Research article Development and disease 2235 Six1 and Six4 homeoproteins are required for Pax3 and Mrf expression during myogenesis in the mouse embryo Raphaelle Grifone1, Josiane Demignon1,*, Christophe Houbron2,*, Evelyne Souil3, Claire Niro1, Mary J. Seller4, Ghislaine Hamard2 and Pascal Maire1,† 1Département Génétique, Développement et Pathologie Moléculaire, Institut Cochin – INSERM 567, CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques 75014 Paris, France 2Plateforme de recombinaison homologue, Institut Cochin – INSERM 567, CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques 75014 Paris, France 3Plateforme d’histologie, Institut Cochin – INSERM 567, CNRS UMR 8104, Université Paris V, 24 Rue du Faubourg Saint Jacques 75014 Paris, France 4Division of Medical and Molecular Genetics, Guy’s Hospital, London SE1 9RT, UK *These authors contributed equally to this work †Author for correspondence (e-mail: [email protected])

Accepted 9 February 2005

Development 132, 2235-2249 Published by The Company of Biologists 2005 doi:10.1242/dev.01773

Summary In mammals, Six5, Six4 and Six1 genes are co-expressed abolished, while it is preserved in Pax3-deficient embryos. during mouse myogenesis. Six4 and Six5 single knockout Within the myotome, absence of Six1 and Six4 impairs the (KO) mice have no developmental defects, while Six1 KO expression of the myogenic regulatory factors myogenin mice die at birth and show multiple organ developmental and Myod1, and Mrf4 expression becomes undetectable. defects. We have generated Six1Six4 double KO mice and Myf5 expression is correctly initiated but becomes show an aggravation of the phenotype previously reported restricted to the caudal region of each somite. Early for the single Six1 KO. Six1Six4 double KO mice are syndetomal expression of scleraxis is reduced in the characterized by severe craniofacial and rib defects, and Six1Six4 embryo, while the myotomal expression of Fgfr4 general muscle hypoplasia. At the limb bud level, Six1 and and Fgf8 but not Fgf4 and Fgf6 is maintained. These results Development Six4 homeogenes control early steps of myogenic cell highlight the different roles played by Six proteins during delamination and migration from the somite through the skeletal myogenesis. control of Pax3 gene expression. Impaired in their migratory pathway, cells of the somitic ventrolateral dermomyotome are rerouted, lose their identity and die by Key words: Six/sine oculis homeoproteins, Pax3, Myogenesis, apoptosis. At the interlimb level, epaxial Met expression is Hypaxial lip, Migration, Myotome, Syndetome

Introduction and Bovolenta, 1999; Oliver et al., 1995; Ozaki et al., 2001; Six1 and Six4 genes belong to the sine oculis/Six gene family Bessarab et al., 2004; Fougerousse et al., 2002; Laclef et al., that includes six members in vertebrates (from Six1 to Six6), 2003a; Schlosser and Ahrens, 2004). This co-expression may Drosophila Caenorhabditis reflect co-regulation of both genes, which are only separated three members in , four members in –/– elegans and at least two members in basal metazoans by 100 kb (Boucher et al., 1996). Six4 mouse present with (Kawakami et al., 2000; Pineda et al., 2000; Seo et al., 1999; no major developmental defects (Ozaki et al., 2004), while –/– Dozier et al., 2001; Bebenek et al., 2004). Originally, sine Six1 mouse neonates do not survive and have defects in many oculis was identified in Drosophila, where it belongs to a organs, including kidney (Xu et al., 2003), thymus, parotid network of genes that drives eye development synergistically glands and ear (Laclef et al., 2003b; Zheng et al., 2003; Ozaki with eyeless (Pax orthologs), eyes absent and dachshund et al., 2004), and rib, craniofacial and muscle deficiencies (Cheyette et al., 1994; Pignoni et al., 1997; Serikaku and (Laclef et al., 2003a; Li et al., 2003). Six1 is also expressed at O’Tousa, 1994). Absence of sine oculis leads to massive high levels in adult skeletal muscles where it participates, in apoptosis of precursor cells in the developing compound eye synergy with Eya1, in the establishment of the fast/glycolytic (Cheyette et al., 1994). In Drosophila, two other sine oculis phenotype of the myofiber (Grifone et al., 2004). With respect related genes have been identified: optix, which is expressed to muscle development, it has been shown that Six1–/– fetuses in the eye imaginal disc (Toy et al., 1998); and dSIX4, which have a selective loss of muscles including diaphragm, proximal is required for myoblast fusion (Kirby et al., 2001). In and distal forelimb muscles, distal ventral hindlimb muscles vertebrates embryos, Six1 and Six4 expression have been and abdominal muscles (Laclef et al., 2003a). detected in overlapping territories: neural placodes, Rathke’s Back skeletal muscles are derived from somitic progenitors pouch, DRG, dermomyotome, myotome, limb bud originating from the epaxial dermomyotome. At the interlimb mesenchyme and in myogenic migrating precursors (Esteve level, the lateral myotome and dermomyotome produce the 2236 Development 132 (9) Research article hypaxial muscles, including thoracic intercostal, abdominal Materials and methods and limb muscles (Buckingham, 2001). Extension of the Construction of Six1-Six4 gene targeting vector lateral dermomyotome is under the control of Pax3 (Williams λ –/– We isolated several Six4 genomic FixII DNA clones from a 129Sv and Ordahl, 1994), and in Pax3 mouse most hypaxial genomic library. We subcloned a 14 kb NotI-NotI genomic fragment migrating myogenic precursors are lacking (Tremblay et al., comprising the entire Six4 gene into pBluescript KS+ (Stratagene). A 1998). Except for tongue muscles, which have a somitic 3′ 3.1 kb Nar-EcoRI fragment comprising the end of the first intron origin, head muscle progenitors originate from head paraxial and the beginning of the second Six4 exon was then ligated in a mesoderm and migrate into the pharyngeal arches to give rise plox2hygro plasmid leading to p3′Six4lox2hygro. A 5′ DNA region to head and neck muscles (Hacker et al., 1998). Limb muscles (NotI-Eco47III 4.2 kb fragment) was ligated in frame into a pEGFP ′ are formed by cells of the dermomyotome that delaminate expression vector (Clontech). This 5 genomic fragment possesses 3.2 kb DNA upstream of Six4 transcription initiation sites as well as the from somites and migrate into the limb bud where they first 144 amino acids. The 5′Six4-GFP fragment was further cloned in further proliferate before activating Mrf genes (Rees et al., p3′Six4lox2hygro, leading to the final invalidation plasmid. 2003). Mrf activation, and more particularly Myf5, is under Homologous recombination with this disruption vector was expected the control of different signaling pathways, depending on the to lead to the deletion of the last 370 amino acids of the first Six4 position of the myogenic precursors cells in the embryo exon, including most of the Six domain and the entire homeodomain, (Hadchouel et al., 2003; Tzahor et al., 2003). Delamination which, together, are responsible for the specific DNA-binding activity of myogenic precursors from the dermomyotome is under the of Six4 protein. control of the Met tyrosine kinase receptor and scatter ES cell screening and chimeric mouse production factor/hepatocyte growth factor (SF/HGF) produced by the Specific Six4 DNA fragment digested by NotI digestion (35 µg), to limb mesenchyme (Bladt et al., 1995; Dietrich et al., 1999). eliminate plasmid DNA sequences, was electroporated (250V; 500 F) Invasion of the limb by myogenic progenitors is also under into 1.5107 Six1-lacZ embryonic stem cells (Laclef et al., 2003a). the control of the Lbx1 homeogene (Alvares et al., 2003). ES cells were selected with 150 µg/ml hygromycin 24 hours after Both Lbx1 and Met expressions are under the control of Pax3, electroporation. The DNA of 310 Six1-Six4 resistant clones was as delamination and myoblast migration into the limb bud is analyzed by Southern blot after PstI digestion. A 5′ fragment and a prevented in Pax3–/– mice (Bober et al., 1994; Epstein et al., 3′ fragments were used as external probes. Ten to 12 cells of three 1996; Goulding et al., 1994). Pax3 expression is not impaired Six1-Six4 independent recombinant ES clones were microinjected in Six1–/– embryos, allowing myoblast migration and limb into C57BL6 blastocysts, which were further implanted into muscle formation (Laclef et al., 2003a). We suggested that pseudopregnant mice. Heterozygous progenies were obtained by backcrosses to C57BL6 and 129/SvJ females. All three clones were ontogenesis of most remaining axial and limb muscles –/– recombined on the Six1-lacZ allele, as F1 animals from the three present in Six1 embryos are under Six4 or Six5 control, as clones were either wild type or heterozygous for both Six1 and Six4. we detected Six5, Six4 and Pax3 expression in migrating F1 progeny was then crossed with EIIaCre animals expressing the Cre –/– Development myoblasts of Six1 embryos at the limb level (Laclef et al., recombinase ubiquitously under the control of E2A promoter 2003a) (data not shown). (Holzenberger et al., 2000). Deletion of the PGK-hygromycin cassette The sclerotome, another somitic compartment, gives rise to was ascertained by Southern blot analysis. All homozygous embryos the axial skeleton and ribs. Interactions between the incipient and fetuses have been genotyped by Southern blot analysis. ribs and growing myotomes at the intercostal level might occur X-gal staining, whole-mount skeletal staining, histology and through Fgf and Pdgf molecules produced by the myotomes immunohistochemistry of the embryos were performed as described previously (Laclef et al., 2003a). Vibratome section (120 µm) were (Huang et al., 2003). In this context, inhibition of Fgf signaling performed after inclusion of the embryos in 4% agarose. Sections causes deletion of developing ribs (Huang et al., 2003b). The were then mounted in Kaiser’s glycerol gelatin solution (Merck). Gel syndetome, which is derived from the sclerotome, gives rise to mobility shift assays were performed as described (Grifone et al., the axial tendons. Scleraxis is one of the earliest genetic marker 2004). characterized for this somitic lineage (Schweitzer et al., 2001). Induction and individualization of the syndetome requires the dermomyotome/myotome contact involving Fgf signaling Results (Brent et al., 2005). Generation of Six1Six4-deficient mice To test the hypothesis that Six4 acts in common with Six1 Owing to the genetic proximity (about 100 kb) of Six1 and during myogenesis, we produced double Six1Six4 knockout Six4 genes on chromosome 12, invalidation of the Six4 gene (dKO) mice. We show here that Six1–/–Six4–/– embryos develop was achieved in ES cells already invalidated at the Six1 locus a more severe muscle phenotype than did the Six1–/– embryo. (Laclef et al., 2003a). Inactivation of the Six4 gene was No muscle is detected in the limbs because of the achieved by replacing the Six domain and homeodomain of the downregulation of Pax3 in the ventral dermomyotomal lips of Six4 protein, both of which are required for specific DNA the somites from which hypaxial progenitors arise. There is no binding, by the EGFP cDNA (Fig. 1A). To identify the proliferation defects but most of these precursors migrate recombination events at the Six4 locus that took place on the aberrantly, lose their myogenic identity and die by apoptosis. Six1 recombined and wild-type allele, we analyzed the DNA Epaxial and non migrating hypaxial musculature is affected by of the progeny of chimeric animals for both Six1 and Six4. In severely compromised expression of Mrf genes within the the three independent ES clones where recombination events myotome. Our results finally suggest that the rib phenotype at the Six4 locus were identified, F1 DNA analysis showed that developed by Six1–/–Six4–/– fetuses could be the result of a loss all three recombination events occurred on the recombined of Mrf4 expression and a downregulation of Fgf production in Six1 allele, leading to a Six1LacZSix4gfp/Six1+Six4+ genotype, the myotome. hereafter referred to Six1+/–Six4+/–. Southern blot analysis with Development and disease Impaired myogenesis in Six1Six4 null embryos 2237

