muscular dystrophy begins early in embryonic …of muscle development, but this has not been studied...

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INTRODUCTIONDuchenne muscular dystrophy (DMD) is the severest of a familyof debilitating congenital diseases associated with disruption offunction of the trans-sarcolemmal dystrophin-glycoproteincomplex (DGC). Classic DMD, which affects the entire skeletalmusculature, arises from loss of an essential intracellularcomponent of the DGC, dystrophin (Hoffman et al., 1987). Thecondition is characterised by early (~3 years age) and progressivedisruption of skeletal muscle function, which leads to extensivemuscle damage and causes early death (usually in the 20s). Deathcommonly results from complications arising from failure of therespiratory muscles, however, 95% of patients with DMD developa cardiomyopathy, which is the primary cause of death in 10-30%of cases (Cox and Kunkel, 1997). The mdx mouse, a model of DMDthat is deficient in dystrophin owing to a point mutation in theDmd gene, exhibits many of the postnatal features of DMD,

including cardiomyopathy; skeletal muscular dystrophy associatedwith bouts of fibre regeneration; fibrosis; hyperproliferation; andapoptosis of skeletal muscle myoblasts (Harper et al., 2002; Smithet al., 1995). The phenotype is less severe than human DMD,although the life span of mdx mice is curtailed with respect to wild-type (WT) mice (Chamberlain et al., 2007). Loss of caveolin-3results in a milder form of muscular dystrophy (MD), known aslimb-girdle muscular dystrophy type 1C (LGMD-1c), in which theaffected muscle groups are predominantly the limb girdle and heart;caveolin-3-deficient mice (cav-3–/–) lack the Cav3 gene and exhibitLGMD, T-tubule defects, cardiomyopathy and skeletal muscleapoptosis (Hagiwara et al., 2000; Minetti et al., 2002).

Embryonic skeletal muscles originate from the dermamyotomalregion of the somites (Cossu et al., 1996; Hollway and Currie, 2003).In the chick embryo, all regions of the dermamyotome appear tocontribute myogenic precursors to this process in a time-dependentmanner (Gros et al., 2004). Dystrophin is first expressed inembryonic somites from embryonic day (E)9.5, and could, thus,have a function in early myogenesis (Huang et al., 2000; Ilsley etal., 2002). Under the control of inducing factors that are secretedby surrounding tissues, the myotome produces cells that becomethe (Myf5 positive) stem cell populations, which then generate(from E10.5) the epaxial (deep muscles of the back) and (from E11.5)

Disease Models & Mechanisms 2, 374-388 (2009) doi:10.1242/dmm.001008Published by The Company of Biologists 2009

Muscular dystrophy begins early in embryonicdevelopment deriving from stem cell loss and disruptedskeletal muscle formationDeborah Merrick1,2,*, Lukas Kurt Josef Stadler1,*, Dean Larner1 and Janet Smith1,‡

SUMMARY

Examination of embryonic myogenesis of two distinct, but functionally related, skeletal muscle dystrophy mutants (mdx and cav-3–/–) establishesfor the first time that key elements of the pathology of Duchenne muscular dystrophy (DMD) and limb-girdle muscular dystrophy type 1C (LGMD-1c) originate in the disruption of the embryonic cardiac and skeletal muscle patterning processes. Disruption of myogenesis occurs earlier in mdxmutants, which lack a functional form of dystrophin, than in cav-3–/– mutants, which lack the Cav3 gene that encodes the protein caveolin-3; thisfinding is consistent with the milder phenotype of LGMD-1c, a condition caused by mutations in Cav3, and the earlier [embryonic day (E)9.5] expressionof dystrophin. Myogenesis is severely disrupted in mdx embryos, which display developmental delays; myotube morphology and displacementdefects; and aberrant stem cell behaviour. In addition, the caveolin-3 protein is elevated in mdx embryos. Both cav-3–/– and mdx mutants (from E15.5and E11.5, respectively) exhibit hyperproliferation and apoptosis of Myf5-positive embryonic myoblasts; attrition of Pax7-positive myoblasts in situ;and depletion of total Pax7 protein in late gestation. Furthermore, both cav-3–/– and mdx mutants have cardiac defects. In cav-3–/– mutants, thereis a more restricted phenotype comprising hypaxial muscle defects, an excess of malformed hypertrophic myotubes, a twofold increase in myonuclei,and reduced fast myosin heavy chain (FMyHC) content. Several mdx mutant embryo pathologies, including myotube hypotrophy, reduced myotubenumbers and increased FMyHC, have reciprocity with cav-3–/– mutants. In double mutant (mdxcav-3+/–) embryos that are deficient in dystrophin(mdx) and heterozygous for caveolin-3 (cav-3+/–), whereby caveolin-3 is reduced to 50% of wild-type (WT) levels, these phenotypes are severelyexacerbated: intercostal muscle fibre density is reduced by 71%, and Pax7-positive cells are depleted entirely from the lower limbs and severelyattenuated elsewhere; these data suggest a compensatory rather than a contributory role for the elevated caveolin-3 levels that are found in mdxembryos. These data establish a key role for dystrophin in early muscle formation and demonstrate that caveolin-3 and dystrophin are essential forcorrect fibre-type specification and emergent stem cell function. These data plug a significant gap in the natural history of muscular dystrophy andwill be invaluable in establishing an earlier diagnosis for DMD/LGMD and in designing earlier treatment protocols, leading to better clinical outcomefor these patients.

1School of Biosciences, University of Birmingham, Edgbaston, Birmingham B152TT, UK2Present address: School of Biomedical Sciences, Nottingham University MedicalSchool, Queen’s Medical Centre, Nottingham NG7 2UH, UK*These authors contributed equally to this work‡Author for correspondence (e-mail: [email protected])

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Disease Models & Mechanisms 375

The embryonic basis of muscular dystrophy RESEARCH ARTICLE

the hypaxial (limb, abdominal, diaphragm) muscles (reviewed byHollway and Currie, 2003).

In mammalian embryonic skeletal muscle differentiation, thereare thought to be two myogeneic ‘waves’ that generate the hypaxiallineage, producing a scaffold of primary myotubes and,subsequently, large numbers of secondary myotubes, whichcomprise the bulk of newly formed muscle (Cossu et al., 1996).Secondary myotubes are a morphologically distinct subset ofmyotubes, which are longer and thinner than primary myotubes,and that form in clusters around a single, larger primary myotube(Cho et al., 1994).

Myotube differentiation is dependent on the presence offunctional muscle stem cell populations. Myf5 is expressed in thesomite at E8.5 making it the earliest of the myogenic regulatoryfactors (MRF); in addition, it is the only MRF that is found beforemuscle differentiation and that persists in all embryonic musclegroups throughout both primary and secondary myogenesis (Cossuet al., 1996). Myf5 is therefore a good marker for the embryonicmyoblast population. Lineage analysis suggests that a majority ofsatellite cells derive from the somite (Armand et al., 1983). Extensiveevidence now links the Pax7-positive cell population, in particular,as being essential to the correct functioning of the adult skeletalmuscle stem cell (the ‘satellite’ cell) population and particularly toits repair function (reviewed by Zammit et al., 2006). Pax7 is atranscription factor, a repressor of myogenesis, and plays importantroles in the maintenance and specification of the adult satellite cellpopulation; it is expressed in undifferentiated muscle stem cellsthat emerge from the somite from E11.5 (Merrick et al., 2007; Relaixet al., 2006; Seale et al., 2000).

The specificity of function of individual skeletal muscle groupsdepends on the appropriate localisation of fast myosin isoforms todifferent myotubes; a process initiated during the late stages ofgestation (Merrick et al., 2007). In mammalian embryos,developmental (embryonic and neonatal) myosin isoforms are co-expressed in newly formed secondary myotubes with adult fastmyosin isoforms (Cho et al., 1994). In later stages, developmentalmyosin isoforms are downregulated and replaced by adult myosinheavy chain (MyHC) isoforms in a muscle-specific pattern, a processthat is not completed until several weeks after birth (Agbulut et al.,2003; Merrick et al., 2007). The embryonic heart expresses twomyosin isoforms (cardiac myosin α and slow/cardiac myosin β) in atemporally regulated manner. Mutation of β-cardiac myosin isimplicated in cardiomyopathy (Geisterfer-Lowrance et al., 1990).

Dystrophin associates with the β-subunit of dystroglycan on theintracellular side of the sarcolemma and is an essential componentof the DGC, a multifunctional protein complex, which links theextracellular matrix to the actin cytoskeleton in skeletal musclemyotubes (Ervasti and Campbell, 1993). In postnatal muscle,dystrophin deficiency results in complete breakdown of the DGCand the secondary downregulation of a majority of the DGCproteins (Ohlendieck et al., 1993). Caveolin-3 localises to bothskeletal muscle caveolae and the DGC where, by means of a specificWW domain, it binds to the same PPXY motif in the β-dystroglycanc-terminus that recognises and binds to dystrophin, thus blockingthe interaction between dystrophin and β-dystroglycan (Jung et al.,1995; Sotgia et al., 2000).

