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INTRODUCTION
The limb myogenic progenitors arise from the ventrolateral
dermomyotome of the somite in response to signals from the
adjacent lateral plate mesoderm (reviewed by Buckingham et
al., 2003; Francis-West et al., 2003). Following delamination,
the premyogenic cells, which express the transcription factors
Pax3, Lbx1 and Msx1, migrate towards the distal tip of the
limb bud, where they become committed to myogenic
differentiation, as shown by the onset of the expression of the
myogenic determination helix-loop-helix factors, MyoD and
Myf5 (reviewed by Buckingham, 2003; Francis-West et al.,
2003). The premyogenic (Pax3 expressing) and early
myogenic cells (MyoD/Myf5 expressing) form the pre-muscle
masses, which are loose collections of cells scattered within
the subectodermal mesenchyme (Christ and Ordahl, 1995;
Amthor et al., 1998).
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
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Development 130, 3503-3514
© 2003 The Company of Biologists Ltd
doi:10.1242/dev.00538
The limb musculature arises by delamination of
premyogenic cells from the lateral dermomyotome. Initially
the cells express Pax3 but, upon entering the limb bud, they
switch on the expression of MyoD and Myf5 and undergo
terminal differentiation into slow or fast fibres, which have
distinct contractile properties that determine how a muscle
will function. In the chick, the premyogenic cells express
the Wnt antagonist Sfrp2, which is downregulated as the
cells differentiate, suggesting that Wnts might regulate
myogenic differentiation. Here, we have investigated the
role of Wnt signalling during myogenic differentiation in
the developing chick wing bud by gain- and loss-of-function
studies in vitro and in vivo. We show that Wnt signalling
changes the number of fast and/or slow fibres. For example,
in vivo, Wnt11 decreases and increases the number of slow
and fast fibres, respectively, whereas overexpression of
Wnt5a or a dominant-negative Wnt11 protein have the
opposite effect. The latter shows that endogenous Wnt11
signalling determines the number of fast and slow
myocytes. The distinct effects of Wnt5a and Wnt11 are
consistent with their different expression patterns, which
correlate with the ultimate distribution of slow and fast
fibres in the wing. Overexpression of activated calmodulin
kinase II mimics the effect of Wnt5a, suggesting that it uses
this pathway. Finally, we show that overexpression of the
Wnt antagonist Sfrp2 and ∆Lef1 reduces the number of
myocytes. In Sfrp2-infected limbs, the number of Pax3
expressing cells was increased, suggesting that Sfrp2
blocks myogenic differentiation. Therefore, Wnt signalling
modulates both the number of terminally differentiated
myogenic cells and the intricate slow/fast patterning of the
limb musculature.
Key words: Wnt, Limb, Myogenic differentiation, Fibre type, Chick
SUMMARY
Wnt signalling regulates myogenic differentiation in the developing avian
wing
Kelly Anakwe1,*, Lesley Robson2,*, Julia Hadley1, Paul Buxton1, Vicki Church1, Steve Allen1,
Christine Hartmann3,‡, Brian Harfe3, Tsutomu Nohno4, Anthony M. C. Brown5, Darrell J. R. Evans6
and Philippa Francis-West1,§
1Department of Craniofacial Development, King’s College, London SE1 9RT, UK
2Department of Neuroscience, Bart’s and The London, Queen Mary’s School of Medicine and Dentistry, London E1 4NS, UK
3Department of Genetics, Harvard Medical School, Boston, MA 02115, USA
4Department of Molecular Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki, 701-0192, Japan
5Department of Cell and Developmental Biology, Weill Medical College of Cornell University and Strang Cancer Prevention Center,
New York, NY 10021, USA
6School of Biosciences, Cardiff University, Cardiff CF10 3US, UK
*These authors contributed equally to this work
‡Present address: Institute of Molecular Pathology, Dr Bohr-Gasse 7, A-1030 Vienna, Austria
§Author for correspondence (e-mail: pfrancis@hgmp.mrc.ac.uk)
Accepted 8 April 2003
3504
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 β-
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.
MATERIALS AND METHODS
Embryos
Fertilized Ross White chicken eggs were supplied by Poyndon Farm
(Goff’s Oak, UK) or SPF-free eggs were obtained from Lohman
K. Anakwe and others
3505Wnts and limb muscle development
Tierzucht, Germany. The eggs were incubated at 38±1°C and the
embryos were staged according to Hamburger and Hamilton
(Hamburger and Hamilton, 1951).
