1 Department of Craniofacial Development, King's College, London SE1 9RT,
UK
2 Department of Neuroscience, Bart's and The London, Queen Mary's School of
Medicine and Dentistry, London E1 4NS, UK
3 Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
4 Department of Molecular Biology, Kawasaki Medical School, 577 Matsushima,
Kurashiki, 701-0192, Japan
5 Department of Cell and Developmental Biology, Weill Medical College of Cornell
University and Strang Cancer Prevention Center, New York, NY 10021, USA
6 School of Biosciences, Cardiff University, Cardiff CF10 3US, UK
Author for correspondence (e-mail:
pfrancis{at}hgmp.mrc.ac.uk)
Accepted 8 April 2003
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SUMMARY |
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Key words: Wnt, Limb, Myogenic differentiation, Fibre type, Chick
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INTRODUCTION |
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The onset of myogenic differentiation is repressed by a number of growth
factors, allowing the expansion of the premyogenic pool and ultimately the
number of terminally differentiated myoblasts within the limb bud. These
repressive signals include scatter factor, which is expressed by the
mesenchyme, and fibroblast growth factors (FGFs), which are expressed by the
apical ectodermal ridge and ectoderm. In addition, bone morphogenetic protein
(BMP) signalling from both the ectoderm and mesenchyme plays a repressive
role, as demonstrated by the ability of the BMPs to maintain Pax3
expression in the developing limb bud
(Amthor et al., 1998;
Scaal et al., 1999
;
Edom-Vovard et al., 2001
).
Sonic hedgehog (Shh) also maintains the ventral muscle precursors in an
undifferentiated state, possibly acting via the maintenance of BMP expression
(Duprez et al., 1998
;
Krüger et al., 2001
;
Bren-Mattison and Olwin, 2002
).
It is not yet totally clear whether the onset of myogenic differentiation
within the limb bud is just a default or passive state following the release
of the premyogenic cells from their inhibitory cues, or whether positive
inductive factors are needed. However, recent work has suggested that
inductive signals from the FGF family are required for differentiation
(Marics et al., 2002
). Thus,
FGF signalling is initially repressive but is later inductive or permissive
for myogenic differentiation, emphasizing the complexity of the molecular
regulatory network that controls myogenesis in the limb bud.
Myoblasts subsequently coalesce to form the dorsal and ventral muscle
masses, which are the template of the future muscles
(Schramm and Solursh, 1990).
Myoblasts also start to differentiate terminally by switching on the
expression of the terminal differentiation factors, the myosin heavy chains
(MyHCs). These terminally differentiated myoblasts then fuse, forming
multinucleated fibres that can contract
(Hilfer et al., 1973
;
Sweeney et al., 1989
). This
period of primary fibre development is followed by secondary fibre formation.
The secondary fibres align on the surface of the primary fibres, starting at
day 7 in the chick embryo, and grow to constitute the bulk of skeletal muscle
at birth (Fredette and Landmesser,
1991
).
Each muscle is characterized by a unique profile of slow and fast fibre
types that will determine how that muscle will function
(Miller and Stockdale, 1986a;
Miller and Stockdale, 1986b
).
Fast fibres express one of the fast MyHC isoforms and usually use glycolytic
metabolism. They can generate high force but fatigue easily. By contrast, slow
fibres use oxidative metabolism and express slow isoforms of the MyHC
(Hughes and Salinas, 1999
).
These fibres contract slowly and are able to maintain a contraction for longer
without fatigue.