The competitive interaction between caveolin-3 and dystrophinfor the β-dystroglycan binding site may be crucial for some aspects

of muscle development, but this has not been studied extensively.All three genes are expressed early in development in the chick,mouse, zebrafish and Xenopus laevis embryos (Biederer et al., 2000;Houzelstein et al., 1992; Nixon et al., 2005; Razani et al., 2002; Shinet al., 2003; Anderson et al., 2007). In zebrafish, loss of any of theseproteins has been shown to disrupt myogenesis and cause grossmuscle abnormalities, whereas in the mouse, dystroglycandeficiency leads to early embryonic lethality (Bassett et al., 2003;Nixon et al., 2005; Parsons et al., 2002; Williamson et al., 1997).

In this study, we examine the impact of the loss of caveolin-3and dystrophin on skeletal muscle development in order to establisha functional role for these two proteins during myogenesis.

RESULTSBranching fibre misalignment and malformed myotubescharacterise the embryonic phenotype of MDWT, cav-3–/– and mdx mice were immunostained with a pan-myosinantibody (MF20) to reveal muscle fibre architecture (Fig. 1).Branching and fibre splitting is found in mdx epaxial, hypaxial andfacial muscles from E13.5 to E17.5, and shows a dynamic temporaland spatial pattern that is consistent with it being an early eventassociated with myotube formation (Fig. 1A-C,G). In matchedmuscle groups, at matched stages, mdx myotubes are hypotrophic,whereas cav-3–/– myotubes are hypertrophic, and myotube widthvaries more in dystrophic embryos with respect to WT embryos (Fig.1C-E, green bars). Maximum morphological defects occur earlier inmdx intercostals (E13.5) than proximal limb muscles (E15.5), whichis consistent with the later development of the proximal limbmuscles (Fig. 1A). There is no fibre splitting in cav-3–/– and WTembryonic myotubes (Fig. 1A,C,E,F,H). Myotube alignment is alsodisrupted early in mdx myogenesis (Fig. 1I). At E13.5, up to an eighthof mdx myotubes are displaced from the median fibre alignment inthe proximal limb, intercostal and facial muscles (Fig. 1I). This isstatistically different from E13.5 WT and cav-3–/– muscle, in whichfewer than 5% of myotubes were displaced. When myotubes weredivided into those which deviate by more than 25° from the median(misalignment) and those that are displaced by less than 25°(tangential fibres), a significant proportion of the displaced mdxmyotubes in individual muscles were misaligned (Fig. 1K), the restbeing tangential, whereas all displaced myotubes in WT or cav-3–/–

muscles were tangentially displaced. In the intercostal muscles ofE13.5 mdx embryos, 7-9% of all myotubes (i.e. more than half of thetotal displaced myotubes) were misaligned. This was also true of theother stages examined (data not shown). The proportion of displacedmyotubes declines with gestational age and is muscle groupdependent (Fig. 1I). At E13.5, 12% of myotubes in mdx intercostalmuscles were aligned incorrectly, this declines with gestational stagebut we found misalignment and tangential displacement of myotubesin mdx intercostal muscles at all gestational stages (E13.5-E17.5, Fig.1I,M). At the same stages, WT and cav-3–/– intercostal myotubeswere aligned correctly to the median and there were no deviatingmyotubes in WT or cav-3–/– intercostals, suggesting that thealignment mechanism in these muscles is regulated tightly anddisrupted severely in mdx mutants (Fig. 1I-N).

Morphological defects in the dystrophic embryonic heartCardiomyopathy is a significant clinical consequence of bothLGMD (1C) and DMD. We established the morphology and

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The embryonic basis of muscular dystrophyRESEARCH ARTICLE

myosin localisation pattern of dystrophic and WT embryonic heartsusing MF20 and the cardiac β-myosin specific antibody N2.261 (Fig.2). Consistent with the literature, skeletal fast myosin isoforms(FMyHC) could not be detected in embryonic hearts between E13.5and E17.5 (My32 antibody, data not shown). Cardiac β-myosin ispresent in WT and dystrophic ventricular and atrial myocytesbetween E13.5 and E17.5 (Fig. 2A-F,I-P). Both mutants exhibitventricular wall thickening (Fig. 2A-F) and cardiac myocytedisorganisation, which is substantially worse in cav-3–/– mutants,where myocytes form in a criss-crossed fashion, overlapping oneanother (Fig. 2E-H). Atrial trabecular formation is also impairedin dystrophic embryos; the trabeculae in E14.5 cav-3–/– embryosare short and stubby, whereas in mdx embryos they are long andhook shaped (Fig. 2I-J). At E17.5, distension or separation of themyocardial and endocardial cell layers of dystrophic atria occursin the mutant embryos, and loss of cardiac β-myosin is alsoobserved (Fig. 2K-P); this is in contrast to WT atria, where stainingis uniform and the two layers are adjoined tightly (Fig. 2K,N). Thereare distinct differences between the two dystrophic hearts; in cav-3–/– mutants at E17.5, we observed patchiness and a reduction in

staining in the myocardial wall, which was most evident in thetrabeculae (Fig. 2O). In the mdx mutants, the loss of staining wasextensive in the intertrabecular regions of the myocardium, butlargely retained in the tips of trabeculae (Fig. 2P).

Myonuclei misplacement and fusion abnormalitiesIn E15.5 WT embryos, myonuclei are spaced evenly along thelength of the myotube and, except for in newly formed myotubes,are arranged evenly and helically around the edge (Fig. 3A, whiteasterisks). In mdx embryos, myonuclei are more frequently locatedcentrally and slightly further apart than in WT embryos (Fig. 3B).By contrast, cav-3–/– myonuclei are found closer together andexhibit severely disrupted myonuclei spacing and nuclei ‘bunching’,which is particularly evident at the ends of myotubes (Fig. 3C-D).Transverse sections demonstrate the presence of peripheral andcentral nuclei in WT muscle at this stage (E15.5) and show thatonly central nuclei are present in mdx and cav-3–/– muscles (Fig.3E-G). To quantify these phenotypes, we scored total myonucleiand myofibres over a fixed area, to establish that cav-3–/– myotubescontained twice as many myonuclei as those of mdx or WT

Fig. 1. Branching and displacement defects in mdx embryonicmyotubes. (A) Skeletal myotube branching in mdx myotubes. IC,intercostal; PL, proximal limb; F, facial muscle groups. The zeroindicates that no abnormalities were detected. (B) Myotubebranching in E15.5 mdx biceps as shown by haematoxylin and eosin(H&E) staining. The insert shows an enlarged section. (C-H) MF20immunostaining of proximal muscle in WT myotubes (C,F), mdxhypotrophic myotubes (D,G), and cav-3–/– hypertrophic myotubes(E,H). The green bars indicate the width of a WT myotube and theblack arrowheads point to myotube splitting in mdx myotubes. (F-H) Variable myotube diameters in mdx (G) and cav-3–/– (H) musclefibres compared with in WT (F) muscle fibres. Bc, biceps.(I) Displaced myotubes (combined tangential and misalignedmyotubes) as a proportion of the total counted. The zeros indicatethat no fibres were displaced. (J) Displaced myotube scoringstrategy: tangential myotubes (T) were classed as <25° from themedian, misaligned myotubes (M) were >25° from the median.(K) Proportion of misaligned mdx myotubes at E13.5. (L-N) MF20immunostaining of intercostal muscles at E13.5. Misalignedmyotubes (M) were observed in mdx muscle (M, black arrow), but nomyotube displacment was seen in WT (L) or cav-3–/– (N) muscles.Error bars indicate s.d. Student’s t-test: *P<0.01, **P<0.001 whencompared with WT value. Bars, 20 μm.

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myotubes; this difference is statistically significant at E13.5, E15.5and E17.5 (Fig. 3K) (P<0.05). There is also a substantial excess ofmyotubes in cav-3–/– mutants at these stages (P<0.05) and asmaller, but significant, reduction (P<0.05) in myotube number inmdx mutants (Fig. 3H-J,L). The disproportion in myotube numbersin cav-3–/– and mdx embryos lessens between E13.5 and E17.5, butis statistically different to WT embryos at all stages.

