1 Département Génétique, Développement et Pathologie
Moléculaire, Institut Cochin INSERM 567, CNRS UMR 8104,
Université Paris V, 24 Rue du Faubourg Saint Jacques, 75014 Paris,
France
2 Plateforme Recombinaison Homologue, Institut Cochin INSERM 567, CNRS
UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques, 75014
Paris, France
3 Plateforme Histologie, Institut Cochin INSERM 567, CNRS UMR 8104,
Université Paris V, 24 Rue du Faubourg Saint Jacques, 75014 Paris,
France
* Author for correspondence (e-mail: maire{at}mail.cochin.inserm.fr)
Accepted 14 February 2003
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SUMMARY |
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Key words: Six/sine oculis homeoproteins, Myogenesis, MyoD, Myogenin, Myf5, Pax3
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INTRODUCTION |
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Studies in Drosophila have revealed that sine oculis
(so), the first Six family gene identified, acts within a
synergistic regulatory network that includes eyeless (Pax
family), eyes absent (Eya family) and dachshund
(Dach family), to trigger compound eye organogenesis. Subsequent
genetic analyses revealed that direct interactions of So and
Eya proteins underlie the functional synergy between these proteins
in inducing ectopic eye development
(Pignoni et al., 1997).
However, the molecular basis for this cooperativity is not fully understood,
and no direct target gene of so and eya has been identified
in Drosophila. In contrast, we have previously shown that the Mef3
site, present in the 184 bp myogenin promoter, is needed to confer a pattern
of lacZ reporter gene expression mimicking that of the endogenous
myogenin gene during mouse embryogenesis
(Spitz et al., 1998
). Since
Six1, Six4 and Six5 proteins specifically bind the Mef3 site and are present
in the embryo when myogenin is activated, we proposed that Six homeoproteins
could act as key regulators of myogenin activation. Indeed, misexpression of
Six1 together with Eya2 can induce myogenic genes such as MyoD,
myogenin and myosin heavy chain in chicken somite explants
(Heanue et al., 1999
). Taken
together, these results strongly suggest that Six homeoproteins, acting in
collaboration with an Eya co-activator, might directly transactivate skeletal
muscle target genes. In further agreement with this idea, the Six1, Six4 and
Six5 genes have all been shown to be expressed in somites during embryogenesis
(Oliver, 1995; Ozaki, 2001; Fougerousse, 2002) (our unpublished data).
However, mice lacking either Six4 or Six5 develop normally and show no muscle
defects, suggesting the possibility of mutual compensation among Six
homeoproteins (Klesert et al.,
2000
; Ozaki et al.,
2001
; Sarkar et al.,
2000
).
The skeletal body muscles of vertebrates are derived from somitic
progenitors originating from the epithelial dermomyotome, which in turn gives
rise to the myotome. The medial myotome produces epaxial muscles, which yield
the intrinsic back muscles. The lateral myotome and the lateral portion of the
dermomyotome produce the hypaxial muscles, which includes thoracic intercostal
and abdominal muscles, limb muscles and superficial back muscles, as well as
the diaphragm and the tip of the tongue
(Ordahl and Le Douarin,
1992).
Markers of myogenic specification belong to the family of basic
helix-loop-helix (bHLH) transcription factors composed of Myf5, MyoD, myogenin
and Myf6 (MRF4). The different roles played in vivo by the myogenic regulatory
factors (MRF) have been elucidated from gene targeting experiments. While mice
lacking either Myf5 or MyoD have normal skeletal muscle
(Braun et al., 1992;
Rudnicki et al., 1992
), mice
lacking both Myf5 and MyoD exhibit a complete absence of myogenic cells
(Rudnicki et al., 1993
), thus
indicating that Myf5 and MyoD have redundant functions
(Rudnicki et al., 1993
).