When and where fibre-type commitment occurs has been a running debate. A
recent elegant study in which the somitic precursors of the quail pectoralis
muscle were grafted into the equivalent position in a chick host suggested
that commitment occurs within the somite
(Nikovits et al., 2001). In
these studies, the slow/fast patterning of the pectoralis muscle was
characteristic of the donor and not the host. Clonal analysis studies have
also shown that myogenic cells are heterogeneous in their slow/fast MyHC
expression and are committed to their different fibre-type fates by stage
24/25 in the quail (DiMario et al.,
1993
) (reviewed by Stockdale,
1990
). This is in contrast to fate-labelling studies in which
individual premyogenic clones were marked with a specific nucleotide tag
(Kardon et al., 2002
). These
studies showed that a single premyogenic cell could give rise to both slow and
fast myoblasts in addition to a distinct lineage (endothelial cells). These
latter data suggest that environmental cues, presumably within the limb bud,
control fibre-type patterning and are consistent with other data in which
clones of foetal or satellite myogenic cells were shown to differentiate or to
modify their fate when grafted into a new host
(Hughes and Blau, 1992
;
DiMario and Stockdale, 1997
;
Robson and Hughes, 1999
). One
way of reconciling this data is to argue that different muscles in the limb
can be governed by a different set of signalling interactions. An alternative,
and equally plausible, argument is that the premyogenic cells are biased to
one fibre-type fate as they leave the somite but that they exhibit plasticity
(i.e. that they are not committed) and their ultimate fate is determined or
modified by local environmental signals (reviewed by
Francis-West et al.,
2003
).
Factors that specify limb myogenic fibre-type differentiation are unknown.
In chick somites and zebrafish adaxial musculature, Shh or hedgehog signalling
promotes and is essential for slow fibre-type formation. Therefore, loss of
Shh signalling inhibits slow fibre development, whereas excess Shh promotes
slow fibre formation (Currie and Ingham,
1996; Blagden et al.,
1997
; Cann et al.,
1999
; Lewis et al.,
1999
; Barresi et al.,
2000
). However, in the limb bud, Shh does not appear to determine
myogenic cell fate but does initially prevent differentiation of a
subpopulation of the presumptive slow muscle precursors, maintaining them in a
proliferative state and, ultimately, increasing the number of slow fibres
(Bren-Mattison and Olwin,
2002
).
The role of the Wnt family of secreted factors during limb myogenic
development has to date been neglected, yet members of this family initiate
myogenic differentiation in the epaxial and hypaxial musculature, substituting
for the neural tube and ectodermal signals, respectively
(Ikeya and Takada, 1998;
Cossu et al., 1996
;
Tajbakhsh et al., 1998
). In
addition, overexpression of the Wnt antagonist Sfrp3 blocks myogenic
differentiation in mouse somites (Borello
et al., 1999
). The Wnt family consists of 19 members, which can
act through one of three pathways that might depend on the Frizzled receptor
profile of the receiving cell - first, through the classical ß-catenin
pathway, second, through a calcium protein kinase C (PKC)-mediated pathway
and, finally, through a novel Jun kinase pathway (reviewed by
Church and Francis-West,
2002
).
Several members of this family are expressed in the limb, where they
control patterning, outgrowth and/or differentiation (reviewed by
Church and Francis-West,
2002). Wnt5a, Wnt11 and Wnt14 are expressed in
the mesenchyme, whereas Wnt4, Wnt6 and Wnt7a are expressed
in the ectoderm, the last of these being restricted to the dorsal surface,
where it controls dorsal ventral patterning (reviewed by
Church and Francis-West,
2002
). In addition, Wnt3a is expressed in the apical
ectodermal ridge (AER). Therefore, within the limb bud the pre- and
differentiating myogenic cells are within range of Wnt signalling, which is
thought to propagate over 11-12 cell diameters, from the ectoderm and
mesenchyme. Thus, it is possible that, as in the somites, Wnts might regulate
myogenic differentiation. Finally, Sfrp2 is expressed in the
migrating muscle precursors in the chick, whereas Sfrp1 is expressed
in the lateral dermomyotome in the mouse, again suggesting that modulation of
Wnt signals might control limb myogenic differentiation
(Ladher et al., 2000b
;
Lee et al., 2000
). Here, we
show by gain- and loss-of-function studies that different members of the Wnt
family have distinct effects on limb muscle development, controlling the
number of terminally differentiated cells and the number expressing either
slow or fast MyHCs. Thus, we identify novel functions of Wnt signalling during
limb myogenic differentiation.
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MATERIALS AND METHODS |
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In situ hybridization
In situ hybridization to whole embryos was carried out as described by
Francis-West et al. (Francis-West et al.,
1995). cDNA and ribroprobes were made as described previously:
MyoD (Lin et al., 1989
),
cWnt5a (Kawakami et al., 1999
)
and cWnt11 (Tanda et al.,
1995
).
