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First published online 23 March 2005
doi: 10.1242/dev.01773
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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 de recombinaison homologue, Institut Cochin – INSERM 567,
CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques
75014 Paris, France
3 Plateforme d'histologie, Institut Cochin – INSERM 567, CNRS UMR 8104,
Université Paris V, 24 Rue du Faubourg Saint Jacques 75014 Paris,
France
4 Division of Medical and Molecular Genetics, Guy's Hospital, London SE1 9RT,
UK
Author for correspondence (e-mail:
maire{at}cochin.inserm.fr)
Accepted 9 February 2005
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SUMMARY |
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 TOP SUMMARY IntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Key words: Six/sine oculis homeoproteins, Pax3, Myogenesis, Hypaxial lip, Migration, Myotome, Syndetome
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Introduction |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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;
Pineda et al., 2000;
Seo et al., 1999;
Dozier et al., 2001;
Bebenek et al., 2004).
Originally, sine oculis was identified in Drosophila, where
it belongs to a network of genes that drives eye development synergistically
with eyeless (Pax orthologs), eyes absent and
dachshund (Cheyette et al.,
1994; Pignoni et al.,
1997; Serikaku and O'Tousa,
1994). Absence of sine oculis leads to massive apoptosis
of precursor cells in the developing compound eye
(Cheyette et al., 1994). In
Drosophila, two other sine oculis related genes have been
identified: optix, which is expressed in the eye imaginal disc
(Toy et al., 1998); and dSIX4,
which is required for myoblast fusion
(Kirby et al., 2001). In
vertebrates embryos, Six1 and Six4 expression have been detected in
overlapping territories: neural placodes, Rathke's pouch, DRG, dermomyotome,
myotome, limb bud mesenchyme and in myogenic migrating precursors
(Esteve and Bovolenta, 1999;
Oliver et al., 1995;
Ozaki et al., 2001;
Bessarab et al., 2004;
Fougerousse et al., 2002;
Laclef et al., 2003a;
Schlosser and Ahrens, 2004).
This co-expression may reflect co-regulation of both genes, which are only
separated by 100 kb (Boucher et al.,
1996). Six4–/– mouse present with
no major developmental defects (Ozaki et
al., 2004), while Six1–/– mouse
neonates do not survive and have defects in many organs, including kidney
(Xu et al., 2003), thymus,
parotid glands and ear (Laclef et al.,
2003b; Zheng et al.,
2003; Ozaki et al.,
2004), and rib, craniofacial and muscle deficiencies
(Laclef et al., 2003a;
Li et al., 2003). Six1 is also
expressed at high levels in adult skeletal muscles where it participates, in
synergy with Eya1, in the establishment of the fast/glycolytic phenotype of
the myofiber (Grifone et al.,
2004). With respect to muscle development, it has been shown that
Six1–/– fetuses have a selective loss of
muscles including diaphragm, proximal and distal forelimb muscles, distal
ventral hindlimb muscles and abdominal muscles
(Laclef et al., 2003a).
Back skeletal muscles are derived from somitic progenitors originating from
the epaxial dermomyotome. At the interlimb level, the lateral myotome and
dermomyotome produce the hypaxial muscles, including thoracic intercostal,
abdominal and limb muscles (Buckingham,
2001). Extension of the lateral dermomyotome is under the control
of Pax3 (Williams and Ordahl,
1994), and in Pax3–/– mouse most hypaxial
migrating myogenic precursors are lacking
(Tremblay et al., 1998).
Except for tongue muscles, which have a somitic origin, head muscle
progenitors originate from head paraxial mesoderm and migrate into the
pharyngeal arches to give rise to head and neck muscles
(Hacker et al., 1998). Limb
muscles are formed by cells of the dermomyotome that delaminate from somites
and migrate into the limb bud where they further proliferate before activating
Mrf genes (Rees et al., 2003).
Mrf activation, and more particularly Myf5, is under the control of different
signaling pathways, depending on the position of the myogenic precursors cells
in the embryo (Hadchouel et al.,
2003; Tzahor et al.,
2003). Delamination of myogenic precursors from the dermomyotome
is under the control of the Met tyrosine kinase receptor and scatter
factor/hepatocyte growth factor (SF/HGF) produced by the limb mesenchyme
(Bladt et al., 1995;
Dietrich et al., 1999).
