The lack of myostatin promotes growth of skeletal muscle, and blockade of its activity has been proposed as a treatment for various muscle-wasting disorders. Here, we have examined two independent mouse lines that harbor mutations in the myostatin gene, constitutive null ( Mstn −/− ) and compact (Berlin High Line, BEH c/c ). We report that, despite a larger muscle mass relative to age-matched wild types, there was no increase in maximum tetanic force generation, but that when expressed as a function of muscle size (specific force), muscles of myostatin-deficient mice were weaker than wild-type muscles. In addition, Mstn −/− muscle contracted and relaxed faster during a single twitch and had a marked increase in the number of type IIb fibers relative to wild-type controls. This change was also accompanied by a significant increase in type IIB fibers containing tubular aggregates. Moreover, the ratio of mitochondrial DNA to nuclear DNA and mitochondria number were decreased in myostatin-deficient muscle, suggesting a mitochondrial depletion. Overall, our results suggest that lack of myostatin compromises force production in association with loss of oxidative characteristics of skeletal muscle.

Satellite cells represent the stem cell population of adult skeletal muscle. The molecular mechanisms that control the proliferation of satellite cells are not well understood. In this study, we show that in response to injury, myofibres activate Wnt ligand transcription and activate a reporter cell line that is sensitive to the canonical Wnt-signalling pathway. Activated satellite cells on isolated cultured myofibres show robust expression of activated-β-catenin (Act-β-Cat), a key downstream transcriptional coactivator of canonical Wnt signalling. We provide evidence that the Wnt family of secreted glycoproteins act on satellite cells in a ligand-specific manner. Overexpression of Wnt1, Wnt3a or Wnt5a protein causes a dramatic increase in satellite-cell proliferation. By contrast, exposure of satellite cells to Wnt4 or Wnt6 diminishes this process. Moreover, we show that the prolonged satellite-cell quiescence induced by inhibitory Wnt is reversible and exposing inhibited satellite cells to stimulatory Wnt signalling restores their proliferation rate. Stimulatory Wnt proteins induce premature satellite cell BrdU incorporation as well as nuclear translocation of Act-β-Cat. Finally, we provide evidence that the Act-β-Cat translocation observed in single fibres during in vitro culture also occurs in cases of acute and chronic skeletal muscle regeneration in rodents and humans. We propose that Wnt proteins may be key factors that regulate the rate of satellite-cell proliferation on adult muscle fibres during the wound-healing response.

SUMMARYIn vertebrates, body musculature originates from somites, whereas head muscles originate from the cranial mesoderm. Neck muscles are located in the transition between these regions. We show that the chick occipital lateral plate mesoderm has myogenic capacity and gives rise to large muscles located in the neck and thorax. We present molecular and genetic evidence to show that these muscles not only have a unique origin, but additionally display a distinct temporal development, forming later than any other muscle group described to date. We further report that these muscles, found in the body of the animal, develop like head musculature rather than deploying the programme used by the trunk muscles. Using mouse genetics we reveal that these muscles are formed in trunk muscle mutants but are absent in head muscle mutants. In concordance with this conclusion, their connective tissue is neural crest in origin. Finally, we provide evidence that the mechanism by which these neck muscles develop is conserved in vertebrates.