Nonetheless, it is clear that Myf5 and MyoD have different roles in the
determination of epaxial and hypaxial myogenic progenitors
(Kablar et al., 1997
). The
development of hypaxial muscles in sites distant from the somites depends on a
multistep process including specification of progenitors in the lateral
dermomyotome, delamination, migration through different pathways towards
correct sites, proliferation of the migrating precursor cells and then
differentiation. These different steps are controlled by Pax3, the c-Met
tyrosine kinase receptor, its ligand SF/HGF and the homeobox factor Lbx1
(Birchmeier and Brohmann,
2000
). The homeobox factor Mox2 is also essential for normal limb
muscle formation, although it is not required for the migration of myogenic
precursors (Mankoo et al.,
1999
). Furthermore, in addition to the spatial distinction of the
different myogenic compartments, two sequential waves of myofiber formation
can be distinguished. In the mouse, a primary wave of muscle differentiation
begins on about E12.5 and a secondary wave begins at approximately E15.5
(Kelly and Zacks, 1969
). Mice
lacking myogenin or both MyoD and Myf6 display a severe muscle hypoplasia
resulting from defects of secondary myogenesis
(Hasty et al., 1993
;
Nabeshima et al., 1993
;
Rawls et al., 1998
;
Valdez et al., 2000
;
Venuti et al., 1995
),
suggesting the existence of different myogenic populations dependent either on
different thresholds of MRF, or dependent on myogenin alone or on both MyoD
and Myf6 for normal differentiation.
Here, we describe the fetal and embryonic phenotype of
Six1-deficient mice and demonstrate that Six1 is required
for primary myogenesis of most body muscles, particularly those of hypaxial
origin. The Six1 phenotype is partially reminiscent of the myogenic
alterations due to the Pax3 mutation in Splotch embryos
(Tremblay et al., 1998).
However, we show that in contrast to Pax3, Six1 is not required for
delamination and migration of muscle precursor cells. Instead, Six1
appears necessary for MyoD and myogenin activation in distal
territories. Thus, the Six1 homeoprotein is required later than Pax3 during
hypaxial muscle differentiation and plays a role distinct from those of other
hypaxial determinants such as cMet, Lbx1 and Mox2.
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MATERIALS AND METHODS |
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ES cell screening and chimeric mouse production
DNA linearized by NotI digestion (35 µg) was electroporated
(250V; 500 µF) into 1.5x107 MPI-II embryonic stem (ES)
cells. ES cells were selected with 250 µg/ml of G418 48 hours after
electroporation, and with 0.5 µg/ml ganciclovyr, 72 hours after
electroporation. The DNA of 279 resistant clones was analysed by Southern blot
after NcoI digestion. A 5' NotI-SpeI fragment
and a 3' EcoRI-Asp718 fragments were used as external
probes. Three independent homologous recombinant clones were identified. For
the three recombinant clones, 10-12 cells were microinjected into C57BL6
blastocysts, which were further implanted into pseudopregnant mice. Chimaeric
males were obtained for the three clones and yielded germline transmission.
Heterozygous progenies were generated by backcrosses to C57BL6 and 129/SvJ
females, and mice were genotyped by PCR analysis. The forward primer in exon1
was 5'GGGAGAACAGAAACCAAGT3', and the reverse primer in the
lacZ allele was 5'TCATCGCGAGCCATGCGG3'. All homozygous
embryos were genotyped by Southern blot analysis as described above.
X-gal staining of mouse embryo
Embryos were staged, taking the appearance of the vaginal plug as embryonic
day (E) 0.5. Embryos were dissected in PBS, fixed in 4% paraformaldehyde (PFA)
for 3 hours at 4°C, washed twice in PBS, and then stained in
5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal) staining
solution (1 mg/ml X-gal, 5 mM K3Fe(CN)6, 5 mM
K4Fe(CN)6 and 2 mM MgCl2 in PBS) at 37°C.
Genotyping of the embryos was carried out by Southern blot using DNA extracted
from the yolk sac. For section analysis, stained embryos were dehydrated in
increasing concentrations of ethanol, cleared in xylene and embedded in
paraffin. Transverse sections (10-20 µm thickness) were dewaxed in xylene
and mounted in Eukitt.
