1 Department of Biomedical Science, University of Sheffield, Firth Court,
Western Bank, Sheffield S10 2TN, UK
2 Department of Cell and Developmental Biology, University of Pennsylvania,
Philadelphia, PA 19104, USA
3 Department of Molecular and Medical Genetics, University of Toronto and
Program in Developmental Biology, The Hospital for Sick Children, 555
University Avenue, Toronto, Ontario M5G 1X8, Canada
4 Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472,
USA
* Author for correspondence (e-mail: a.g.borycki{at}sheffield.ac.uk)
Accepted 18 October 2004
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SUMMARY |
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Key words: Myf5, Sonic hedgehog, Gli, Skeletal muscle, Somite, Mouse
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Introduction |
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Genetic studies have established that the myogenic regulatory factor (MRF)
Myf5 plays a key role in the specification of mesodermal cells to the
different myogenic lineages, because Myf5-deficient mouse embryos
display a significant delay in myogenesis until the onset of Myod1
expression (Braun et al.,
1994; Tajbakhsh et al.,
1997
) and in the absence of three of the four MRFs, Myf5,
Myod1 and Mrf4 (Myf6 Mouse Genome Informatics)
no skeletal muscle progenitor cell is formed
(Kassar-Duchossoy et al.,
2004
; Rudnicki et al.,
1993
). Furthermore, Myf5-deficient muscle progenitor
cells at the DML fail to enter the epaxial myotome and migrate aberrantly in
the dermatome and the sclerotome
(Tajbakhsh et al., 1996
). The
temporal and spatial expression pattern of Myf5 is consistent with
its primary role in myogenic specification. Myf5 is first found in
the DML progenitor cells for epaxial muscles at E8.0 (embryonic day 8.0),
followed by the VLL progenitor cells for hypaxial muscles at E9.75
(Ott et al., 1991
;
Tajbakhsh and Buckingham,
2000
).
As expected for a protein acting upstream in the cascade of gene activation
leading to skeletal muscle differentiation, Myf5 transcriptional
regulation is particularly complex and has only been comprehensively described
recently. Regulatory elements have been found over a region covering nearly
140 kb upstream and downstream of the Myf5 transcriptional start
(Buchberger et al., 2003;
Carvajal et al., 2001
;
Hadchouel et al., 2003
;
Hadchouel et al., 2000
;
Summerbell et al., 2000
). Each
element appears to function specifically in the control of Myf5
expression at discrete sites in the embryo, indicating that Myf5
regulation is controlled independently in individual skeletal muscle
progenitor domains. Consistent with this observation, distinct signals
produced by tissues surrounding the somite have been found to activate
Myf5 expression in epaxial and hypaxial muscle progenitor cells
(Borycki and Emerson, 2000
).
Notably, Wnt signals, secreted by surface ectoderm and neural tube cells,
Sonic hedgehog (Shh), secreted by notochord and floor plate cells, and Bmp4,
secreted by lateral plate cells, cooperate to induce and establish the somitic
pattern of Myf5 expression
(Borycki et al., 1999
;
Dietrich et al., 1998
;
Ikeya and Takada, 1998
;
Pourquie et al., 1996
;
Tajbakhsh et al., 1998
). In
previous work, we have shown that Shh is required for Myf5 activation
in epaxial, but not in hypaxial muscle progenitor cells
(Borycki et al., 1999
;
Kruger et al., 2001
).
Furthermore, Shh signalling appears to directly act on Myf5
transcription in epaxial progenitor cells. Indeed, transgenic mice expressing
a reporter gene under the control of the epaxial somite (ES) enhancer [also
referred to as EEE, Early Epaxial Enhancer
(Teboul et al., 2002
)],
located at 6.1 kb of the Myf5 transcriptional start,
recapitulates endogenous epaxial Myf5 expression
(Gustafsson et al., 2001
;
Teboul et al., 2002
). In the
context of a heterologous promoter, expression of this transgene is abolished
in a Shh/ background or following mutation
of an internal Gli-binding site
(Gustafsson et al., 2001
),
suggesting that Gli proteins, which mediate Shh signalling
(Ingham, 1998
;
Ruiz i Altaba, 1997
), directly
control Myf5 expression in epaxial muscle progenitor cells.