Invasion of the limb by myogenic progenitors is also under the control of the
Lbx1 homeogene (Alvares et al.,
2003). Both Lbx1 and Met expressions are under the control of
Pax3, as delamination and myoblast migration into the limb bud is prevented in
Pax3–/– mice
(Bober et al., 1994;
Epstein et al., 1996;
Goulding et al., 1994). Pax3
expression is not impaired in Six1–/– embryos,
allowing myoblast migration and limb muscle formation
(Laclef et al., 2003a). We
suggested that ontogenesis of most remaining axial and limb muscles present in
Six1–/– embryos are under Six4 or Six5
control, as we detected Six5, Six4 and Pax3 expression in migrating myoblasts
of Six1–/– embryos at the limb level
(Laclef et al., 2003a) (data
not shown).
The sclerotome, another somitic compartment, gives rise to the axial
skeleton and ribs. Interactions between the incipient ribs and growing
myotomes at the intercostal level might occur through Fgf and Pdgf molecules
produced by the myotomes (Huang et al.,
2003). In this context, inhibition of Fgf signaling causes
deletion of developing ribs (Huang et al., 2003b). The syndetome, which is
derived from the sclerotome, gives rise to the axial tendons. Scleraxis is one
of the earliest genetic marker characterized for this somitic lineage
(Schweitzer et al., 2001).
Induction and individualization of the syndetome requires the
dermomyotome/myotome contact involving Fgf signaling
(Brent et al., 2005).
To test the hypothesis that Six4 acts in common with Six1 during myogenesis, we produced double Six1Six4 knockout (dKO) mice. We show here that Six1–/–Six4–/– embryos develop a more severe muscle phenotype than did the Six1–/– embryo. No muscle is detected in the limbs because of the downregulation of Pax3 in the ventral dermomyotomal lips of the somites from which hypaxial progenitors arise. There is no proliferation defects but most of these precursors migrate aberrantly, lose their myogenic identity and die by apoptosis. Epaxial and non migrating hypaxial musculature is affected by severely compromised expression of Mrf genes within the myotome. Our results finally suggest that the rib phenotype developed by Six1–/–Six4–/– fetuses could be the result of a loss of Mrf4 expression and a downregulation of Fgf production in the myotome.
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Materials and methods |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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FixII DNA clones from a
129Sv genomic library. We subcloned a 14 kb NotI-NotI
genomic fragment comprising the entire Six4 gene into pBluescript KS+
(Stratagene). A 3' 3.1 kb Nar-EcoRI fragment comprising the end
of the first intron and the beginning of the second Six4 exon was then ligated
in a plox2hygro plasmid leading to p3'Six4lox2hygro. A 5' DNA
region (NotI-Eco47III 4.2 kb fragment) was ligated in frame
into a pEGFP expression vector (Clontech). This 5' genomic fragment
possesses 3.2 kb DNA upstream of Six4 transcription initiation sites
as well as the first 144 amino acids. The 5'Six4-GFP fragment
was further cloned in p3'Six4lox2hygro, leading to the final
invalidation plasmid. Homologous recombination with this disruption vector was
expected to lead to the deletion of the last 370 amino acids of the first Six4
exon, including most of the Six domain and the entire homeodomain, which,
together, are responsible for the specific DNA-binding activity of Six4
protein.
ES cell screening and chimeric mouse production
Specific Six4 DNA fragment digested by NotI digestion (35 µg),
to eliminate plasmid DNA sequences, was electroporated (250V; 500 F) into
1.5x107 Six1-lacZ embryonic stem cells
(Laclef et al., 2003a). ES
cells were selected with 150 µg/ml hygromycin 24 hours after
electroporation. The DNA of 310 Six1-Six4 resistant clones
was analyzed by Southern blot after PstI digestion. A 5'
fragment and a 3' fragments were used as external probes. Ten to 12
cells of three Six1-Six4 independent recombinant ES clones were microinjected
into C57BL6 blastocysts, which were further implanted into pseudopregnant
mice. Heterozygous progenies were obtained by backcrosses to C57BL6 and
129/SvJ females. All three clones were recombined on the Six1-lacZ
allele, as F1 animals from the three clones were either wild type or
heterozygous for both Six1 and Six4. F1 progeny was then crossed with EIIaCre
animals expressing the Cre recombinase ubiquitously under the control of E2A
promoter (Holzenberger et al.,
2000). Deletion of the PGK-hygromycin cassette was ascertained by
Southern blot analysis. All homozygous embryos and fetuses have been genotyped
by Southern blot analysis.
X-gal staining, whole-mount skeletal staining, histology and
immunohistochemistry of the embryos were performed as described previously
(Laclef et al., 2003a).
Vibratome section (120 µm) were performed after inclusion of the embryos in
4% agarose. Sections were then mounted in Kaiser's glycerol gelatin solution
(Merck). Gel mobility shift assays were performed as described
(Grifone et al., 2004).