Whole-mount skeletal staining
To stain cartilage, E18.5 fetuses were skinned and eviscerated prior to
fixation. Embryos were fixed in 95% ethanol for 3 days, and then placed for 24
hours in Alcian Blue solution (15 mg Alcian Blue 8GX (Sigma) in 80 ml 95%
ethanol and 20 ml glacial acetic acid) at 4°C. To stain bone, embryos were
rinsed twice in 95% ethanol and placed for 2 days in 95% ethanol, prior to
clearing in 1% KOH for 2 hours at 4°C, and counterstaining in Alizarin Red
solution (5 mg Alizarin Red (Sigma) in 100 ml of 1% KOH) for 3 hours at
4°C. Clearing of embryos was completed in the following ratios of 1% KOH
to glycerol: 80:20, 60:40, 40:60, 20:80.
Histology, immunohistochemistry and embryos extracts
E18.5 fetuses were snap frozen in isopentane (30°C) cooled in
liquid nitrogen and sliced into 14 µm sagittal cryostat sections. For
histological staining, sections were fixed for 15 minutes in 4% PFA, and
stained with Haematoxylin and Eosin, quickly dehydrated and mounted in Eukitt.
For ß-galactosidase detection, sections were fixed for 5 minutes in 1%
formaldehyde (1x PBS; 5 mM EGTA; 2 mM MgCl2; 0.02% NP40),
incubated in X-gal staining solution at 37°C overnight, and then
counterstained with Eosin, quickly dehydrated and mounted in Eukitt. For fast
or slow myosin heavy chain (MHC) immunodetection, sections are dried for 30
minutes at room temperature, incubated overnight with 1/2000 antibody (MY32
and NOQ7.4.2.D; Sigma) in PBS, washed twice in PBS and treated according to
the Vectastain ABC Kit protocol (Vector Laboratories). Immunostained sections
were mounted in aqueous Vectashield (Vector Laboratories). Counting of the
respective slow and fast myofibers, determination of the cross-section areas
of dorsal intercostal muscles, ventral intercostal muscles, tibialis anterior,
plantaris and median gestrocnemius muscles, and determination of individual
fiber areas were performed using the computer-assisted morphometric
measurements logiciel Image Tool 3.0
(http://ddsdx.uthscsa.edu/dig/download.html).
Fixed embryos were incubated overnight in 20% sucrose before being frozen in isopentane and sectioned (10 µm). Dried sections are incubated for 20 minutes in 1x PBS, 0.1% Triton X-100, blocked for 1 hour in saturation solution (1x PBS, 1.6% goat serum, 2% BSA, 0.1% Triton X-100), incubated overnight with primary antibodies in a saturation solution [Myf5 (Santa Cruz) 1/800, MyoD (DAKO) 1/20, myogenin (DAKO) 1/30, Pax3 1/2000, ß-gal (Rockland) 1/500]. After three washes in PBT (1x PBS, 0.1% Tween 20), slides were incubated for 1 hour with secondary antibodies [1/200 mouse-FITC (Jackson Laboratories), 1/100 rabbit-FITC (DAKO), 1/500 rabbit-Texas red (Vector Laboratories)] and washed in PBT prior to mounting in Vectashield. Apoptosis was detected with the Fluorescein In Situ Cell Death Detection Kit, according to the protocole provided by the manufacturer, Roche.
Preparation of adult muscle nuclear extracts and total embryo extracts, as
well as gel-mobility shift assays (GMSA) were performed as described
previously (Spitz et al.,
1998).
Whole-mount in situ hybridization
Embryos were collected and treated according to the protocol described by
Jowett (Jowett and Lettice,
1994), and adapted for whole-mount in situ hybridization of mouse
embryos. Embryos were dissected in PBS, fixed in 4% PFA for 3 hours at
4°C, washed twice in PBS, dehydrated in sequentially increasing
concentrations of methanol in PBT (25%, 50%, 75%, 2x 100%) and stored at
20°C in 100% methanol. They were subsequently rehydrated following
the reverse procedure up to the PBT stage. Embryos were bleached in 6%
H2O2 in PBT for 1 hour, washed twice in PBT, treated
with proteinase K solution (1 µg proteinase K per ml of 100 mM Tris, 50 mM
EDTA) for 30 minutes at room temperature (RT), washed twice in PBT, refixed in
4% PFA + 0.2% glutaraldehyde for 30 minutes. After two washes in PBT, embryos
were placed for at least 2 hours in hybridization buffer (50% formamide;
5x SSC; 0.5% Chaps; 0.1% Tween 20, 20 µg/ml yeast tRNA, heparin, pH
adjusted to 4.5 with citric acid), before overnight hybridization with 1 µg
digoxigenin (DIG)-labeled antisense RNA probe at 70°C. Embryos were then
washed twice in hybridization buffer and twice in MABT (100 mM maleic acid pH
7.5; 150 mM NaCl; 0.1% Tween 20) at hybridization temperature. Following this
they were incubated for 1 hour in MABT supplemented with 2% blocking powder
(MABTB) at RT, and 2 hours in MABTB containing 20% goat serum (heated to
60°C before use), and overnight at 4°C in MABTB, 20% goat serum
containing 1/2000 alkaline phosphatase anti-DIG Fab fragments (Boehringer).
