Myod function is required for early hypaxial
myogenesis, which is probably dependent on Pax3
(Kablar et al., 1997;
Tajbakhsh et al., 1997).
Consistent with this, Myod is the only MRF thought to be able to drive
myogenesis in the absence of any other MRF, but is not efficient
(Kassar-Duchossoy et al.,
2004; Rawls et al.,
1995; Valdez et al.,
2000). The consensus view remains that the combined activity of
several MRFs promotes myogenesis
(Weintraub, 1993).
Null mutation in Myog, however, is perinatal lethal owing to a
failure of terminal differentiation of a large proportion of myoblasts in some
muscles. Mutants have significant myogenesis at early stages of development
and there is efficient terminal differentiation of Myog-null
myoblasts in cell culture (Hasty et al.,
1993; Nabeshima et al.,
1993; Venuti et al.,
1995). Early reports presumed that Mrf4, being
structurally related to Myog and abundantly expressed in
differentiated muscle fibres, permitted this differentiation. However,
pairwise ablation of Myog and either Mrf4, Myod or
Myf5/Mrf4 in mice did not worsen the Myog-null phenotype
(Rawls et al., 1995;
Rawls et al., 1998). The role
and relationship of Myog to the other MRFs remains ambiguous.
Here, we turn to zebrafish in the hope of finding shared and divergent
roles for individual MRFs in myogenesis. Each zebrafish somite generates at
least four populations of muscle fibres within the first 15 hours of
somitogenesis, a timing and diversity that are akin to amniote somite
myogenesis (Kahane et al.,
2001; Kassar-Duchossoy et al.,
2004). Two Hedgehog (Hh)-dependent medial mononucleate slow muscle
fibre types derive from the myf5- and myod-expressing
adaxial cells lying adjacent to the notochord
(Blagden et al., 1997;
Coutelle et al., 2001;
Weinberg et al., 1996;
Wolff et al., 2003).
Approximately three muscle pioneer fibres remain at the dorsoventral midline,
whereas 
20 superficial slow fibres migrate to the lateral myotome surface
(Devoto et al., 1996). More
lateral paraxial cells within the somite also express the MRFs myf5
and myod (Coutelle et al.,
2001; Weinberg et al.,
1996) and give rise to two kinds of multinucleate fast fibre: the
Fgf8-dependent lateral fast fibres and the Fgf8-independent medial fast
fibres, a subset of which becomes the fast Engrailed-expressing cells as a
result of later Hh signalling (Groves et
al., 2005; Wolff et al.,
2003). Both slow and fast lineage cells also express myog
and mrf4 (Hinits et al.,
2007; Weinberg et al.,
1996). Using predicted null mutations in myf5 and
mrf4 and morpholino antisense oligonucleotides (MOs) to knockdown
myod and myog function, we show that distinct muscle cell
lineages use different combinations of MRFs to drive myogenesis. myf5
mutants die during larval growth, whereas mrf4 mutants are viable.
Medial slow and fast fibres require either Myf5 or Myod to undergo myogenesis.
Lateral myogenesis is driven by Myod alone. No Mrf4-initiated myogenesis was
observed. Our data show that Myf5 and Myod are likely to have been the
ancestral triggers of vertebrate myogenesis. However, neither Myf5/Myod- nor
Myod/Myog-requiring cell types are restricted to epaxial or hypaxial somite
domains. We speculate that the common ancestor of teleosts and amniotes had
several kinds of somite skeletal muscle cell that utilised Myf5 and Myod
differently.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
Tg(mylz2:GFP) (Moore et al.,
2007) were maintained on King's College wild-type background, and
staging and husbandry were as described
(Westerfield, 1995).
myf5hu2022 and mrf4hu2041 were on a Tl
background and were genotyped by sequencing of PCR products amplified from fin
clip or embryo genomic DNA using primers
5'-GCAACTTGCGCTTCGTCTCC-3' and
5'-CATCGGCAGGCTGTAGTAGTTCTC-3' for myf5, and
5'-TGAATCTGAAGCCCCGCAAC-3' and
5'-ATTGCTCTGCTCCTGCTCATCC3'-for mrf4.