In the mouse, three Gli proteins, Gli1, Gli2, and Gli3 have been
characterised (Hui et al.,
1994). As is the case for Cubitus interruptus (Ci), the fly
homologue of Gli, Gli2 and Gli3 can both activate and repress Shh target gene
transcription in vitro (Dai et al.,
1999
; Sasaki et al.,
1999
). However, in vivo analyses of mouse mutants have shown that
Gli2 acts primarily as a transcriptional activator
(Ding et al., 1998
;
Matise et al., 1998
), whereas
Gli3 retains a bipotential activity, acting as transcriptional activator in
motorneuron and ventral interneuron specification and as transcriptional
repressor in dorsal interneuron specification
(Bai et al., 2004
;
Litingtung and Chiang, 2000
;
Meyer and Roelink, 2003
;
Motoyama et al., 2003
;
Persson et al., 2002
). Gli1
functions as a transcriptional activator of Shh target genes in vitro and in
overexpression studies (Dai et al.,
1999
; Lee et al.,
1997
; Ruiz i Altaba,
1999
; Sasaki et al.,
1999
). However, mouse mutants show that Gli1 is dispensable
(Park et al., 2000
), although
it can mediate Shh signalling in the absence of Gli2
(Bai and Joyner, 2001
;
Park et al., 2000
). Together,
these observations indicate a complex interplay between Gli proteins in
vivo.
Thus, a prediction from our previous studies of myogenesis in Shh/ mice and ES trangenic mice would be that Gli proteins are required during somite myogenesis to mediate Shh signalling. To address this question and to investigate whether Myf5 activation and somite patterning require the activator or the repressor function of Gli proteins, we have examined the expression pattern and the regulation of Gli1, Gli2, and Gli3 during somite formation in the mouse embryo. We also carried out genetic studies using Gli and Shh mutant mice, as well as transgenic mice expressing lacZ under the control of the Myf5 epaxial enhancer (ES), to test the redundant function and the transcriptional activity of Gli proteins in Myf5 activation and somite patterning. We found that Gli2 or Gli3 is required for epaxial muscle progenitor cell specification and that Shh plays an essential role in this process to convert Gli3, but not Gli2 into a transcriptional activator. In addition, our study reveals an unexpected differential role for Gli2 and Gli3 along the antero-posterior axis in patterning the epaxial, hypaxial and myotomal compartments. Together, these data establish that Gli genes have essential specific and redundant functions during skeletal myogenesis.
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Materials and methods |
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Whole-mount in situ hybridization
Embryos were collected at various stages and fixed overnight at 4°C in
4% paraformaldehyde, washed in PTW (0.1% Tween 20 in PBS) and processed for in
situ hybridisation according to the protocol described elsewhere
(Henrique et al., 1995). To
allow for quantitative comparisons, embryos were treated together in a single
tube and staining allowed to develop for the same length of time. Digoxigenin
(DIG)-labelled antisense riboprobes were generated from linearised plasmids
containing inserts for Myf5 (Ott
et al., 1991
), Myod1
(Sassoon et al., 1989
),
Pax1 (Deutsch et al.,
1988
), Pax3 (Goulding
et al., 1991
), Pax7
(Jostes et al., 1991
),
Gli1 (Hui et al.,
1994
), Gli2 (Hui et
al., 1994
), Gli3 (Hui
et al., 1994
), myogenin
(Sassoon et al., 1989
),
scleraxis (Cserjesi et al.,
1995
), paraxis (Burgess et
al., 1995
), Lbx1
(Jagla et al., 1995
),
Sim1 (Fan et al.,
1996
) and Noggin
(McMahon et al., 1998
).
Stained embryos were photographed under a LEICA MZ12 stereomicroscope using a
Spot digital camera (Diagnostic Instruments). Embryos were then embedded in 2%
agarose in PBS and 80 µm transverse sections were performed using a
vibratome. Sections were mounted on slides using Glycergel (Dako), and
photographed under Nomarski optics on a LEICA DM-R microscope using a LEICA
digital camera.
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Results |
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In interlimb and anterior somites of E9.5
Gli2/Gli3/
(Fig. 3T,X) and
Gli2/Gli3+/
(Fig. 3R,V), but not
Gli2+/Gli3/
(Fig. 3S,W) embryos,
Myf5 expression is upregulated and shifted ventrally. Because
Myf5 becomes activated in the myotome and in the hypaxial muscle
progenitor cells at E9.5, it is most likely that this observation indicates a
function for Gli2 and Gli3 in the formation and/or patterning of the myotome
and the hypaxial somitic domain. Consistent with this possibility,
Myf5 transcripts take a central position in
Gli2/Gli3/ somites
compared to wild-type somites (compare Fig.