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Results |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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). Inactivation of the Six4 gene was achieved by
replacing the Six domain and homeodomain of the Six4 protein, both of which
are required for specific DNA binding, by the EGFP cDNA
(Fig. 1A). To identify the
recombination events at the Six4 locus that took place on the Six1 recombined
and wild-type allele, we analyzed the DNA of the progeny of chimeric animals
for both Six1 and Six4. In the three independent ES clones where recombination
events at the Six4 locus were identified, F1 DNA analysis showed that all
three recombination events occurred on the recombined Six1 allele, leading to
a Six1LacZSix4gfp/Six1+Six4+
genotype, hereafter referred to
Six1+/–Six4+/–. Southern
blot analysis with 5' and 3' external probes confirmed these
events (Fig. 1B).
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Skeletal malformations in Six1–/–Six4–/– newborn animals
Examination of the skeleton of E18.5
Six1–/–Six4–/–
fetuses revealed a more severe phenotype than was observed in
Six1–/– fetuses
(Laclef et al., 2003b). This
was most severe at the rib/sternum level, where the distal ribs were reduced
to small protrusions (Fig.
2B,C). None of the six
Six1–/–Six4–/–
fetuses analyzed in this study, on the two genetic mutant backgrounds, shows
rib attachment to the sternum (Fig.
2), whereas such attachments were observed occasionally in
Six1–/– mutants
(Laclef et al., 2003a). At the
cranial level, the mandible bone is drastically shortened and the fetuses have
small orbits with protruding eyes. Meckel cartilage is absent, jugal, nasal
and premaxilla bones are absent, the palatal process of the maxilla is
lacking, the squamosal bone is reduced, and the cartilages of the inner ear
and the ectotympanic bone are lacking (Fig.
2D,E, and data not shown). It thus appears that absence of Six4 in
a Six1–/– background leads to a more severe
craniofacial and rib phenotype. In both forelimbs and hindlimbs, a clinodactly
(digit curvature) with a curved fifth digit was observed in all E18.5 fetuses
analyzed (n=6, Fig.
2F,G).
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;
Gallardo et al., 1999;
Laclef et al., 2003b;
Lemyre et al., 1998;
Xu et al., 2003). It has been
shown that in these individuals the SIX6 gene was deleted, suggesting
that anophtalmia and pituitary hypoplasia could be caused by SIX6
haploinsufficiency (Gallardo et al.,
1999), which agrees with analysis of Six6 KO mice
(Li et al., 2002). We
identified hemizygosity for both SIX1 and SIX4 genes in a
human 21-week-old fetus (Fig.
2H). This fetus had hypoplastic kidneys and clinodactily
(Bennett et al., 1991),
suggesting that Six1 and Six4 haploinsufficiency could be responsible for
these developmental defects.
General muscle deficiencies in double Six1Six4 new born animals
We have shown previously that Six1–/–
fetuses present a severe but selective muscle hypoplasia, while
Six4–/– fetuses are normal
(Laclef et al., 2003a;
Ozaki et al., 2001). As Six1
and Six4 are co-expressed during myogenesis, we suspected a compensatory role
for Six4 in the genesis of the remaining muscles of
Six1–/– fetuses. X-gal staining of
Six1–/–Six4–/–
E18.5 fetuses revealed a more severe muscle phenotype than observed previously
in Six1–/– fetuses
(Fig. 3A-L). All muscles of the
distal forelimb and hindlimb are missing, as revealed by the absence of any
fast or slow myosin positive fibers (Fig.
3I-L; data not shown), and all muscles of the proximal limbs are
missing as revealed by X-gal/Eosin staining
(Fig. 3A-D for the forelimb;
Fig. 3G,H for the hindlimb).
Hypaxial musculature at the trunk level is also severely diminished
(Fig. 3A,B) and most ventral
muscles are missing (see enlargement of
Fig. 3G,H). Back muscle masses
at the forelimb level and more rostrally are incorrectly shaped, while back
muscles at the interlimb level and at the hindlimb level show less
disorganization (Fig. 3E,F).
This suggests that Six1 and Six4 are dispensable for the formation of
remaining back muscle fibers because all of these muscles express
lacZ (Fig. 3C-F),
myosin heavy chains and desmin proteins (data not shown). Tendon formation did
not appear altered in these remaining muscles (data not shown). Most of the
head muscles, including the masseter, temporalis, pterygoid and extra-ocular
muscles are present at E18.5 (Fig.
