Occipital foramina development involves localised regulation of mesenchyme proliferation and is independent of apoptosis.

Cranial foramina are holes within the skull, formed during development, allowing entry and exit of blood vessels and nerves. Once formed they must remain open, due to the vital structures they contain, i.e. optic nerves, jugular vein, carotid artery, and other cranial nerves and blood vessels. Understanding cranial foramina development is essential as cranial malformations lead to the stenosis or complete closure of these structures, resulting in blindness, deafness, facial paralysis, raised intracranial pressure and lethality. Here we focus on describing early events in the formation of the jugular, carotid and hypoglossal cranial foramina that form in the mesoderm-derived, endochondral occipital bones at the base of the embryonic chick skull. Whole-mount skeletal staining of skulls indicates the appearance of these foramina from HH32/D7.5 onwards. Haematoxylin & eosin staining of sections shows that the intimately associated mesenchyme, neighbouring the contents of these cranial foramina, is initially very dense and gradually becomes sparser as development proceeds. Histological examination also revealed that these foramina initially contain relatively large-diameter nerves, which later become refined, and are closely associated with the blood vessel, which they also innervate within the confines of the foramina. Interestingly cranial foramina in the base of the skull contain blood vessels lacking smooth muscle actin, which suggests these blood vessels belong to glomus body structures within the foramina. The blood vessel shape also appears to dictate the overall shape of the resulting foramina. We initially hypothesised that cranial foramina development could involve targeted proliferation and local apoptosis to cause 'mesenchymal clearing' and the creation of cavities in a mechanism similar to joint cavitation. We find that this is not the case, and propose that a mechanism reliant upon local nerve/blood vessel-derived restriction of ossification may contribute to foramina formation during cranial development.

Symmorphosis through dietary regulation: a combinatorial role for proteolysis, autophagy and protein synthesis in normalising muscle metabolism and function of hypertrophic mice after acute starvation.

Animals are imbued with adaptive mechanisms spanning from the tissue/organ to the cellular scale which insure that processes of homeostasis are preserved in the landscape of size change. However we and others have postulated that the degree of adaptation is limited and that once outside the normal levels of size fluctuations, cells and tissues function in an aberant manner. In this study we examine the function of muscle in the myostatin null mouse which is an excellent model for hypertrophy beyond levels of normal growth and consequeces of acute starvation to restore mass. We show that muscle growth is sustained through protein synthesis driven by Serum/Glucocorticoid Kinase 1 (SGK1) rather than Akt1. Furthermore our metabonomic profiling of hypertrophic muscle shows that carbon from nutrient sources is being channelled for the production of biomass rather than ATP production. However the muscle displays elevated levels of autophagy and decreased levels of muscle tension. We demonstrate the myostatin null muscle is acutely sensitive to changes in diet and activates both the proteolytic and autophagy programmes and shutting down protein synthesis more extensively than is the case for wild-types. Poignantly we show that acute starvation which is detrimental to wild-type animals is beneficial in terms of metabolism and muscle function in the myostatin null mice by normalising tension production.

Propeptide-mediated inhibition of myostatin increases muscle mass through inhibiting proteolytic pathways in aged mice.

Mammalian aging is accompanied by a progressive loss of skeletal muscle, a process called sarcopenia. Myostatin, a secreted member of the transforming growth factor-ß family of signaling molecules, has been shown to be a potent inhibitor of muscle growth. Here, we examined whether muscle growth could be promoted in aged animals by antagonizing the activity of myostatin through the neutralizing activity of the myostatin propeptide. We show that a single injection of an AAV8 virus expressing the myostatin propeptide induced an increase in whole body weights and all muscles examined within 7 weeks of treatment. Our cellular studies demonstrate that muscle enlargement was due to selective fiber type hypertrophy, which was accompanied by a shift toward a glycolytic phenotype. Our molecular investigations elucidate the mechanism underpinning muscle hypertrophy by showing a decrease in the expression of key genes that control ubiquitin-mediated protein breakdown. Most importantly, we show that the hypertrophic muscle that develops as a consequence of myostatin propeptide in aged mice has normal contractile properties. We suggest that attenuating myostatin signaling could be a very attractive strategy to halt and possibly reverse age-related muscle loss.

Exercise training attenuates the hypermuscular phenotype and restores skeletal muscle function in the myostatin null mouse.

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.

Neuromuscular junction morphology, fiber-type proportions, and satellite-cell proliferation rates are altered in MyoD(-/-) mice.