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 β-catenin, Sfrp2 and dominant-negative
Lef1 (∆Lef1) retroviruses are as described previously: Wnt3a,
activated β-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 (>108pfu) 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 (γspecific) conjugated to FITC (Vector; 1:400) and
donkey anti-mouse IgM (µspecific) 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 ttest.
RESULTS
Correlation of Wnt expression with myogenic
differentiation
To determine potential roles of Wnt signalling during limb
myogenic differentiation, we analysed the expression of Wnts
by whole-mount in situ hybridization and compared their
expression, both temporally and spatially, to that of the muscle
determination factor MyoD. As the limb myogenic precursors
enter and differentiate in the limb bud, they come into contact
with the mesenchymal signals Wnt5a and Wnt11. Wnt5a is
initially expressed throughout the mesenchyme at stage 18,
later becoming predominantly confined to the distal tip with
lower expression levels proximally (Fig. 1A-C,J-M and data
not shown) (see also Dealy et al., 1993; Kawakami et al.,
1999). Between stages 25 and 27, Wnt5a expression is also
found in the central core next to the developing cartilage
elements and muscle masses (Fig. 1C,M). By contrast, Wnt11
is not expressed until after the onset of MyoD expression and
hence myogenic commitment (Fig. 1D,G). Wnt11 is first
expressed in the proximal dorsal subectodermal mesenchyme
Fig. 1. Correlation of Wnt5a and Wnt11 expression with myogenic
differentiation. The expression of Wnt5a (A-C,J-M), MyoD (D-F,N)
and Wnt11 (G-I,N) in stage 22 (A,D,G), 25 (B,E,H) and 27 (C,F,I)
wing buds was determined by whole-mount in situ hybridization.
(J-N) Transverse vibratome sections of wing buds expressing Wnt5a
(J-M) and Wnt11 and MyoD (N) at stages 22 (J,K), 26 (N) and 27
(L,M). In (A-M), expression is shown in purple/red. In (N), MyoD
expression is shown in red and Wnt11 expression is shown in purple.
In (A-N), anterior is uppermost and, in (A-I), distal is to the right. In
the vibratome sections, (J) is more distal than (K), and (L) is more
distal than (M). In (M), developing cartilage is circled in red,
whereas developing muscle is circled in black. DM, dorsal muscle
mass; R, radius; U, ulna; VM, ventral muscle mass. Scale bars: 100
µm in A,D,G; 150 µm in B,C,E,F,H,I; 125 µm in J,K; 150 µm in
L,M.
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overlying the myogenic cells at late stage 22 in the wing (data
not shown) (see Tanda et al., 1995). By stage 24, Wnt11
transcripts are clearly detectable in the dorsal mesenchyme and
are also found in the ventral subectodermal mesenchyme, again
overlying the developing myogenic precursors (Fig. 1E,H and
data not shown). Between stages 24 and 30, Wnt11 expression
extends distally over the developing myogenic cells and is
slightly more advanced on the dorsal side in correlation with
the advanced rate of myogenic differentiation (Fig. 1E,F,H,I,N
and data not shown). At stage 26, Wnt14 is also expressed in
the mesenchyme around the developing cartilage elements and
forming joint regions, where it might influence primary muscle
fibre development, which is not yet complete (V.C. and P.F.-
W., unpublished) (Hartman and Tabin, 2001).
In addition to the mesenchymal signals, particularly at early
stages of differentiation, the myogenic precursors also come
into close proximity with ectodermal signals, which are known
to influence the rate of myogenic differentiation (Amthor et al.,
1998). Wnt4, Wnt6 and Wnt7a are expressed in the ectoderm,
the latter being confined to the dorsal surface, whereas Wnt3a
is expressed in the AER (data not shown) (reviewed in Church
and Francis-West, 2002). The relationship between the
myogenic cells and Wnt expression in a stage 24 wing is
illustrated in Fig. 2A. In summary, the premyogenic cells are
within range of Wnt signals from the ectoderm and are in
contact with or in close proximity to the mesenchymal signals
Wnt5a and Wnt11. Overall, the different expression of Wnt5a
and Wnt11 between stages 23 and 27 correlates with the
ultimate distribution of slow and fast fibres in the primary
muscle fibres in the wing. In general, slow fibres are
concentrated towards the centre of the limb bud, where Wnt5a
transcripts are found, whereas the fast fibres are found closer
to the ectoderm, where Wnt11 transcripts are located (Fig.