Retroviral constructs and culture
Concentrated retroviral stocks and retrovirally infected chicken embryonic
cells for grafting were prepared as described by Logan and Francis-West
(Logan and Francis-West,
1999). The Wnt3a, Wnt5a, Wnt7a, Wnt14, activated ß-catenin,
Sfrp2 and dominant-negative Lef1 (
Lef1) retroviruses are as described
previously: Wnt3a, activated ß-catenin and
Lef1
(Kengaku et al., 1998
), Wnt5a
(Kawakami et al., 1999
), Wnt7a
(Rudnicki and Brown, 1997
),
Wnt14 (Hartmann and Tabin,
2001
) and Sfrp2 (Ellies et
al., 2000
). The other retroviruses were constructed in RCAS(BP)
and encode Xenopus Wnt4, mouse Wnt6, a partial chick Wnt11 cDNA
equivalent to the Xenopus Wnt11 construct described by Tada and Smith
(Tada and Smith, 2000
), which
acts as a dominant-negative, activated rat calmodulin kinase II
(Kühl et al., 2000a
),
Xenopus Dsh, which lacks the PDZ domain (Dsh
PDZ)
(Tada and Smith, 2000
),
enhanced-green fluorescent protein (eGFP; Clontech), or in RCAS-L14, which
encodes chicken Wnt11 (Tanda et al.,
1995
).
Retroviral misexpression studies
Grafting of retrovirally infected cells into stage 18-21 limb buds was as
described in Francis-West et al.
(Francis-West et al., 1999).
Stage 19/20 and 21/22 wing bud micromass cultures were prepared as described
in Francis-West et al. (Francis-West et
al., 1999
) except that they were plated in the presence of high
titre (>108 pfu) RCAS(BP) retroviruses and were cultured in the
absence of ascorbate. The micromasses were cultured for three days.
Immunohistochemistry
Embryos were dissected and placed into 20% sucrose in PBS at 4°C. They
were embedded in OCT compound (BDH Lab Supplies) and cryosectioned at 15
µm. Micromass cultures were fixed in methanol for 2 minutes and were washed
twice for 5 minutes with PBS. Muscle development was analysed using the
following primary antibodies diluted in PBS: A4.1025 (1 in 100), which
recognizes all terminally differentiated muscle cells and A4.840 (1 in 50),
which recognizes cells expressing the slow MyHC isoforms SM3 and SM1 (from the
developmental hybridoma bank) (Webster et
al., 1988; Hughes and Blau,
1992
). The Pax3 antibody (1 in 100) was a gift from C. Ordahl, C.
Marcelle and M. Bronner-Fraser, and is described by Baker et al.
(Baker et al., 1999
). The GAG
antibody (1 in 5) is as described in Logan and Francis-West
(Logan and Francis-West,
1999
). Incubation with the primary antibodies was followed by
incubation with horse anti-mouse IgG (
specific) conjugated to FITC
(Vector; 1:400) and donkey anti-mouse IgM (µ specific) conjugated to Cy3
(Jackson; 1:800) for at least 1 hour at room temperature. Cultures and
sections were mounted under coverslips with PBS:glycerol (1:9) with 0.1%
phenylenediamine as an antifade reagent. They were then viewed and the images
were captured using a Leica DMRD microscope and the HiPic32 program. The data
was analysed using Student's t test.
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RESULTS |
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Control cultures typically possessed 1060±64 MyHC-expressing cells, of which 94% were mononucleate. As for the control micromass cultures, 92% or greater of MyHC-expressing cells in Wnt-infected cultures were mononucleate (Wnt3a, 100%; Wnt4, 92%; Wnt5a, 96%; Wnt6, 94%; Wnt7a, 99%; Wnt11, 92%; Wnt14, 97%). Overexpression of different members of the Wnt gene family had two distinct effects on myogenic differentiation. First, Wnt signalling could change the number of terminally differentiated myogenic cells. Second, a change in the number of slow and/or fast-MyHC-expressing cells was observed (Figs 3, 4, Table 1).