Gene compensation by members of the myogenic regulatory factor (MRF) family has been proposed to explain the apparent normal adult phenotype of MyoD(-/-) mice. Nerve and field stimulation were used to investigate contraction properties of muscle from MyoD(-/-) mice, and molecular approaches were used to investigate satellite-cell behavior. We demonstrate that MyoD deletion results in major alterations in the organization of the neuromuscular junction, which have a dramatic influence on the physiological contractile properties of skeletal muscle. Second, we show that the lineage progression of satellite cells (especially initial proliferation) in the absence of MyoD is abnormal and linked to perturbations in the nuclear localization of beta-catenin, a key readout of canonical Wnt signaling. These results show that MyoD has unique functions in both developing and adult skeletal muscle that are not carried out by other members of the MRF family.

A hypoplastic model of skeletal muscle development displaying reduced foetal myoblast cell numbers, increased oxidative myofibres and improved specific tension capacity.

The major component of skeletal muscle is the myofibre. Genetic intervention inducing over-enlargement of myofibres beyond a certain threshold through acellular growth causes a reduction in the specific tension generating capacity of the muscle. However the physiological parameters of a genetic model that harbours reduced skeletal muscle mass have yet to be analysed. Genetic deletion of Meox2 in mice leads to reduced limb muscle size and causes some patterning defects. The loss of Meox2 is not embryonically lethal and a small percentage of animals survive to adulthood making it an excellent model with which to investigate how skeletal muscle responds to reductions in mass. In this study we have performed a detailed analysis of both late foetal and adult muscle development in the absence of Meox2. In the adult, we show that the loss of Meox2 results in smaller limb muscles that harbour reduced numbers of myofibres. However, these fibres are enlarged. These myofibres display a molecular and metabolic fibre type switch towards a more oxidative phenotype that is induced through abnormalities in foetal fibre formation. In spite of these changes, the muscle from Meox2 mutant mice is able to generate increased levels of specific tension compared to that of the wild type.

Molecular, cellular and physiological investigation of myostatin propeptide-mediated muscle growth in adult mice.

Inhibition of myostatin signalling or its biological activity has recently emerged as a potential remedial approach against muscle wasting and degenerative diseases such as muscular dystrophies. In the present study we systemically administered a recombinant AAV8 vector expressing a mutated myostatin propeptide (AAV8ProMyo) to healthy mice in order to assess its impact on the histological, cellular and physiological properties of the skeletal muscle, exploiting the fact that myostatin is naturally inhibited by its own propeptide. We report that a single intravenous administration of AAV8ProMyo leads to increases in muscle mass of tibialis anterior, extensor digitorum longus and gastrocnemius muscles 8 weeks post-injection and tibialis anterior, gastrocnemius and rectus femoris muscles 17 weeks post-injection. Moreover, treatment resulted in muscle fibre hypertrophy but not hyperplasia, with IIB myofibres responding to the greatest extent following propeptide-induced myostatin inhibition. Additionally, myofibre nuclear:cytoplasmic ratio was decreased in the AAV8ProMyo treated animals. Importantly, the hypertrophic EDL muscle 8 weeks after AAV8ProMyo treatment did not show the dramatic decrease in specific force displayed by the germline myostatin null mice.

Muscle hypertrophy driven by myostatin blockade does not require stem/precursor-cell activity.

Myostatin, a member of the TGF-beta 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.

Evidence that a maternal "junk food" diet during pregnancy and lactation can reduce muscle force in offspring.

BACKGROUND: Obesity is a multi-factorial condition generally attributed to an unbalanced diet and lack of exercise. Recent evidence suggests that maternal malnutrition during pregnancy and lactation can also contribute to the development of obesity in offspring. We have developed an animal model in rats to examine the effects of maternal overeating on a westernized "junk food" diet using palatable processed foods rich in fat, sugar and salt designed for human consumption. Using this model, we have shown that such a maternal diet can promote overeating and a greater preference for junk food in offspring at the end of adolescence. The maternal junk food diet also promoted adiposity and muscle atrophy at weaning. Impaired muscle development may permanently affect the function of this tissue including its ability to generate force. AIMS: The aim of this study is to determine whether a maternal junk food diet can impair muscle force generation in offspring. METHODS: Twitch and tetanic tensions were measured in offspring fed either chow alone (C) or with a junk food diet (J) during gestation, lactation and/or post-weaning up to the end of adolescence such that three groups of offspring were used, namely the CCC, JJC and JJJ groups. RESULTS: We show that adult offspring from mothers fed the junk food diet in pregnancy and lactation display reduced muscle force (both specific twitch and tetanic tensions) regardless of the post-weaning diet compared with offspring from mothers fed a balanced diet. CONCLUSIONS: Maternal malnutrition can influence muscle force production in offspring which may affect an individual's ability to exercise and thereby combat obesity.

