1 Laboratoire de Génétique et de Physiologie du
Développement (LGPD), Developmental Biology Institute of Marseille
(IBDM), CNRS UMR 6545, University Aix-Marseille II, Campus de Luminy, case
907, 13288 Marseille Cedex 09, France
2 Department of Physiology and Developmental Biology, Brigham Young University,
Provo, UT 84602, USA
Author for correspondence (e-mail:
marcelle{at}ibdm.univ-mrs.fr)
Accepted 30 June 2003
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SUMMARY |
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Key words: MyoD, Myf5, Wnt5b, Shh, Bmp, Somite, Presomitic mesoderm
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Introduction |
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A number of studies have addressed the molecular mechanisms that regulate
myogenesis. In vitro and in vivo studies have shown that the neural tube, the
surface ectoderm and the notochord/ventral neural tube have the ability to
promote myogenic differentiation (Borman
and Yorde, 1994; Buffinger and
Stockdale, 1994
; Christ et
al., 1992
; Kenny-Mobbs and
Thorogood, 1987
; Rong et al.,
1992
; Stern et al.,
1995
; Teillet and Le Douarin,
1983
; Vivarelli and Cossu,
1986
; Cossu et al.,
1996a
; Kuratani et al.,
1994
; Buffinger and Stockdale,
1994
; Stern and Hauschka,
1995
; Münsterberg et al.,
1995
; Münsterberg and
Lassar, 1995
). The search for the molecular signals that mediate
the activities of these tissues has led to the identification of Sonic
Hedgehog (Shh, expressed in the notochord and the neural plate) and Wnt family
members (expressed in the dorsal neural tube and ectoderm). The combination of
Wnt and Shh activates robust muscle marker expression in presomitic mesoderm
explants, whereas Shh alone has no effect and Wnt alone has a low level effect
(Fan and Tessier-Lavigne,
1994
; Kos et al.,
1998
; Munsterberg et al.,
1995
; Tajbakhsh et al.,
1998
; Stern et al.,
1995
; Maroto et al.,
1997
; Reshef et al.,
1998
; Munsterberg and Lassar,
1995
). These data led to the proposal that largely naive cells
adopt myogenic fate through tissue induction mediated by the combined action
of Wnt and Shh emanating from surrounding tissues
(Munsterberg et al., 1995
).
Clearly, this is the most popular model for initiation of myogenesis in
vertebrates.
Despite the evidence that Shh and Wnt can induce the myogenic program in
naive mesoderm, other experiments argue that the initiation of myogenesis may
take place independent of these factors. For example, removal of the notochord
in vivo fails to alter activation of myogenesis in somites, despite the fact
that this operation results in the removal of the source of Shh
(Bober et al., 1994). Moreover,
analyses of the zebrafish and mouse Shh and Smoothened knockouts, and
experimental studies performed in chick have shown that myogenesis is
initiated, but not maintained, in the absence of Hedgehog signaling,
suggesting that it might be dispensable for the initiation of myogenesis
(Chiang et al., 1996
;
Krüger et al., 2000
;
Duprez et al., 1998
;
Fan et al., 1995
;
Marcelle et al., 1999
;
Teillet et al., 1998
;
Zhang et al., 2001
;
Coutelle et al., 2001
). More
surprising is the observation that myogenesis is the preferred pathway of
presomitic mesoderm cells, when they are dissociated to produce a single cell
suspension and cultured in serum-free medium
(George-Weinstein et al.,
1996
; George-Weinstein et al.,
1997
). These data support a model where the initiation of
myogenesis is at least partially independent of environmental cues. Such a
model is difficult to reconcile with the inductive model mentioned above.