5′ and 3′ external probes confirmed these events (Fig. 1B). Six1+/–Six4+/– animals are viable and fertile, suggesting that one allele of Six1 and Six4 are sufficient for correct development, either on 129SV or C57black6N backgrounds (F8 generation). Indeed, Six1–/–Six4–/– animals die at birth and can be easily distinguished from their littermates. They are smaller, have an enlarged abdomen and present severe craniofacial malformations (Fig. 1C). In contrast to Six1–/–, Six1–/–Six4–/– are earless, have open eyelids, a very reduced maxillary and mandible, abnormally bent hindlimbs and forelimbs, and a general bent posture as if dorsal musculature is inadequate. 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 Development 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 Fig. 1. Targeted disruption of the Six4 gene. (A) Schematic representation of the cartilages of the inner ear and the ectotympanic wild-type Six4-Six1 locus, with the restriction sites used in the present study. The 4.2 bone are lacking (Fig. 2D,E, and data not shown). kb 5′ and 3.1 kb 3′ homologous fragments are indicated by double headed arrows. It thus appears that absence of Six4 in a Six1–/– The Six4-loxP targeting fragment is shown, and the two potential targeted Six4 background leads to a more severe craniofacial mutant alleles after Cre excision are depicted below. Schematic representations are and rib phenotype. In both forelimbs and not to scale. (B) Southern blot analysis of genomic DNA digested by PstI and hindlimbs, a clinodactly (digit curvature) with a hybridized with a 5′ external probe (above) and a 3′ external probe (below), before Cre recombination leading to a Six4hygrogfp allele, or after Cre recombination leading curved fifth digit was observed in all E18.5 gfp –/– –/– fetuses analyzed (n=6, Fig. 2F,G). to a Six4 allele. (C) Phenotype of wild type (left) and Six1 Six4 (right) The phenotype of the transgenic mice was newborn mice. compared with a human condition connected to the deletion of 14q22. This region comprises the SIX1, SIX4 et al., 1991), suggesting that Six1 and Six4 haploinsufficiency and SIX6 gene cluster of about 200 kb. We have previously could be responsible for these developmental defects. found that some of the human symptoms correspond to what can be observed in Six1 homo- and heterozygous null mutant General muscle deficiencies in double Six1Six4 new mice, such as hypotonia and hypoplastic kidneys, while others born animals (such as anophtalmia and pituitary hypoplasia) were not We have shown previously that Six1–/– fetuses present a severe observed in the Six1–/– null (Bennett et al., 1991; Gallardo et but selective muscle hypoplasia, while Six4–/– fetuses are al., 1999; Laclef et al., 2003b; Lemyre et al., 1998; Xu et al., normal (Laclef et al., 2003a; Ozaki et al., 2001). As Six1 and 2003). It has been shown that in these individuals the SIX6 gene Six4 are co-expressed during myogenesis, we suspected a was deleted, suggesting that anophtalmia and pituitary compensatory role for Six4 in the genesis of the remaining hypoplasia could be caused by SIX6 haploinsufficiency muscles of Six1–/– fetuses. X-gal staining of Six1–/–Six4–/– (Gallardo et al., 1999), which agrees with analysis of Six6 KO E18.5 fetuses revealed a more severe muscle phenotype than mice (Li et al., 2002). We identified hemizygosity for both observed previously in Six1–/– fetuses (Fig. 3A-L). All muscles SIX1 and SIX4 genes in a human 21-week-old fetus (Fig. 2H). of the distal forelimb and hindlimb are missing, as revealed by This fetus had hypoplastic kidneys and clinodactily (Bennett the absence of any fast or slow myosin positive fibers (Fig. 3I- 2238 Development 132 (9) Research article

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. Primary myogenesis is severely disorganized in Six1–/–Six4–/– embryos A severe muscle hypoplasia was already apparent in embryonic forelimbs by E13.5, as revealed by β- gal staining (Fig. 3M-N) and desmin expression (Fig. 3S-T). There is a total absence of desmin- positive myogenic cells in limbs. There is also a disorganization of muscle masses at the back level, which is more obvious rostrally to the forelimb (compare Fig. 3T with 3V), and a total absence of myogenic cells in the abdominal region (Fig. 3U- Development V). In the head, most muscles are lacking at the eye level (Fig. 3O-P) and the tongue muscles are absent (Fig. 3Q-R), while other muscles like the masseter are present (Fig. 3Q-R). In the head, these defects seem transient, as most head muscles are present by E18.5.