3P to 3M), indicating that anterior and posterior somitic
compartments are more severely affected. Furthermore, myogenin, whose
transcripts mark myotomal cells as they delaminate from the DML and enter the
epaxial myotome (Fig. 5A)
(Sassoon et al., 1989), is
mispatterned and found in a more ventral domain in
Gli2/Gli3+/ and
Gli2/Gli3/ somites
(Fig.
5A',A'''). Notably, myogenin transcripts are
found in an overlapping domain with Myf5, along the ventral
epithelial extension that characterizes the double mutant somite (compare
Fig. 5A''' and
Fig. 3L). Finally,
Myod1 expression is also precociously activated and misexpressed in a
ventral domain of the epaxial myotome in
Gli2/Gli3/ embryos
(Fig. 5E'''). These
data are consistent with the idea that Gli2 and Gli3 are essential for the
activation of Myf5 in the epaxial muscle progenitor cells and the
subsequent formation of the epaxial myotome, and are required for the normal
dorsoventral patterning of the myotome.
To clarify the identity of the epithelial cells extending from the dorsal
medial lip along the neural tube, we used the dermamyotomal markers paraxis,
Pax3 and Pax7 (Burgess
et al., 1995; Jostes et al.,
1991
). Paraxis, Pax3 and Pax7 are all expressed
in the dermamyotome of wild-type embryos
(Fig. 5B-D) and their
expression is unchanged in
Gli2/Gli3+/ and
Gli2+/Gli3/ embryos
(Fig.
5B',B'',C',C'',D',D''). In
contrast, cells in the ventral epithelial extension of
Gli2/Gli3/ somites
express paraxis Pax3 and Pax7, although at lower levels than
dorsal dermamyotomal cells (Fig.
5B''',C''',D'''). To investigate
whether these cells express DML markers, we performed an in situ hybridisation
using Noggin, which specifically labels DML cells from E9.5 onward
(Fig. 5G) (McMahon et al., 1998
).
Noggin transcripts remain localised to the dorso-medial domain of
Gli2/Gli3/ somites
and are not found in the epithelial cells that extend ventrally
(Fig. 5G'''),
indicating that these cells do not have the molecular identity of DML cells,
although they do express dermamyotomal markers. Noggin expression has
been proposed to provide a permissive environment for Myf5 expression
in DML cells (Hirsinger et al.,
1997
; Marcelle et al.,
1997
). However, we find here that Myf5 expression in
Gli2/Gli3/ mice
occurs in a non-Noggin-expressing domain, indicating that
antagonising Bmp4 signals is possibly necessary but not sufficient for
Myf5 activation in the dorsomedial somite.
We then examined the specification, differentiation, and patterning of
hypaxial muscle progenitor cells in Gli mutant embryos using Pax3, Sim1,
Myod1 and Lbx1 expression. Hypaxial muscle progenitor cells
originate from the Pax3-positive, Sim1-positive lateral
dermamyotomal cells (Fig.
5D,H). As they delaminate from the dermamyotome to enter the
hypaxial myotome or migrate to the limbs, they activate Myod1 or
Lbx1, respectively (Fig.
5E,F). Thus, Myod1 is detected at E10.0 in the hypaxial
muscle progenitor cells of interlimb somites
(Fig. 5E) and this expression
is immediately preceded by that of Myf5
(Tajbakhsh and Buckingham,
2000). By E10.5, Myod1 is detected in all hypaxial
progenitor cells, including those of the body wall, limb and tongue muscles
(Sassoon et al., 1989
). In
contrast, Lbx1 is activated at E9.5 at the level of occipital and
limb somites in a subset of hypaxial muscle progenitor cells
(Fig. 5F)
(Jagla et al., 1995
;
Uchiyama et al., 2000
), which
are committed to migrate to the limb bud or to the head
(Brohmann et al., 2000
;
Dietrich et al., 1998
;
Gross et al., 2000
). As
expected from our previous analysis of Shh mutant mice
(Borycki et al., 1999
), none
of the mutant combinations studied shows a defect in the onset of hypaxial
muscle progenitor cell specification (Fig.