Apparently contradictory findings are often due to differing experimental protocols. Experiments which have shown that myogenesis is regulated by environmental cues typically analyzed the myogenic differentiation of rostral presomitic mesoderm explants (considered as naive tissue) placed in the presence of putative inductive tissues or factors for 24, 48 and sometimes 72 hours. However, it is now clear that in the embryo, inductive processes take place in hours or less: during embryogenesis the rostral presomitic mesoderm (i.e. the tissue that was tested in these experiments) initiates myogenesis as somites form, only hours later. It is thus possible that past experiments have identified molecules implicated in later, rather than earlier stages of myogenesis. Therefore, it seemed important to re-examine the initial steps of vertebrate myogenesis both in vivo and in vitro. The chick embryo is particularly well-suited for these experiments, as embryonic microsurgery and ectopic expression of various molecules can be readily evaluated within hours of manipulation. Here, we first show that the activation of Myf5 and MyoD expression is mesoderm-autonomous. Reception of a Wnt signal is required for the initiation of MyoD, but not Myf5 expression. However, ablation experiments demonstrate that the source of the endogenous Wnt signal is neither the ectoderm nor the dorsal neural tube but the mesoderm itself, where Wnt5b is expressed. Our data indicate that Wnt5b is likely to be the MyoD activating signal, suggesting that the activation of MyoD expression is a mesoderm `auto-induction'. Expression of Wnt5b precedes that of MyoD by several hours and this delay is controlled by a Bmp signal that inhibits MyoD expression in the rostral presomitic mesoderm. Although the initial activation of MyoD expression does not require extrinsic signals, we show that Shh emanating from axial structures is able to enhance MyoD expression. We believe our results reconcile previously conflicting data by showing that myogenesis is a complex process that is initiated prior to somite formation in a mesoderm-autonomous fashion; extrinsic influences are likely to be required for the enhancement and/or maintenance of these initial events, thereby leading mesodermal cells further along the myogenic differentiation pathway.
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Materials and methods |
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Construction of a secreted form of Fz7, a full size Wnt5b and a
dominant negative form of Wnt5b
The extracellular region of the chick Frizzled 7 was amplified by PCR,
using the full size Fz7 clone as template, and the primers
5'-GGATTCACCATGGGGCCCGCGGCGGGAGAAGCG-3' and
5'-GTCGACCGGCTGTCGGCTGCGCCGTG-3'. The amplified fragment was fused
to a 6x Myc Tag sequence, subcloned into the Slax13 shuttle vector, and
finally transferred into the RCAS-BP(A) retroviral vector
(Hughes et al., 1987). The
human alkaline phosphatase gene cloned into the RCAS-BP (A) vector
(Morgan and Fekete, 1996
)
served as control.
Dominant-negative Wnt5b was constructed by a deletion of the C terminus of
the mouse Wnt5b protein. PCR amplification of amino acids 1 to 301 (including
the signal peptide) was performed using a cDNA clone for full size mouse
Wnt5b as template (kindly provided by A. McMahon) and the primers
5'-CTCGAGCCACCATGGTGGTCCCAGGGCAT-3' that includes a consensual
Kozak's sequence and a 5'-GAATTCTCAGCAGTAGTCAGGACTGGG-3'
containing a TCA stop codon after cysteine residue 301 from the mouse
Wnt5b coding sequence. The first amino acid of the mouse
Wnt5b was changed from a valine to a leucine to conserve a Kozak
consensus sequence. This fragment was then transferred into the pCLAG2
electroporation vector (Marics et al.,
2002).
The full coding region of mouse Wnt5b (kindly provided by A. McMahon) was also inserted into RCAS-BP(B) retroviral vector, as described for S-Fz7.
Cell transfection and injection
Chicken embryonic fibroblast cell line UMNSAH/DF-1
(Himly et al., 1998) were
transfected with the lipofectamine reagent (Gibco BRL) according to the
manufacturer's instructions. Five to 7 days after transfection, cell infection
was tested by immunohistochemistry reaction against the viral core protein p27
(using a rabbit anti p27 polyclonal antibody: SPAFAS). We verified that
infected cells efficiently secreted S-Fz7-Myc by an immunohistochemistry
reaction against the Myc tag (using a monoclonal mouse anti-human Myc) (Santa
Cruz Biotechnology); Wnt5b expression was detected using in situ
hybridization on transfected cells in culture. The infected fibroblasts stably
produced the S-Fz7 and mouse Wnt5b molecules for at least 2 months in culture.
Bmp4-producing cells were obtained in a similar way by transfection of
UMNSAH/DF-1 fibroblasts with a mouse Bmp4 construct cloned into RCAS(A),
provided by Dr P. Brickell.
Transfected cells were injected into embryos. To inhibit endogenous Fz7 signaling, S-Fz7 expressing cells were pressure-injected into somites IV, rostral, middle and caudal PSM. These embryos were analyzed after overnight incubation. Injections were performed using a Picospritzer pressure injector (General Valve Corporation) and glass needles. As MyoD expression was abolished after S-Fz7 injection, this served as a positive control when the effect of S-Fz7 on Myf5 expression was analyzed. Injections of mixed cells were prepared as follows: S-Fz7- and mouse Wnt5b-transfected cells were trypsinized, counted and mixed at a ratio 1:5, then injected as described before. Alkaline phosphatase-transfected cells were used as control and mixed in the same ratio with S-Fz7. Noggin cell (kindly provided by Dr R. Harland) were injected in the middle presomitic mesoderm and embryos were analyzed 6-8 hours later, CHO dhfr cells were used as control.