–/– –/– Severe myotomal disorganization in Fig. 2. Skeletal defects in Six1 Six4 newborn mice as revealed by Alizarin Red Six1–/–Six4–/– embryos (bones) and Alcian Blue (cartilage) staining. (A,D,F) Six1+/–Six4+/– fetuses, (B,C,E,G) Six1–/–Six4–/– fetuses. Lateral and ventral view (A-C) of the trunk Six1 and Six4 expression has been monitored by X- skeleton, showing truncation of the ribs, fusion and branching (black arrow in B). Gal staining and GFP expression at different The sternum is shortened (black arrow in C). (D,E) Lateral view at the head level embryonic stages ranging from E9.5 to E12.5 on showing a shortened mandible bone (*), shortened squamosal bone devoid of wild-type and null backgrounds (Fig. 4). In E9.5- retrotympanic process (arrow) and lack of Meckel’s cartilage (arrowhead) in the E10.5 Six1–/–Six4–/– embryos, Six1 and Six4 genes dKO fetuses, and complete absence of ear structure (**). (F,G) Dorsal view of are still highly expressed in somites, suggesting that hands of forelimbs at the left or hindlimbs at the right of Six1+/–Six4+/– (F) and –/– –/– Six1 and Six4 proteins are not required for their Six1 Six4 fetuses (G) showing a clinodactily in the dKO. The fifth finger is own transcription in these structures. Conversely, curved (arrows in G). (H) Human DNA samples from one individual with a 14q22 their gene expression is severely reduced at the deletion (1), and two control DNA samples (2,3). DNA was digested with BamHI and hybridized with SIX1 cDNA (a, left panel) or SIX5 cDNA (c, right panel), or cranial level in the trigeminal placode and in the digested with HindIII and hybridized with SIX4 cDNA (b, middle panel). M.W., otic vesicle, where X-Gal staining becomes barely standard molecular weights. The signal observed for SIX1 and SIX4 in the detectable at E10.5 (Fig. 4E-H). These results individual with the 14q22 deletion is half that one observed in the control DNA, suggest either that in these cells Six1 and Six4 while the signal for SIX5 is comparable in all samples. control positively their own transcription, or that apoptosis occurs in these placodal precursors because they require Six1 and Six4 to survive (see L; data not shown), and all muscles of the proximal limbs are later). At E9.5, Six1 expression was greatly reduced in the otic missing as revealed by X-gal/Eosin staining (Fig. 3A-D for the vesicle of Six1–/–Six4–/– embryo (Fig. 4, compare A,C with forelimb; Fig. 3G,H for the hindlimb). Hypaxial musculature B,D) probably reflecting a loss of Six1–/–Six4–/– cells through at the trunk level is also severely diminished (Fig. 3A,B) and apoptosis (Ozaki et al., 2004; Zheng et al., 2003). At E9.5, the Development and disease Impaired myogenesis in Six1Six4 null embryos 2239

Fig. 3. Absence of most skeletal muscles in Six1–/–Six4–/– E18.5 fetuses (A-L) and in Six1–/–Six4–/– E13.5 embryos (M-V). (A,C,E,G,I,K,M,O,Q,S,U) Six1–/+Six4–/+ animals; (B,D,F,H,J,L,N,P,R,T,V) Six1–/–Six4–/– animals. General view of E18.5 heterozygous (A) and double dKO (B) fetuses, as revealed by X-gal staining. Skeletal muscles are absent from the limbs, abdominal ventral muscles are missing and, at the shoulder and hip levels, most muscles are lacking. X-gal Eosin staining on transversal sections (C-H) or immunostaining performed with fast-type myosin heavy chain antibody My32 (I-L) confirmed the general axial muscle hypoplasia and that limbs have no muscle. (C,D) Shoulder level, with the humerus (hu), thymus (thy) and vertebra (v) indicated. Most muscles are lacking in the dKO fetuses, except epaxial muscles, which appear incorrectly shaped, near the vertebral column. (E,F) Hip level revealing deep back muscles in the control and dKO fetus. (G,H) Hip level revealing that proximal muscles around the femur (fe) are lacking in the dKO fetus, and that ventral-most muscles are also lacking (see enlargement of the boxed ventral region. int, intestine. (I,J) Forelimb and (K,L) hindlimb levels, revealing the absence of muscles in the dKO fetuses. r, radius; u, ulna; t, tibia; f, fibula. General view of E13.5 heterozygous (M) and dKO (N) fetuses, as revealed by X-gal staining. Abdominal ventral muscles are missing and, at the shoulder level, most muscles are lacking. Transverse sections

Development (O-V) revealing desmin expression after immunostaining. (O,P) Eye level, showing that most muscles are absent in the dKO embryo. Eo, extraocular muscles; pt, pterigoid muscles; ca, capitalis muscles; nt, neural tube (Q,R) Tongue level, revealing absence of tongue muscle (to) in the dKO embryo. Ma, masseter. (S,T) Forelimb level, revealing absence of any desmin-positive cells in the forelimb of dKO embryo. fl, forelimb; he, heart; db, deep back muscles; st, spinotrapezius; cm, cutaneus maximus; ii, intercostals interni; bb, biceps brachii, pe, pectoralis; tt, transversus thoracis. (U,V) Diaphragm level, showing that ventral muscles and diaphragm are lacking in dKO embryos. li, liver; dia, diaphragm; sm, scalenus medius; ie, intercostales externi; ra, rectus abdominis; ta, transversus abdominis.

maxillary primordium and mandibular primordium of the first well as in myotome-like structures of each somite, where their and second branchial arches appeared fused (compare Fig. 4C expression patterns appear more diffuse rostrocaudally and with 4D). mediolaterally, and severely diminished in their ventral extent At E10.5, Six1 and Six4 gene expression within each somite (Fig. 4U,V; data not shown). Posterior domains of limb buds is largely diffuse rostrocaudally and severely reduced ventrally display strong expression of both genes (Fig. 4Q-T) but these (Fig. 4I-P). Vibratome sections through the dKO embryo show cells are probably not myogenic, as they are also detected here that β-gal-positive cells are located ventromedially at the in Pax3–/– (splotch) mice, which are devoid of myogenic cells hindlimb level and ventrolaterally beneath the ectoderm at in limb buds (Oliver et al., 1995). Most notably, Six1 and Six4 interlimb or forelimb levels (Fig. 4L,N,P), instead of extending gene expression is not detected in the ventral and dorsal lips ventrally or migrating into limb buds as shown in control of the dermomyotome (Fig. 4Q,R,U,V). At E12.5, β-gal- heterozygous embryos (Fig. 4K,M,O). These mislocated β-gal- positive cells are dispersed in the Six1–/–Six4–/– somites when positive cells do not express Pax3, desmin or Myf5, and are compared with the heterozygous (Fig. 4W-Z). β-Gal cells also probably no longer myogenic precursor cells (see later). Six1 appear less abundant at this stage in the homozygote. It is also gene expression in the DRG is not affected (Fig. 4M-P). At evident that most of cells are blocked in their migration E11.5, Six1 and Six4 remain highly expressed in the DRGs, as pathway, and fail to enter the limb bud (Fig. 4L,N,P). 2240 Development 132 (9) Research article