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|
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Wnt5a and Wnt11 can modulate fibre-type development in vivo
We next investigated whether misexpression of Wnts can change fibre-type
differentiation in vivo, focusing on Wnt5a and Wnt11, which are expressed
around the developing muscles as they start to differentiate. These Wnts were
also initially chosen because their presence gives dramatic effects on
slow/fast differentiation in vitro and their expression correlates with the
slow/fast fibre distribution in the wing
(Fig. 2A-C). Thus, we
misexpressed these Wnts in the developing chick limb using the RCAS(BP)
retrovirus. Following in vivo infection between stages 18 and 20, the embryos
were subsequently allowed to develop until day 7 or 8, when they were fixed
and the virally infected and unmanipulated limbs were sectioned in parallel to
obtain equivalent sections along the proximodistal axis. This route of
retrovirus infection resulted in all the muscles being infected with no
consistent or obvious dorsoventral or proximodistal bias
(Fig. 5A,B) [see also Duprez et
al. (Duprez et al., 1996) for
further analyses of the rate of viral spread]. The muscles were then analysed
for fast and slow MyHC expression in the primary muscle fibres using the
A4.1025 and A4.840 antibodies. At this stage, some of the secondary fibres
have also formed, using the primary myotubes as a scaffold. These small fibres
are easily distinguished morphologically from the much larger primary fibres
and were not included in this analysis. The number of terminally
differentiated primary myogenic cells and those expressing slow MyHCs was
analysed every 75 µm along the proximodistal axis in each muscle in the
autopod and zeugopod. The total number of fibres recognized by either of the
antibodies in each section was analysed and compared with the control
contralateral limb.
|
We also tested the effect of Wnt6 and Wnt14, because these members of the
Wnt family had a significant effect in vitro. Furthermore, in addition to the
earlier expression adjacent to developing muscle cells, Wnt14 has
been shown to be expressed in developing muscle cells at day 15 in the chick
embryo (Hartmann and Tabin,
2001). As in vitro, Wnt6 significantly increased the number of
slow-MyHC-expressing cells (n=3, P<0.05) while not
significantly affecting the total number of terminally differentiated myogenic
cells (data not shown). By contrast, Wnt14 did not give a significant effect
on muscle development in vivo (n=3, data not shown). We did not test
other Wnts such as Wnt3a and Wnt7a as these would give changes in patterning
and/or outgrowth, which would have secondary consequences on muscle
development in vivo.
Loss of Wnt function alters muscle differentiation
Effect of the secreted Wnt antagonist Sfrp2
The overexpression studies showed that ectopic Wnt signalling can affect
myogenic differentiation, although this did not prove that there is an
endogenous role in vivo. To further determine the role of Wnt signalling and
to confirm the role of endogenous Wnt signalling, we took a loss-of-function
approach. We first focused on Sfrp2 because, as we previously reported,
Sfrp2 is expressed in the migratory premyogenic limb cells and
appears to be downregulated as myogenic cells differentiate
(Ladher et al., 2000b). Thus,
we misexpressed Sfrp2 using the RCAS(BP) retrovirus in the developing limb and
in micromass culture.
Following grafting of virally infected cells into stage 18-20 limb buds in vivo, there was a 56% decrease in the number of terminally differentiated myogenic cells and some muscles were reduced to remnants or were entirely absent (Fig. 6A-D, n=3, P<0.001). The percentage of slow-MyHC-expressing cells was similar to that in the controls: 36% in Sfrp2-transfected limbs compared to 41% in the uninfected contralateral limb. Sfrp2 might reduce the number of terminally differentiated muscle cells by changing cell proliferation and/or survival, or it might block myogenic differentiation. To investigate the latter, we determined the number of Pax3-expressing cells (i.e. the number of premyogenic cells) in Sfrp2-transfected limbs and found an average increase of 49% compared with the contralateral control limb (n=3, P<0.05, data not shown). Similarly, Sfrp2 reduced the number of terminally differentiated myoblasts in stage 19/20 micromass cultures (n=9, P<0.001) and, as observed in vivo, the ratio of fast to slow myocytes was not significantly affected (control slow 40%; Sfrp2 slow 43%, data not shown).