Surgery
Fertilized chick eggs were obtained from a commercial source. Glass needles
were used to separate ectoderm from paraxial mesoderm; contact was permanently
impeded by placing a Tantalum foil of appropriate size between the two tissues
(GoodFellow Cambridge Limited). To extirpate the dorsal part of the neural
tube, an incision in the ectoderm was made using a glass needle, then neural
tube and paraxial mesoderm were pulled apart and the dorsal region of the
right side of the neural tube was sectioned out. Embryos were re-incubated for
3-5 hours.
Presomitic mesoderm explants and cell dissociation
Presomitic mesoderm cultures were prepared as follows: dispase solution (1
mg/ml) was briefly applied in ovo onto presomitic mesoderm of 13 HH stage
embryos where ectoderm has been mechanically removed. The presomitic mesoderm,
starting at the Hensen's node until its rostral most part, was isolated and
cultured for 3-4 hours in the following media: L-15 Leibovitz medium (Life and
Technology) with 10% fetal calf serum, 2 ng/ml of bFGF (Sigma), 50 µg/ml
sodium bicarbonate (Gibco Life and Technology).
For cell dissociation, presomitic mesoderms were isolated as described, and
incubated in dispase during 10 minutes. Mechanical dissociation was performed
through a Pasteur pipette and cells were cultured, in the media described
above, for 6 hours. To test the effect of Bmp4 on MyoD activation, dissociated
presomitic mesoderm cells were cultured in the presence of supernatant from
Bmp4-transfected UMNSAH/DF-1 cells. The supernantant was collected from
subconfluent cultures grown ON in the L-15 medium described above. This medium
was concentrated 7x on Ultrafree-15 Millipore filters (molecular weight
limit: 10K). As control, 7x concentrated medium collected from
non-transfected UMNSAH/DF-1 cells was used. To test the effect of Shh on
isolated presomitic cells, bacterially produced Shh protein (R&D) was
added to the cell culture medium to a concentration of 3 µg/ml. While
concentrations of Shh ranging from 1 to 50 µg/ml have been used in the
literature, 3 µg/ml corresponds to the average concentration that was found
to be efficient in most experimental conditions
(Lai et al., 2003;
Detmer et al., 2000
;
Norris et al., 2000
;
Nakamura et al., 1997
;
Kanda et al., 2003
).
In situ hybridization
Whole-mount in situ hybridization on chick embryos were performed as
described (Henrique et al.,
1995). The probes used in this study were: a chick MyoD
probe corresponding to the complete 1518 bp MyoD cDNA
(Wright et al., 1989
); chick
Myf5 corresponding to 336 bp spanning the 5'UTR to the 5'
coding region (Kiefer and Hauschka,
2001
); chick Wnt4 and Wnt5b probes corresponding
to 400 bp of the coding region (Hollyday
et al., 1995
); and chick Delta-1 probe corresponding to
900 bp of the coding sequence (Henrique et
al., 1995
) (kindly provided by Dr D. Henrique).
Double in situ hybridization was carried out in the same way as single in
situ, but fluorescein-labeled and digoxigenin-labeled probes were added. The
first probe was revealed by an anti-fluorescein alkaline
phosphatase-conjugated antibody (Roche) and the INT/BCIP substrate (Roche)
giving a red precipitate. Embryos were fixed in 4% formaldehyde PBS overnight.
Anti-fluorescein antibody was inactivated by incubating embryos in EDTA 100 mM
in MABT buffer (Henrique et al.,
1995) at 65°C for 1 hour. Embryos were then incubated with
anti-digoxigenin alkaline phosphatase-conjugated antibody (Roche), and
revealed with NBT/BCIP reagent (Gibco Life Technologies).
In vivo electroporation
In vivo electroporation were performed as described
(Marics et al., 2002).
Briefly, Qiagen EndoFree purified DN-Wnt5b plasmid was injected into
the lumen of the neural tube of stage 13 HH embryos. Electrodes were placed on
both sides of the embryo and pulsed five times at 80V, 20 mseconds length with
a Intracell TSS 10 electroporator. By placing the positive electrode on the
right side of the embryo, we electroporated this half of the neural tube.