5D-D''',E-E''',F-F''',H-H''').
Likewise, no change in Myod1 and Lbx1 expression pattern is
observed in Gli2/,
Gli3/ and
Gli2/Gli3+/ embryos at
E9.5 and E10.0 (Fig.
5E',F' and data not shown). However, Myod1 is
upregulated in the hypaxial domain of
Gli2+/Gli3/ and
Gli2/Gli3/ somites,
and both Myod1 and Lbx1 are upregulated and expand into the
ventromedial somitic domain of
Gli2/Gli3/ embryos
(Fig.
5E'',E''',F'',F'''). Noticeably,
no such upregulation occurs for Pax3 and Sim1 dermamyotomal
expression in Gli2+/Gli3/
and Gli2/Gli3/
somites (Fig.
5D'',D''',H'',H'''). This
indicates that in the absence of Gli2 and Gli3, although hypaxial muscle
progenitor cells form and are specified normally in the dermamyotome, they
become unproperly patterned as they enter the hypaxial myotome or migrate into
the limbs, as is the case for Shh mutant mouse embryos.
Gli3, but not Gli2 acts as a transcriptional repressor in the absence of Shh signalling
Gli2 and Gli3 proteins contain recognition sites for protein kinase
A-dependent phosphorylation, a process that leads to the proteolytic cleavage
of the full-length protein into a shorter N-terminal polypeptide containing a
repressor domain in Drosophila
(Chen et al., 1998;
Epstein et al., 1996
;
Jia et al., 2002
;
Price and Kalderon, 2002
). In
vitro, both Gli2 and Gli3, but not Gli1 have been shown to act as
transcriptional repressors (Dai et al.,
1999
; Sasaki et al.,
1999
). Shh signalling prevents this cleavage and converts Gli2 and
Gli3 into transcriptional activators
(Aza-Blanc et al., 2000
). In
vivo, only Gli3 was found to have repressor function and to require Shh
signalling to be converted into a transcriptional activator
(te Welscher et al., 2002
;
Wang et al., 2000
). This
implies that defects observed in dorsoventral patterning of the neural tube of
Shh mutant mice can be partially rescued in Shh/Gli3
compound mutant mice, as the result of the loss of a Gli3 repressor function
(Litingtung and Chiang,
2000
).
To test whether some of the somitic defects observed in Shh mutant
mice are due to a repressor activity of Gli2 or Gli3, we analysed by in situ
hybridisation the expression of Myf5 and myogenin in
Shh/,
Gli3+/Shh/ and
Gli3/Shh/ embryos,
as well as in Gli2+/Shh/
and Gli2/Shh/
embryos. As shown before (Borycki et al.,
1999), Myf5 expression is not activated in the epaxial
muscle progenitor cells of the DML in Shh/
embryos (Fig. 6F,J), although
its activation comes on schedule in the hypaxial muscle progenitor cells
(Fig. 6F,J,N,V,V'). In
addition, hypaxial Myf5 expression expands into the ventromedial
somite (compare Fig. 6M and
6N,V'), and Myf5 and myogenin expression
is greatly reduced in occipital somites of
Shh/ embryos
(Fig. 6B,F,R). Removal of one
Gli3 allele in the Shh/ background
restores Myf5 expression in the DML epaxial muscle progenitor cells
of interlimb somites, but not posterior somites
(Fig. 6G,K). However, removal
of both Gli3 alleles in the Shh/
background fully restores Myf5 expression in DML muscle progenitor
cells along the entire anteroposterior axis
(Fig. 6H,L,P,T). Unlike for
Gli3, loss of Gli2 alleles in the
Shh/ background does not restore epaxial
Myf5 expression (Fig.
6U-X'). This indicates that Shh signalling is required to
convert Gli3, but not Gli2 into a transcriptional activator for Myf5
expression in DML cells. These data also suggest that DML cells of posterior
and occipital somites are more sensitive to a ratio of Gli3 activator versus
Gli3 repressor
1 than are DML cells of interlimb somites.