2B,C).
Wnt signalling affects myogenic differentiation in
vitro
To analyse the potential roles of Wnt signalling during
myogenic development, we first overexpressed these factors
using the replication-competent retrovirus RCAS(BP) in an
in vitro micromass culture that recapitulates myogenic
differentiation in vivo (Swalla and Solursh, 1986; Archer et al.,
1992). In addition, it has the advantage that the effect of Wnts
on patterning are uncoupled from their effects on myogenic
development. For example, overexpression of Wnt7a
dorsalizes the ventral limb, whereas Wnt3a overexpression
induces ectopic AER formation and hence additional regions
of outgrowth, which would induce secondary changes in
muscle patterning and differentiation. Changes in muscle
differentiation were assessed by double-labelling using the
antibodies A4.1025, which is a pan-MyHC marker, and
A4.840, which recognizes the slow MyHC SM3 and an
embryonic MyHC, SM1. SM1 is initially expressed by most
myogenic cells but is downregulated rapidly in fast myogenic
cells while being maintained in slow-MyHC-expressing cells
(Webster et al., 1988; Hughes and Blau, 1992). The A4.840
antibody will recognize slow MyHCs present in mixed
slow/fast fibres and fibres that exclusively express slow MyHC;
for ease of reading, these myogenic cells will be referred to as
‘slow’. To determine the number of myoblasts expressing
exclusively fast MyHCs, the number of myogenic cells
recognized by the antibody A4.840 was subtracted from the
number recognized by the pan-A4.1025 antibody. For each
Wnt, at least three independent experiments were carried out,
consisting of at least three micromass cultures. Control
micromasses were infected with a retrovirus encoding green
fluorescent protein (GFP).
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).
Wnt5a and Wnt6 had no significant effect on the number of
myocytes, whereas Wnt3a significantly decreased the number
of terminally differentiated myogenic cells (Fig. 3A,B,D,E,
Fig. 4, Table 1). By contrast, Wnt4, Wnt7a, Wnt11 and Wnt14
overexpression increased the number of MyHC-expressing
cells (Fig. 3A,C,F-H, Fig. 4, Table 1). These changes in
number were linked with different changes in the number of
fast and/or slow myocytes. In Wnt3a transfected cultures, the
K. Anakwe and others
Fig. 2. Correlation of Wnt expression with muscle differentiation. (A) A sketch through the dorso-ventral axis of a stage 24 wing bud showing
the relationship between Wnt expression and the developing myogenic cells. Initially, Wnt7a is expressed throughout the dorsal ectoderm but,
by stage 24, its expression is restricted to the dorsal ectoderm overlying the progress zone. (B) Transverse section through an 8-day limb
showing the expression of slow MyHC (orange) versus fast MyHC (green). (C) Diagrammatic sketch of (B). ANC, anconeus; EDC, extensor
digitorum communis; EIL, extensor indicis longus; EMR, extensor metacarpi radialis; EMU, extensor metacarpi ulnaris; Ent,
entepicondyloulnaris; FCU, flexor carpi ulnaris; PP, pronator profundus; PS, pronator superficialis; SUP, supinator.
3507Wnts and limb muscle development
decrease in the total number of MyHC-expressing cells was
linked to a reduction in the number of slow myocytes, whereas
the number of fast-MyHC-expressing cells was not
significantly changed (Fig. 3A,B,I,J, Fig. 4, Table 1). By
contrast, the increase in the number of terminally differentiated
cells in Wnt4- and Wnt7a-transfected micromasses was
associated with a significant increase in the number of slow
MyHC-expressing cells (Fig. 3I,K,N, Fig. 4, Table 1). In the
Wnt7a micromasses there was a small reduction in the number
of fast myocytes, whereas the number of fast myocytes was
unaffected in Wnt4-transfected micromasses (Fig. 4, Table 1).