3A,B; data not shown), showing that Six1 and Six4 are not required
for the formation of head muscles. However, while the mylohyoid is present,
the genioglossus muscle is absent and intrinsic tongue muscle is reduced (data
not shown) in dKO E18.5 fetuses.
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Severe myotomal disorganization in Six1–/–Six4–/– embryos
Six1 and Six4 expression has been monitored by X-Gal staining and GFP
expression at different embryonic stages ranging from E9.5 to E12.5 on
wild-type and null backgrounds (Fig.
4). In E9.5-E10.5
Six1–/–Six4–/–
embryos, Six1 and Six4 genes are still highly expressed in
somites, suggesting that Six1 and Six4 proteins are not required for their own
transcription in these structures. Conversely, their gene expression is
severely reduced at the cranial level in the trigeminal placode and in the
otic vesicle, where X-Gal staining becomes barely detectable at E10.5
(Fig. 4E-H). These results
suggest either that in these cells Six1 and Six4 control positively their own
transcription, or that apoptosis occurs in these placodal precursors because
they require Six1 and Six4 to survive (see later). At E9.5, Six1 expression
was greatly reduced in the otic vesicle of
Six1–/–Six4–/–
embryo (Fig. 4, compare A,C
with B,D) probably reflecting a loss of
Six1–/–Six4–/–
cells through apoptosis (Ozaki et al.,
2004; Zheng et al.,
2003). At E9.5, the maxillary primordium and mandibular primordium
of the first and second branchial arches appeared fused (compare
Fig. 4C with 4D).
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). Most notably, Six1 and Six4 gene expression is not
detected in the ventral and dorsal lips of the dermomyotome
(Fig. 4Q,R,U,V). At E12.5,
ß-gal-positive cells are dispersed in the
Six1–/–Six4–/–
somites when compared with the heterozygous
(Fig. 4W-Z). ß-Gal cells
also appear less abundant at this stage in the homozygote. It is also evident
that most of cells are blocked in their migration pathway, and fail to enter
the limb bud (Fig. 4L,N,P).
Mislocated cells in Six1–/–Six4–/– embryos die by apoptosis
Whether Six1 and Six4 are important for the proliferation of myogenic
progenitors was first examined by the analysis of BrdU incorporation and
phospho-histone H3 expression. No significant difference of proliferation was
observed in the somites of E10-E11 dKO versus control embryos (data not
shown). We next analyzed confocal sections to evaluate the total number of
ß-gal-positive cells in one somite of E10.5 heterozygous and dKO embryos.
Analysis of seven serial sections at the hindlimb level showed that the number
of ß-gal-positive cells in the dKO (78±28) was significantly
greater (P<0.0005, Student's t-test) than in
heterozygotes (32±14), which reflects their failure to migrate to the
limb bud. At the abdominal level, we found 229 positive cells (±39) in
a heterozygous and 302 in a dKO (±89) (P<0.025), showing no
proliferation defects of
Six1–/–Six4–/–
cells at this level. At the forelimb level, we found 55 positive cells
(±19) in a heterozygous and 152 in a dKO (±39)
(P<0.0005). These results combined with the observation of
comparable levels of ß-gal and GFP accumulation in heterozygous and dKO
embryos (Fig. 4I,J,U,V)
indicated that at least until E10.5, the absence of Six1 and Six4 in vivo does
not prevent proliferation of the somitic myogenic precursors.
As Six1–/–Six4–/– fetuses were smaller than their littermate without proliferation defect in somitic structures, at least between E10 and E11, we decided to examine the extent of apoptosis in the dKO embryos. In contrast to control heterozygous embryos, activated caspase 3-positive cells were detected at the ventral lip level of dKO embryos at E10.5 (Fig. 5A-D), and later at E12.5 in cells which were rerouted ventrally (Fig. 5E,F). These apoptotic events were not specific to the somitic compartment as caspase 3-positive cells were also found at E10.5 in branchial arches (Fig. 5G,H).
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Lbx1 expression is not detected at forelimb and hypoglossal chord levels in dKO embryos when compared with the control (Fig. 6, part III, A-D). Lbx1 is weekly detectable in three out of the six Lbx1-expressing somites facing the hindlimb bud (Fig. 6, part III, E,F), corresponding to sacral somites where Met and Pax3 are expressed ventrally. Lbx1 was not detected in occipital somites and at the forelimb level in E9.5 dKO embryos (data not shown).