In situ mRNA hybridisation, immunohistochemistry and western analysis
In situ mRNA hybridisation and immunohistochemistry were performed as
described (Hinits and Hughes,
2007). The fluorescein- or digoxigenin-tagged probes used were
mef2ca and mef2d (Ticho
et al., 1996), mrf4
(Hinits et al., 2007),
myf5 (Groves et al.,
2005), myod and myog
(Weinberg et al., 1996),
eng1a and eng2a (Ekker
et al., 1992), mylz2, tpma and myhz1
(Xu et al., 2000),
pax3 (Hammond et al.,
2007), meox1 and lbx2
(Neyt et al., 2000),
smyhc1 (Bryson-Richardson et al.,
2005), actin (actc1, IMAGE 7284336), hsp90a
(IMAGE 7259827), prdm1 (Baxendale
et al., 2004), twist2
(Morin-Kensicki and Eisen,
1997) and ptc1
(Concordet et al., 1996).
Antibodies were against Myog (Devoto et
al., 2006), Myod (Hammond et
al., 2007), Mef2 (Hinits and
Hughes, 2007), MyHC (A4.1025), fast MyHC (EB165)
(Blagden et al., 1997), slow
MyHC [F59 or S58 (Devoto et al.,
1996)] and GFP (Torrey Pines). Western analysis was performed as
described (Blagden et al.,
1997).
Morpholino knockdown
MOs (Gene-Tools) were injected into 1- to 2-cell stage embryos unless
otherwise stated. MO sequences are: myog MO-1,
5'-GCTGGTTTAGAGTCCACCCGCTGTG-3'; myog MO-2,
5'-GGGTTGGTCTTCGAAAAGCTCCATGT-3'; myod,
5'-ATATCCGACAACTCCATCTTTTTTG-3'; and myf5,
5'-GATCTGGGATGTGGAGAATACGTCC-3'.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Hinits et al., 2007;
Maves et al., 2007)
(Fig. 1). As myog is
expressed in adaxial cells, we tested its requirement for slow myogenesis in
morphants. Two non-overlapping myog MOs blocked Myog protein
accumulation as detected by western analysis and ablated nuclear Myog
immunoreactivity (see Fig. S1 in the supplementary material). At the 15-somite
stage (15 ss), myog morphants showed no change in any of the adaxial
markers examined (Fig. 1). A
myod MO also reduced Myod accumulation (see Fig. S1 in the
supplementary material) (Hammond et al.,
2007). Injection of sufficient doses of myod MO reduced
myog expression and mildly delayed, but did not prevent,
smyhc1 expression and slow muscle differentiation
(Fig. 1). Expression of the
thin filament genes 
-actin and -tropomyosin
(tpma) was not affected [Fig.
1; as for desmin
(Maves et al., 2007)]. Myf5
knockdown did not affect any of the markers examined
(Fig. 1). Although
mrf4 is Myf5/Myod-dependent, it is not expressed until late in slow
muscle differentiation and, therefore, does not trigger slow myogenesis
(Hinits et al., 2007) (see
below). Pairwise loss-of-function of myog with either myf5
or myod did not prevent slow fibre formation (see below). We conclude
that either Myf5 or Myod alone can drive slow myogenesis, but that Myod
activity might be rate limiting in the wild-type condition.
Myf5 is dispensable for embryonic and larval development
Injection of both myf5 and myod MOs blocked expression of
late myogenesis markers and diminished, but did not entirely eliminate,
expression of mRNAs encoding the earlier-expressed thin filament proteins
(Fig. 1)
(Hinits et al., 2007). As we
have no antibody to Myf5, we were concerned that the myf5 MO might
not ablate production of all Myf5 protein. The zebrafish strain
myf5hu2022, in which cysteine 59 of the myf5 gene
has changed to a stop codon, was obtained from the Sanger Centre TILLING
screen (Stemple, 2004) (see
Fig. S2 in the supplementary material). myf5hu2022 is a
predicted null, truncating Myf5 upstream of the basic helix-loop-helix (bHLH)
domain (Fig. 2A). Heterozygous
myf5+/hu2022 incrosses yielded clutches of normal size and
the survival of embryos and larvae was good throughout early development. No
gross morphological defects were observed and larvae moved and fed normally
(Fig. 2B). The similarity of
myf5hu2022/hu2022 (hereafter called
myf5hu2022) and myf5 morphants suggests that the
latter represent strong loss-of-function (see below).