Embryos were then washed at least 5x 1 hour in PBT, 1% BSA, prior to
incubation for 30 minutes in NTMT (100 mM Tris pH 9.5, 50 mM MgCl2
and 0.1% Tween 20) and staining overnight in NTMT solution containing NBT/BCIP
substrates (Gibco). Stained embryos were refixed in 4% PFA overnight, and
transferred into 100% glycerol.
DIG-labeled antisense RNA probes were prepared from linearized plasmids with DIG RNA Labeling mixture (Boehringer) and T3 (MyoD) or T7 (myogenin) RNA polymerase according to the instructions provided by the manufacturer.
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RESULTS |
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Altered primary myogenesis of most body muscles in
Six1/ embryos
Staining for ß-galactosidase of Six1+/
embryos between E8 and E13.5 revealed that this recombinant allele behaves as
the endogenous one: lacZ expression recapitulates the spatiotemporal
expression of Six1 already published (Oliver, 1995) (C.L., E.S., J.D.
and P.M., unpublished). ß-galactosidase expression is missing in cells in
the anterior region of the forelimb bud (asterisks
Fig. 4A,B) of
Six1/ embryos and the ventral extension of
dermomyotome at the interlimb level is significantly reduced (arrows
Fig. 4A,B) compared with that
of the heterozygotes. Primary myofiber formation begins at approximately E12.5
in the mouse by the fusion of embryonic myoblasts and is complete by E15.5.
After E15.5, a second population of myoblasts begin to fuse to form secondary
myofibers, using the primary myofibers as a scaffold. Major differences
between Six1/ and normal littermates appear
between E12.5 and E13.5 while muscle organogenesis is progressing
(Fig. 4A-H). Whereas head
muscles appear correctly differentiated
(Fig. 4C,D,J), most body
muscles are strikingly reduced and disorganized in
Six1/ embryos, especially shoulder
(arrowhead, Fig. 4G,H),
thoracic and abdominal (double arrow, Fig.
4G,H) muscles as well as the superficial back muscle, latissimus
dorsi (double arrowhead, Fig.
4G,H). However, some deep back muscles such as the longissimus
dorsi seem correctly developed (arrow, Fig.
4G-H). Transverse sections at the interlimb level
(Fig. 4E-F) show that the
external myogenic layer (cutaneus maximus) is absent. In addition, the
internal myogenic layer is reduced and disorganized, and specific muscle areas
(spinotrapezius) are missing. Nevertheless, a few primary myofibers are
present at shoulder level (Fig.
4I). These results clearly show that in the absence of
Six1, the primary myogenesis of most body muscles is strikingly
impaired. Therefore, it is reasonable to speculate that the reduced number of
primary myofibers differentiated at this stage might be the cause of muscle
hypoplasia in Six1/ fetuses.
|
To gain further insight into the role of Six1 during these different steps of myogenesis, we studied the expression of myogenic factors in early embryos. At E9.5, the expression of Myf5 and myogenin is no different in the somites of Six1/ embryos compared with those of normal littermates (Fig. 5A,B). At this stage, Pax3 is also correctly expressed in the dermomyotome (Fig. 5C). Thus, absence of Six1 does not impair early specification of myogenic cells in the somites.
|
Transcription of the lacZ gene inserted at the Six1 locus
is not affected in Six1/ embryos,
(Fig. 5E,F) showing that Six1
does not regulate its own transcription and that the inserted
nls-lacZ gene and PGK-neomycin cassette do not impair
transcription from the Six1 locus. Six1 is located between
the Six6 and Six4 genes on chromosome 12 in the mouse
(Gallardo et al., 1999). Six6
expression is restricted to developing retina, hypothalamic and pituitary
regions (Jean et al., 1999
).