Myostatin, a member of the TGF-␤ family, has been identified as a powerful inhibitor of muscle growth. Absence or blockade of myostatin induces massive skeletal muscle hypertrophy that is widely attributed to proliferation of the population of muscle fiber-associated satellite cells that have been identified as the principle source of new muscle tissue during growth and regeneration. Postnatal blockade of myostatin has been proposed as a basis for therapeutic strategies to combat muscle loss in genetic and acquired myopathies. But this approach, according to the accepted mechanism, would raise the threat of premature exhaustion of the pool of satellite cells and eventual failure of muscle regeneration. Here, we show that hypertrophy in the absence of myostatin involves little or no input from satellite cells. Hypertrophic fibers contain no more myonuclei or satellite cells and myostatin had no significant effect on satellite cell proliferation in vitro, while expression of myostatin receptors dropped to the limits of detectability in postnatal satellite cells. Moreover, hypertrophy of dystrophic muscle arising from myostatin blockade was achieved without any apparent enhancement of contribution of myonuclei from satellite cells. These findings contradict the accepted model of myostatin-based control of size of postnatal muscle and reorient fundamental investigations away from the mechanisms that control satellite cell proliferation and toward those that increase myonuclear domain, by modulating synthesis and turnover of structural muscle fiber proteins. It predicts too that any benefits of myostatin blockade in chronic myopathies are unlikely to impose any extra stress on the satellite cells. muscle growth ͉ muscular dystrophy ͉ TGF-beta ͉ muscle stem cells ͉ myonuclear domain L oss of muscle mass and strength is a major clinical feature of inherited myopathies such as Duchenne muscular dystrophy (DMD) and also of more common acquired atrophies associated with disuse, aging, and cancer. This loss has fostered widespread interest in the powerful inhibitory effect of myostatin, a member of the TGF-␤ family of signaling molecules, on muscle growth (1) with specific focus on the prospect of modulating this system to counteract atrophic processes. Indeed, muscle fiber hypertrophy arising from absence or blockade of myostatin has been reported to be associated with therapeutic benefits in the mdx mouse model of DMD (2, 3). This hypertrophy has been attributed to proliferation of satellite cells (4, 5), the principal cellular source for growing and regenerating skeletal muscle (6-10), consequent upon their release from myostatin inhibition (5,11,12).Here, we have investigated the contribution of satellite cells in 2 myostatin-null mouse models, constitutive (mstn Ϫ/Ϫ ) and compact (BEH c/c ), and following myostatin blockade by AAVmediated overexpression of myostatin propeptide. These data, together with our results from in vitro studies on the effect of presence or absence of myostatin on satellite cells contradict co...

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the "In-Out" mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb.

Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN −/− ) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force-generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN −/− mice that can be induced by training. To this end, we subjected MSTN −/− mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross-sectional area of hypertrophic myofibres from MSTN −/− mice decreased towards wild-type values in response to exercise and, in this context, Bnip3, a key autophagy-related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear-to-cytoplasmic ratio towards wild-type values. Importantly, both training regimes increased muscle force in MSTN −/− mice. We conclude that impaired skeletal muscle function in myostatin-deficient mice can be improved through endurance exercise-mediated remodelling of muscle fibre size and metabolic profile.

Satellite cells are essential for postnatal growth and repair of skeletal muscle. The paired-box transcription factors Pax3 and Pax7 are expressed in emerging muscle precursors. Recent studies have traced the origin of satellite cells to the embryonic dermomyotome, however, their developmental regulation throughout embryogenesis remains unclear. We show the overlying surface ectoderm and lateral plate are essential for Pax3 expression, and that the overlying surface ectoderm and neural tube are necessary for Pax7 expression within the dorsal somite. Furthermore we show that the notochord acts to down regulate the expression of both genes. Moreover, we identify diffusible factors within these tissues that act to maintain expression of Pax3 ( + ) and Pax7 (+) muscle precursors. We show that Wnt1, 3a, 4 and 6 proteins are able to up regulate and expand the expression of Pax3 and Pax7 within the dorsal somite. Finally, we show that Wnt6 can mimic the effect of the dorsal ectoderm to maintain Pax3 and Pax7 expression.

Adult skeletal muscle possesses a resident stem cell population called satellite cells, which are responsible for tissue repair following damage. Satellite cell migration is crucial in promoting rapid tissue regeneration, but it is a poorly understood process. Furthermore, the mechanisms facilitating satellite cell movement have yet to be elucidated. This study investigates the process of satellite cell migration, revealing that they undergo two distinct phases of movement, first under the basal lamina and then rapidly increasing their velocity when on the myofiber surface. Most significantly, we show that satellite cells move using a highly dynamic blebbing or amoeboid-based mechanism and not via lamellipodia-mediated propulsion. We show that nitric oxide and noncanonical Wnt signaling pathways are necessary for regulating the formation of blebs and the migration of satellite cells. In summary, we propose that the formation of blebs and their necessity for satellite cell migration has significant implications in the future development of therapeutic regimes aimed at promoting skeletal muscle regeneration.