myf5hu2022 homozygotes survive to adulthood less well
than their siblings; no adults have been found
(Fig. 2C). Despite the lack of
early muscle defects, in situ mRNA hybridisation of myf5 at 15 ss
revealed a reduction in myf5 mRNA accumulation, presumably reflecting
nonsense-mediated decay, which was not observed in myf5 morphants
(Isken and Maquat, 2007)
(Fig. 2D). We conclude that a
wild-type zygotic myf5 locus is not essential for early myogenesis,
but might confer a selective advantage during growth and maturation of fish
into adulthood.
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; Kassar-Duchossoy et al.,
2004; Kaul et al.,
2000). Analysis of zebrafish myf5hu2022
mutants revealed a 1- to 2-hour delay in early adaxial myog
expression and a 30-minute delay in expression in fast muscle precursors
(Fig. 2E). Thus, Myf5
contributes to the rate of myogenesis.
We used myf5hu2022 to examine the role of Myf5 in
muscle formation. All embryos from myf5hu2022 heterozygote
crosses expressed myod, myog, mrf4, mef2ca, pax3, prdm1, ptc1,
smyhc1, -actin and tpma genes normally at 15 ss
(see Fig. S2C in the supplementary material; data not shown). Where analysed,
injection of myf5 MO gave the same result
(Fig. 1; data not shown). When
myod MO was injected into a myf5+/hu2022 incross,
25% of the resulting embryos lacked expression of slow terminal
differentiation markers, confirming the previous conclusion based on
myf5 MO (Fig. 3A;
59/243, for all quantitative data see Table S1 in the supplementary material).
Thus, Myf5 and Myod cooperate to drive adaxial slow myogenesis in
zebrafish.
MRFs are not required for adaxial prdm1 expression
Which step in slow myogenesis is affected by loss of Myf5 and Myod
function? Expression of most regulatory genes implicated in early myogenesis,
such as myog and mef2d (presomitic adaxial markers) and
mrf4 and mef2ca (adaxial slow fibre markers), is ablated in
double morphants or myf5 mutant;myod morphants
(Fig. 1,
Fig. 3A; data not shown)
(Hinits and Hughes, 2007;
Hinits et al., 2007;
Weinberg et al., 1996). By
contrast, expression of prdm1 and ptc1 mRNAs, which encode
proteins required for proper Hh-dependent slow fibre differentiation
(Baxendale et al., 2004;
Concordet et al., 1996), was
unaffected by loss of Myf5 and Myod function, showing that the slow muscle
precursor cells were still present (Fig.
3A). Thus, Myf5 and Myod drive a specific module of gene
expression within slow muscle precursors.
Myod, but not Myf5, drives cranial and pectoral fin myogenesis
It has been reported, based on MO knockdown of myf5, that Myf5 is
required for myogenesis of specific head muscles and brain morphogenesis
(Chen and Tsai, 2002;
Lin et al., 2006). We analysed
cranial, fin and hypaxial myogenesis in myf5hu2022 embryos
and larvae and did not detect any defects, consistent with the viability of
some 3-week-old fry (Fig.
3B-D). Moreover, using the myf5 ATG MO, with a sequence
identical to that reported previously (Lin
et al., 2006), we only obtained head defects at high, probably
toxic, MO concentrations (data not shown). As maternally expressed
myf5 was not detected (Chen et
al., 2001; Coutelle et al.,
2001), myf5 is dispensable for non-somitic myogenesis in
zebrafish embryos.
|
). We
conclude that Myod is the primary MRF driver of myog expression and
early myogenesis in cranial muscles.