Six4, however, is a putative myogenic regulator despite the fact that knockout
experiments did not lead to muscle defects
(Ozaki et al., 2001
;
Spitz et al., 1998
). Indeed,
the Six4 expression pattern during mouse development is very similar to that
of Six1 (Oliver et al., 1995
;
Ozaki et al., 2001
), and our
data indicate that Six4 expression in somites is not altered in
Six1/ embryos
(Fig. 5J,L). While Six4 is
mainly expressed in the dermomyotome (arrowheads
Fig. 5J,L), in the myotome,
Six4 appears to be colocalised with myogenin (double arrowheads
Fig. 5J-M), suggesting that in
these myotomal cells Six4 might compensate for the absence of Six1.
No increase of cell death within the myogenic progenitor population
in Six1/ embryos
Given that Pax3-expressing cells migrate correctly into the limb buds in
Six1/ embryos and that Pax3 is required for
myoblast proliferation (Borycki et al.,
1999), the Six1/ myogenic
precursor cells do not seem to be impaired in their proliferation potential at
least until E11. To determine whether Six1/
myogenic progenitors undergo apoptosis, we performed TUNEL staining on
sections of E11 Six1/ and
Six1+/ embryos at the forelimb level
(Fig. 6). These experiments did
not provide evidence for an increase in cell death by apoptosis in
Six1/ embryos
(Fig. 6A,B) compared with
heterozygous littermates (Fig.
6D,E). Hence, the ß-galactosidase-expressing cells that
congregate into dorsal and ventral muscle masses in the limb bud are not
stained strongly by the TUNEL reaction
(Fig. 6B,C,E,F).
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DISCUSSION |
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Six1 is not necessary for MRF activation in somite
Our results show that Six1 is not needed for proper
transcriptional activation of Myf5, MyoD and myogenin genes
in the myotome. These results are in apparent conflict with our previous
finding showing that the MEF3 binding site present in the 184 bp myogenin
promoter was absolutely required for its expression in a transgenic mouse
model (Spitz et al., 1998).
Two different hypotheses could explain this apparent discrepancy. The first
possibility that we favour involves a functional redundancy between Six1, Six4
and Six5. These proteins are co-expressed in somites, show similar binding
specificities to the myogenin MEF3 site
(Spitz et al., 1998
) and are
known to be able to activate the myogenin promoter in transient
transfection assays (Ohto et al.,
1999
). The Six4 expression pattern is almost identical to that of
Six1 (Ohto et al., 1999
) and
we show that Six4 is correctly expressed in somites of
Six1/ embryos at E11. Thus, Six4 might
partially compensate for the absence of Six1 to activate myogenin in
the myotomal cells. Conversely, the absence of muscle defects in mice lacking
Six4 or Six5 may be due to partial genetic redundancy and compensation by Six1
(Klesert et al., 2000
;
Ozaki et al., 2001
;
Sarkar et al., 2000
).
Nevertheless, as MyoD and myogenin expression is delayed and
reduced in limb buds of Six1/ embryos,
Six4 and Six5 cannot substitute for Six1 functions
in all myoblast populations. According to this hypothesis the selective muscle
hypoplasia described in Six1/ mice could
result either from insufficient levels of Six4 and Six5 to compensate for Six1
in the affected myogenic precursor cells or from the existence of specific
Six1 target genes.
The second possibility is that the endogenous myogenin gene does
not behave as the 184 bp promoter fragment used in the transgenic study
(Spitz et al., 1998). While
this promoter fragment is efficient in recapitulating the embryonic expression
of myogenin in a transgenic animal model, enhancer elements upstream
of the 184 pb promoter have been characterized that are active during
embryonic development (Cheng et al.,
1993
; Yee and Rigby,
1993
). According to this second hypothesis, Six1 would not occupy
the MEF3 site of the endogenous myogenin gene in
Six1/ embryos. Nevertheless, enhancers at
the myogenin locus would override this absence and allow
myogenin transcription in the somites. Absence of MEF3 site occupancy
of the native myogenin locus could thus have a less severe
repercussion than mutation of the MEF3 site on the 184 bp fragment used in our
previous transgenic investigations. Analysis of double Six1/Six4 and
Six1/Six5 knockout mice will distinguish between these two
hypotheses.