|
Misexpression of intracellular components of the Wnt pathway
To further investigate the role of Wnt signalling during limb myogenic
differentiation and to determine the intracellular pathways involved, we
misexpressed activated calmodulin kinase II (CamKII), which is implicated in
the Wnt5a signal transduction pathway, and ß-catenin, which mediates
Wnt3a function in the limb (Kengaku et
al., 1998, Kühl et al.,
2000a
; Kühl et al.,
2001
). In addition, we blocked endogenous Wnt signalling with
mutated Lef1 (
Lef1) and Dsh proteins (Dsh
PDZ), which block the
ß-catenin signalling pathway
(Slusarski et al., 1997a
;
Slusarski et al., 1997b
;
Kengaku et al., 1998
;
Kühl et al., 2000a
;
Kühl et al., 2000b
). The
mutated Dsh protein also blocks the planar cell polarity pathway, which is
activated by Wnt11 signalling. As before, we performed this assay at least
three times with at least three micromasses per experiment and determined the
number of terminally differentiated myogenic cells and those recognised by the
A4.840 antibody. In none of these assays was there a significant change in the
percentage of mononucleate cells (GFP, 94%; activated CamKII 95%;
Lef1,
95%; Dsh
PDZ, 96%).
Overexpression of the activated components in stage 21/22 cultures in general mimicked the effect of the Wnts proposed to signal through them. Thus, activated CamKII promoted slow myocyte formation while decreasing the number of the fast-MyHC-expressing myocytes (Fig. 7A,D,E,H, Fig. 8, Table 1). The latter also resulted in a significant reduction in the number of myocytes, which was not observed in the Wnt5a-transfected cultures (Fig. 3D,L, Fig. 4, Fig. 7D,H, Fig. 8, Table 1). Like Wnt3a, activated ß-catenin significantly reduced the number of terminally differentiated myoblasts and those expressing slow MyHCs at stage 21/22 (Fig. 8, Table 1 and data not shown). However, ß-catenin also reduced the number of fast-MyHC-expressing cells. A similar result was obtained with stage 19/20 micromasses except that, at this stage, the number of slow myocytes was not significantly changed (Table 1 and data not shown).
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DISCUSSION |
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A striking observation in these studies was that modulation of the Wnt
signalling pathway changes the number of slow and/or fast fibres both in vivo
and in vitro. Of particular importance are the mesenchymal signals Wnt5a and
Wnt11, which are expressed adjacent to the developing muscle cells. As
premyogenic cells enter the developing limb, they encounter Wnt5a, which is
produced throughout the mesenchyme. Later, Wnt5a expression becomes
predominantly restricted to the progress zone, just distal to the
differentiating muscle masses, which are now expressing MyoD.
However, Wnt5a expression is also maintained at higher levels around
the developing cartilaginous core adjacent to the developing muscle masses,
where the muscles containing most of the slow fibres develop in the chick
wing, such as the extensor indicis longus and pronator profundus
(Fig. 2B,C). By contrast,
Wnt11 expression is switched on in the subectodermal mesenchyme
overlying the developing muscles after the onset of myogenic commitment.
Overexpression of these Wnts both in vivo and in vitro had opposing effects,
with Wnt5a and Wnt11 enhancing and reducing the number of slow myocytes,
respectively. The total number of terminally differentiated cells was
relatively unchanged from the controls, suggesting that (as in neural crest
development) Wnt signalling acts as a cell fate switch, although this is as
yet unproven (Jin et al.,
2001). If this was the case, it would be similar to hedgehog
signalling in zebrafish adaxial muscle development, in which hedgehog has been
proposed to act as a binary switch specifying slow versus fast fibre-type fate
(Norris et al., 2000
). Whether
Wnt5a and Wnt11 act directly on the myogenic cells themselves or signal as a
relay via other mesenchymal signals is currently unknown but the close
proximity of Wnt5a- and Wnt11-expressing cells with developing myogenic cells
suggests that the former is highly likely.
In vivo, all of the limb muscles analysed in the zeugopod and autopod could
be affected, although none was completely transformed to an exclusively slow
or fast fate. This might be related to the variability in and timing of viral
spread, such that the Wnts are not misexpressed at a stage when they can
affect myogenic differentiation. Alternatively, there might be local extrinsic
environmental signals, such as FGFs and BMPs or opposing members of the Wnt
family, which modulate the effect of ectopic signalling. Indeed, FGFs have
been shown to modulate Wnt activity during limb, otic and neural development
in the chick, and BMPs have been shown to affect Wnt regulation of neural
crest differentiation (Farrell and
Münsterberg, 2000; Jin et
al., 2001
; Ladher et al.,
2000a
; Wilson et al.,
2001
). Furthermore, in Xenopus, different members of the
Wnt family have been shown to have antagonistic actions
(Du et al., 1995
;
Torres et al., 1996
;
Kühl et al., 2001
).