Bandshift assays using a potential MEF3 site (TCAGGTTTC) found in the human and mouse Lbx1 promoter (TCAGGTTggC) as a probe together with nuclear extracts or in vitro synthesized Six1 protein, demonstrated a specific interaction of Six proteins with the Lbx1 promoter (Fig. 6, part III, G; data not shown). Hence, Lbx-1 may be under the control of Six proteins, at least in a population of myogenic migrating precursors. Nonetheless, the presence of a few Lbx-1 positive cells in the hindlimb suggests that some specific myogenic precursors can activate Lbx1 and Met in the absence of Six1 and Six4 homeogenes.
Finally, at hindlimb and interlimb levels, no major difference in Pax7 expression, another dermomyotomal marker, is detected between control and dKO embryos (Fig. 6, part IV, C,D). However, at the forelimb level, Pax7 is slightly reduced in the ventral and dorsal dermomyotome in the dKO embryo (Fig. 6, part IV, I,J).
Pax3 expression in the dermomyotome is under the control of Six1 and Six4
We next performed double immunofluorescence on transverse sections of E10.5
embryos to detect both Pax3 and lacZ expression, the latest
reflecting the presence of cells that turned on Six1 gene expression
(Fig. 7). Caudally to the
hindlimb in both heterozygous and homozygous embryos, Pax3 and Six1 are
co-expressed in the entire epithelial dermomyotome
(Fig. 7A-D). At the hindlimb
level in heterozygous embryos, Pax3 and Six1 are still co-expressed in the
entire dermomyotome, in migrating myoblasts, and in laminin- and
desmin-positive myotomal cells (Fig.
7E-F,I-J). In caudalmost somites facing the hindlimb of dKO
embryos, Pax3 and Six1 genes are co-expressed ventrally in a region where
there was no lateral migration to the limb
(Fig. 7G-H) and in some
laminin- and desmin-positive myotomal cells
(Fig. 7K-L). In rostralmost
somites facing the hindlimb, Pax3 expression becomes lower ventrally, whereas
many Six1-positive cells are still present
(Fig. 7M,N). Without Six
proteins, these cells lose Pax3 expression. They subsequently lose their
identity and migrate medially instead of invading the bud or differentiating
in the myotome (Fig. 7G,H,M,N),
even when Met and Lbx1 are transiently activated
(Fig. 6, parts II,III). Thus,
Six proteins are required to activate Pax3 expression and to impose a myogenic
fate to dermomyotomal cells of the somites. Interestingly, in the most caudal
somites of dKO embryos, the weak level of Lbx1 observed in hypaxial cells
expressing Pax3 suggests that Pax3 requires Six1 and Six4 to activate
Lbx1 gene to high level (Fig.
6, part III). At the thoracic level in dKO embryos, Pax3
expression is severely reduced and remains only detectable in a medial domain
of the somite (Fig. 7O-R).
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Six homeoproteins control early Mrf genes expression
We have previously shown that Six proteins were directly required for
myogenin transactivation during embryogenesis
(Spitz et al., 1998). Although
no alteration of myogenin expression has been reported in
Six4–/– embryos, we showed that Six1 was
required for early expression of myogenin in limbs but not in the myotome
(Laclef et al., 2003a). We
show here that 90% of myogenin level was lost in the absence of Six1 and Six4
at E9.5 (Fig. 8, part II, A,B).
A few specific cells can nevertheless bypass Six signaling to activate
myogenin and probably give rise to the remaining epaxial muscles present in
Six1–/–Six4–/–
embryos at older stages. Their central localization within the myotome,
combined to the remaining Pax3 expression, suggest that these cells only
originate from the caudal lip of the dermomyotome. At E10.5, myogenin
expression remains very weak (Fig.
8, part III, A,B) at interlimb level, barely detectable in more
rostral somites and is faintly detectable on vibratome sections
(Fig. 8, part III, C-F). We
next examined the three myogenic determination genes Myf5, Myod1 and
Mrf4. In absence of Six1 and Six4, most of the
early Myod1 activation is blocked at E10.5
(Fig. 8, part IV).
Myod1 remains expressed in ventral–hypaxial myotome of
interlimb somites (Fig. 8, part
IV, C-H). Early Myf5 expression at E9.25 (20-somite stage) is lower in dKO
embryos, even if correctly initiated in their epaxial compartment
(Fig. 8, part I). At E10.5, the
ventral and dorsal expression of Myf5 was lower, especially in rostral
somites, (Fig. 8, part V, A-H),
showing again the impaired ability of dermomyotomal lips in producing myogenic
cells. Myf5 is restricted to the center of the myotome when examined on
transverse sections (Fig. 8,
part V, C-H). At E9.5 and E10.5, Mrf4 is completely undetectable in the dKO
(Fig. 8, part VI; data not
shown), placing Six proteins upstream of this determination gene. At E11.5
Myod1 expression remain low in dKO embryo, and is undetectable in the most
ventral and dorsal positions (Fig.