Six1 is needed for MyoD and myogenin
activation in limb buds at E11.5
We have demonstrated that in Six1/ mice,
hypaxial progenitors are correctly specified in somites, migrate normally into
the limb buds and do not undergo apoptosis. However, there is a failure of
these cells to activate MyoD and myogenin at E11.5. These
observations argue in favour of a direct role of Six1 in
MyoD and myogenin gene regulation, as previously suggested
by misexpression experiments in chicken somite explants
(Heanue et al., 1999).
Accordingly, Six1 could bind to enhancers of these genes that are
specific for their transcriptional activation in limb buds and in the
ventrolateral extension of the dermomyotome.
We have already demonstrated that Six homeoproteins can directly control
myogenin expression (Spitz et
al., 1998). Regulatory elements controlling expression of
MyoD in different territories have been characterized
(Goldhamer et al., 1995
;
Kablar et al., 1999
) and
consist of two regulatory regions upstream of the transcription start site: a
core enhancer at 20 kb and a distal enhancer at 11 kb. The
distal enhancer alone is not sufficient to drive transcription in embryonic
limb buds at E11.0. However, at E12.0 this enhancer is functional, showing
that the other regulatory elements present in the core enhancer are required
to activate MyoD between E11.0 and E12.0
(Asakura et al., 1995
). This is
reminiscent of our observations in Six1/
limb buds: while undetectable at E11.5, some MyoD-positive cells are
present at E12.5, suggesting that Six1 could control MyoD
transcription through regulatory elements present in the distal enhancer.
Careful analysis of this distal enhancer revealed the presence of a putative
MEF3 site (box17), for which mutations lead to a reduced expression of
MyoD (Kucharczuk et al.,
1999
). However, a delay of 1 day in MyoD activation in
the limb buds of Six1/ embryos is unlikely
to be sufficient to impair primary myogenesis, or to provoke severe muscle
hypoplasia in fetuses, since a delay of 2.5 days in myogenic differentiation
in limb buds of MyoD/ embryos does not lead
to subsequent muscle alterations (Kablar
et al., 1997
). Therefore, it appears that Six1 is not
only involved in MyoD and myogenin gene activation in limb
buds, but also acts at later steps of the myogenic differentiation
process.
Six1 is crucial for primary myogenesis of body muscles
Primary myogenesis is strikingly impaired in E13.5
Six1/ embryos. Between E12.5 and E13.5
myoblasts fuse into multinucleated fibers and individual muscles adopt their
characteristic shapes and positions
(Baumeister et al., 1997). The
morphogenesis characterizing primary myogenesis is altered in E13.5
Six1/ embryos, even if early steps of
myogenic determination have been correctly initiated in myotomal cells,
showing that Six1 plays a crucial role in these morphogenetic events. Although
a number of proteins are known to regulate events required for myogenesis in
the early embryo, far less is known about the molecular factors needed during
primary myogenesis. A delayed onset of primary myogenesis of hypaxial and
epaxial muscles has been described in MyoD/
and Myf5/ mutants, respectively
(Kablar et al., 1997
).
However, such a delay does not lead to subsequent muscle hypoplasia as found
in Six1 mutants. Although severe muscle hypoplasia is found in mice
lacking either myogenin alone or both MyoD and Myf6
(Hasty et al., 1993
;
Nabeshima et al., 1993
;
Rawls et al., 1998
;
Venuti et al., 1995
), a
reduction in the number of myofibers in these mutants results mainly from an
altered secondary rather than primary myogenesis. While mice lacking NFATC3
also have reduced muscles as a consequence of altered primary myogenesis
(Kegley et al., 2001
), these
mice are viable and do not show the profound and selective muscle hypoplasia
observed in Six1 knockout mice.