Embryos were then re-incubated for 8-10 hours.
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Results |
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We first tested whether the neural tube regulates MyoD expression.
We removed the dorsal half of the neural tube at the rostral level of the
presomitic mesoderm of 2.5-day-old embryos (stage 13 HH)
(Hamburger and Hamilton,
1992). At the level of the microsurgery, MyoD expression
is not detectable by in situ hybridization. Embryos were allowed to form four
or five additional somites (incubation of 6-8 hours) after ablations. Among
the Wnts expressed in the neural tube (Wnt1, Wnt3a, Wnt4),
Wnt4 extends most ventrally. Therefore, a Wnt4 probe was
incorporated in the in situ hybridization to ensure that we had ablated the
entire Wnt-expressing domain of the neural tube. No difference in
MyoD expression was observed between the operated and the control
side (Fig. 1A-C), indicating
that the neural tube containing Wnt1, Wnt3a and Wnt4 is not
required for the initial induction of MyoD expression.
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Finally, to exclude the possibility that Wnts from the neural tube and the
ectoderm provide synergistic signal(s) to activate MyoD expression,
we ablated both structures simultaneously using the same protocol described
above. Again, MyoD expression was not altered in the absence of
ectoderm and neural tube (Fig.
1G-I). These experiments indicate that the initiation of
MyoD expression is independent of the presence of dorsal neural tube
and ectoderm. The possibility remained, however, that the MyoD
expression we observed after surgery is the consequence of an inductive event
mediated by surrounding tissues prior to surgery. To address this possibility,
it was important to test the stability of MyoD mRNA in paraxial
mesoderm. When MyoD-expressing somites are surgically separated from
axial structures, which normally maintain its expression
(Marcelle et al., 1999),
MyoD expression disappears. We repeated these experiments, monitoring
the time that it takes for MyoD expression to disappear from a somite
that already expresses this molecule. We observed that MyoD was
undetectable in less than 1 hour (Fig.
1J). This indicates that MyoD mRNA is degraded very
rapidly in vivo, and suggests that the expression we observed after ablation
of the dorsal neural tube and/or ectoderm is a de novo mRNA production
resulting from a continuous activation process that took place in the
presomitic mesoderm during the time of the experiment.
Frizzled receptors are expressed in somites at the time that
myogenesis is initiated
For Wnt signaling to play a role in myogenesis, members of the Wnt
signaling pathway must be present in the myogenic region of the somite at the
time and place where MyoD expression is initiated. Wnts mediate their
activities through seven transmembrane domain Frizzled receptors (Fz), which
in turn activate ß-catenin-dependent and/or -independent signaling
pathways (reviewed by Huelsken and
Birchmeier, 2001). Using mRNA extracted from somites, we have
amplified by RT-PCR fragments of Fz receptors (see Materials and methods).
Three of the amplified fragments, corresponding to chick Fz1, Fz2 and
Fz7 display an expression patterns consistent with a possible role
during myogenesis. The three genes display similar expression patterns in the
paraxial mesoderm: they are observed in most of the presomitic mesoderm; their
expression increases as somites form (Fig.
2A-C). In sections, we observed that the three genes are expressed
throughout the epithelial undifferentiated somites, overlapping the region
where MyoD expression is first observed (i.e. the medial wall of the
somite; Fig. 2D). Therefore,
Fz1, Fz2 and Fz7 expression patterns, which precede and
overlap that of MyoD, have the proper spatiotemporal distribution
pattern to play a role in the initiation of its expression.
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Developing chick embryos were screened with a number of Wnt probes
(Wnt1, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8c, Wnt11);
of these, we found that Wnt5b is expressed in the paraxial mesoderm
prior to initiation of MyoD expression. In the rostral presomitic
mesoderm, Wnt5b is expressed in two transversal bands which
correspond to the prospective caudal borders of somite 0 and I
(Fig. 2E). Therefore,
Wnt5b expression is initiated 3 hours before MyoD
expression in somites. Soon after somites have formed, Wnt5b
expression gradually shifts medially, such that from somite stage II onwards,
the medial border of all somites expresses Wnt5b
(Fig. 2E). As somites
differentiate into a ventral sclerotome and a dorsal dermomyotome,
Wnt5b expression becomes restricted to the dorsomedial lip (not
shown), which contains progenitors for epaxial myotomal cells
(Denetclaw et al., 2001
;
Denetclaw et al., 1997
).