Therefore, in vivo, the effect of Wnt signalling will probably be modulated by
other factors.
Wnt5a and Wnt11 are expressed in very similar domains in the developing leg bud but the ultimate arrangement of slow and fast fibres in the leg is distinct. In the leg, as in the wing, slow fibres are found centrally but they are also found at the periphery (for example, in the sartorius and anterior iliotibialis muscles, which are almost exclusively slow). At first glance, this might suggest that our model is wrong. However, we have found that Wnts have distinct effects in leg and wing micromasses. As in the wing, Wnt5a promotes slow myocyte formation and Wnt11 increases the total number of myocytes (data not shown). However, in contrast to the wing, Wnt11 has no significant effect on the ratio of slow to fast myocytes in the leg (data not shown).
The role of Wnt11 in the regulation of limb fibre-type differentiation was
supported by misexpression of Wnt11 in vivo, which gave the opposite
effect to Wnt11 misexpression: increasing the number of slow fibres while
decreasing the number of fast myogenic cells. This shows that endogenous Wnt
signalling can change the number of fast and slow fibres. Taken together with
the gain-of-function studies, this suggests two possible mechanisms of action.
First, Wnt11 signalling might specify myoblasts to a fast fibre-type fate at
the expense of slow myoblasts. In this case, Wnt11 would be instructive and it
is assumed that all myoblasts are equivalent to respond. This would be
consistent with the overall similarity of the numbers seen in the
gain-of-function studies. Alternatively, as proposed by others, there might be
at least two populations of presumptive myoblasts prespecified to become
either slow or fast myocytes (reviewed by
Stockdale, 1990
). In this
scenario, Wnt11 would be permissive for fast myocytes, promoting their
differentiation and/or proliferation while inhibiting the development of the
slow myoblast populations.
The in vitro data were slightly different but, as in vivo, the number of
fast myocytes was decreased, indicating that Wnt11 signalling is required for
fast fibre-type differentiation. In contrast to the in vivo data, the numbers
of slow myocytes was also slightly decreased, suggesting that endogenous Wnt11
signalling is not inhibitory, and might even be required, for their
development, at least in a micromass assay. The same effect on slow myocyte
development was observed in stage 19/20 micromasses following overexpression
of DshPDZ, which blocks both the ß-catenin and the JNK pathways,
but not following overexpression of
Lef1, which only blocks
ß-catenin signalling. This implicates the JNK pathway in the initial
regulation of slow myocyte development. A possible explanation for the
different effects on slow myocytes in vivo and in vitro is that, as discussed
above, limb environmental signals might modulate the effect of Wnt signalling.
In micromass culture, these will be different to those present in vivo: the
ectodermal signals are absent and this is also associated with the
downregulation of mesenchymal signals such as Shh and BMPs
(Krüger et al.,
2001
).
Other members of the Wnt family also changed the number of fast and slow
myocytes. In Wnt4- and Wnt7a-transfected micromass cultures, the increase in
myogenic cell number was linked to a significant increase in the number of
slow myocytes, whereas, in Wnt14-transfected micromasses, there was a
significant increase in both slow and fast myocytes. Like Shh, Wnt4, Wnt7a and
Wnt14 might delay myogenic differentiation and, in the case of Wnt4 and Wnt7a,
have distinct effects on different subpopulations of proliferating myogenic
precursors, which would ultimate increase the number of slow and/or fast
myocytes (Duprez et al., 1998;
Bren-Mattison and Olwin,
2002
).
Our results do not resolve the problem of when and where the slow and fast
fibre types are specified, nor whether Wnts are acting as permissive or
instructive signals. However, they clearly indicate that the number of fast or
slow fibres is controlled within the limb bud, as have other recent studies in
which Shh has been shown to act selectively on the presumptive slow myoblast
population (Bren-Mattison and Olwin,
2002). When and where fibre-type specification occurs is still
being debated. The results of Nikovits et al.