Myogenic phenotype of Six1/ embryos
is partly reminiscent of Splotch mutants
The muscle phenotype found in Six1 knockout mice most closely
resembles the myogenic defects described in Splotch mutants in which
the Pax3 gene is mutated (Fig.
8A). Pax3 is required for specification and migration initiation
of hypaxial progenitors (Bober et al.,
1994; Daston et al.,
1996
; Goulding et al.,
1994
; Tremblay et al.,
1998
). In Splotch mutants, the migration process is
impaired and consequently no myoblasts reach the most distal regions. As a
result, most of the hypaxial muscles, such as limb, tongue, diaphragm and the
ventral thoracic and abdominal muscles fail to form
(Tremblay et al., 1998
). The
similarity in the phenotypes caused by Six1 and Pax3 mutations suggest the
possibility of a functional link between these two homeodomain transcription
factors.
|
It has been shown recently that the homeoprotein Six1 may be localised
either in the cytoplasm or in the nucleus of myogenic cells during human
embryogenesis (Fougerousse, 2002), suggesting that Six1 activity may
depend on environmental signals controlling Six1 protein translocation into
the nucleus. In addition, Six proteins can recruit Eya co-factors to activate
the transcription of their target genes, and Eya proteins may also be
localized either in the cytoplasm or in the nucleus
(Buller et al., 2001;
Fan et al., 2000
;
Heanue et al., 1999
;
Ohto et al., 1999
). The
nuclear localisation of Six1 protein has been documented in adult skeletal
muscles, where it controls expression of the muscle promoter of the
aldolase A gene (Spitz et al.,
1998
; Spitz et al.,
1997
). Thus, it will be interesting to establish a correlation
between the Six1 nucleo-cytoplasmic shuttle, the wide expression of
this protein, and the phenotype of the Six1/
mice.
Specific myogenic features of Six1/
mice compared with cMet-, Gab1-, Lbx1- and Mox2-deficient
mice
Whether Pax3 and Six1 can cooperate to activate genes
required for hypaxial lineage determination remains to be clarified.
Interestingly, as in Six1 knockout animals, mice lacking c-Met,
Gab1, Lbx1 or Mox2 have, with certain important differences,
impaired differentiation of the hypaxial lineage
(Fig. 7).
The c-Met-tyrosine kinase receptor, whose expression is directly regulated
by Pax3 (Epstein et al.,
1996), plays an essential role in the migration initiation of
myogenic precursor cells (Bladt et al.,
1995
; Maina et al.,
1996
). Its specific ligand SF/HGF (scatter factor/hepatocyte
growth factor) is expressed in limb mesenchyme and provides the signal for
migration (Dietrich et al.,
1999
; Scaal et al.,
1999
) that is mediated by c-Met and subsequently relayed by
intracellular signalling pathway requiring Gab1
(Sachs et al., 2000
). The
myogenic phenotype of mice deficient for the c-Met gene is similar to
the myogenic alteration described in Splotch mutants
(Bladt et al., 1995
).
Gab1/ embryos also display impaired
migration of myogenic precursor cells into the limb anlagen, leading to lack
of the diaphragm and extensor muscles of the forelimb
(Sachs et al., 2000
).
Pax3 is also necessary for Lbx1 expression in myogenic
precursor cells of the limb (Mennerich et
al., 1998). Lbx1 expression is restricted to the lateral
part of the somites located at occipital, cervical and limb levels, where
myogenic precursor cells delaminate and subsequently migrate over large
distances along characteristic paths
(Dietrich et al., 1998
;
Uchiyama et al., 2000
). In
Lbx1/ embryos, precursor cells delaminate
but fail to migrate laterally into the limb buds to form the dorsal muscle
masses (Gross et al., 2000
).
At birth, inactivation of Lbx1 leads to the lack of dorsal extensor
muscles in forelimbs and to the absence of muscles in hindlimbs
(Brohmann et al., 2000
;
Gross et al., 2000
;
Schafer and Braun, 1999
).