Unlike most cranial muscle, fin muscle is somite derived and composed
exclusively of fast fibres that express the lbx2 gene (previously
known as lbx1 or lbx1h) at early stages
(Neyt et al., 2000;
Patterson et al., 2008;
Wotton et al., 2008). Myod
knockdown prevented myog mRNA and myosin accumulation but not
lbx2 expression in fin buds, whereas myf5hu2022
mutants appeared normal (Fig.
3E). Therefore, Myod is the major MRF required for fast pectoral
fin myogenesis.
Myf5 drives hsp90a expression in fast muscle precursors
We have shown that Myod is important for the formation of certain fast
fibres in cranial, fin and somitic muscle. Yet myf5 is the first MRF
expressed in early presomitic mesoderm and tailbud and persists in fast muscle
precursors of the posterior somite (Chen
et al., 2001; Coutelle et al.,
2001; Stellabotte et al.,
2007). Loss of myf5 function decreased expression of
hsp90a in the anterior presomitic mesoderm, but other markers of this
tissue, such as meox1 and myod, were unaffected
(Fig. 3F; see Fig. S2C in the
supplementary material). Anteriorly, in the nascent somites, hsp90a
mRNA expression was upregulated, dependent on Myod activity
(Fig. 3F). No defect in late
muscle gene expression was found in myf5 mutants. Hsp90a is required
for proper sarcomere assembly (Du et al.,
2008; Hawkins et al.,
2008). Thus, early Myf5 function may prime the somite for fast
myogenesis, but is not essential for it.
Myf5 and Myod are essential for fast myogenesis
myf5 and myod double morphants lacked myog mRNA
at 10 ss and muscle at 15 ss (Hammond et
al., 2007; Maves et al.,
2007) (Fig. 1,
Fig. 4A). By 24 hpf, however,
small amounts of fast muscle formed in the medial somite
(Fig. 4B,C). Knockdown of Myod
in myf5hu2022 homozygotes further reduced fast muscle
(Fig. 4B,D). The addition of
myf5 MO did not worsen the phenotype of myod MO;
myf5hu2022 mutants. Thus, the myf5 mutant
revealed that myf5 morphants are hypomorphic.
It is likely that myod morphants also retain low levels of Myod function, even though we detected little Myod protein (see Fig. S1 in the supplementary material). In the myf5hu2022 homozygote background, myod MO either completely ablated or greatly reduced fast muscle in individual somites. However, embryos entirely lacking fast muscle were never observed; a few residual fast muscle cells were present in the medial region of many somites, but lateral to undifferentiated cells that presumably represent adaxial cells or sclerotome (Fig. 4D). Residual fast muscle cells expressed myog by 20 ss, and went on to differentiate and accumulate myhz1, tpma and mylz2 mRNA at 24 hpf, but did not elongate or fuse (Fig. 4A,D). Lower doses of myod MO left more fast muscle in each somite. Therefore, the level of Myod in myf5hu2022 mutants determines the quantity of medial fast cells that can express myog and differentiate. Clearly, the medially located residual muscle required less Myf5/Myod activity than did other fast muscle.
|
). Could Mrf4 activity account for the residual muscle in Myf5
plus Myod knockdown fish? TILLING yielded the mrf4hu2041
allele, a predicted null mutation in which a stop codon in the basic domain
eliminates the helix-loop-helix region essential for dimerisation and myogenic
function (Fig. 5A; see Fig. S3
in the supplementary material). mrf4hu2041 mutant fish
were viable and fertile (Fig.
5B). Myosin accumulation in slow, fast, cranial and fin muscles
occurred on time. Among a series of myogenic or muscle-specific genes examined
at various developmental stages, none was altered in
mrf4hu2041 homozygotes. Although we have no proof that the
mrf4hu2041 allele is null, it appears that mrf4
is not an essential gene in zebrafish.