In contrast to Wnt7a, the increase in the number of terminally
differentiated myogenic cells in Wnt11-transfected
micromasses was linked to an increase in the number of fast-
MyHC-expressing cells (Fig. 3A,G, Fig. 4, Table 1). There was
also a dramatic decrease in the number of slow MyHC-
expressing cells (Fig. 3I,O, Fig. 4, Table 1). In contrast to the
other Wnts, Wnt14 increased both the number of slow and fast
myocytes (Fig. 3A,H,I,P, Fig. 4, Table 1). Finally, although
misexpression of Wnt5a and Wnt6 did not change the number
of myocytes, the ratio of fast to slow myocytes was
dramatically altered: there was an increase in the number of
myocytes expressing slow MyHCs with a simultaneous
decrease in the number of terminally differentiated myogenic
cells expressing fast MyHCs (Fig. 3A,D,E,I,L,M, Fig. 4, Table
1).
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
Fig. 3. Effects of Wnt overexpression on fibre-type differentiation in vitro. (A-P) Fluorescent images of stage 21/22 wing micromass cultures
showing terminally differentiated myogenic cells that have been visualized with antibody A4.1025, which recognizes both slow and fast
MyHCs (A-H, green), and antibody A4.840, which specifically recognizes slow MyHCs (I-P, red). The micromass cultures have been infected
with control RCAS(BP) virus (A,I) or retroviruses expressing Wnt3a (B,J), Wnt4 (C,K), Wnt5a (D,L), Wnt6 (E,M), Wnt7a (F,N) Wnt11 (G,O)
or Wnt14 (H,P). Scale bars, 100 µm.
Fig. 4. The effects of Wnt overexpression on fibre-type
differentiation in vitro. The bar chart shows the total number of
differentiated myogenic cells, and the number expressing fast or slow
MyHCs in 3-day limb micromass cultures that have been infected
with either a control RCAS(BP) virus or retroviruses expressing
Wnt3a, Wnt4, Wnt5a, Wnt6, Wnt7a, Wnt11 or Wnt14, as shown in
Fig. 2. The slow population of myoblasts (red) might express either
exclusively slow MyHC or both slow and fast MyHCs, whereas the
fast myogenic population (yellow) only expresses fast MyHC
isoforms. *, P<0.05; **, P<0.01; ***, P<0.001.
Table 1. Summary of the effect of Wnt signalling on limb
myogenic differentiation
Protein Pan MyHC Slow MyHc Fast MyHC
Stage 21/22
Wnt3a f<0.001 f<0.001 –
Wnt4 F<0.010 F<0.050 –
Wnt5a – F<0.001 f<0.001
Wnt6 – F<0.001 f<0.001
Wnt7a F<0.001 F<0.001 f<0.050
Wnt11 F<0.001 f<0.001 F<0.001
Wnt14 F<0.001 F<0.001 F<0.050
∆Wnt11 f<0.001 f<0.050 f<0.001
β-Catenin f<0.001 f<0.050 f<0.001
CamKII f<0.001 F<0.001 f<0.001
∆Lef1 f<0.050 F<0.050 f<0.001
Dsh/∆PDZ f<0.010 F<0.050 f<0.001
Stage 19/20
β-Catenin f<0.001 – f<0.010
∆Lef1 f<0.050 – f<0.050
Dsh/∆PDZ f<0.001 f<0.050 f<0.001
The effect of overexpression or loss of function of the Wnt signalling
pathway on myogenic differentiation in stage 21/22 and stage 19/20
micromass cultures.
3508
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.
This analysis showed that all muscles in the autopod and
zeugopod could be affected by misexpression of Wnt5a and
Wnt11. Overall, the effect was not as dramatic as that observed
in vitro. In general, there were small pockets of ectopic fast or
slow fibres found dotted throughout each muscle (arrowed in
Fig. 5F; compare with 5E). In control GFP-infected limbs, the
number of terminally differentiated fibres and those recognized
by A4.840 was not significantly changed compared to the
contralateral limb (n=3; data not shown). By contrast and
consistent with the effects observed in the micromass cultures,
Wnt5a significantly increased and decreased the number of
slow and fast MyHC-expressing cells, respectively (n=5,
P<0.05) even though the total number of terminally
differentiated fibres was not significantly affected (Fig. 5C-F).