Double in situ hybridization with MyoD and Wnt5b indicate
that cells within the medial somite and the medial lip co-express both genes
(Fig. 2F). Altogether, these
data indicate that Wnt5b is expressed in a pattern compatible with a
role in MyoD induction.
Wnt5b likely represents the MyoD activating cue
To be a candidate in the initiation of MyoD expression,
Wnt5b should oppose the effect of S-Fz7, which we showed above
inhibits MyoD. To test this, pellets of cells expressing the secreted
form of Fz7, mixed with control cells or with cells expressing a mouse
Wnt5b construct, were injected into the paraxial mesoderm. S-Fz7
cells mixed with control (i.e. alkaline phosphatase-transfected) cells (1:5)
inhibited MyoD induction (Fig.
4A). By contrast, injection of S-Fz7 cells mixed with
Wnt5b-expressing cells (1:5) rescued normal MyoD expression
(Fig. 4B), demonstrating that
Wnt5b is able to counter the MyoD-inhibiting activity of
secreted Fz7. As Fz7 and Wnt5b are co-expressed in the chick
presomitic mesoderm and somites, the rescue of the phenotype obtained with
S-Fz7 by Wnt5b further indicates that Wnt5b mediates its
activity, at least in part, through Fz7.
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Expression of MyoD in the presomitic mesoderm is repressed
by a Bmp signal
Although the data described above suggest a likely role for Wnt5b
in the induction of MyoD expression, it is paradoxical that
expression of Wnt5b in the presomitic mesoderm precedes that of
MyoD by at least 3 hours. Such a developmental delay is consistent
with the possibility that initiation of MyoD expression may be
temporarily inhibited in the presomitic mesoderm. A candidate for an inhibitor
is Bmp4. Bmp4, expressed in the lateral plate mesoderm, is believed to repress
MyoD expression in the lateral somite, thereby delaying hypaxial
(i.e. lateral) muscle formation; epaxial (i.e. medial) muscles, which are
further away from the Bmp source, develop earlier
(Pourquié et al.,
1996).
To test the possibility that Bmp might act on the presomitic mesoderm as
well, we injected cells expressing the Bmp-antagonist Noggin at the level of
the middle presomitic mesoderm of developing embryos. In 91% of the embryos,
we observed 6-8 hours later that MyoD was prematurely expressed in
the presomitic mesoderm as one, and sometimes two, transverse bands
(Fig. 5B-D), in an expression
pattern reminiscent to that of Wnt5b. In addition, MyoD was
ectopically expressed at the posterior border of newly formed somites. As
observed previously (Marcelle et al.,
1997), Noggin acts at a considerable distance, as its effect is
observed on the contralateral, uninjected side of the embryo. On sections, we
observed MyoD expression in the dorsal region of the rostral
presomitic mesoderm (Fig. 5D,
arrows) and of the epithelial, undifferentiated somite
(Fig. 5C, arrows). This
observation indicates that rostral presomitic mesoderm cells and the dorsal
portion of undifferentiated somites are competent to initiate MyoD
expression, but are repressed to do so by a Bmp signal. The expression pattern
of MyoD after ectopic expression of Noggin supports the hypothesis
that MyoD-activating signals are initially localized at the caudal
border of the segments, constituting additional supporting evidence for a role
of Wnt5b in MyoD activation. The observation that MyoD
expression was never expressed further caudally (i.e. more than two transverse
bands) in the presomitic mesoderm indicates that the competence to express
MyoD is acquired by presomitic mesoderm cells between somite levels I
and 0.
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|
As previous studies had shown that Shh synergizes with Wnt to
activate myogenesis (Munsterberg et al.,
1995; Tajbakhsh et al.,
1998
), we tested whether addition of Shh protein to presomitic
mesodermal cells in culture modifies MyoD expression. The expression
of Pax1, which served as a positive control, was induced in these
conditions (Fig. 6I).
MyoD expression was enhanced in presence of Shh protein
(Fig. 6J). However, the
expression of Wnt5b and Fz7 was increased as well
(Fig. 6K,L): it is therefore
unclear whether Shh acts directly on MyoD expression or indirectly
through the activation of its putative activating signal, Wnt5b and
of the receptor Fz7.