Mislocated cells in Six1–/–Six4–/– embryos die by total number of β-gal-positive cells in one somite of E10.5 apoptosis heterozygous and dKO embryos. Analysis of seven serial Whether Six1 and Six4 are important for the proliferation of sections at the hindlimb level showed that the number of β-gal- myogenic progenitors was first examined by the analysis of positive cells in the dKO (78±28) was significantly greater BrdU incorporation and phospho-histone H3 expression. No (P<0.0005, Student’s t-test) than in heterozygotes (32±14), significant difference of proliferation was observed in the which reflects their failure to migrate to the limb bud. At the somites of E10-E11 dKO versus control embryos (data not abdominal level, we found 229 positive cells (±39) in a shown). We next analyzed confocal sections to evaluate the heterozygous and 302 in a dKO (±89) (P<0.025), showing no

Fig. 4. X-Gal and GFP expression in heterozygous (A,C,E,G,I,K,M,O,Q,S,U,W,Y) and homozygous mutant animals (B,D,F,H,J,L,N,P,R,T,V,X,Z) at E9.5 (A-D), E10.5 (E-P), E11.5 (Q-V) and E12.5 embryos (W-Z), revealing Six4-GFP expression and Six1- lacZ expression. (A-D) Six1 expression is detected in the head mesenchyme (asterisk in A), otic vesicle (white arrow in A), branchial arches (black arrow in A) and pharyngeal clefts (white arrows in C) in heterozygous embryos. In dKO embryos, Six1 expression at the head level is severely reduced (asterisk in B) and lost in the otic vesicle (white arrow in B); although mesodermal Six1 expression is still detected (D), most Six1 expression is lost in pharyngeal pouch endoderm and surface ectoderm in dKO embryos. Fusion between the first and second branchial arches is observed at this stage (white arrow in D). At the thoracic level, the nephrogenic chord expression of Six1 is detected in the heterozygote (black arrowhead in A) but not detected in the dKO embryo (black arrowhead in B). At the somitic level, Six1 is expressed at comparable levels in the heterozygous (A) and homozygous

Development (B) animals. (E-P) Six1 is still highly expressed in the ventral otic vesicle (white arrow in E,G) and trigeminal ganglion (white arrowhead in E,G) in heterozygotes, but not in the dKO embryos (white arrow and white arrowhead, respectively, in F and H). (G,H) Enlargement of control (G) and KO (H) embryos at the head level. (I,J) Enlargement of control (I) and KO (J) embryo at the interlimb level. At the somitic interlimb level, Six1 expression is detected in the myotome (E,I). In the dKO, Six1 expression is more diffuse, while still expressed in myotomes (F,J). (K-P) Vibratome sections. At the hindlimb level (K,L), Six1 is detected in the nephrogenic chord (arrow in K) but is not in the dKO, while many β-gal-positive cells invade this ventral region of the embryo (L). Six1-positive cells enter the limb bud in the heterozygous embryos (K), but not in the dKO embryo, where somitic β- gal-positive cells are not confined to the somite but are found more ventromedially (L). At the interlimb level (M,N) Six1-positive cells are diffuse and do not extend ventrally in the dKO embryo (N), while they invade ventral region in the heterozygous embryos (M). Six1 expression in DRG is preserved, but a few β-gal-positive cells are also seen in the neural tube in the dKO (N,P; data not shown). At the forelimb level (O,P), Six1-positive cells are present in the limb bud in the heterozygous embryos (O), while in dKO embryos most cells accumulate less ventrally, and are not found in the limb bud, but are also detected beneath the ectoderm more medially (P). (Q-V) Six1 (Q, R) and Six4 (S-V) are mainly detected in limbs, and somites, where they are co-expressed in the DRG, myotomes, and ventral (black arrows) and dorsal dermomyotomal lips. In dKO, somites still express these two genes (R,T). Myotomes are disorganized, their ventral extensions are reduced (white double headed arrow), and GFP and β-gal are no longer detected in the ventral lips (black arrows). A population of GFP and β-gal-positive cells is found in the limbs of dKO embryos but they are not myogenic cells (Bonnin et al., 2005). (W-Z) Six1 is expressed at higher levels in heterozygous (W,Y) than in homozygous mutant embryos (X,Z). At the thoracic level, ventral extension of the dermomyotome, normally marked by Six1 expression, is lost in dKO embryos (white arrows). Posterior expression in the limbs is maintained. Development and disease Impaired myogenesis in Six1Six4 null embryos 2241

proliferation defects of Six1–/–Six4–/– cells at this level. At the myoblast migration as no myogenic precursors are detected in forelimb level, we found 55 positive cells (±19) in a the forelimb of dKO embryos. heterozygous and 152 in a dKO (±39) (P<0.0005). These Lbx1 expression is not detected at forelimb and hypoglossal results combined with the observation of comparable levels of chord levels in dKO embryos when compared with the control β-gal and GFP accumulation in heterozygous and dKO (Fig. 6, part III, A-D). Lbx1 is weekly detectable in three out embryos (Fig. 4I,J,U,V) indicated that at least until E10.5, the of the six Lbx1-expressing somites facing the hindlimb bud absence of Six1 and Six4 in vivo does not prevent proliferation (Fig. 6, part III, E,F), corresponding to sacral somites where 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).

Impaired migration of myogenic precursors in Six1–/–Six4–/– embryos is due to a decrease of Lbx1, Met and Pax3 expression To further investigate the origin of limb muscle defects, the expression of primordial myogenic markers was examined at E10.5 in dKO and heterozygous embryos. In less mature caudal somites, Pax3 is expressed normally in the dorsal neural tube and the dermomyotome of Six1–/–Six4–/– embryos (Fig. 6, part I, A-F). In more rostral somites, at the hindlimb level, the ventralmost expression of Pax3 is lower and no Pax3-positive cells have delaminated from the dermomyotome to invade the limb bud (Fig. 6, part I, G-J). At the thoracic level, Pax3