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Discussion |
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Gli genes are differentially expressed in the somite, with Gli1 restricted to the ventral somite, and Gli2 and to a lesser extent Gli3, initially expressed throughout the somite and then predominantly found in the dorsal somite. This expression pattern is consistent with our findings that Gli2 and Gli3 are required for Gli1 activation and that Gli3 can only compensate for the loss of Gli2 in newly formed somites. However, Gli3 acts as a weak transcriptional activator, because the presence of one Gli3 allele in a Gli2 mutant background is not sufficient to activate Gli1 expression in newly formed somites. The overlapping expression of Gli2 and Gli3 in the dorsal somite also accounts for their functional redundancy during somite myogenesis, although their respective preponderance in the medial and lateral somite is consistent with independent functions in mediolateral patterning of the somite.
Gli2 or Gli3 is required for the specification of epaxial muscle progenitor cells
We provide several lines of evidence that Gli2 or Gli3 is required to
mediate Shh signalling in the specification of epaxial muscle progenitor cells
from the DML. First, as for Shh/ embryos,
Myf5 expression is not activated in DML cells of
Gli2/Gli3/ embryos,
whereas activation occurs, albeit at lower levels, in
Gli2/Gli3+/ and
Gli2+/Gli3/ embryos.
Second, no ß-Gal+ cell is observed in newly formed somites of
Gli2/Gli3/ embryos
crossed into the Myf5 ES/LacZ transgenic mice, which express the
reporter gene in the epaxial muscle progenitor cells
(Gustafsson et al., 2001;
Teboul et al., 2002
). Third,
Shh signalling is necessary to convert Gli3R into Gli3A for Myf5
activation in DML cells. This establishes that Gli2 and Gli3 both act as
transcriptional activators of Myf5 expression in DML cells and are
required for the transcriptional activity of the Myf5 ES enhancer
(Fig. 7). These data are
consistent with our previous findings that Shh signalling is necessary to
initiate epaxial Myf5 expression, and that the Gli binding site
located in the Myf5 ES enhancer is essential for the enhancer
activity in posterior somites (Gustafsson
et al., 2001
). In contrast, the present data do not support
previous findings by Teboul et al., that the Gli binding site in the
Myf5 ES enhancer is required for the maintenance of epaxial
Myf5 expression but not for its initiation
(Teboul et al., 2003
). We
believe this discrepancy reflects the different behaviour of the transgenic
constructs used, which in the latter case, contains upstream of the
Myf5 promoter both the branchial arch enhancer and the Myf5
ES enhancer (Teboul et al.,
2003
). It is possible that cross-talk between enhancers, yet to be
characterised, occurs and alters the activity of the ES enhancer.
|
The ventral position of epaxial Myf5 transcripts in
Gli2/Gli3/
reveals that dermamyotomal and myotomal cells are mislocated in the
ventromedial somite. This observation correlates with the reduction or loss of
Pax1-expressing cells in the ventral somite of
Gli2/Gli3/
mice (Buttitta et al., 2003),
indicating that normal dorsoventral patterning of the somite is disrupted in
the absence of Gli proteins. This defect is reminiscent of the ventral shift
of neuronal cells in the developing central nervous system of
Gli2/Gli3/
mice (Wijgerde et al., 2002
).
This suggests that, as was demonstrated in the neural tube
(Jessell, 2000
), somitic cells
adopt a specific cell fate along the ventro-dorsal axis according to the level
of Hedgehog signalling transduced (Fig.
7).
Dual function of Gli2 and Gli3 in somite myogenesis
Biochemical and in vitro studies have shown that Gli2 and Gli3 can act as
transcriptional activators and repressors
(Ruiz i Altaba, 1999;
Sasaki et al., 1999
), but
until recently it was thought that in vivo, Gli2 had only activator function
and Gli3 had only repressor function (Bai
et al., 2002
; Ding et al.,
1998
; Litingtung and Chiang,
2000
; Matise et al.,
1998
; Wang et al.,
2000
). Our data show that Gli2 and Gli3 have both activator and
repressor function in the dorsal somite depending on the genetic context. For
instance, Gli2 represses hypaxial gene expression in the absence of Gli3, as
illustrated by the gradual increase in hypaxial gene expression in
Gli2+/Gli3/ and
Gli2/Gli3/ embryos
(Fig. 7). Conversely, Gli3
activates epaxial gene expression in the absence of Gli2, as Myf5
expression, although reduced, is still observed in posterior somites of
Gli2/Gli3+/ embryos and is
lost in Gli2/Gli3/
embryos (Fig. 7). These results
are in line with recent studies showing that Gli3 has activator function in
the specification of floor plate and V3 interneurons in the neural tube
(Bai et al., 2004
), and in the
specification of sclerotomal cells in the somite
(Buttitta et al., 2003
).