Hyperproliferation and apoptosis in cultured embryonic musclestem cellsTo characterise the embryonic muscle stem cell population we useddystrophic and WT (E11.5-E17.5) embryonic myoblast cultures(Smith and Merrick, 2008). The somite origin of these cells wasestablished using Myf5 staining (Fig. 4). mdx and cav-3–/– explantsboth have outgrowth rates that deviate from WT explants (Fig. 4A-B). Compared with WT embryos, outgrowth of Myf5-positive cellsis significantly greater at all stages (E11.5–E17.5) in mdx embryosand significantly greater from E15.5-E17.5 in cav-3–/– embryos. Inearlier stages (E11.5–E13.5), the cav-3–/– outgrowth rate isindistinguishable from that of WT embryos, suggesting that defectsoccur later in this mutant. Between E11.5 and E17.5, myoblast

proliferation (determined by Ki67 immunoreactivity) and apoptosis(shown by DAPI staining) were both significantly elevated in mdxmutants, whereas in cav-3–/– mutants, apoptosis and proliferationrates were equal to those of WT embryos until E15.5 when therewas a sharp elevation in both parameters to levels approachingthose of mdx embryos (Fig. 4C,E). In the mdx mutants, proliferation(and apoptosis) of myoblasts declined from E13.3 to E17.5, althoughit remained significantly higher than WT levels; however, theproliferation and apoptotic rates of cav-3–/– myoblasts increaseduntil E15.5 (Fig. 4C-F). At E17.5, the proliferative rate of cav-3–/–

cultures declined slightly, in line with mdx levels, whereas theapoptotic rate is maintained at the level of E15.5 cav-3–/– myoblasts(Fig. 4E). The mdx explant-derived soluble factors were shown toincrease outgrowth of E11.5 WT explants (Fig. 4I).

The Pax7 skeletal muscle stem cell population is attenuated anddisorganised in dystrophic embryosAt E17.5, the WT Pax7-positive stem cell population is interspersedmore sparsely between skeletal muscle myotubes, although levelsof Pax7 protein continue to increase with muscle size (Merrick etal., 2007) (Fig. 5A,B,G,H,M,O). In WT embryos, the Pax7 staining

Fig. 2. Disrupted development of the heart in dystrophic mice.(A-F,I-N) Mid-section hearts immunostained for β-cardiac myosinheavy chain. At E13.5, cav-3–/– (B) and mdx (C) hearts show mild andextensive ventricular wall hypertrophy, respectively. At E14.5, theventricle apex structure is disorganised in cav-3–/– hearts (E) and lesswell developed in mdx hearts (F) when compared with WT hearts(D). (G,H) An MF20-stained E15.5 cav-3–/– heart showingdisorganisation of the ventricular wall (G) and cardiac myocytes thatcriss-cross each other (H). (I,J) E14.5 atrial trabeculae (tb) are shortand stubby in cav-3–/– mutants (I) and hook shaped in mdx mutants(J). (K-P) At E17.5, attenuated N2.261 labelling and distension of celllayers was visible in cav-3–/– trabeculae (L,O) and in the mdx atrialwall (M,P), compared with in WT atria (K,N). (Q) Low-magnificationimage of an MF20-labelled mdx heart to illustrate the orientationand matching of hearts that were sectioned sagitally through themost central portion of the heart. Bars, 20μm.

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intensity is uniform and constant with gestational age (Fig.5A,B,G,H,Q; lower proximal limb). Between E15.5 and E17.5, bothcav-3–/– and mdx embryos undergo attrition of their Pax7-positivecell population throughout their musculature so that, by E17.5,there is a significant reduction of Pax7 staining and Pax7 proteinin both mutants (Fig. 5C-L,M,P; shown for lower proximal limb).This is particularly evident in cav-3–/– proximal hind limb muscles,where Pax7-positive cells are almost absent at E17.5 and those thatremain stain very weakly (Fig. 5J, white arrowhead). At E15.5 (cav-3–/– and mdx) and E17.5 (mdx), Pax7-positive cell fragments arefound in dystrophic muscles (Fig. 5L, indicated by a blackarrowhead), suggesting that these cells may be undergoingapoptosis. Mutant mdxcav-3+/– embryos, which are deficient indystrophin (mdx) and heterozygous for caveolin-3 (cav-3+/–), havea much more severe phenotype in the proximal limb where Pax7-positive cells are very sparsely present at E15.5 (Fig. 5M,N,R);furthermore, Pax7-positive cells are almost entirely absent in thelower proximal limb of these embryos at E17.5 (Fig. 5O-P) and arevery much attenuated throughout the musculature (see Fig. 7).

Whole-embryo immunoblotting demonstrates an increase incaveolin-3 protein in WT (and mdx) embryos at E15.5-E17.5compared with at E11.5 and E13.5, and confirms its absence in cav-

3–/– embryos (Fig. 5M-N). In mdx mutants, caveolin-3 is notdetected at E11.5 (Fig. 5M,N) but there is a several fold increase incaveolin-3 content at E13.5-E17.5 compared with in WT embryos,as measured by densitometry on four separate gel runs (P=0.05 forall three stages) (Fig. 5O). Pax7 content increases in WT embryosduring gestation but is reduced substantially in cav-3–/– and mdxmutant embryos at E15.5-E17.5, with the reduction being greater incav-3–/– embryos than mdx embryos at both stages, suggesting thatcaveolin-3 may regulate Pax7-positive myoblast survival in lategestation. At E15.5, there is a reduction in the intensity of the Pax7bands in mdx and cav-3–/– dystrophic embryos by 15% and 60%,respectively (Fig. 5N,P). At E13.5, Pax7 is elevated slightly in cav-3–/– embryos and reduced in mdx embryos; this may relate to theincrease and decrease in myotube number that is found in cav-3–/–

and mdx embryos, respectively, at this stage (see Fig. 3), and suggeststhat developmental timing may be disrupted in these embryos.

Mislocalisation of fast myosin isoforms in dystrophic embryosfollows a reciprocal pattern in dystrophin- and caveolin-3-deficient embryonic musclesIn WT embryos, FMyHC is present in small numbers of secondarymyotubes by as early as E11.5, and by E15.5 it is localised strongly

Fig. 3. Size, shape and number of myotubes are altered indystrophic mice. (A-C) Arrangement of myonuclei in E15.5 proximalmuscle: (A) WT, helical with even spacing; (B) mdx, central location;(C) cav-3–/–, close central location with irregular spacing. Whiteasterisks indicate myonuclei spacing in one myotube; white arrowspoint to myonuclei. In the WT proximal muscle (A), the white arrowsindicate peripheral nuclei in two separate myotubes. Peripheralnuclei are not evident in mdx and cav-3–/– muscles (B,C).(D) Bunching of myonuclei at myotube ends in cav-3–/– muscle.(E-G) Transverse sections of E15.5 WT (E), mdx (F) and cav-3–/– (G)lower proximal limb myotubes showing that peripheral myonuclei(arrow) are associated with WT, but not with dystrophic embryo,myotubes at this stage. The hypotrophy of mdx and hypertrophy ofcav-3–/– myotubes can also be seen; the green lines in F and Grepresent the WT myotube diameter. (H-J) Lower magnificationimages of the lower proximal limb regions of WT (H), mdx (I) andcav-3–/– (J) embryos showing the reduced and increased musclefibre densities in mdx and cav-3–/– embryos, respectively, whencompared with WT fibres in matched embryo sections.(K) Myonuclei numbers are doubled in cav-3–/– myotubes (E13.5-E17.5) when compared with WT myotubes; *P<0.05. There was aslight, but not statistically significant, reduction in the number ofmdx myonuclei (E13.5-E17.5) when compared with WT myonuclei.The mean myonuclei content of myotubes was constant betweenE13.5 and E17.5. (F) Myotube density was increased in cav-3–/–

mutants (E13.5-E17.5) and decreased in mdx mutants (E13.5) whencompared with WT embryos. Myotubes were counted over a fixed,matched muscle area for each strain and stage; *P<0.05. Bars, 20 μm.

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to around 45% of secondary myotubes across a wide range of musclegroups. Around 80% of myotubes contain FMyHC at E17.5, butstaining intensity is heterogeneous and weak in many myotubes(Merrick et al., 2007). This dynamic and muscle-specific patternof fast myosin localisation is disrupted in cav-3–/– and mdx mutantembryos, which show defects in: (1) the developmental timing ofFMyHC staining, (2) the total number of fast-myosin-positivemyotubes present and (3) the intensity of fast myosin staining (Fig.6; supplementary material Fig. S1). At E13.5, FMyHC is elevatedabove WT levels in some cav-3–/– muscles (notably the respiratorymuscles) but is attenuated substantially in all mdx muscles and incav-3–/– proximal muscles (Fig. 6A-F).