Development expression in the dorsal and ventral extensions of the dermomyotome is undetectable (Fig. 6, part I, M-N) and Pax3 expression is only detected in caudal and rostral lips, where it is conversely higher than in heterozygous embryos (Fig. 6, part I, K,L). At the forelimb level, Pax3 expression remains detectable in the caudal lips but is not detected in dorsal and ventral lips. Hence, Pax3 expression is also restricted to a central domain within the somites and no Pax3-positive cells are detected in the forelimb (Fig. 6, part I, O-R). More rostrally in occipital somites, Pax3 expression is severely reduced (Fig. 6, part I, A,B). In E9.5, dKO embryos, Pax3 expression is also restricted to the caudal lips of the dermomyotome of existing somites (data not shown). Met expression is not affected in the most caudal somites of dKO embryos (Fig. 6, part II, A,B), but it declines more rostrally (Fig. 6, part II: A,B,E,F). In somites facing the hindlimb, Met is poorly detectable in the ventral Fig. 5. Increased apoptosis in Six1–/–Six4–/– embryos. Sections (10 dermomyotome and only a few Met-positive cells are detected µm) of heterozygous (A,C) and dKO (B,D) embryos at E10.5 at the in the limb (Fig. 6, part II, C,D). At the interlimb level, Met is hindlimb level, hybridized with antibodies revealing β-gal protein mainly detected in ectodermal structures. The absence of (A,B) and activated caspase 3 (C,D), showing activated caspase 3- Met in the ventral dermomyotome coincides with the positive cells only in dKO embryos (white arrow in D). Sections (10 downregulation of Pax3, while its downregulation in the µm) of heterozygous (E) or dKO (F) embryos at E12.5 at the epaxial domain is specific to the Six1Six4 dKO. At the forelimb hindlimb level revealed with antibodies against β-gal protein (in red) β level, a weak Met expression is detected in the centre of the and activated caspase 3 (in green), and showing apoptosis of -gal- somite (Fig. 6, part II, H) where Pax3 was also restricted (Fig. positive cells in dKO embryos that are rerouted ventrally (white arrow in F). Sections (10 µm) of heterozygous (G) and dKO (H) 6, part I, R). This Met expression domain is not observed in embryos at E10.5 at the first branchial arch level hybridized with heterozygous or wild-type embryos (Fig. 6, part II, G), antibodies revealing β-gal protein (in red) and activated caspase 3 (in probably because of a low Pax3 expression (Fig. 6, part I, Q). green), and showing apoptosis of β-gal-positive cells in dKO However, this level of Met appears insufficient to allow embryos. 2242 Development 132 (9) Research article

Fig. 6. Detection of Pax3 (part I), Met (part II), Lbx-1 (part III) and Pax7 (part IV) by whole-mount in situ hybridization on E10.5 heterozygous and dKO embryos. (Part I) Pax3 expression in heterozygous (A,C,E,G,I,K,M,O,Q) and dKO embryos (B,D,F,H,J,L,N,P,R). Pax3 is still expressed in the dKO (B). At the caudal level, in youngest somites Pax3 expression is initiated similarly in both control and dKO embryo (C,D) and its dorsoventral expression domain is comparable, as revealed by vibratome sections (E,F). At the hindlimb level, ventral expression of Pax3 is perturbed (G-J) and no Pax3 myogenic precursor delaminate to reach the limb bud. At the interlimb level (K-N), Pax3 expression is restricted to the posterior lip in the dKO embryo (arrow in L); in ventral (arrow in N) and dorsal lips, expression is progressively lost (L,N) when compared with wild-type expression (K,M). More rostrally at the forelimb level Pax3 is only detected in the caudal lip of dKO embryos (P), and completely lost in anterior, dorsal and ventral lips (P,R) when compared with heterozygous embryos (O,Q). No myogenic precursor is detected in the limb bud (R). Dorsal neural tube expression of Pax3 is conserved in dKO embryos along the rostrocaudal level (F,J,N,R). In occipital somites, Pax3 expression is very low in dKO embryos. (Part II) Comparison of

Development Met expression in heterozygous embryos (A,C,E,G), with its caudal expression in the top right-hand corner of A, and in dKO embryos (B,D,F,H), with its caudal expression in the bottom left-hand corner in B. Although Met expression is correct in the two first caudal somites of dKO embryos, most expression is lost in more rostral somites, and ectodermal expression is detected at the interlimb level in dKO embryos (arrow in B). Transverse sections through embryos at hindlimb level (C,D), interlimb levels (E,F) and forelimb bud levels (G,H). At the hindlimb level, Met can be detected in dorsal and ventral dermomyotomal lips (D) and few cells are detected in the hindlimb bud – in caudal-most areas of this limb bud (arrow in D); however, Met expression is lost more rostrally from these hindlimb somites. The central somitic expression of Met is faint and restricted (H) at the forelimb level (arrowhead). Epaxial Met expression (white arrow in A) is lost in dKO embryos (white arrow in B). (Part III) Lbx1 expression in wild-type (A,C,E) or KO (B,D,F) embryos. Lbx1 expressed at the hypoglossal chord (arrow in C and D) and forelimb bud levels (asterisks in C and D) is abrogated in dKO embryos but still faintly detected in a few somites at the hindlimb level (arrowhead in F). (G) Bandshift assays performed with recombinant Eya1, Six1 or Six1+Eya1 proteins on a potential MEF3 site present in the promoter of mouse Lbx1 gene showing formation of a MEF3Lbx-Six1 or a MEF3Lbx-Six1-Eya1 DNA protein complex. (Part IV) Pax7 expression in heterozygous (A,C,E,G,I) and KO (B,D,F,H,J) embryos. Pax7 is expressed in dermomyotome and its expression pattern is mainly unchanged in KO somites (C,D) at the hindlimb level, as revealed by vibratome sections (E,F), and at the interlimb level (G,H). At the forelimb bud level, we noticed that dorsoventral extension of the expression domain of Pax7 was reduced (I,J).

Met and Pax3 are expressed ventrally. Lbx1 was not detected (Fig. 6, part III, G; data not shown). Hence, Lbx-1 may be in occipital somites and at the forelimb level in E9.5 dKO under the control of Six proteins, at least in a population of embryos (data not shown). myogenic migrating precursors. Nonetheless, the presence of Bandshift assays using a potential MEF3 site a few Lbx-1 positive cells in the hindlimb suggests that some (TCAGGTTTC) found in the human and mouse Lbx1 specific myogenic precursors can activate Lbx1 and Met in the promoter (TCAGGTTggC) as a probe together with nuclear absence of Six1 and Six4 homeogenes. extracts or in vitro synthesized Six1 protein, demonstrated a Finally, at hindlimb and interlimb levels, no major difference specific interaction of Six proteins with the Lbx1 promoter in Pax7 expression, another dermomyotomal marker, is Development and disease Impaired myogenesis in Six1Six4 null embryos 2243

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 Development 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). At the forelimb level in dKO embryos, the ventrolateral Fig. 7. Disruption of ventral and dorsal dermomyotomal lips in extension of Pax3 expression is severely reduced when Six1–/–Six4–/– embryos at caudal (A-D), hindlimb (E-N), thoracic compared with the control (Fig. 7S,U). Six1 is uniformly (O-R) and forelimb (S-X) levels. Section (10 µm) of heterozygous expressed dorsoventrally from the ectoderm to more internal (A,B,E,F,I,J,O,P,S,T,W) and dKO (C,D,G,H,K,L,M,N,Q,R,U,V,X) mesenchyme structures, including the myotome (Fig. 7V) E10.5 embryos hybridized with antibodies revealing Pax3 protein in instead of being restricted to myotomal and migrating cells red (A,C,E,G,M,O,Q,S,U), β-gal protein in green (Fig. 7T). In absence of Six1/Six4, cells are unable to express (B,D,F,H,J,L,N,P,R,T,V), desmin (J,L) and laminin (I,K,W,X). Pax3 Pax3, lose their myogenic identity and are consequently expression is similarly expressed in rostral somites of both dispersed and re-located beneath the ectoderm or more heterozygous and dKO embryos, and is co-expressed with Six1 medially. Although laminin expression that delimits the (compare C,D with A,B). At the hindlimb level in the dKO embryos, we checked desmin, laminin, Six1 and Pax3 expression in sacral myotome is robust in heterozygous mice (Fig. 7W), a few somites facing the limb bud (G,H,K,L) and in more rostral somites laminin-positive cells are detected in the dKO at the forelimb facing the limb bud (M,N). Laminin and desmin expression is found in level (Fig. 7X), showing a severe reduction of the myotome the myotomes in Six1-expressing cells on both wild-type and dKO at this rostral level when compared with caudal levels (Fig. sections (white arrows in I-L). At this hindlimb level, the myotome of 7K). dKO embryos is not disorganized. More rostrally, Pax3 expression is progressively lost ventrally in lumbar somites at the hindlimb level, Six homeoproteins control early Mrf genes while most cells are Six1 positive (compare G,H with M,N). At the expression thoracic level, Pax3 expression is restricted in the medial aspect of the We have previously shown that Six proteins were directly dermomyotome of dKO embryos (Q), while Six1 gene expression is required for myogenin transactivation during embryogenesis found in the myotomes (R). More rostrally, at the forelimb bud level, Six1-positive/Pax3-negative cells are disorganized and have lost their (Spitz et al., 1998). Although no alteration of myogenin –/– identity; they also fail to enter the limb bud in the double KO (compare expression has been reported in Six4 embryos, we showed S,T with U,V). At this level, the myotome of dKO embryos is that Six1 was required for early expression of myogenin in disorganized, as revealed by the low laminin expression (white arrow limbs but not in the myotome (Laclef et al., 2003a). We show in X), when compared with laminin expression in control embryos here that 90% of myogenin level was lost in the absence of (white arrow in W). 2244 Development 132 (9) Research article