Together, these data confirm those obtained in vivo, and suggest that MyoD expression is activated in a Wnt-dependent, mesoderm autonomous fashion in the presomitic mesoderm. In the presomitic mesoderm region, external cues are likely to repress, through Bmp signaling, the premature activation of myogenesis. As somites form, Shh, which emanates from axial tissues, is likely to play a role in the enhancement and/or maintenance of inductive events that took place in the presomitic mesoderm.
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Discussion |
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The significance of Myf5 expression for presomitic mesoderm cells
is unclear. Although Myf5 has been previously shown to act as a
muscle master gene when overexpressed in cell culture
(Braun et al., 1989), this
cannot be its role in the presomitic mesoderm, because it is expressed in a
large population of cells, not all bound for the muscle lineage (for a review,
see Brand-Saberi et al., 1996
).
In mice mutant for Myf5 and MyoD, it was shown that it is
not only the presence, but also the dose of MRF that is important for muscle
differentiation (Rudnicki et al.,
1993
). Therefore, it is possible that the low level expression of
Myf5 confers a bias or a competence of the entire rostral paraxial
mesoderm towards the myogenic pathway prior to somitogenesis. The medial
restriction of Myf5 expression that is observed in vivo was not
observed when presomitic mesoderm was cultured in isolation from surrounding
structures. This indicates that extrinsic cues might restrict its expression
medially. At present it is unclear whether, similar to MyoD, Bmp inhibiting
signals participate in the medial restriction of Myf5. It is also possible
that, as was recently shown in mouse, Myf5 expression in the medial somite
would be positively modulated by activating cues, such as Shh, emanating from
axial structures (Gustafsson et al.,
2002
).
As somites form, strong MyoD expression is observed in the medial
somite. Experimental evidence indicate that cells present within the medial
somite are committed to the myogenic lineage, as they differentiate into
myocytes even when exposed to a challenging environment, i.e. a notochord
influence (Williams and Ordahl,
1997). This suggests that the expression of MyoD is a
crucial step in the molecular mechanisms that lead somitic cells to muscle
terminal differentiation. The level of MyoD expression observed in
dissociated presomitic mesodermal cells were lower than those which are
observed in intact embryos. This indicates that additional factor(s) might be
required to obtain a strong expression of MyoD in newly formed
somites. We show here that the addition of Shh to dissociated presomitic
mesoderm cells enhances MyoD expression, thereby providing
experimental evidence for a role of Shh in the amplification of the
mesoderm-autonomous activation of MyoD. Importantly, Shh also enhances the
transcription of the putative MyoD-activating signal, Wnt5b
and its receptor Fz7, indicating that the enhancing activity of Shh
on MyoD might be indirect. In addition, Shh was previously shown to act both
as a cell survival and as a maintenance factor during myogenesis
(Teillet et al., 1998
;
Marcelle et al., 1999
;
Duprez et al., 1998
); this
emphasizes the complexity of Shh role during this process.
The data presented here suggest that intrinsic Wnt and extrinsic
Shh signaling are sequentially required to activate a cascade of
molecular events that lead the presomitic mesodermal cells towards their
terminal myogenic differentiation. Recent evidence indicates that Wnt and Shh
signaling might interact directly during myogenesis, as (1) Wnt signaling in
the presomitic mesoderm regulates the expression of the Gli
effectors, which mediate Shh activity
(Borycki et al., 2000), whereas
(2) GSK3, an effector of the ß-catenin-dependent Wnt signaling, directly
modulates the transcriptional activity of Gli
(Price and Kalderon, 2002
;
Jia et al., 2002
). Thus, it
would be interesting to examine whether Gli molecules could serve as
a molecular node where Wnt and Shh signaling converge and synergize
to regulate the expression of MyoD in somites.
The observation that ectopic Wnt5b opposes the
MyoD-inhibitory effect of S-Fz7 demonstrates that Wnt5b binds Fz7.
Interestingly, Wnt5b, its putative receptor Fz7 and
MyoD are co-expressed in somitic cells. Although this could suggest
that Wnt5b mediates its myogenic activity in an cell autonomous
fashion, the observation (1) that myogenic differentiation is more conspicuous
if presomitic mesoderm cells are cultured at high density
(George-Weinstein et al.,
1997; George-Weinstein et al.,
1996
), and (2) that MyoD expression was observed in the
present work in groups, rather than isolated presomitic cells, suggests on the
contrary that Wnt5b might act in a non cell-autonomous fashion.