The appearance of FMyHC-positive myotubes is delayed in mdxembryos; at E13.5, there are very small numbers only of FMyHC-positive myotubes throughout the musculature of mdx embryos,compared with in WT or cav-3–/– embryos at the same stage(respiratory muscles Fig. 6A-F) [proximal muscles show a similarphenotype (data not shown)]. This finding is supported byimmunoblotting where there is a statistically significant reductionin the intensity of the mdx E13.5 band (Fig. 6T,U) and by myotubecounts (Fig. 6V), which demonstrate a statistically significant

deficiency of FMyHC-positive myotubes in E13.5 mdx embryos(P<0.05). A total of six mdx and four WT E13.5 embryos were usedto establish these data. In mdx embryos, the staining intensity andproportion of FMyHC-positive myotubes increases bydevelopmental stage so that, from E15.5 to E17.5, there is asubstantial excess of My32 staining in dystrophin-deficientmyotubes compared with in WT myotubes, and a progressiveincrease in the staining intensity of FMyHC bands in mdx whole-embryo immunoblots (Fig. 6G-O,T). Densitometry confirmed thisconclusion by revealing an increase in FMyHC content in mdxembryos and a decrease in FMyHC content in cav-3–/– embryos;these results were statistically significant at P<0.001 and P<0.05,respectively (Fig. 6U). Quantification of matched, FMyHC-immunostained muscle sections from WT and dystrophic embryosestablished that the proportion of FMyHC-positive myotubes isperturbed in cav-3–/– and mdx embryos (Fig. 6V). mdx embryosthat were heterozygous for the Cav3 gene (mdxcav-3+/–) also failedto downregulate FMyHC and show increased FMyHC stainingthroughout the musculature at E15.5 (Fig. 6R) and E17.5 (Fig. 6Q,S),even in muscles such as the intercostals where myotubes aredepleted severely (Fig. 7T-U).

Fig. 4. Dystrophic, embryonic Myf5-positive myoblastsare hyperproliferative and prone to apoptosis. (A) Theoutgrowth rate of embryonic myoblasts from muscleexplant cultures is increased both in mdx mutants fromE11.5 and in cav-3–/– mutants at E15.5 and E17.5 whencompared with WT explants cultured in parallel. (B) AMyf5-immunostained explant. (C) Hyperproliferation ofembryonic myoblasts in mdx mutants from E11.5 and incav-3–/– mutants from E15.5, as determined by Ki67-positive immunoreactivity (D). (E) Elevated apoptosis fromE11.5 in mdx embryos and from E15.5 in cav-3–/– embryos,as shown by DAPI staining (F); the arrow in F points to anapoptotic cell. *P<0.05 compared with WT; **P<0.01compared with WT; #P<0.05 when comparing mdx withcav-3–/–. (G,H) E15.5 primary cultured WT embryonicmyoblasts with Myf5 staining (G) and a second antibodycontrol (H). (I) The outgrowth rate of E11.5 WT explantsincreased (*P<0.05) in E11.5 mdx explant-conditionedmedium (CM), but not in cav-3–/– or WT CM. Error barsindicate s.d.

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Extensive intercostal muscle fibre loss and Pax7 depletion inmdxcav-3+/– mutant embryosTo establish whether upregulated caveolin-3 was likely toameliorate, or contribute to, the mdx/DMD phenotype, wegenerated double mutant embryos that were deficient in dystrophin(mdx) and heterozygous for the caveolin-3 null mutation (cav-3+/–).At E15.5, these mdxcav-3+/– embryos have a 50% reduction ofcaveolin-3 compared with WT embryos (Fig. 5). Immunostainingwith MF20 and Pax7 establishes that, at E17.5, mdxcav-3+/–

embryos have a more severe phenotype that, in addition to loss ofhind limb Pax7 myoblasts (see Fig. 5), also includes a severedepletion of their intercostal muscle fibres, which is accompaniedby significant attenuation of Pax7-positive intercostal myoblasts(Fig. 7A-J). Even at low magnification, the fibre-loss phenotype canbe identified by the presence of gaps (‘white space’) between thefibres of mdxcav-3+/– intercostals (Fig. 7A,D,F,I), which are packed

together densely in WT intercostal muscle fibres. At highermagnification (Fig. 7K-P), it is clear that this phenotype is a resultof there being far fewer fibre clusters in mdxcav-3+/– intercostalscompared with in WT, mdx or cav-3+/– intercostal muscles. Thisphenotype is very consistent; three different mdxcav-3+/– embryosare shown (Fig. 7N-P). In the mdx embryos, the intercostal fibresalso appear to be distributed more sparsely than in WT embryos,suggesting a milder form of this phenotype. Quantitation of fibredensity was achieved by counting intercostal fibre number over afixed grid area and established that both mdx and het (mdxcav-3+/–) embryos have reduced fibre densities; this reduction wasstatistically significantly in the het mutant (P<0.05) (Fig. 7R).When expressed as a proportion of WT intercostal myotubes, themdxcav-3+/– mutant embryos have a massive depletion (71.2%) oftheir intercostal myotubes by E17.5 (Fig. 7S) (based on the meanof counts from three different mdxcav-3+/– embryos at E17.5),

Fig. 5. Depletion of Pax7-positive myoblasts inembryonic dystrophic muscles. Pax 7 immunostaining inWT, mdx, cav-3–/– and mdxcav-3+/– lower proximal limbs atE15.5 (A-F,M,N) and E17.5 (G-L,O,P) at low magnification(A,C,E,G,I,K,M,O) and high magnification (B,D,F,H,J,L,N,P).Bars, 10 μm. (Q,R) Whole-region views of WT (Q) andmdxcav-3+/– (R) lower proximal limbs at E15.5 showing themassive reduction in Pax7-immunoreactive cells in thedouble mutant (mdxcav-3+/–). Putative apoptotic(fragmented) Pax7-positive nuclei are visible in mdxcav-3+/–,cav-3–/– and mdx mutant limbs, but not WT limbs, at E15.5and in mdx limbs (L) at E17.5 (black arrowhead). Attenuationof Pax7 staining, as indicated by white arrowheads, was seenin cav-3–/– (C,D) and mdxcav-3+/– (M,N) limbs at E15.5 and incav-3–/– limbs at E17.5 (I-J). At E17.5, mdxcav-3+/– lowerproximal limb muscles are devoid of Pax7-positive cells (O,P).(S) Whole-embryo immunoblotting for caveolin-3 (blots i-iii)and Pax7 (blot iv); exposure times: 10 seconds (i), 1 minute(ii), 4 minutes (iii,iv). Pax7 protein levels are reduced in E15.5and E17.5 dystrophic embryos. At E11.5, following a 4-minute exposure, Pax7 is detected only in WT embryos.(T) Densitometric analysis of the caveolin-3:α-tubulin ratioconfirms the increased caveolin-3 levels in mdx mutantembryos. *P<0.05; mean±s.d. of two separate experiments.(U) The estimated fold increase of caveolin-3 in mdxembryos (over WT embryos), following separate analysis ofthree embryonic stages (four experiments per stage). P=0.05for all stages. (V) Densitometric analysis of the Pax7:α-tubulin ratio demonstrating the reduction of Pax7 protein incav-3–/– and mdx embryos (E15.5-E17.5). E13.5 cav-3–/–

embryos contain significantly more Pax7 than WT embryos.*P<0.05; mean±s.d. of two separate experiments.(W) Immunoblot demonstrating that caveolin-3 protein isreduced in E15.5 mdxcav-3+/– embryos [by 50% with respectto WT embryos, based on densitometric analysis of thecaveolin-3:α-tubulin ratio in WT embryos (track 1)compared with mdxcav-3+/– embryos (tracks 3 and 4)] andsignificantly reduced when compared with E15.5 mdxembryo siblings, in which caveolin-3 levels are increasedwhen compared with WT embryos. Neomycinimmunostaining confirms the presence of the Cav3knockout transgene in cav-3–/– and mdxcav-3+/– embryos.The reduced neomycin staining seen in mdxcav-3+/–

embryos compared with in cav-3–/– embryos reflects theirheterozygosity for the Cav3 knockout transgene.

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whereas mdx embryos have a depletion of a third (37.5%). Themajority of these fibres express FMyHC in both mutants (Fig. 7U).

DISCUSSIONIn this study, we demonstrate the embryonic phenotype of mdx andcav-3–/– dystrophic mice and show that the embryonic phenotype is

significantly more severe in mice that are mutant for both proteins(mdxcav-3+/–). We establish crucial roles for dystrophin and caveolin-3 in murine embryogenesis, and establish that the pathologies thatdefine the adult dystrophic phenotype and clinical picture of DMDand LGMD-1c originate in the failure of embryonic muscle differen-tiation. The key findings of this analysis are summarised in Table 1.