Fig. 8. (Part I) Myf5 expression in E9.25 (20 somites stage) in heterozygous (A) and dKO (B) embryos. Myf5 is expressed in the epaxial somites of heterozygous embryos, and more faintly in epaxial- rostral somites in dKO embryos. (Part II) Myogenin expression in E9.5 embryos in heterozygous (A) and dKO (B) embryos. A few positive myogenin- expressing cells are detected in the most rostral somites of dKO embryos (arrows in B). (Part III) Myogenin expression in E10.5 heterozygous (A,C,E) and dKO (B,D,F) embryos. Vibratome sections at the interlimb level (C,D) and forelimb level (E,F) show a strong decrease of myogenin expression that is faintly detected in the epaxial region of dKO embryos, hypaxial extension expression domain being lost. (Part IV) Myod1 expression in E10.5 heterozygous (A,C,E,G) and dKO (B,D,F,H) embryos. Most Myod1 expression is lost in Six1–/–Six4–/– embryos, and remaining expression can be visualized at the interlimb or forelimb levels in the central and hypaxial somite (F,H). (Part V) Myf5 expression in heterozygous (A,C,E,G) and in dKO (B,D,F,H) E10.5 embryos. Myf5 is mainly detected in caudal lips of homozygous dKO animals at interlimb levels (B, upper right), ventral and dorsal lip expression being lost (compare A with B). This expression loss is well detected on vibratome sections at hindlimb (C,D), interlimb (E,F) and forelimb (G,H) levels. In the dKO, at the forelimb level the myotome is formed only of a central region expressing Myf5 (H). In the top right- hand corners of A and B, magnification of interlimb Myf5 expression can be seen that is restricted to the caudal region of the somites in the dKO embryo (B). (Part VI) At E10.5, MRF4 expression is lost in Six1–/–Six4–/– embryos (B), when compared with

Development control hetererozygous expression (A). (Part VII) Myod1 expression in heterozygous (A,C,E,G) and in Six1–/–Six4–/– (B,D,F,H) E11.5 embryos. A low diffuse Myod1 expression in interlimb somites of dKO embryos is detected (B) with restricted ventral extension (B, and enlargement in the top right-hand corner) when compared with Myod1 expression in heterozygous embryos (A, enlargement in the top right-hand corner). Vibratome sections (C-H) showing a faint Myod1 expression at hindlimb level, with most dorsal and ventral extension being lost (C,D). At the interlimb level, Myod1 expression is restricted to the central region of the myotome, dorsal and ventral expression is lost (E,F). A low myotomal expression is detected at the forelimb level (G,H).

Six1 and Six4 at E9.5 (Fig. 8, part II, A,B). A few specific cells of interlimb somites (Fig. 8, part IV, C-H). Early Myf5 can nevertheless bypass Six signaling to activate myogenin and expression at E9.25 (20-somite stage) is lower in dKO probably give rise to the remaining epaxial muscles present in embryos, even if correctly initiated in their epaxial Six1–/–Six4–/– embryos at older stages. Their central localization compartment (Fig. 8, part I). At E10.5, the ventral and dorsal within the myotome, combined to the remaining Pax3 expression of Myf5 was lower, especially in rostral somites, expression, suggest that these cells only originate from the (Fig. 8, part V, A-H), showing again the impaired ability of caudal lip of the dermomyotome. At E10.5, myogenin dermomyotomal lips in producing myogenic cells. Myf5 is expression remains very weak (Fig. 8, part III, A,B) at restricted to the center of the myotome when examined on interlimb level, barely detectable in more rostral somites and transverse sections (Fig. 8, part V, C-H). At E9.5 and E10.5, is faintly detectable on vibratome sections (Fig. 8, part III, C- Mrf4 is completely undetectable in the dKO (Fig. 8, part VI; F). We next examined the three myogenic determination genes data not shown), placing Six proteins upstream of this Myf5, Myod1 and Mrf4. In absence of Six1 and Six4, most of determination gene. At E11.5 Myod1 expression remain low the early Myod1 activation is blocked at E10.5 (Fig. 8, part in dKO embryo, and is undetectable in the most ventral and IV). Myod1 remains expressed in ventral–hypaxial myotome dorsal positions (Fig. 8, part VII). Development and disease Impaired myogenesis in Six1Six4 null embryos 2245

Fig. 9. Whole-mount in situ hybridization with Fgf4 (part I), Fgf6 (part II), Fgf8 (part III), Fgfr4 (part IV), scleraxis (parts V,VI) on heterozygous or dKO E10.5 embryos and vibratome sections. (Part I) Fgf4 expression in heterozygous (A,C,E) or dKO (B,D,F) embryos, showing a decrease of Fgf4 somitic expression, AER expression is maintained (A,B). At the interlimb level (C-F), Fgf4 expression is restricted to the central region of the myotome. (Part II) Fgf6 expression in heterozygous (A,C) or dKO (B,D) embryos, showing a severe reduction of Fgf6 in the somites (A,B) of the dKO embryo. Vibratome sections at the interlimb level reveal a strong myotomal expression in the heterozygous embryo (C), while a low signal can be detected in the dKO embryo (D). (Part III) Fgf8 expression in heterozygous (A,D,F) or dKO (B,C,E,G) embryos showing that somitic Fgf8 expression is maintained in the dKO embryos. Fgf8 is expressed in the AER, presomitic mesoderm, otic placode and branchial arches in wild-type or heterozygous embryo (A,D,F). At the somitic level, we detect Fgf8 mRNA, mainly in the caudal lip and more faintly in rostral lip (A,D), allowing Fgf8 to accumulate preferentially in the central myotome (F). In the dKO embryos, presomitic, AER and somitic expression (C,E,G) are preserved, while branchial arches expression (arrows in A and B) is lost (B). (Part IV) Fgfr4 expression in heterozygous (A) or dKO (B) embryos at the interlimb level showing a decrease of Fgfr4 expression in Six1–/–Six4–/– embryos. (Part V) Scleraxis expression in

Development heterozygous (A) or dKO (B) embryos. Scleraxis is expressed in tendon progenitors at the limb level (A), this expression is preserved in the dKO embryo (B), while early somitic syndetomal expression of scleraxis is lost in the dKO embryo (B). (Part VI) Scleraxis is not expressed in Six1 somitic cell population. Double Six1-lacZ (blue) and scleraxis (purple) heterozygous embryo showing the different cell populations of the somites, which express exclusively scleraxis (white arrow) or Six1 (black arrow).