In Wnt11-transfected limbs and as in vitro, the total number of
terminally differentiated cells was significantly increased (Fig.
5G,H; n=3, P<0.05). Again, as in vitro, there was an significant
increase and decrease in the number of fast MyHC and slow
MyHC-expressing cells respectively (Fig. 5G-J; n=3, P<0.05).
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
K. Anakwe and others
Fig. 5. Effects of overexpression of Wnt5a and Wnt11 on fibre-type differentiation in vivo. Fluorescent images of transverse cryosections of
day 8 (A-F) and stage 29 (G-J) chick wings that were infected with retroviruses expressing GFP (B), Wnt5a (D,F) or Wnt11 (H,J) at stage 18-
20, and of the contralateral control wings (A,C,E,G,I). Wings have been visualized with antibody A4.1025, which recognizes slow and fast
MyHCs (C,D,G,H, green), and antibody A4.840, which recognizes slow MyHC (E,F,I,J, red). (B) The embryo was pathogen free and viral
spread has been visualized with an anti-GAG antibody. The arrows in (B) indicate viral spread throughout the limb bud and muscles (compare
with A). The arrows in (E) and (F) compare equivalent muscles, showing an increase in slow MyHC expression in the manipulated limb (F). In
the Wnt11-transfected limb, there are fewer slow fibres in the dorsal and ventral muscle masses (J) than in the contralateral limb (I). d, dorsal
muscle mass; EDC, extensor digitorum communis; EIL, extensor indicis longus; EMR, extensor metacarpi radialis; EMU, extensor metacarpi
ulnaris; FCU, flexor carpi ulnaris; FDP, flexor digitorum profundus; R, radius; u, ulna; v, ventral muscle mass. Scale bars: 200 µm in A-D; 100
µm in E-H.
3509Wnts and limb muscle development
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).
Effect of ∆Wnt11
We also constructed a retrovirus encoding a truncated Wnt11
protein, which has been shown to act as a dominant negative,
to test its effect on myogenic differentiation (Tada and Smith,
2000). Following overexpression of RCAS(BP)/∆Wnt11 in
vivo, the number of terminally differentiated cells was not
significantly changed, although the limbs appeared much
thinner upon fixing (Fig. 6E-H; n=3). Closer analysis showed
that the muscles were smaller and the individual fibres were
more closely packed than those in the contralateral control limb
(compare Fig. 6E and F). The number of fast- and slow-MyHC-
expressing myogenic cells was significantly decreased and
increased, respectively, which is the opposite of the effect of
misexpressing Wnt11 in vivo (Fig. 6G,H, n=3, P<0.05).
Similarly, in vitro misexpression of RCAS(BP)/∆Wnt11
significantly reduced the number of fast myocytes in stage
21/22 micromass cultures (n=9, Table 1, P<0.001 and data not
shown). However, there was also a small but significant
reduction in the number of slow myocytes (Table 1, P<0.0.5,
and data not shown). The overall reduction in the number of
terminally differentiated cells was not due to fusion of
myocytes because all were mononucleate.
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 β-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 β-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%).
Fig. 6. Effects of loss-of-function of Wnt signalling in vivo.
Fluorescent images of transverse cryosections of day 8 chick wings
that were infected with retroviruses expressing Sfrp2 (B,D) and
∆Wnt11 (F,H) at stage 18-20, and the contralateral control wings
(A,C,E,G), which have been visualized with antibody A4.1025,
which recognizes slow and fast MyHCs (A,B,E,F, green), and
antibody A4.840, which recognizes slow MyHC (C,D,G,H, red).
(A-D) Sections through the whole wing. (E-H) High power pictures
of the EMU and EDC muscles. The arrows in (A,B,G,H) compare
equivalent muscles, showing the changes in muscle development. In
(B), the muscles are absent (EMU, SUP) or are decreased in size
(EMR, FCU). In H, the EMU muscle has more slow fibres. EDC,
extensor digitorum communis; EIL, extensor indicis longus; EMR,
extensor metacarpi radialis; EMU, extensor metacarpi ulnaris; FDP,
flexor digitorum profundus; SUP, supinator. Scale bars: 200 µm in A-
D; 100 µm in E-L.
3510
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 β-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, β-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).