8, part VII).
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), and
presumably in proliferating cells of dKO embryos as well
(Fig. 9, part IV). Finally,
scleraxis expression in the syndetome has been shown to be controlled by Fgf
signaling provided by the adjacent myotome
(Brent and Tabin, 2004).
Interestingly, no scleraxis expression is detected in the somites of
Six1–/–Six4–/–
embryos (Fig. 9, part V).
Moreover Six1 and Six4 proteins are not produced by normal syndetomal cells
(Fig. 9, part VI; data not
shown). These results suggest that some specific Fgf molecules emanating from
the somite are absent in dKO embryos, leading to the loss of early scleraxis
expression.
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|
Discussion |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussionREFERENCES |
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Met is the crucial factor necessary for myogenic cells of the VLL to
undergo an epithelial to mesenchymal transition prior to migration in the limb
bud (Bladt et al., 1995). In
E10.5
Six1–/–Six4–/–
embryos, Met expression is correct in the most caudal somites but severely
reduced and highly diffuse more rostrally, thus completely preventing
migration. As Met has been demonstrated to be directly controlled by Pax3
(Relaix et al., 2003), this
caudorostral downregulation of Met in
Six1–/–Six4–/–
embryos may be a direct consequence of the loss of Pax3. This could be
effectively the case for rostral somites where Pax3 expression is lost, but
not for the most caudal somites facing the hindlimb, where Pax3 seems
correctly activated, while Met is only faintly detected. These results
coincide with previous results showing that Met activation in cells migrating
from somite to limb was not entirely dependent on Pax3
(Mennerich et al., 1998).
Thus, Six1 and Six4 control early steps of a genetic network involved in
ventral lip formation and that coordinate the expression of a set of genes
required for migration, including Pax3 and Met. Furthermore, while epaxial Met
expression is not altered in splotch mice
(Mennerich et al., 1998) and
not increased in Pax3-FKHR mice (Relaix et
al., 2003), it is abolished in all E10.5
Six1–/–Six4–/– somites, even in
caudal somites where Pax3 expression is not yet extinguished. This result
suggests a direct control of Met by Six homeoproteins, independently of Pax3.
In the light of Six1 metastatic properties
(Ford et al., 1998;
Yu et al., 2004), these
results suggest that Six1 could control the metastatic behavior of
rhabdomyosarcoma cells through direct Met transactivation, as this
proto-oncogene has been implicated in the development of several human
cancers, including melanomas, breast cancer and rhabdomyosarcomas
(Sharp et al., 2002).
Finally, our analysis provides strong evidence that Six1 and Six4
homeoproteins are also required for the activation of the Lbx1 gene
in the hypaxial myogenic precursors. In E10.5 dKO embryos, Lbx1 expression is
reduced but detectable in sacral somites facing the hindlimb where Pax3 is not
affected by the lack of Six homeoproteins. Lbx1 expression is completely
impaired in more rostral somites and hypoglossal chord. So far, Lbx1 has been
regarded as a Pax3 target because Lbx1 transcripts were not detectable in
splotch mice (Dietrich et al.,
1999). These conclusions were compromised by the fact that cells
that would normally express Lbx1 are lost by the lack of Pax3
(Borycki et al., 1999).
Furthermore Lbx1 expression is detectable in occipital somites of splotch
embryos contrary to what observed in
Six1–/–Six4–/–
embryos (Dietrich et al.,
1999). These results suggesting a direct control of Lbx1 by Six
proteins are further supported by bandshift assays demonstrating the capacity
of Six1, Six4 and Six5 homeoproteins to bind the potential MEF3 site
identified in human and mouse Lbx1 promoter.
The absence of either Six1 or Six4 did not block Pax3 expression and cell
migration into the limb bud (Laclef et
al., 2003a; Ozaki et al.,
2001). Therefore, the double knockout analysis clearly
demonstrates the overlapping functions shared by Six1 and Six4 homeoproteins
to activate the myogenic migration program in somites through the control of
the expression of Pax3, Met and Lbx1genes.
Absence of Six1 and Six4 homeoproteins impaired induction of Pax3 and Mrfs leading to a severe trunk musculature hypoplasia
Epaxial myogenesis is more affected at the rostral level than at the
interlimb and caudal levels in
Six1–/–Six4–/–
embryos. This is observed from E10.5 and persisted throughout embryogenesis.