Fgf signaling is altered in Six1Six4 KO embryos (Fig. 9, part V). Moreover Six1 and Six4 proteins are not The hypothesis that rib defects observed in dKO fetuses was produced by normal syndetomal cells (Fig. 9, part VI; data not caused by an insufficient Fgf myotomal production was tested shown). These results suggest that some specific Fgf molecules in dKO E10.5 embryos. Fgf4 expression is relatively reduced emanating from the somite are absent in dKO embryos, leading in the myotome of Six1–/–Six4–/– embryos, especially in more to the loss of early scleraxis expression. rostral somites, and Fgf6 expression seems more severely diminished than Fgf4 in dKO embryos (Fig. 9, parts I, II). Fgf8 expression is detected in the myotomes, AER, presomitic Discussion mesoderm and branchial arches of heterozygous embryos, and An important finding from this study is that the delamination this expression is generally unaffected in Six1–/–Six4–/– and migration of the hypaxial precursors from the ventral lip embryos, except in the third and fourth branchial arches (Fig. to the limb buds are under the control of Six homeoproteins. 9, part III, A-E). Fgfr4 is expressed by the proliferating In absence of Six1 and Six4, cells that normally give rise to myotomal cells (Marcelle et al., 1995), and presumably in the ventral lip of the dermomyotome lose Pax3 expression and proliferating cells of dKO embryos as well (Fig. 9, part IV). lose their identity; some of them are misrouted dorsally and Finally, scleraxis expression in the syndetome has been shown detected beneath the ectoderm, others are misrouted medially. to be controlled by Fgf signaling provided by the adjacent Met is the crucial factor necessary for myogenic cells of the myotome (Brent and Tabin, 2004). Interestingly, no scleraxis VLL to undergo an epithelial to mesenchymal transition prior expression is detected in the somites of Six1–/–Six4–/– embryos to migration in the limb bud (Bladt et al., 1995). In E10.5 2246 Development 132 (9) Research article

Six1–/–Six4–/– embryos, Met expression is correct in the most is observed from E10.5 and persisted throughout caudal somites but severely reduced and highly diffuse more embryogenesis. Pax3, Mrfs and other myotomal-specific genes rostrally, thus completely preventing migration. As Met has expression is more severely altered in rostral somites than in been demonstrated to be directly controlled by Pax3 (Relaix et more caudal ones, giving rise to a more severe disorganization al., 2003), this caudorostral downregulation of Met in of back muscle masses at the shoulder level than at the Six1–/–Six4–/– embryos may be a direct consequence of the loss interlimb and hip levels in dKO fetuses. This suggests that the of Pax3. This could be effectively the case for rostral somites regulatory myogenic pathways operating in rostral somites are where Pax3 expression is lost, but not for the most caudal distinct from those operating more caudally, and is reminiscent somites facing the hindlimb, where Pax3 seems correctly of the complex activation of the Mrf4/Myf5 locus in different activated, while Met is only faintly detected. These results precursor populations (Carvajal et al., 2001; Hadchouel et al., coincide with previous results showing that Met activation in 2003). E13.5 Six1–/–Six4–/– embryos develop more serious cells migrating from somite to limb was not entirely dependent defects in the body musculature than splotch embryos, which on Pax3 (Mennerich et al., 1998). Thus, Six1 and Six4 control are essentially affected in their most dorsal and ventral muscles early steps of a genetic network involved in ventral lip (Tremblay et al., 1998). Thus, the digenesis of more profound formation and that coordinate the expression of a set of genes thoracic and abdominal muscles in Six1–/–Six4–/– embryos required for migration, including Pax3 and Met. Furthermore, appears to rely on an impaired determination and while epaxial Met expression is not altered in splotch mice differentiation of myotomal precursors. E10.5 Six1–/–Six4–/– (Mennerich et al., 1998) and not increased in Pax3-FKHR mice embryos, indeed, present a more severe decrease of Myf5 and (Relaix et al., 2003), it is abolished in all E10.5 Six1–/–Six4–/– Myod1 expression in the myotome than what observed in somites, even in caudal somites where Pax3 expression is not splotch embryo (Tajbakhsh et al., 1997), suggesting that Six yet extinguished. This result suggests a direct control of Met homeoproteins can activate both genes in subpopulations of by Six homeoproteins, independently of Pax3. In the light of myogenic precursors independently of Pax3. The genetic link Six1 metastatic properties (Ford et al., 1998; Yu et al., 2004), between Myod1 expression and Six1 during limb myogenesis these results suggest that Six1 could control the metastatic (Laclef et al., 2003a) suggests a direct role for Six1 and Six4 behavior of rhabdomyosarcoma cells through direct Met in the transactivation of Myod1 in myogenic precursors, while transactivation, as this proto-oncogene has been implicated in it has been established that the activation of Myod1 by Pax3 is the development of several human cancers, including indirect (Relaix et al., 2003). It is also possible that impaired melanomas, breast cancer and rhabdomyosarcomas (Sharp et Myod1 expression is dependent upon the decrease of Myf5 al., 2002). expression and loss of Mrf4 expression (see below). Finally, our analysis provides strong evidence that Six1 and Most remaining myofibers in the dKO embryos arose from Six4 homeoproteins are also required for the activation of the caudal dermomyotomal lips, as epaxial and hypaxial lip Lbx1 gene in the hypaxial myogenic precursors. In E10.5 dKO structure is lost. These dermomyotomal caudal lips precursors Development embryos, Lbx1 expression is reduced but detectable in sacral have been shown to contribute to myotome growth in its somites facing the hindlimb where Pax3 is not affected by the dorsoventral extent (Kahane et al., 1998), are known to express lack of Six homeoproteins. Lbx1 expression is completely specifically Delta1, and are able to give rise to epaxial and impaired in more rostral somites and hypoglossal chord. So far, hypaxial myocytes (Gros et al., 2004; Kahane et al., 1998). Lbx1 has been regarded as a Pax3 target because Lbx1 Myogenin expression is dramatically reduced in the transcripts were not detectable in splotch mice (Dietrich et al., myotome of Six1–/–Six4–/– embryos at E9.5 and E10.5. Only a 1999). These conclusions were compromised by the fact that few positive cells are detected in the more central-epaxial part cells that would normally express Lbx1 are lost by the lack of of the myotome. The comparative analysis of myogenin Pax3 (Borycki et al., 1999). Furthermore Lbx1 expression is expression between Six1–/–Six4–/– and splotch mutants embryos detectable in occipital somites of splotch embryos contrary to (Tajbakhsh et al., 1997) tends to demonstrate the dependence what observed in Six1–/–Six4–/– embryos (Dietrich et al., 1999). of myogenin activation by Six proteins, as expected by our These results suggesting a direct control of Lbx1 by Six previous finding (Spitz et al., 1998). The observation that proteins are further supported by bandshift assays somitic expression of myogenin was preserved in Six1–/– or in demonstrating the capacity of Six1, Six4 and Six5 Six4–/– embryos, and that limbs expression of myogenin was homeoproteins to bind the potential MEF3 site identified in only delayed in Six1–/– embryos (Laclef et al., 2003a; Ozaki et human and mouse Lbx1 promoter. al., 2001) probably reflected again the compensation The absence of either Six1 or Six4 did not block Pax3 mechanism that exists between Six1 and Six4. The remaining expression and cell migration into the limb bud (Laclef et al., myogenin and β-gal-positive cells in the center of the 2003a; Ozaki et al., 2001). Therefore, the double knockout myotomes suggest that a specific population of myogenic cells analysis clearly demonstrates the overlapping functions shared can activate myogenin and an alternative myogenic program in by Six1 and Six4 homeoproteins to activate the myogenic the absence of Six proteins, as already proposed (Laclef et al., migration program in somites through the control of the 2003a). expression of Pax3, Met and Lbx1genes. Surprisingly, Mrf4 expression is completely lost in Six1–/–Six4–/– embryo. MRF4 has been recently identified as a Absence of Six1 and Six4 homeoproteins impaired key determination gene controlling the activation of Myod1 in induction of Pax3 and Mrfs leading to a severe trunk the myotome, in parallel to Myf5 (Kassar-Duchossoy et al., musculature hypoplasia 2004). The lack of Mrf4 in Six1–/–Six4–/– embryo may thus Epaxial myogenesis is more affected at the rostral level than at participate in the downregulation of myotomal Myod1 the interlimb and caudal levels in Six1–/–Six4–/– embryos. This expression. Myod1 expression has been shown to be under two Development and disease Impaired myogenesis in Six1Six4 null embryos 2247