Blocking the β-catenin pathway by misexpression of ∆Lef1
and Dsh∆PDZ proteins reduced the total number of myocytes,
and this was associated with a significant decrease in the
number expressing fast MyHCs (Fig. 7A-C,E-G, Fig. 8, Table
1). By contrast, the number of slow-MyHC-expressing cells
was increased (Fig. 7E-G, Fig. 8, Table 1 and data not shown).
The morphology of the myocytes in ∆Lef1-infected cultures
was also affected: they were much smaller and had a rounded
appearance, lacking the typical elongated processes, indicating
either a delay in development or a requirement for β-catenin
signalling in assembly of the myocyte cytoskeleton (compare
Fig. 7I,J). A reduction of the total number of myocytes was
also observed with stage 19/20 micromasses in ∆Lef1- and
Dsh∆PDZ-transfected cultures (Table 1 and data not shown).
As at stage 21/22, this reduction was linked to lower numbers
of fast myocytes in both cases (Table 1 and data not shown).
However, in contrast to the stage 21/22 micromasses, the
number of slow myocytes was not significantly affected in the
∆Lef1-transfected micromasses and was decreased in the
Dsh∆PDZ-transfected cultures (Table 1 and data not shown).
DISCUSSION
The Wnt family of secreted factors has been shown to be
essential for differentiation of the epaxial musculature (reviewed
by Buckingham et al., 2003). Here, we have analysed the
potential roles of Wnt signalling during limb myogenic
differentiation, which to date have been unexplored. We have
shown by gain- and loss-of-function studies that members of the
Wnt family have distinct effects on myogenic differentiation,
controlling the number of terminally differentiated myoblasts
and the ratio and/or number of slow and fast myocytes/fibres.
Thus, Wnt signalling within the limb bud might initiate the onset
of myogenic differentiation as in the somite and, in particular,
regulate fibre-type specification and development.
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
K. Anakwe and others
Fig. 7. The effect of overexpression of
components and dominant-negative
components of the Wnt signalling pathway
on fibre-type differentiation in vitro.
(A-J) Fluorescent images showing
terminally differentiated myogenic cells
that have been visualized using antibody
A4.1025, which recognizes slow and fast
MyHCs (A-D,I,J, green), and antibody
A4.840, which recognizes slow MyHC
(E-H, red) in stage 21/22 micromass
cultures that have been infected with
control RCAS virus (A,E,I) or retroviruses
expressing Dsh∆PDZ (B,F), ∆Lef1
(C,G,J) or activated CamKII (D,H). Scale
bars: 100 µm in A-H; 25 µm in I,J.
3511Wnts and limb muscle development
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 Dsh∆PDZ,
which blocks both the β-catenin and the JNK pathways, but
not following overexpression of ∆Lef1, which only blocks β-
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
Fig. 8. The effect of overexpression of activated components and
dominant-negative components of the Wnt signalling pathway on
fibre-type differentiation in vitro. The bar chart shows the total
number of differentiated myogenic cells and the number expressing
fast or slow MyHCs in 3-day limb micromass cultures that have been
infected with either a control RCAS(BP) virus or retroviruses
expressing Dsh∆PDZ, ∆Lef, β-catenin or activated CamKII. The
slow population of myoblasts (red) might express either exclusively
slow MyHC or both slow and fast MyHCs, whereas the fast
myogenic population indicated by the yellow bar only expresses fast
MyHC. *, P<0.05; **, P<0.01; ***, P<0.001.
3512
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 β-catenin,
or blocking the β-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 β-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
β-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 β-catenin signalling regulating myogenic differentiation.
For example, it has been found that, in C2 cells, both
overexpression and inhibition of β-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 β-catenin decreases the number of slow
myocytes at stage 21/22 but has no effect at stage 19/20.
Similarly, blocking β-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.
We thank the MRC, the ARC, BBSRC, NIH (grant number
CA47207), the Japanese Ministry of Education, Science, Sports and
Culture, and Kawasaki Medical School for funding, M. Chaperlin for
technical support, C. Healy, S. Hughes, M. Kühl, R. Moon, B.
Paterson, C. Tabin, M. Tada and J. Smith for the gift of the cDNA
clones and retroviruses, and M. Bronner-Fraser, C. Marcelle and C.
Ordahl for the gift of the Pax3 antibody.
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K. Anakwe and others