Fig. 6. Fast myosin fibre-type specificationis disrupted in dystrophic embryos. (A-F) FMyHC immunostaining in the diaphragm(A-C) and intercostals (D-F) at E13.5. WT (A,D),mdx (B,E) and cav-3–/– (C,F) sections showingreduced FMyHC in mdx embryos (B,E) andincreased FMyHC in the respiratory muscles ofcav-3–/– embryos (C,F). (G-L) FMyHCimmunostaining in the diaphragm (G-I) andintercostals (J-L) of WT (G,J), mdx (H,K) andcav-3–/– (I,L) embryos at E15.5. An increase inFMyHC staining was observed in mdx embryos(N) and a decrease in FMyHC staining wasobserved in cav-3–/– embryos (O) whencompared with FMyHC staining in WTproximal limb (M) at E17.5. (P-S) FMyHC is alsoincreased in mdxcav-3+/– embryos comparedwith in WT embryos at E17.5 (Q,S) (proximallimb shown) and E15.5 (R) (intercostal anddiaphragm shown). Furthermore, comparisonof a cav-3–/– embryo (P) with an mdxcav-3+/–

embryo (Q) at E17.5 establishes thatdystrophin deficiency suppresses thedownregulation of FMyHC that is seen in cav-3–/– and WT embryos. (T) Immunoblotting ofWT, cav-3–/– and mdx embryos shows that,with respect to WT embryos, FMyHC isdelayed in both cav-3–/– and mdx embryosuntil E13.5. In the later stages, FMyHC isattenuated in cav-3–/– embryos andoverproduced in mdx embryos (T). (U) FMyHCdensitometry. The FMyHC:α-tubulin ratiodemonstrates a statistically significant increaseand reduction in FMyHC content in mdx andcav-3–/– embryos, respectively, at E17.5. AtE13.5, there is a significant excess of FMyHC incav-3–/– embryos; mean±s.d. of two separateexperiments. (V) FMyHC-positive myotubesexpressed as a proportion of the total countedin WT, mdx and cav-3–/– embryos (at E13.5,E15.5 and E17.5). Error bars indicate s.d.*P<0.05, **P< 0.01. Bars, 20 μm.

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Early muscle patterning is disrupted in dystrophin-deficientembryosThe growth behaviour of Myf5-positive mdx embryonic myoblastsis disrupted from E11.5, and mdx Myf5-positive myoblasts arehyperproliferative and apoptotic. Myf5 marks the embryonicmyoblast population; it is expressed in myotome (E8.5) beforemyotube differentiation is initiated and is found in all muscle groupsthroughout myogenesis (Hadchouel et al., 2003; Ott et al., 1991).Dystrophin is expressed at a crucial stage in myotomedifferentiation (E9.5), one day later than Myf5 expression at E8.5,and 24 hours before the appearance of the first fully differentiatedmyotomal (epaxial) myotubes and the first expression of myosinheavy chain (MyHC) (E10.5) (Houzelstein et al., 1992; Ott et al.,

1991; Schofield et al., 1993). Early hypaxial and secondarymyogenesis are severely disrupted and delayed in mdx embryos,as shown by the late appearance of the FMyHC, Pax7 and caveolin-3 proteins and by the incomplete formation and disorganisation of mdx musculature at between E11.5 and E13.5 (Figs 1, 6;supplementary material Fig. S1). These data suggest that dystrophinhas an essential role in myotome differentiation, which is disruptedin mdx embryos with severe consequences for patterning andfunction of the entire musculature.

Also at E10.5, Myf5-positive myoblasts migrate from themyotome to initiate hypaxial muscle formation (Cusella-De Angeliset al., 1992). Myoblasts migrate under the control of Hox genes(Pax3 and Lbx) and differentiate in response to growth factors

Fig. 7. Substantial muscle fibre lossaccompanied by depletion of Pax7-positivemyoblasts in double mutant embryos. WT mice(A-E) and mdx mice that are heterozygous forcaveolin-3 (mdxcav-3+/–) (F-J), immunostained withMF20 (B,E,G,J) or Pax7 (A,C,D,F,H,I) at E17.5 toillustrate the extensive loss of Pax7 myoblasts andmuscle fibre density in the muscles of lategestation embryos. (K-P) Higher magnificationimages of MF20-labelled intercostal musclesections (matched fourth intercostal for eachembryo) showing the difference in myotubedensity between WT (K), mdx (L), cav-3–/– (M) andthree different mdxcav-3+/– embryos (N-P) at E17.5.(R) Fibre density assessed over a fixed grid areaand (S) percentage reduction in fibre density inWT, mdx and mdxcav-3+/– (het) embryonicintercostal muscle at E17.5. WT (T) and mdxcav-3+/– (U) E17.5 intercostal muscle, labelled withMy32 antibody, showing that almost all mdxcav-3+/– fibres are strongly FMyHC positive; by contrast,FMyHC is undergoing downregulation in many WTintercostal fibres. Bars, 20 μm

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(Wnt1 and Shh) secreted by adjacent tissues (Gross et al., 2000;Hadchouel et al., 2003). The cues that trigger embryonic myoblastmigration are not known. In other tissues, stem cell migration isregulated by guidance cues from originating and target tissues; thisprocess is essential for the correct patterning of embryonicstructures and can be disrupted easily (Lehmann, 2001). It isprobable, therefore, that E10.5-E11.5 epaxial myotubes providesignalling cues that trigger migration of Myf5-positive myoblastsfrom the myotome, and initiate hypaxial and secondary myogenesis.In the absence of dystrophin, these cues are absent and both themigration and initiation processes are impaired. The aberrantbehaviour of E11.5 WT explants cultured in E11.5 mdx CM (Fig.4I) supports the view that the myotome releases secreted factorsthat modify the behaviour of the embryonic myoblast populationat E11.5. Disruption of early myogenesis in mdx embryos suggeststhese early role(s) for dystrophin are distinct from those of utrophin.This conclusion is consistent with published mRNA in situ patternsfor dystrophin and utrophin, which differ in the early embryonicstages (Houzelstein et al., 1992; Schofield et al., 1993). In the laterstages of gestation (see below), there appears to be a ‘catch-upprocess’ in mdx myogenesis, which could be mediated by thesubsequent overproduction of caveolin-3 in these embryos or by

compensatory expression of another protein, for example a-utrophin. a-utrophin is upregulated in postnatal, dystrophin-deficient muscles and may take over some functions of dystrophinin mdx embryos (Weir et al., 2004). The increased severity of thedouble mutant phenotype (mdxcav-3+/–) in late gestation indicatesthat there may be a compensatory effect of caveolin-3 in lategestation mdx embryos.

Caveolin-3 is regulated by muscle regulatory factors (MRFs),activated during myotube differentiation, and expressed later thandystrophin in the E11.5 myotome (Biederer et al., 2000). This isconsistent with our detection of caveolin-3 at E11.5 in WTembryos. MRFs also regulate Pax7, which also emerges at E11.5(Merrick et al., 2007). In cav-3–/– embryos, the early musclepatterning process appears intact, Myf5-positive myoblastbehaviour is not disrupted, and the localisation and timing ofhypaxial muscle formation is comparable to that seen in WTembryos. Caveolin-3 does not, therefore, seem to be required forthese early stages of myogenesis. In mdx embryos, the appearanceof both caveolin-3 and Pax7 is delayed; this could be as aconsequence of the developmental delay in myogenesis in theseembryos or, more specifically, a downstream result of the failureto activate dystrophin.

Table 1. A summary of the phenotypic differences between dystrophic and wild type embryos

Phenotype compared with WT embryo mdx cav-3–/– mdxcav-3+/–

Myotube size Hypotrophy Hypertrophy Hypotrophy

Total myotube number/muscle Reduced Increased Reduced

Intercostal fibre density (E17.5) 37.5% reduction ND 71.2% reduction

(P<0.05)

Fibre density (limb) Decreased Increased (P<0.05) ND

High number of centrally located

myonuclei (E17.5)Yes No Yes

Myotube misalignment Yes (P<0.01) No Yes

Branching and fibre splitting Yes (P<0.001) No ND

Increased myonuclei No Yes (P<0.05) ND

Myonuclei displacement (bunching) No Yes (P<0.05) ND

Cardiac left ventricular wall thickening Yes (minimal) Yes ND

Morphometry

Cardiac atrium-trabecular defects Yes, short and stubby Yes, long and hooked ND

Proliferation Hyperproliferation (from

E11.5) (P<0.01)

Hyperproliferation (from

E15.5) (P<0.01)ND

Apoptosis Elevated from E11.5

(P<0.01)

Elevated from E15.5

(P<0.01)ND

Pax7-positive cell loss Yes (+) Yes (++) Yes (+++)

Stem cell

behaviour

Total Pax7 protein (WB) 15% reduction (E15.5)(P<0.05)

60% reduction (E15.5)(P<0.05)

ND

Developmental delay Yes No Yes

Increased FMyHC-positive myotubes (%) Yes No (reduced) Yes

Fast myosin

mislocalisation

Total FMyHC protein (WB) Increase (P<0.001) Decrease (P<0.05) ND

Total caveolin-3 protein 1.3–10-fold increase

(P<0.05)Absent Reduced (50%) at E15.5Caveolin-3

Developmental delay Yes (E13.5) Not applicable ND

Summary of the phenotypic differences between dystrophic mdx, cav-3–/– and mdxcav-3+/– embryos compared with WT embryos. The evidence is sub-dividedunder the key headings: morphometry, stem cell behaviour and fast myosin mislocalisation, which together establish a significant phenotypic difference

between dystrophic and WT embryos, and demonstrate the embryonic basis for the Duchenne (DMD) and limb girdle (LGMD) forms of muscular dystrophy.