complementary genetic pathways involving Myf5 and Pax3 (Laclef et al., 2003a), are observed in several other KO that (Tajbakhsh et al., 1997). Whether Myod1 activation by Six prevent axial myogenesis. Fgf and Pdgfa signaling is required proteins follows the Myf5 or Pax3 network, or both, remains for correct rib growth and both signaling pathways are to be determined. diminished in the Myf5 KO that is devoid of early myotome We have demonstrated that in the absence of Six1 and Six4 and Mrf4 expression (Grass et al., 1996; Kassar-Duchossoy et myogenic factors, the formation of the myotome is first al., 2004; Patapoutian et al., 1995; Tallquist et al., 2000), and compromised by a loss of Pax3 and second by an impaired one can hypothesize that it is the case for the other KO in which activation of the Mrf proteins in the myogenic precursors correct hypaxial myogenesis is impaired. Absence of early already present in the myotome. It also shows that although ventrolateral differentiated myotome producing Fgf signaling, impaired at multiple levels – absence of ventral and dorsal lips, as observed in Six1–/–Six4–/– embryos should preclude growing and decrease of Mrf protein expression – primary myogenesis of the sternal region of the ribs (Evans, 2003; Huang et al., can nevertheless take place, mainly owing to contribution of 2003). Six1 and Eya1 have been shown already to control caudal lips in which Pax3 is still expressed independently of Fgf3 and Fgf10 signaling during kidney and otic development Six1 and Six4. (Xu et al., 1999; Zheng et al., 2003), suggesting that one signaling pathway controlled by the Pax-Six-Eya network may Increased apoptosis in somites of Six1–/–Six4–/– that of the Fgf signaling affecting different types of embryos organogenesis. Although there is some evidence that Six1 could control cell proliferation (Yu et al., 2004), proliferation deficiencies were Pax-Six-Eya genetic loop not detected in the myogenic lineage of Six1–/–Six4–/– embryos. The demonstration of an epistatic relationship between However, Six1 and Six4 appeared necessary to prevent cells Six1/Six4 genes and Pax3 gene in myogenic precursors from apoptosis and to induce myogenic differentiation through originating from the lateral dermomyotome of the somites is myogenin induction and the accompanying cell cycle consistent with the genetic link characterized during early withdrawal (Zhang et al., 1999). Cells in the branchial arches, kidney development in the mouse embryo, where Pax2 in the DRG and in the ventrolateral dermomyotome of the dKO expression has been shown to be markedly reduced in the embryos were found to lose their identity and die by apoptosis. metanephric mesenchyme of Six1 mutant mice (Xu et al., Interestingly, this phenotype has been reported in Drosophila, 2003). The genetic hierarchy placing sine oculis downstream where absence of sine oculis does not prevent cell proliferation eyeless in Drosophila (Halder et al., 1998) does not seem to be but induces apoptosis of those cells that are unable to progress conserved during myogenic development nor during the early in their differentiation (Cheyette et al., 1994). Interestingly, organogenesis of kidney, as Six1 expression is not altered in apoptosis has been also reported in Pax3–/– embryos at the the metanephric mesenchyme of Pax2 mutant mice embryo somitic level (Borycki et al., 1999). It has been suggested that (Xu et al., 2003). Interestingly, although Six1/Six4 control Development overexpression or misexpression of one protein of the Pax-Six- Pax3 expression in the lateral dermomyotome of the occipital, Eya-Dach network triggered a default apoptotic program cervical, thoracic and lumbar somites, Pax3 is activated (Clark et al., 2002). Apoptosis has also been detected in Six1–/– independently of Six proteins in the lateral dermomyotome of embryos in the metanephric mesenchyme (Xu et al., 2003). sacral and caudal somites, and in the anterior and posterior lips This suggests that different cell types adopt the same strategy of the dermomyotome of all somites along the anteroposterior facing the absence of Six homeoproteins, or that Six proteins axis. Altogether, these results are reminiscent to the recent are important actors for cell survival. observation that Lbx1 gene is activated by Hox proteins in the somites facing the limb buds (Alvares et al., 2003), and suggest Six1 and Six4 control Fgf production in the that Hox proteins that control the axial identity of somites myotomes (Burke, 2000) may, in cooperation with Six proteins, control Several Fgf molecules are produced by the somites (Karabagli Pax3, Lbx1 and, more generally, hypaxial myogenesis. et al., 2002). We find in fact that Fgf4 and Fgf6 ventrolateral somitic expression is greatly diminished in Six1–/–Six4–/– E10.5 We thank A. K. Voss and P. Gruss for the gift of ES cells, S. embryos. We can hypothesize that as Fgf molecules produced Tajbakhsh, C. Marcelle, C. Birchmeier, G. Martin, H. H. Arnold, E. Olson and C. Ponzetto for probes and antibodies, and D. Metzger for by the ventrolateral myotome, i.e. Fgf6 and Fgf4, are lacking –/– –/– the plox2hygro plasmid. We thank F. Relaix and M. Lagha for their in E10.5 Six1 Six4 embryos, scleraxis transcription is helpful support concerning in situ analysis, C. Laclef and D. Duprez delayed. In fact, while early scleraxis activation is inhibited, in for helpful discussions, and A. Baudinière for technical assistance E12.5 embryos, scleraxis expression becomes detectable (D. during Six4 genomic cloning. We thank R. Schweitzer and D. Duprez Duprez, personnal communication). Our results support the for communicating results before publication. We thank M. hypothesis of Fgf signaling by the myotome is required to Buckingham, D. Daegelen, M. Morgan and S. Tajbakhsh for helpful induce scleraxis and, hence, the syndetomal compartment, in critical reading of the manuscript. R.G. was supported by a fellowship agreement with recent findings (Brent et al., 2005). As axial from the Ministère de la Recherche et de l’Education nationale, from tendon formation can take place during mouse embryogenesis the Association Française contre les Myopathies (AFM) and from the even in the absence of scleraxis (Ronen Schweitzer, personal Fondation pour la Recherche Médicale (FRM). Financial support for this work has been provided by the Institut National pour la Santé et communication), the tendons observed at the axial level in la Recherche Médicale (INSERM), the Centre National de la E18.5 fetuses is not in conflict with a default early induction Recherche Scientifique (CNRS), the AFM, and by the Action –/– –/– of scleraxis in Six1 Six4 embryos. Concertée Incitative 0220514. The contribution of the Région Ile de We already reported that Six1–/– mice had severe rib and France to the Institut Cochin animal care facility is also skeletal craniofacial defects. Rib defects, as discussed already acknowledged. 2248 Development 132 (9) Research article

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