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Both epaxial and hypaxial somitic compartments have two modes of fast myogenesis
In mouse somites, Mrf4 drives some lateral/hypaxial myogenesis, but Myod is
also involved in this region
(Kassar-Duchossoy et al.,
2004). In fish, Myod is the major myogenic gene in the lateral
somite. Knockdown of Myod alone depletes lateral muscle in both epaxial and
hypaxial somites (Fig. 4B,D)
(Hammond et al., 2007).
Expression of Myod was first observed in the lateral somite in posterior
border cells (pbcs), but these cells only accumulated myog and
mef2d mRNAs at 15 ss, just prior to becoming fast muscle
(Fig. 1,
Fig. 4E). Myod is required for
pbc myog expression (Maves et
al., 2007). However, to ablate mef2d mRNA, Myf5, which is
transiently expressed in pbcs, also had to be removed
(Fig. 4E). Rostrally, however,
myod morphants lacked lateral mef2d mRNA, suggesting that
without Myod lateral cells cannot express myog, maintain
mef2d or undergo terminal differentiation
(Fig. 4E).
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Myod and Myog drive fast muscle differentiation
Whereas myog is expressed at low levels in slow muscle precursors,
it is abundant just before terminal differentiation in both medial and lateral
fast precursors, both in wild-type and manipulated embryos (Figs
1 and
4). For example, myog
mRNA accumulated prior to mylz2 mRNA in residual medial fast fibres
of myod MO-injected myf5hu2022 mutant embryos
(Fig. 4D). Upon injecting both
myog and myod MOs into myf5hu2022 mutant
embryos, all muscle was ablated (Fig.
6A). We conclude that Myog accumulation is essential for residual
medial fast fibre differentiation.
Could activation of myog expression in the medial somite of myf5 mutant;myod morphant embryos occur independent of residual Myod activity? We think not, for three reasons. First, although myog mRNA appears in medial fast fibres prior to their differentiation, Myog knockdown alone has no detectable effect on fast myogenesis (Fig. 1, Fig. 6D). Second, knockdown of Myf5 and Myod prevents all early myog expression, including that in the medial somite region that is destined to make the first fast muscle. Third, the occurrence of individual somites entirely lacking muscle in myf5 mutant;myod morphant embryos argues against a specific myog-dependent population in the medial region of each somite. We propose that residual Myod activity drives myog expression, but proof will await a null myod mutant.
We next tested whether Myod or Myf5 alone can drive fast myogenesis.
Injection of myog MO into myf5hu2022 mutant
embryos did not detectably reduce muscle
(Fig. 6B). By contrast, when
myog and myod MOs were co-injected into
-actin:GFP transgenic embryos, essentially all fast muscle was
ablated (Fig. 6C,D). Slow
muscle fibres were still present in normal numbers and accumulated GFP, slow
Myosin heavy chain (MyHC) and Mef2. However, the somewhat U-shaped somites had
shorter, disorganised slow fibres and less Engrailed, a marker of muscle
pioneer cells (Fig. 6C,
Fig. 7C). Both myhz1
transcript and fast MyHC were essentially absent from myog;myod
morphants (Fig. 6D). Therefore,
Myod drives fast muscle differentiation in the absence of Myog. Moreover, Myf5
drives significant fast muscle differentiation in the absence of Myod through
activation of myog expression.
Hedgehog signalling can drive medial fast myogenesis
A unique population of medial fast fibres, defined by weak Engrailed
expression and previously designated MFFs, are dependent on midline Hh
signalling at relatively late stages
(Wolff et al., 2003). We
therefore examined the Hh dependence of our residual medial fast cells. The
Hh-signal-blocking drug cyclopamine completely ablated residual myhz1
expression in myf5hu2022 mutants injected with
myod MO (Fig. 7A).
Thus, Hh can promote medial fast cell differentiation, perhaps by enhancing
residual Myod activity.
Hh is not required for most medial fast myogenesis. Treatment of myod morphants with cyclopamine produced little if any change in myhz1 expression (Fig. 7A). Thus, Myf5 and Myog can trigger fast muscle differentiation without Hh.