A fourth category demonstrates the disruption of embryonic caveolin-3 protein. Phenotypic analyses that includes double mutant embryos (mdxcav-3+/–) areshown in bold, as are probability values for parameters with a statistical significance of at least P<0.05.

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Hypaxial musculature and secondary myogenesisInterpretation of the role of dystrophin in later embryonic eventsis complicated by the upregulation of caveolin-3 in mdx embryosfrom E13.5. In postnatal tissues, overexpression of caveolin-3causes dystrophin downregulation and a DMD-like phenotype(Galbiati et al., 2000). Loss of dystrophin causes breakdown of theDGC and suppression of dystrophin-associated proteins(Ohlendieck et al., 1993; Vaghy et al., 1998). However, the musclesof DMD patients and adult mdx embryos have a 1.5–2.4-fold excessand a 2–3-fold excess, respectively, of caveolin-3 (Vaghy et al., 1998).In E13.5-E17.5 mdx embryos, we found, on average, a 3–5-foldexcess of caveolin-3 (depending on the embryonic stage), suggestingthat disruption of caveolin-3 expression is an early and significantconsequence of dystrophin deficiency. Embryonic mRNAexpression patterns of dystrophin, but not utrophin, overlap thoseof dystroglycan in WT embryos and this pattern is unchanged inmdx embryos (Houzelstein et al., 1992; Schofield et al., 1995).Therefore, dystrophin may negatively regulate caveolin-3 bycompetitive binding to β-dystroglycan (Ilsley et al., 2002).

In zebrafish embryos, caveolin-3 mutants have cytoskeletal andfusion defects, and dystrophin mutants have unstable muscleattachments, suggesting a role for caveolin-3 in the placement andorientation of embryonic myotubes (Bassett et al., 2003; Nixon etal., 2005). Myogenesis is disrupted in cav3–/– and dmd zebrafishmutants at E13.5; the resulting phenotypes suggest that murinecaveolin-3 and dystrophin have similar roles to their zebrafishcounterparts. In E13.5 mdx embryos, we found myotubedisplacement, splitting and branching. Myotube orientation defectsare not found in caveolin-3-deficient mutants. Instead, embryoniccav-3–/– myonuclei are bunched at the myotube ends, indicative ofthe T-tubule defect that has been reported postnatally for cav-3–/–

mice (Minetti et al., 2002). Excessive cav-3–/– myotube productionwas also found between E13.5 and E17.5, in addition to a twofoldincrease in myonuclei content and hypertrophy. These data matchthose of an in vitro study that used cultured cav-3–/– myoblastsand found a reciprocal phenotype (i.e. reduced myotube number,fewer myonuclei and hypertrophy) in caveolin-3-overexpressingmyoblasts, suggesting that caveolin-3 suppresses myoblast fusion(Volonte et al., 2003). E13.5 mdx embryos partially reproduce theoverexpression phenotype because they have hypotrophicmyotubes and reduced myotube numbers, although the numberof myonuclei is not affected. However, although caveolin-3 levelsremain elevated in later stages (E15.5-E17.5), mdx myotubenumbers are comparable to those of WT embryos. This suggeststhat caveolin-3 may regulate myotube size, but that the E13.5myotube deficit is the result of delayed hypaxial myogenesis in mdxembryos (see section above), rather than an excess of caveolin-3.

Reciprocal disruption of fast fibre specification in cav-3–/– and mdxembryosFMyHC-positive myotubes appear first in WT epaxial muscles ataround E11.5 and herald the start of secondary myogenesis(Merrick et al., 2007). Fibre-type switching begins at E15.5, whensome myotubes switch between slow and fast myosin expression,and the process of establishing adult fibre-type ratios begins (Choet al., 1994; Merrick et al., 2007). FMyHC-positive myotubedifferentiation is significantly perturbed in mdx embryos (seesections above) and fast fibre-type specification is disrupted in both

mdx and cav-3–/– embryos (Fig. 6). Between E15.5 and E17.5, thereis a reciprocal phenotype where mdx embryos have a significantexcess of the FMyHC protein and a higher proportion of FMyHC-positive myotubes compared with WT embryos; there is substantialreduction of FMyHC in cav-3–/– mutants. The overproduction ofFMyHC in mdx embryos could result from a ‘catch-up process’ inE13.5 mdx muscles, facilitated by a compensatory increase incaveolin-3. However, the loss of FMYHC-positive myotubes in cav-3–/– mutants at E15.5-E17.5 suggests that caveolin-3 is also requiredfor fast fibre specification, and that overproduction of caveolin-3in mdx embryos is pathogenic for this phenotype and causes anoverproduction of FMyHC fibres in E15.5-E17.5 mdx mutants.Postnatal fast muscle fibres degenerate preferentially in DMD andin mdx mice, with severe consequences for muscle function(Webster et al., 1988). In mutants that are heterozygous forcaveolin-3, but entirely deficient for dystrophin (mdxcav-3+/–),FMyHC-positive fibres are still over-represented at E17.5 comparedwith in WT embryos, suggesting that it is the balanced relationshipbetween dystrophin and caveolin-3 that is crucial for correct fastfibre proportion rather than caveolin-3 levels alone.

In adult tissues, caveolin-3 and dystrophin interact directly bycompetitive binding to β-dystroglycan in the DGC (Sotgia et al.,2000). These data establish distinct roles for dystrophin andcaveolin-3 in myogenesis, but suggest that both proteins and afunctional DGC may be required for fibre-type specification.

Loss of Pax7 myoblastsAt E15.5-E17.5, cav-3–/– and mdx embryos exhibit Pax7-positivemyoblast attrition and a significant depletion of Pax7 protein. Lossof Pax7 occurs rapidly in cav-3–/– mutants at E15.5, a time pointwhen caveolin-3 is strongly upregulated in WT embryos, and theloss is greater than in E15.5 mdx embryos. Caveolin-3 can elicitsurvival signalling in muscle, as can Pax7. Our data suggest a directrole for caveolin-3 in the rapid loss of Pax7-positive myoblasts atE15.5 in cav-3–/– mutants, and suggest that the attrition of Pax7-positive myoblasts could be partially compensated for in mdxmuscles by their increased caveolin-3 levels (Relaix et al., 2006;Smythe et al., 2003). This conclusion is supported by the findingthat, in mdxcav-3+/– mutants, Pax7 is depleted substantially morethan in either single mutant, such that, by E17.5, Pax7 cells areentirely absent in mdxcav-3+/– lower proximal limb muscles andare very much reduced in other muscles. Pax7-positive myoblastsare crucial for normal postnatal satellite cell emergence (Relaix etal., 2006; Seale et al., 2000). In the absence of Pax7 (Pax7–/– mice),satellite cells are reduced in number and apoptosis is elevated.However, owing to the presence of Pax3-positive myoblasts, satellitecells are not lost entirely, and there appear to be sufficient satellitecells to establish and sustain (postnatal) juvenile muscledevelopment in these mice, although regeneration is impaired inadults (Oustanina et al., 2004; Relaix et al., 2006; Seale et al., 2000).Our findings that E17.5 mdxcav-3+/– embryonic intercostal musclesare severely depleted of myotubes, as well as of Pax7-positive cells,suggests that Pax7 may also play an important role in embryonicmyogenesis. The findings suggest that dystrophic embryos have areduced capacity for generating muscle fibres from late gestation,which may be carried over to postnatal life. Dystrophic muscleregeneration is known to be abnormal, being associated withmyoblast apoptosis, hyperproliferation, irregular fibre size and

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progressive fibrotic deposition. Both mdx and cav-3–/– musclesexperience bouts of degeneration and regeneration in the earlypostnatal weeks, which are associated with elevated levels ofapoptosis (Hagiwara et al., 2000; Smith et al., 1995). In mdx mutants,there is progressive failure of muscle regeneration from 4 monthsof age, which, in mdx mice but not DMD patients, is amelioratedby a concomitant reduction of degenerative processes that may, inpart, be mediated by upregulation of utrophin (Reimann et al., 2000;Roig et al., 2004; Roma et al., 2004). Regeneration has not beenstudied in detail in adult cav-3–/– mice. Our data suggest that theregenerative machinery of dystrophic mutants is abnormal atbirth, because these mutants are deficient in Pax7; this early losscould underlie the progressive impairment of the regenerativeprocess in adulthood. The significant loss of muscle fibre densityin the respiratory muscles of E17.5 mdxcav-3+/– embryos, togetherwith a massive loss of Pax7-positive cells, further strengthens thisconclusion and suggests that increased caveolin-3 levels play animportant compensatory role in the degenerative phenotype seenin mdx mice (and potentially in the early stages of DMD).