Although the in vitro and in vivo data presented here indicate that
Wnt5b might play a necessary role in initiation of myogenesis, this
does not rule out the possibility that other signaling molecules may also be
involved. In Drosophila, the Notch/Delta pathway has been
shown to play a fundamental role in this process
(Baylies and Michelson, 2001;
Frasch, 1999
). In chick,
overexpression of Delta inhibits MyoD, but not Myf5
expression in differentiated muscle cells
(Delfini et al., 2000
;
Hirsinger et al., 2001
).
Although the relevance of these observations to the process of muscle
determination in vivo is not yet understood, it is clear that there are still
a number of avenues to be explored in order to fully understand how mesodermal
cells progress towards myogenesis in vertebrates.
An unanswered question is whether Wnts in the neural tube or in the
ectoderm have a role in myogenic differentiation. Wnt1 and Wnt3a in
the neural tube regulate the formation and the maintenance of the dorsomedial
lip (DML), a structure that specifically expresses Wnt11, and that drives the
growth and the morphogenesis of both the epaxial myotome and the dermomyotome
(Marcelle et al., 1997;
Ikeya and Takada, 1998
;
Ordahl et al., 2001
). Thus, if
the source of Wnt1 and Wnt3a (i.e. the dorsal neural tube) is surgically
removed, myogenesis is initiated (as shown in the present work), but the DML
is not formed, and therefore muscle growth is arrested. A similar result is
obtained if the DML itself is surgically removed
(Ordahl et al., 2001
). This
results in embryos where dorsal muscles are missing, not because they are not
induced, but because they do not grow. In mice mutant for Wnt1 and
Wnt3a, a similar phenotype is observed
(Ikeya and Takada, 1998
). This
suggests that Wnt1 and Wnt3a from the dorsal neural tube act
similar to Shh as maintenance and growth factors for the epaxial myotome. Wnts
are also expressed in the ectoderm where they are believed to play a role in
the specification of the dorsal compartment of the somite, the dermomyotome
(Fan et al., 1997
;
Maroto et al., 1997
;
Capdevila et al., 1998
;
Wagner et al., 2000
). As the
dermomyotome is the source of muscle cells, it is likely that a change in its
specification would lead indirectly to modifications in myotome formation.
Thus, neither the mutant mice nor experiments performed in vivo have
demonstrated that these or other Wnts secreted from the tissues surrounding
the somites play a direct role in myogenesis.
Premature initiation of myogenesis in the presomitic mesoderm is abrogated
by Bmp. Lateral plate mesoderm is a plausible source for this Bmp signal
(Pourquie et al., 1996). But
it is also possible that Bmp-like molecules expressed in other tissues,
including the presomitic mesoderm itself, would play this role in vivo. We and
others have shown that the initiation of Noggin expression in the presomitic
mesoderm, slightly before somites form, is likely to play a role in the
release of the Bmp inhibitory activity
(Hirsinger et al., 1997
;
Marcelle et al., 1997
;
Reshef et al., 1998
).
Interestingly, Bmp4 in the lateral plate mesoderm was recently shown to
activate the expression of its own inhibitor noggin in the presomitic
mesoderm, thereby uncovering an unexpected active role of Bmp in the
initiation of MyoD expression
(Sela-Donenfeld and Kalcheim,
2002
). Other possible players in antagonising Bmp activity are the
Bmp inhibitors Follistatin and Flik (Follistatin-like protein), which are both
expressed in the dermomyotome (Amthor et
al., 1996
; Patel et al.,
1996
; Zimmerman et al.,
1996
). Interestingly, Fgf8 expressed in the caudal presomitic
mesoderm was recently shown to inhibit myogenesis
(Dubrulle et al., 2001
). This
observation is consistent with a model where myogenesis in the presomitic
mesoderm is actively repressed by multiple molecular mechanisms until somites
form.
In conclusion, the experiments and observations described here suggest that at the time that somitogenesis is taking place, paraxial mesoderm is not entirely naive, as myogenesis is initiated, but actively repressed, by Bmp signaling. Myogenesis, which has been examined in the past largely as a one-step event, is likely to be the result of a number of intrinsic and extrinsic signals that synergize in a highly organized choreography to form the primitive vertebrate skeletal muscle.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: University College London, Department of Anatomy and
Developmental Biology, Gower Street, London WC1E 6BT, UK
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