(Nikovits et al., 2001
) have
shown that the fibre types are specified within the somite, at least for the
pectoralis muscle, but whether this is true for all limb muscles is currently
unclear. Recent fate labelling studies of individual myogenic precursors have
strongly suggested that there is no inherent specification of fast and slow
muscle precursors as they leave the somite
(Kardon et al., 2002
) [see
Francis-West et al. (Francis-West et al.,
2003
) for further discussion].
CamKII gave a similar phenotype to Wnt5a, suggesting that, as in
Xenopus, Wnt5a signals via the PKC pathway in the developing limb bud
(Kühl et al., 2000a).
Increases in calcium signalling have also been linked to slow fibre formation
in adult muscles, suggesting that patterning mechanisms that occur in the
distinct adult muscle populations also occur during specification/development
of embryonic myoblasts (Chin et al.,
1998
; Bigard et al.,
2000
; Delling et al.,
2000
; Naya et al.,
2000
; Serrano et al.,
2001
). Wnt6 had the same effect as Wnt5a, suggesting that Wnt6
might also use the PKC pathway. However, at present, no signalling pathway has
been identified for Wnt6, and an equally likely and alternative explanation is
that Wnt6 might induce and mediate its effects via Wnt5a expression.
We also found that overexpression of Wnt3a or ß-catenin, or blocking
the ß-catenin pathway with Lef1 decreased the number of terminally
differentiated myocytes. In addition, misexpression of the Wnt antagonist
Sfrp2, which is expressed by uncommitted myogenic precursors, also decreased
myocyte number both in vivo and in vitro. The loss-of-function data show that
endogenous Wnt signalling determines the number of terminally differentiated
cells but does not identify a mechanism. The increase in the number of
Pax3-expressing cells observed following misexpression of Sfrp2 in vivo
suggests that Wnt signalling is needed for the onset of MRF expression. This
proposal is consistent with the data in the embryonic carcinoma cell line P19,
in which it has been shown that ß-catenin can initiate and is required
for myogenic commitment (Petropoulos and
Skerjanc, 2002
). Furthermore, in somites, overexpression of the
secreted Wnt antagonist Sfrp3 blocks myogenesis without affecting
Pax3 expression, suggesting that Wnt signalling acts downstream of
Pax3 to induce myogenic commitment (Borello
et al., 1999
). However, if this proposal is correct, the ligand
responsible for this activation is currently unknown. It is unlikely to be
Wnt3a, which is restricted to the AER and, in vivo, activates Fgf8
expression in the ectoderm (FGF8 is an inhibitor of myogenic differentiation)
(Kengaku et al., 1998
). Wnt3a
is also not antagonized by Sfrp2, which must be the candidate molecule that
prevents initiation of myogenic differentiation
(Ladher et al., 2000b
;
Lee et al., 2000
). An
alternative mechanism is that ß-catenin might repress myogenic
differentiation in the developing limb bud. This has been suggested from
studies in the myogenic cell lines L8, C2 and its derivative C2C12
(Goichberg et al., 2001
;
Martin et al., 2002
). However,
the situation might be much more complex, with a fine balance of
ß-catenin signalling regulating myogenic differentiation. For example, it
has been found that, in C2 cells, both overexpression and inhibition of
ß-catenin signalling suppress myogenic differentiation
(Goichberg et al., 2001
). This
complexity is also emphasized by our in vitro data, in which we have found
that ß-catenin decreases the number of slow myocytes at stage 21/22 but
has no effect at stage 19/20. Similarly, blocking ß-catenin signalling
has distinct effects on slow myocyte development at these two stages. The
reasons for this are currently unclear and are under investigation.
Here, we have shown a role for endogenous Wnt signalling during limb myogenic development, showing that Wnts modulate both the number of terminally differentiated myocytes and the number expressing either slow or fast MyHCs. Different members of the Wnt family have very distinct and even antagonistic effects on muscle development. The next challenge will be to dissect out how these opposing effects are mediated and how other signalling factors modulate the effect of Wnt signalling to produce the intricate pattern of slow and fast fibres within each muscle, which is responsible for co-ordinated movement and the maintenance of posture.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030
Vienna, Austria
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REFERENCES |
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