Pax3, Mrfs and other myotomal-specific genes expression is more severely
altered in rostral somites than in more caudal ones, giving rise to a more
severe disorganization of back muscle masses at the shoulder level than at the
interlimb and hip levels in dKO fetuses. This suggests that the regulatory
myogenic pathways operating in rostral somites are distinct from those
operating more caudally, and is reminiscent of the complex activation of the
Mrf4/Myf5 locus in different precursor populations
(Carvajal et al., 2001;
Hadchouel et al., 2003). E13.5
Six1–/–Six4–/–
embryos develop more serious defects in the body musculature than splotch
embryos, which are essentially affected in their most dorsal and ventral
muscles (Tremblay et al.,
1998). Thus, the digenesis of more profound thoracic and abdominal
muscles in
Six1–/–Six4–/–
embryos appears to rely on an impaired determination and differentiation of
myotomal precursors. E10.5
Six1–/–Six4–/–
embryos, indeed, present a more severe decrease of Myf5 and Myod1 expression
in the myotome than what observed in splotch embryo
(Tajbakhsh et al., 1997),
suggesting that Six homeoproteins can activate both genes in subpopulations of
myogenic precursors independently of Pax3. The genetic link between Myod1
expression and Six1 during limb myogenesis
(Laclef et al., 2003a)
suggests a direct role for Six1 and Six4 in the transactivation of Myod1 in
myogenic precursors, while it has been established that the activation of
Myod1 by Pax3 is indirect (Relaix et al.,
2003). It is also possible that impaired Myod1 expression is
dependent upon the decrease of Myf5 expression and loss of Mrf4 expression
(see below).
Most remaining myofibers in the dKO embryos arose from caudal dermomyotomal
lips, as epaxial and hypaxial lip structure is lost. These dermomyotomal
caudal lips precursors have been shown to contribute to myotome growth in its
dorsoventral extent (Kahane et al.,
1998), are known to express specifically Delta1, and are
able to give rise to epaxial and hypaxial myocytes
(Gros et al., 2004;
Kahane et al., 1998).
Myogenin expression is dramatically reduced in the myotome of
Six1–/–Six4–/–
embryos at E9.5 and E10.5. Only a few positive cells are detected in the more
central-epaxial part of the myotome. The comparative analysis of myogenin
expression between
Six1–/–Six4–/–
and splotch mutants embryos (Tajbakhsh et
al., 1997) tends to demonstrate the dependence of myogenin
activation by Six proteins, as expected by our previous finding
(Spitz et al., 1998). The
observation that somitic expression of myogenin was preserved in
Six1–/– or in
Six4–/– embryos, and that limbs expression of
myogenin was only delayed in Six1–/– embryos
(Laclef et al., 2003a;
Ozaki et al., 2001) probably
reflected again the compensation mechanism that exists between Six1 and Six4.
The remaining myogenin and ß-gal-positive cells in the center of the
myotomes suggest that a specific population of myogenic cells can activate
myogenin and an alternative myogenic program in the absence of Six proteins,
as already proposed (Laclef et al.,
2003a).
Surprisingly, Mrf4 expression is completely lost in
Six1–/–Six4–/–
embryo. MRF4 has been recently identified as a key determination gene
controlling the activation of Myod1 in the myotome, in parallel to Myf5
(Kassar-Duchossoy et al.,
2004). The lack of Mrf4 in
Six1–/–Six4–/–
embryo may thus participate in the downregulation of myotomal Myod1
expression. Myod1 expression has been shown to be under two complementary
genetic pathways involving Myf5 and Pax3
(Tajbakhsh et al., 1997).
Whether Myod1 activation by Six proteins follows the Myf5 or Pax3 network, or
both, remains to be determined.
We have demonstrated that in the absence of Six1 and Six4 myogenic factors, the formation of the myotome is first compromised by a loss of Pax3 and second by an impaired activation of the Mrf proteins in the myogenic precursors already present in the myotome. It also shows that although impaired at multiple levels – absence of ventral and dorsal lips, and decrease of Mrf protein expression – primary myogenesis can nevertheless take place, mainly owing to contribution of caudal lips in which Pax3 is still expressed independently of Six1 and Six4.
Increased apoptosis in somites of Six1–/–Six4–/– embryos
Although there is some evidence that Six1 could control cell proliferation
(Yu et al., 2004),
proliferation deficiencies were not detected in the myogenic lineage of
Six1–/–Six4–/–
embryos. However, Six1 and Six4 appeared necessary to prevent cells from
apoptosis and to induce myogenic differentiation through myogenin induction
and the accompanying cell cycle withdrawal
(Zhang et al., 1999). Cells in
the branchial arches, in the DRG and in the ventrolateral dermomyotome of the
dKO embryos were found to lose their identity and die by apoptosis.