We could not detect Engrailed in residual fast fibres in
myf5hu2022 mutants injected with myod MO or in
myf5;myod double morphants (Fig.
7B; data not shown). Thus, the residual cells are not MFFs, as
originally defined. However, the continued presence of unmigrated adaxial
cells expressing ptc1 in myf5;myod morphants might reduce Hh
signal strength in the region of the residual fast fibres, consistent with the
original proposal that MFF Engrailed expression is triggered by enhanced Hh
signalling once slow fibres migrate (Wolff
et al., 2003). Interestingly, myod MO alone prevented
eng2a expression and reduced Engrailed protein in the somite, without
obviously affecting eng1a mRNA
(Fig. 7B). Moreover,
myod;myog double morphants lacked eng2a and had little
eng1a mRNA (Fig.
7B).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Chen et al.,
1994). In deuterostomes, MRFs have greater importance: the single
Ciona MRF drives myogenesis
(Meedel et al., 2007). The
duplicated MRFs in mice and zebrafish are essential for skeletal myogenesis,
but can partially compensate for one another. There are two major modes of myogenesis in the early zebrafish (Fig. 7D). In the `medial' mode, Myf5 and Myod act together to drive slow myogenesis and the first fast myogenesis in the medial somite. In the `lateral' mode, Myod is uniquely required for lateral muscle precursor myogenesis, including that in the pectoral fin. We also show that myog expression is dependent on Myf5 and Myod and contributes to both medial and lateral modes of fast muscle differentiation. Zebrafish cranial muscles develop by the Myod-dependent mode, in contrast to murine head muscle. The data reveal strong similarities, but also significant differences, in the myogenic programmes regulating specific fibre populations across vertebrate phylogeny.
Role of zebrafish MRFs
Predicted null mutations in myf5 and mrf4 demonstrate
that these genes are not essential for early myogenesis. Loss of Myf5 delayed
myog and hsp90a expression in slow and fast muscle
precursors, but this soon recovered due to Myod action. In mice, Mrf4, not
Myod, appears to permit recovery of epaxial muscle in
Myf5loxP/loxP mutants, after a longer delay
(Kassar-Duchossoy et al.,
2004). In fish, unlike mice, ablation of both myf5 and
myod led to loss of essentially all muscle formation. Interpretation
of murine Myf5 knockouts has been plagued by cis effects on the
linked Mrf4 gene
(Kassar-Duchossoy et al.,
2004). Because our myf5-null allele is a single base
change and can be phenocopied by MO knockdown, we can rule out cis effects on
mrf4, which is likewise linked to myf5 in zebrafish. Perhaps
unsurprisingly, mutants are more effective than morphants. In
myf5hu2022 mutants injected with myod MO, medial
Hh signalling permits low levels of residual Myod to activate myog
and thereby drive some myogenesis. Mrf4 does not account for residual muscle.
We conclude that either Myf5 or Myod is required for myog expression
and muscle differentiation throughout the early zebrafish embryo.
|
). Our
mutation is predicted to disrupt both mrf4 transcripts upstream of
the HLH domain required for myogenesis
(Hinits et al., 2007;
Wang et al., 2008). Murine
Mrf4 initiates myogenesis in certain hypaxial cells in embryonic somites
(Kassar-Duchossoy et al.,
2004). Fish mrf4 expression is restricted to
differentiated muscle fibres (Hinits et
al., 2007). Even though ectopic Mrf4 expression is potently
myogenic (see Fig. S3B in the supplementary material), we have no evidence for
an early myogenic role of Mrf4 in zebrafish.
Zebrafish lacking myf5 function fail to thrive during larval
phases. Although it has been reported that Myf5 is required for the formation
of most head muscles (Lin et al.,
2006), we observe no such defects in myf5 mutants. Head,
fin and trunk muscles appear to form normally in myf5 mutants, as in
our myf5 morphants. We suspect, therefore, that the defects in brain
and head musculature previously reported after Myf5 knockdown
(Chen and Tsai, 2002;
Lin et al., 2006) reflect MO
toxicity. To date, the cause of death in myf5hu2022
mutants is unknown. Because the myf5hu2022 allele was
found by TILLING in mutagenised individuals, mutation of a linked gene could
account for the phenotype. The mrf4 coding sequence is, however,
wild-type in myf5hu2022 mutants (data not shown), and
mrf4 is expressed normally.