Our data clearly indicate that the failure to activate Myf5 in the
epaxial somitic domain of Shh/ embryos is
due to the repressor activity of Gli3, as we observe a progressive restoration
of Myf5 expression in
Gli3+/Shh/ and
Gli3/Shh/ embryos.
Moreover, Gli3 repressor clearly acts in a concentration-dependent manner on
Myf5 activation (Fig.
7), and accounts for the more severe phenotype observed in
Shh/ mice compared to
Gli2/Gli3/ mice.
Similar effects have been reported in the dorso-ventral patterning of the
neural tube and in the anteroposterior patterning of the limb
(Litingtung and Chiang, 2000;
Litingtung et al., 2002
;
te Welscher et al., 2002
),
revealing that repression of target genes by Gli3 in the absence of Shh
signals constitutes a fundamental mechanism in tissue patterning. This
remarkable result also indicates that Myf5 activation can occur in
the absence of both Shh and Gli signalling, as Gli1 and Gli2 proteins are
thought to not be active in the absence of Shh. This observation is in line
with previous studies showing that combinatorial Wnt and Shh signalling
activate Myf5 expression
(Munsterberg et al., 1995
;
Tajbakhsh et al., 1998
), and
suggests that in the absence of Gli activity, other transcription factors
activate the Myf5 ES enhancer and Myf5 transcription.
Crossing Gli3/Shh mutant mice into the ES/lacZ transgenic
background would confirm that this is the case. Nevertheless, the fact that
the Myf5 ES enhancer has a single Gli site suggests that the variable
phenotype observed in our compound mutants can only be explained if activator
and repressor forms of Gli, alone or in association with partners, bind the
Gli binding site with different affinities.
Gli2 and Gli3 have a central role in the patterning of the myotome and hypaxial muscle progenitor cells
In a previous report, we showed that in the absence of Shh, hypaxial
Myf5 expression expanded into the ventromedial somite
(Borycki et al., 1999). Here,
we find that expression of Myf5 and myogenin in epaxial myotomal
cells is upregulated and expands ventrally in
Gli2/Gli3+/ embryos, and
expression of Myf5, Myod1 and Lbx1 in hypaxial muscle cells
is upregulated and expands medially in
Gli2+/Gli3/ and
Gli2/Gli3/ embryos,
indicating that in addition to their role in epaxial muscle cell
determination, Gli2 and Gli3 function also in dorsoventral and mediolateral
patterning of the somite. Moreover, this somite patterning defect persists in
Gli3/Shh/ interlimb
somites, in agreement with the idea that Gli3 and possibly Gli2, act
synergistically in the lateral somite to repress the hypaxial programme. The
exact mechanism of Shh/Gli function in mediolateral patterning remains to be
determined, and in particular, whether Gli repressors act directly or
indirectly on the hypaxial/myotomal genes. Evidence for a direct role would
require that similar studies to that presented here are performed using the
hypaxial Myf5 enhancer. In the avian somite, evidence for an indirect
role proposes that mediolateral patterning is established via counteracting
activities between a lateral Bmp4 gradient originating from the lateral
mesoderm and a medial Shh gradient originating from the axial mesoderm
(Hirsinger et al., 1997
;
Marcelle et al., 1997
). It is
still not clear how Bmp4 and Shh counteract each other, but one possibility
could be that Gli3R interacts with Smad proteins, which mediate Bmp4
signalling in the control of hypaxial gene expression
(Fig. 7)
(Liu et al., 1998
). Finally,
mispatterning could result from the deregulated cell division of specific
muscle progenitor cell subpopulations (i.e. epaxial myotome and hypaxial
cells). In this view, Shh signalling has previously been shown to control cell
cycle progression via the regulation of cyclins
(Bai et al., 2004
;
Barnes et al., 2001
;
Kenney and Rowitch, 2000
), and
is involved in the balance of proliferation/differentiation of limb muscle
cells (Amthor et al., 1999
;
Duprez et al., 1998
). Future
experiments are required to investigate the molecular factors controlling the
activity of the enhancers of hypaxial and myotomal genes and the possible
cooperation of Smad and Gli proteins, as well as the relationship between Shh
signalling and cell cycle regulators in skeletal muscle progenitor cells.
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ACKNOWLEDGMENTS |
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