Interestingly, this phenotype has been reported in Drosophila, where
absence of sine oculis does not prevent cell proliferation but
induces apoptosis of those cells that are unable to progress in their
differentiation (Cheyette et al.,
1994). Interestingly, apoptosis has been also reported in
Pax3–/– embryos at the somitic level
(Borycki et al., 1999). It has
been suggested that overexpression or misexpression of one protein of the
Pax-Six-Eya-Dach network triggered a default apoptotic program
(Clark et al., 2002).
Apoptosis has also been detected in Six1–/–
embryos in the metanephric mesenchyme (Xu
et al., 2003). This suggests that different cell types adopt the
same strategy facing the absence of Six homeoproteins, or that Six proteins
are important actors for cell survival.
Six1 and Six4 control Fgf production in the myotomes
Several Fgf molecules are produced by the somites
(Karabagli et al., 2002). We
find in fact that Fgf4 and Fgf6 ventrolateral somitic expression is greatly
diminished in
Six1–/–Six4–/–
E10.5 embryos. We can hypothesize that as Fgf molecules produced by the
ventrolateral myotome, i.e. Fgf6 and Fgf4, are lacking in E10.5
Six1–/–Six4–/–
embryos, scleraxis transcription is delayed. In fact, while early scleraxis
activation is inhibited, in E12.5 embryos, scleraxis expression becomes
detectable (D. Duprez, personnal communication). Our results support the
hypothesis of Fgf signaling by the myotome is required to induce scleraxis
and, hence, the syndetomal compartment, in agreement with recent findings
(Brent et al., 2005). As axial
tendon formation can take place during mouse embryogenesis even in the absence
of scleraxis (Ronen Schweitzer, personal communication), the tendons observed
at the axial level in E18.5 fetuses is not in conflict with a default early
induction of scleraxis in
Six1–/–Six4–/–
embryos.
We already reported that Six1–/– mice had
severe rib and skeletal craniofacial defects. Rib defects, as discussed
already (Laclef et al.,
2003a), are observed in several other KO that prevent axial
myogenesis. Fgf and Pdgfa signaling is required for correct rib growth and
both signaling pathways are diminished in the Myf5 KO that is devoid of early
myotome and Mrf4 expression (Grass et al.,
1996; Kassar-Duchossoy et al.,
2004; Patapoutian et al.,
1995; Tallquist et al.,
2000), and one can hypothesize that it is the case for the other
KO in which correct hypaxial myogenesis is impaired. Absence of early
ventrolateral differentiated myotome producing Fgf signaling, as observed in
Six1–/–Six4–/–
embryos should preclude growing of the sternal region of the ribs
(Evans, 2003;
Huang et al., 2003). Six1 and
Eya1 have been shown already to control Fgf3 and Fgf10 signaling during kidney
and otic development (Xu et al.,
1999; Zheng et al.,
2003), suggesting that one signaling pathway controlled by the
Pax-Six-Eya network may that of the Fgf signaling affecting different types of
organogenesis.
Pax-Six-Eya genetic loop
The demonstration of an epistatic relationship between
Six1/Six4 genes and Pax3 gene in myogenic precursors
originating from the lateral dermomyotome of the somites is consistent with
the genetic link characterized during early kidney development in the mouse
embryo, where Pax2 expression has been shown to be markedly reduced in the
metanephric mesenchyme of Six1 mutant mice
(Xu et al., 2003). The genetic
hierarchy placing sine oculis downstream eyeless in
Drosophila (Halder et al.,
1998) does not seem to be conserved during myogenic development
nor during the early organogenesis of kidney, as Six1 expression is not
altered in the metanephric mesenchyme of Pax2 mutant mice embryo
(Xu et al., 2003).
Interestingly, although Six1/Six4 control Pax3 expression in the lateral
dermomyotome of the occipital, cervical, thoracic and lumbar somites, Pax3 is
activated independently of Six proteins in the lateral dermomyotome of sacral
and caudal somites, and in the anterior and posterior lips of the dermomyotome
of all somites along the anteroposterior axis. Altogether, these results are
reminiscent to the recent observation that Lbx1 gene is activated by Hox
proteins in the somites facing the limb buds
(Alvares et al., 2003), and
suggest that Hox proteins that control the axial identity of somites
(Burke, 2000) may, in
cooperation with Six proteins, control Pax3, Lbx1 and, more generally,
hypaxial myogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOPSUMMARYIntroductionMaterials and methodsResultsDiscussion |
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