Correlation between dystrophic mouse phenotypes and theclinical pathology of MDDMD and LGMD-1c are early onset, progressive skeletal musclediseases of children affecting cardiac and skeletal muscle functionand muscle stability (Hoffman et al., 1987; Minetti et al., 2002). InDMD, there is widespread, progressive malfunction of the entiremusculature, abnormal caveolin-3 expression, myoblast apoptosis,cardiomyopathy, and regeneration defects (Cox and Kunkel, 1997;Smith et al., 1995; Vaghy et al., 1998).

Patients with LGMD-1c exhibit similar, but restricted, myopathicchanges that particularly affect the muscles of the limb, diaphragmand heart (Galbiati et al., 2001; Hagiwara et al., 2000; Smythe etal., 2003). In most respects, the mdx and cav-3–/– postnatalphenotypes are sufficiently similar to the clinical pathologies ofDMD and LGMD to be used widely as disease models(Chamberlain et al., 2007; Chan et al., 2007; Galbiati et al., 2001;Hagiwara et al., 2000; Roig et al., 2004; Vaghy et al., 1998). Theembryonic phenotypes of mdx and cav-3–/– mutants strengthenthe validity of these mice as models for LGMD and DMD,respectively; provide new insight into the mechanisms underlyingMD and the mode of function of dystrophin and caveolin-3; andsuggest new approaches to dissecting the differences between thehuman and murine forms of the disease. We identify importantdevelopmental stages, myotome differentiation (E11.5) andsecondary myogenesis (E13.5), at which dystrophin and caveolin-3, respectively, play key roles. In addition, we reveal two pathologiesin late gestation, fast fibre specification and attrition of Pax7myoblasts, that provide insight into the mechanism underlying MDpathology, and that suggest routes for therapeutic intervention andearlier diagnosis of MD.

Cardiomyopathy, particularly left ventricular failure, is asignificant clinical consequence of DMD and many other MDs, andan established pathology of cav-3–/–, mdx and caveolin-3overexpressing mice (Aravamudan et al., 2003; Cox and Kunkel,1997; Hayashi et al., 2004; Quinlan et al., 2004; Woodman et al.,2002; Yue et al., 2003). Dystrophin and caveolin-3 are expressed inthe mouse embryonic heart at E9.5 and E10.5, respectively (Biedereret al., 2000; Houzelstein et al., 1992). At E13.5, mdx mice have

moderate thickening of the ventricular apex and atrial trabeculardefects. In cav-3–/– mutants, there is a severe thickening of theventricle, as well as atrial defects and a progressively worseningdisruption of myocyte organisation that is characteristic ofcardiomyopathy and consistent with the early-onset postnatalcardiomyopathy seen in caveolin-3-deficient mice at 3-4 monthsof age. These data suggest that caveolin-3 is required for normalheart development and has role(s) in cardiomyopathy. β-cardiacmyosin is mislocalised in mdx and cav-3–/– embryos at E17.5, andtrabeculae are abnormal. β-cardiac myosin defects underlie somefamiliar cardiomyopathies; therefore, the disrupted expression ofβ-cardiac myosin in dystrophic hearts is significant (Cuda et al.,1993). Atrial defects have not previously been reported for eithermouse. The finding in mdx mice suggests a developmental originfor a recent report of hypertrabeculation in a 28-year-old DMDpatient (Finsterer et al., 2005). Although postnatal mdx hearts arereported to have WT levels of β-cardiac myosin and increased levelsof utrophin, which may compensate for the dystrophin deficiency,these mice have a progressive cardiomyopathy (Quinlan et al., 2004).Localisation of β-cardiac myosin has not yet been establishedpostnatally; its disrupted atrial localisation could, therefore, persistinto the adult or may be lost in the perinatal or juvenile period(Wilding et al., 2005).

Hyperproliferation and elevated muscle cell apoptosis are well-established, widespread features of postnatal MD muscle pathology,which characterise both mouse and human forms of mdx/DMDand cav-3–/–/LGMD-1c, as well as most other MD types(Baghdiguian et al., 1999; Smith et al., 1995; Smith et al., 2000;Smythe et al., 2003). Dystrophin, dystroglycan and caveolin-3 haveroles in survival signalling (Glass, 2005; Smythe et al., 2003). Inmdx muscles, high levels of myoblast apoptosis and myoblasthyperproliferation are established from 1 week postbirth toadulthood (Smith, 1996; Smith et al., 1995; Spencer et al., 1997),and, in this study, during embryonic stages from E11.5 to E17.5.These data suggest that disrupted stem cell behaviour and apoptosisare an early consequence of loss of dystrophin and an importantcontributor to the pathology of DMD. Myoblast apoptosis ariseslater in cav-3–/– mice, at E15.5, suggesting that this phenotype maybe an accurate predictor of severity of disease.

New insights: significance for MDThese data offer a new perspective on the aetiology of MD;establish embryonic roles for dystrophin and caveolin-3 thatprogress our understanding of myogenesis; and suggest that Pax7myoblast replacement, and therapeutic strategies that seek tomodify fibre-type specification, might be productive in the earlytreatment of MD. Earlier diagnosis and therapeutic interventionare likely to improve the quality of life of MD patients.

METHODSMouse modelsWT (C57BL/10) and isogenic mdx and cav-3–/– mouse strains wereused. cav-3–/– dystrophic mice on a C57BL/10 background werefrom Yoshito Hagiwara (Tokyo) (Hagiwara et al., 2000). mdx andC57BL/10 mice were generated in-house (Merrick et al., 2007).Double mutant mice (mdxcav-3+/–) were null for dystrophin (mdx)and heterozygous for caveolin-3 (cav-3+/–), and were generated byintercrossing mdx and cav-3–/– mice using a strategy described

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previously to generate dystrophin-deficient mutants that areheterozygous for an Igf2 transgene (mdxIgf-2+/–) (Smith et al., 2000).Genotyping was achieved by PCR for neomycin (to detect the Cav3knockout transgene) and caveolin-3 (expressed by WT, mdx andcav-3+/– mice, but not by cav-3–/– mice) (Fig. 7). The data shownare derived from two separate litters, each of E15.5 and E17.5embryos, containing approximately a 50:50 ratio of mdx andmdxcav-3+/– embryos. Litter size (7-9 embryos per litter) wascomparable to those obtained from WT and mdx mice.

Preparation of mouse embryosStaged WT, cav-3–/–, mdx and mdxcav-3+/– embryos were fixedand processed for paraffin wax embedding, as described previously(Smith and Merrick, 2008). The morning of plug detection wasestimated as E0.5. All sections were sagittal (5 μM). Plane and depthof cut were established at the midline of the embryo (bisectionpoint) and by using Kaufman’s atlas of embryology (Kaufman, 1995).Matched WT, cav-3–/–, mdx and mdxcav-3+/– sections from at leastthree separate embryos per strain were used for all analyses.

ImmunohistochemistryImmunostaining and conditions for pan-myosin (MF20), FMyHC(My32) and Pax7 antibodies have been described previously(Merrick et al., 2007). An extended peroxidase blocking step wasincluded in all staining runs. Secondary antibody controls showedno staining. β-cardiac myosin was detected using the N2.261antibody (1:1000; DHSB, Iowa City). FMyHC staining wasquantified, as shown previously, by counting the proportion ofFMyHC-positive myotubes over a fixed area, and analysed usingthe Student’s t-test and analysis of variance (ANOVA) (Merrick etal., 2007). At least 3000 myotubes were counted for each data point.