MRFs and slow myogenesis
MRFs are an essential part of the slow muscle programme. Knockdown of Myf5
and Myod ablates early slow myogenesis
(Hammond et al., 2007;
Maves et al., 2007)
(Fig. 1). The myf5
mutation confirms this result. Loss-of-function of either myf5 or
myod alone delays but ultimately does not prevent slow (and medial
fast) myogenesis. Myog is not essential, supporting the original view that the
combined activity level of various MRFs drives muscle formation
(Weintraub, 1993).
Several genes important for slow myogenesis require little or no MRF. Like
myf5 and myod themselves, prdm1 and ptc1
are Hh-dependent genes involved in adaxial slow myogenesis
(Baxendale et al., 2004;
Concordet et al., 1996;
Coutelle et al., 2001;
Koudijs et al., 2008;
Weinberg et al., 1996). In MRF
knockdown embryos, adaxial prdm1 expression is normal at 15 ss,
apparently recovering from an earlier defect
(Liew et al., 2008). As Prdm1
is not required for MRF expression
(Baxendale et al., 2004) (our
unpublished observations), Hh signalling may bifurcate, promoting MRF
expression to trigger muscle formation and prdm1 to drive slow
character. The reduction of Engrailed in myod morphants
(Fig. 7) suggests that MRF
activity might also contribute to aspects of differentiated slow fibre
character.
Differential MRF activity in medial and lateral fast myogenesis
In wild-type fish, terminal differentiation of fast fibres is delayed until
well after somite border formation and follows myog expression in
fast precursors (Blagden et al.,
1997; Devoto et al.,
1996; Weinberg et al.,
1996; Xu et al.,
2000). Maves et al. (Maves et
al., 2007) showed that Myod promotes timely myog and
myhz1 expression in fast muscle precursors in vivo, which we confirm.
However, knockdown of Myod alone does not prevent fast muscle differentiation
(Hammond et al., 2007). To
ablate fast muscle it is necessary to knockdown both Myod and Myf5, which
prevents myog expression. Indeed, knockdown of Myod and Myog ablates
fast muscle differentiation, but leaves slow fibres intact. As myf5
mRNA accumulates only transiently as somites form, it seems that myog
is a target of both Myf5 and Myod that amplifies MRF activity during fast
myogenesis, as first speculated for specific muscle lineages by Weintraub
(Weintraub, 1993).
Lateral somite myogenesis is distinct from medial based on (1) a specific
requirement for Myod to drive myog expression and trigger
differentiation (Hammond et al.,
2007; Maves et al.,
2007), (2) the differential requirement for Fgf signalling
(Groves et al., 2005;
Hamade et al., 2006) and (3)
the finding that myf5 and myod sequentially activate
mef2d expression in lateral cells just prior to their
differentiation. Mef2d is not essential for fast myogenesis
(Hinits and Hughes, 2007). We
hypothesise that, together with other Myod targets, Mef2d works to promote
lateral myogenesis in fish, as suggested from studies in culture
(Penn et al., 2004).
Expression of both myog and mef2d persists in medial
somite cells after Myod knockdown, but is lost if Myf5 is also removed. Medial
fast fibre precursors require the lowest levels of Myf5 and Myod. The Hh
dependence of residual myogenesis in myf5hu2022;myod
morphants suggests that Hh promotes Myod-driven myog expression. Hh
signalling is required in several aspects of fast fibre differentiation
(Henry and Amacher, 2004;
Wolff et al., 2003). Perhaps
some medial fast fibres depend on two phases of Hh action for their
differentiation, analogous to the two phases of Hh action in slow myogenesis
(Wolff et al., 2003).