These muscular alterations differ from those of
Six1/ fetuses, in which forelimb muscles are
more affected than hindlimb muscles. Moreover, in distal hindlimbs of
Six1/ fetuses the dorsal extensor muscles
are reduced whereas most ventral flexor muscles are lacking. These results
suggest that Six1 and Lbx1 genes have distinct functions
during hypaxial muscle development, which could involve actions in
complementary myogenic limb compartments.
Mox2 is another crucial gene controlling limb muscle development
(Mankoo et al., 1999). In the
distal forelimb of Mox2-deficient mice, several muscles of the flexor
compartment are absent and the extensor muscles are severely reduced. In the
hindlimb, although no specific muscle is absent, the overall muscle mass, in
particular that of the gastrocnemius, is greatly reduced. These limb muscle
alterations are similar to the phenotype of
Six1/ fetuses. However, whereas
Six1/ mice have no diaphragm, a very reduced
tongue and disorganized body-wall muscles,
Mox2/ mice do not display such muscle
defects (Mankoo et al.,
1999
).
Rib defects might be a consequence of the myogenic alterations
Rib and sternum defects are also important features of
Six1/ mutants. This skeletal phenotype is
reminiscent of the rib defects initially reported in homozygous Myf5
mutant mice (Braun et al.,
1992; Tajbakhsh et al.,
1996
). It has been shown more recently that these rib defects
could result from the residual presence of the PGKneo cassette at the
Myf5 locus (Kaul et al.,
2000
). As a number of potential inductive signals expressed in
myotome such as FGFs and PDGF
are absent in Myf5 mutant mice
(Grass et al., 1996
;
Tallquist et al., 2000
), it
has been proposed that the rib phenotype could result from secondary events
resulting from myotome defects. This hypothesis was further supported by the
generation of three different alleles of the Myf6 gene, which is
located 8 kb upstream of the Myf5 gene on mouse chromosome 10
(Braun and Arnold, 1995
;
Patapoutian et al., 1995
;
Zhang et al., 1995
).
Nevertheless, the generation of two other Myf5 alleles, which do not
produce any malformations of the ribs seems to rule out a direct involvement
of the Myf5 and/or Myf6 proteins in the generation of the rib phenotype
(Kaul et al., 2000
). This does
not necessarily mean, however, that cross-talk between different somitic
layers is not required for rib formation, since the knockout of the
myogenin gene and the mutation of the Pax3 gene that reside
on different chromosomes also result in a rib phenotype
(Dickman et al., 1999
;
Hasty et al., 1993
;
Henderson et al., 1999
;
Nabeshima et al., 1993
;
Vivian et al., 2000
).
In Six1/ mice, only the sternal region of
the ribs is affected. The distal rib primordium arises from the lateral
portion of the somite (Olivera-Martinez et
al., 2000), but its precise origin is still controversial. While
some data demonstrate that both the proximal and the distal parts of the ribs
originate from the sclerotomal mesenchyme
(Huang et al., 2000
), other
results suggest that the sternal segment of the ribs originate from the
ventrolateral part of the dermomyotome
(Kato and Aoyama, 1998
). The
ventrolateral sclerotome marker, Mfh-1 (FoxC2), is closely associated with
Pax3 in the somitic bud that invades the lateral plate mesoderm at the
thoracic level, suggesting that interactions might occur between the incipient
ribs and intercostal muscles during their migration and differentiation
(Brent and Tabin, 2002
;
Sudo et al., 2001
). In
Six1/ mice, the rib defects restricted to
the distal segments are correlated with the muscle defects that are more
severely affected in the ventral region than in the dorsal anlagen, suggesting
that these skeletal defects are secondary to adjacent muscle defects.
Finally, it seems that in vertebrates the genetic markers of the hypaxial
compartment are more diverse than initially suspected, and that different
myogenic programmes can be activated, thereby leading to muscular diversity.
Six1 appears as a new genetic marker whose function is unique for the building
of specific body and limb muscles. Presently, no human pathology has been
associated with SIX1 mutations, but deletions in 14q (q22q23)
overlapping the SIX1 locus lead to multiple abnormalities including
muscle hypotonia (Bennett et al.,
1991; Gallardo et al.,
1999
; Lemyre et al.,
1998
), which may be due to SIX1 haploinsufficiency.
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ACKNOWLEDGMENTS |
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