Analysis of myotube morphology and quantitation in MF20-stained sectionsSagittally cut, MF20-stained WT, cav-3–/– and mdx embryos werematched for stage, plane and angle of section. To avoid artefacts,we counted only splits and branches that were entirely within theplane of cut; this was carefully controlled between sections. Thisanalysis may underestimate splitting/branching events. Sagittalsections from E13.5, E15.5 and E17.5 embryos were matchedcarefully against a standard published mouse atlas (Kaufman,1995), using the midline point of the embryo (to facilitate matching),to determine the location of morphological features. Sections werecounted blind, by two separate observers, for both misalignmentand branching phenotypes, and were subject to statistical analysis.For each mutant and WT embryo scored, branching was scoredover a fixed area using a grid graticule in the same longitudinallypresenting muscles in the intercostal, upper and lower limb, andfacial muscle regions. Scoring of misaligned fibres was achieved byorienting the direction of the muscle fibres in longitudinal sectionsto a grid graticule and scoring a fixed area for fibres that deviatedby more than a 25 degree angle. We are confident our data are areliable indicator of splitting/branching: when two experimenterscounted slides, the second being unaware of the strain,splitting/branching was only identified in mdx mutants and wasstatistically significant. For myotube counts, we did not attempt toestablish the ends of every myotube (an impossible task in sectionedembryos), but instead counted carefully matched sections of

proximal, distal, intercostal and deep back muscles in three differentembryos per strain over a fixed area (total myotubes counted perstrain: 2000-3000). This enabled us to estimate the proportion ofmyonuclei to myotubes and the average number of myotubes persection. Data was analysed using the Student’s t-test and ANOVA.

Embryo explantsE11.5-E17.5 embryos were dissected to isolate areas that were richin skeletal muscle cells. The head, spinal cord and internal organswere removed from all embryos. In older embryos (E15.5-E17.5),the skin and cartilage/bone were also removed. Muscle-rich tissueswere microdissected into microexplants and cultured in microwells(Smith and Merrick, 2008; Smith and Schofield, 1994). WT, mdxor cav-3–/– E11.5 explant-conditioned medium (CM) was removedfrom confluent cultures, filtered (with a 0.2 μm Acrodisc syringefilter; VWR International, UK), replenished with standard mediasupplements (20% FCS and 2 mM glutamine), and added to freshE11.5 WT explants before being cultured for 18 days (Smith andSchofield, 1997). 180 explants from three embryos were analysedfor each of the three strains included in this study. Data analysiswas by ANOVA.

Outgrowth analysisOutgrowth is a reliable and highly reproducible measure of thegrowth rate of skeletal muscle explants (Smith and Schofield, 1994).Explants were cultured for 3 weeks and scored according to thelevel of confluence of cells in each well. Myf5 immunostaining(rabbit anti-Myf5 C-20, 1:5000; Santa Cruz Biotechnology) was usedto establish the muscle origin of E11.5-E17.5 WT, mdx and cav-3–/– explant cultures; more than 85% of cells were Myf5 positive(Smith and Merrick, 2008). The secondary antibody controls thatwere performed with each section always stained negatively. Myf5C-20 is used extensively to establish myogenicity (Frock et al., 2006;Lindon et al., 1998).

Measurement of apoptosis and proliferationConfluent explant cultures with myoblast morphological featureswere subcultured with dispase (Smith and Schofield, 1994) andplated onto coverslips (at 5�103 cells/cm2) for 6 hours beforefixation. Apoptotic nuclei cells were stained with 10 μg/ml of DAPIfor 3 minutes (Smith et al., 1995). Proliferative cells wereimmunostained with Ki67 (rabbit anti-Ki67, 1:1000; NovocastraLaboratories, UK), as discussed above. For antigen retrieval (usinga pressure cooker), coverslips were first firmly attached onto glassslides using standard paper clips. Three separate experiments wereperformed in duplicate for each strain (WT, cav-3–/–, mdx).

Isolation of protein and ImmunoblottingProtein was extracted from embryos directly into a glasshomogeniser (1-5 ml; VWR International) containing RIPA buffer.Immunoblotting was carried out using standard protocols anddetected by ECL (enhanced chemiluminescence) (Pierce EndogenHyclone). The antibodies used were: fast myosin (My32, 1:1000;Sigma), α-tubulin (1:1000; Sigma), Pax7 (1:1000; DevelopmentalStudies Hybridoma Bank, Iowa City, IA), caveolin-3 (1:1000; SantaCruz Biotechnology) and goat anti-mouse IgG-HRP (1:2000; SantaCruz Biotechnology). Protein concentration was determined usingan ELISA form of the Bradford assay (Merrick et al., 2007).

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ACKNOWLEDGEMENTSWe thank the Wellcome Trust (VS/05/BIR/A8) for funding L.K.J.S. and the BBSRC fortheir support of D.M. and D.L. The Royal Society (RSRG19484), Muscular DystrophyCampaign (RA2/592/2) and SPARKS (02BHM04) also supported this work. We thankYoshito Hagiwara for supplying cav-3–/– mice. Deposited in PMC for release after 6months. This article is freely accessible online from the date of publication.

COMPETING INTERESTSThe authors declare no competing financial interests.

AUTHOR CONTRIBUTIONSJ.S. wrote the paper and designed the study; she also contributed to thephotography, interpretation and analysis of data and to the construction of allfigures. D.M. contributed data and analysis to Figs 1, 3-6. L.K.J.S. contributed dataand analysis to Figs 2, 5, 6. Both D.M. and L.K.J.S. contributed to the writing of themanuscript. D.L. contributed data to Figs 5, 6, 7.

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.001008/-/DC1

Received 20 June 2008; Accepted 17 February 2009.

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Galbiati, F., Volonte, D., Chu, J. B., Li, M., Fine, S. W., Fu, M., Bermudez, J.,Pedemonte, M., Weidenheim, K. M., Pestell, R. G. et al. (2000). Transgenicoverexpression of caveolin-3 in skeletal muscle fibers induces a Duchenne-likemuscular dystrophy phenotype. Proc. Natl. Acad. Sci. USA 97, 9689-9694.

Galbiati, F., Engleman, J., Volonte, D., Zhang, X., Minetti, C., Li, M., Hou, H., Kneitz,B., Edelmann, W. and Lisanti, M. (2001). Caveolin-3 null mice show a loss ofcaveolae, changes in the microdomain distribution of the dystrophin-glycoproteincomplex, and T-tubule abnormalities. J. Biol. Chem. 276, 21425-21433.

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Glass, D. (2005). A signalling role for dystrophin: inhibiting skeletal muscle atrophypathways. Cancer Cell 5, 351-352.

Gros, J., Scaal, M. and Marcelle, C. (2004). A two-step mechanism for myotomeformation in chick. Dev. Cell 6, 875-882.

Gross, M. K., Moran-Rivard, L., Velasquez, T., Nakatsu, M. N., Jagla, K. andGoulding, M. (2000). Lbx1 is required for muscle precursor migration along a lateralpathway into the limb. Development 127, 413-424.

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TRANSLATIONAL IMPACTClinical issueThe skeletal muscle dystrophies (MD) are skeletal muscle diseases thatcause progressive weakness and muscle degeneration. Duchennemuscular dystrophy (DMD), the most severe form of MD, results inrespiratory muscle failure and/or cardiomyopathy leading to death. DMDresults from the loss of the protein dystrophin from the dystrophin-associated glycoprotein complex (DGC). Deficiency of another DGCprotein, caveolin-3, causes a milder limb girdle form of MD (LGMD-1c).

The function of dystrophin and caveolin-3 is not clearly understood.Interestingly, although these proteins are normally present in the embryo,DMD is usually not diagnosed until ages 2-5 years, by which time thepathology of the disease is well established. A possible explanation is thatthese proteins have a key functional role in the genesis of skeletal muscleand the heart. Understanding the role of these proteins duringembryogenesis could be exploited for the benefit of earlier diagnosis andtreatment of DMD children.

ResultsIn this study, the authors use two mouse models of MD, mdx (deficient indystrophin) and cav-3–/– (lacking caveolin-3) to examine the impact ofthese proteins on skeletal muscle development. In mdx embryos, skeletalmuscle formation is delayed and stem cell behaviour is perturbed. In cav-3–/– embryos there is a later and less severe disruption of stem cellbehaviour and muscle formation, consistent with the milder pathology ofLGMD-1c compared with DMD. These data establish a key role fordystrophin in early muscle formation and demonstrate that caveolin-3and dystrophin are essential for correct fibre-type formation and foremergent stem cell function.

Implications and future directionsIt is now clear that early treatment significantly improves life expectancyand quality of life for DMD children. However, diagnosis is often delayeduntil the disease is well underway. This study suggests that understandingthe role of dystrophin and its associated proteins may also lead to earlierdiagnoses and treatment for DMD/LGMD patients, which would in turnlead to enhanced quality of life for individuals affected by these diseases.

doi:10.1242/dmm.003657

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RESEARCH ARTICLE The embryonic basis of muscular dystrophy

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muscular dystrophy begins early in embryonic …of muscle development, but this has not been studied...

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