Myod drives fin and head myogenesis
Myod has an important role in abaxial pectoral fin and head muscle
formation in zebrafish. Mice lacking Myod are viable and fertile without
reported head muscle defects, but do show a delay in limb myogenesis, similar
to, but less severe than, that in fish
(Kablar et al., 1998;
Kablar et al., 1997;
Rudnicki et al., 1992). In
both species, both Myf5 and Myod are expressed in fin and
head muscle precursors. However, in fish myf5 becomes downregulated
in head muscle anlage prior to the expression of muscle structural genes
(Lin et al., 2006). Myod is
essential at these locations for myog expression, which itself is
required for normal head myogenesis. Interestingly, Myog knockdown failed to
phenocopy Myod knockdown in primaxial muscle, despite affecting mef2d
expression, perhaps indicating greater Myod activity in the somite.
Pectoral fin muscle derives from the rostral somites
(Neyt et al., 2000). As with
lateral fast primaxial muscle, Myod is required for early fin muscle, which is
entirely fast (Patterson et al.,
2008). Thus, fin muscle fibres may have an origin, molecular
mechanism of myogenesis and fate similar to those of the lateral somitic
fibres lacking in myod morphants
(Hammond et al., 2007). As
compared with mammals, teleosts retain a bodyplan more similar to that of
primitive teleostomi (bony fish and tetrapods), so we suggest that lateral
Myod-driven myogenesis was an ancestral trait of teleostomi.
Modes of myogenesis provide insight into somite evolution
Medial and lateral somite myogenesis are fundamentally distinct
(Fig. 7D). Our findings do not
reveal differences in MRF function that correspond either to the traditional
epaxial/hypaxial division of the somite, or to the more recent
primaxial/abaxial proposal (Burke and
Nowicki, 2003). Zebrafish have retained an ancestral mode of
somite myogenesis in which midline-derived Hh signalling induces slow muscle
fibres that contribute to both dorsal/epaxial and ventral/hypaxial regions,
which differ in innervation (Devoto et
al., 2006; Eisen,
1991; Lacalli,
2002). Similarly, Hh-dependent MFFs
(Wolff et al., 2003) and
additional `medial mode' fast fibres are present in both epaxial and hypaxial
regions. These medial fibre populations are primaxial and form through either
Myf5 or Myod activity. However, Myod-dependent `lateral mode' fibres [which
appear Fgf8-dependent (Hammond et al.,
2007)] also form primaxially, again in both epaxial and hypaxial
portions. We speculate that duplication of the ancestral myf5/myod
gene in an invertebrate might have facilitated the evolution of novel lateral
myogenesis under distinct myod regulation.
Muscle in the pectoral fin, hypaxial yolk region and possibly sternohyoid,
arises from migratory muscle precursors that originate in the lateral somite
(Haines et al., 2004;
Neyt et al., 2000). The
majority of such migratory precursors, reminiscent of the abaxial division of
amniote somites (Burke and Nowicki,
2003), are Myod-dependent. Moreover, the intrinsic head muscles
share Myod dependence with these abaxial populations. As the abaxial division
arises from the lateral somite, Myod dependence appears to be the common theme
in zebrafish lateral somitic muscle.
Are homologues of adaxial cells and/or medial fast fibres present in
amniotes? These medial mode populations are unified by Myf5/Myod and Hh
dependence. Unlike in fish, mouse Myod does not contribute to early
epaxial/medial myogenesis in the trunk or tail
(Kassar-Duchossoy et al.,
2004). However, other tetrapods, such as birds and frogs, have
retained Myod expression in Hh-dependent medial cells
(Borycki et al., 1998;
Grimaldi et al., 2004;
Hacker and Guthrie, 1998;
Hopwood et al., 1991). In
mice, nevertheless, epaxial myogenesis is dependent on Hh signalling from the
ventral midline (Borycki et al.,
1999). Thus, changes in MRF utilisation were selected during
evolution. Perhaps mammals have lost adaxial cells, leaving Myf5- and
Hh-dependent homologues of medial fast fibres to generate the early
myotome.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/3/403/DC1
|
Footnotes |
|---|
* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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