Lymph heart in chick--somitic origin, development and embryonic oedema.

The lymph heart is a sac-like structure on either side of avian tail. In some adult birds, it empties the lymph from the copulatory organ; however, during embryonic development, it is thought to circulate extra-embryonic lymph. Very little is known about the origin, innervation and the cellular changes it undergoes during development. Using immunohistochemistry and gene expression profiling we show that the musculature of the lymph heart is initially composed solely of striated skeletal muscle but later develops an additional layer composed of smooth myofibroblasts. Chick-quail fate-mapping demonstrates that the lymph heart originates from the hypaxial compartments of somites 34-41. The embryonic lymph heart is transiently innervated by somatic motoneurons with no autonomic input. In comparison to body muscles, the lymph heart has different sensitivity to neuromuscular junction blockers (sensitive only to decamethonium). Furthermore, its abundant bungarotoxin-positive acetylcholinesterase receptors are unique as they completely lack specific acetylcholinesterase activity. Several lines of evidence suggest that the lymph heart may possess an intrinsic pacing mechanism. Finally, we assessed the function of the lymph heart during embryogenesis and demonstrate that it is responsible for preventing embryonic oedema in birds, a role previously thought to be played by body skeletal muscle contractions.

Lack of myostatin results in excessive muscle growth but impaired force generation.

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.

Myostatin imposes reversible quiescence on embryonic muscle precursors.

We have previously shown that Myostatin, a member of the transforming growth factor beta (TFG-beta) family of signalling molecules, is expressed in developing muscle, and that treatment with recombinant Myostatin inhibited the expression of key myogenic transcription factors during chick embryogenesis. In this study, we followed the fate of muscle precursors after exposure to Myostatin. We report that in contrast to the down-regulation in expression of Pax-3, Myf-5, MyoD, and Myogenin, expression of Pax-7 was maintained. However, Myostatin completely inhibited cell division in the Pax-7-expressing cells. The inhibitory effect of Myostatin was reversible, as upon withdrawal myogenic cells re-initiated cell proliferation as well as expression of Pax-3 and MyoD. These results led us to investigate the temporal and spatial distribution of quiescent muscle precursors during development. To this end, we analysed distribution and mitotic behaviour of Pax-7-expressing cells during muscle development. Our studies revealed two populations of Pax-7-expressing cells, one that proliferated and incorporated BrdU, whilst the other did not. At early developmental stages, a high proportion of Pax-7-expressing cells proliferated, but there was a significant number of non-dividing Pax-7-expressing cells intermingled with differentiated muscle. Proliferating precursors became less frequent as development proceeded and at late fetal stages all Pax-7-expressing cells were mitotically quiescent. We suggest that Myostatin is an important signalling molecule responsible for imposing quiescence upon myogenic precursors during embryonic and foetal development.

Activin A inhibits formation of skeletal muscle during chick development.

In this study we investigated the effect of recombinant activin A on the differentiation of limb muscle precursors of chick embryos. We show that treatment with activin resulted in a downregulation of Pax-3 and MyoD expression within 6 h after treatment, whereas expression of Myf-5 and Pax-7 was largely unaffected. The effect on gene expression was transient because 1 day after activin exposure the development of the premuscle masses had proceeded, and Pax-3 and MyoD expression was reexpressed at normal levels. Unlike other transforming growth factors-beta, activin did not induce programmed cell death in limb mesenchyme, thus myogenic cells were not permanently lost. In high-density cultures of embryonic chick limb mesenchyme (micromass cultures), activin repressed the generation of Pax-7-expressing muscle precursors. Furthermore, in the presence of activin, fewer muscle precursors differentiated, and the population of differentiating cells failed to fuse and form myotubes. Our data suggest that activin reversibly inhibited expression of two transcription factors, Pax-3 and MyoD, and thus transiently inhibited proliferation and differentiation of limb muscle precursors. However, myogenic cells were not lost as they continued to express Pax-7 and Myf-5, and this may have allowed precursors to commence development after the activin effect faded. We suggest that activin acts in conjunction with a closely related signalling molecule, myostatin, to prevent excessive growth of skeletal muscle.

Decamethonium bromide-mediated inhibition of embryonic muscle development.

This study determined the effect of decamethonium bromide (DMBr), a non-competitive blocker of the neuromuscular junction, on skeletal muscle development during chick embryogenesis. Decamethonium bromide caused generalized edema and high mortality with treated embryos rarely surviving beyond day 16 of incubation. Muscle degeneration was grossly evident on the muscles of abdomen, pectoral girdle, and leg. Semi-thin sections showed a high infiltration of macrophages in treated embryos and a massive degenerative process. Electron microscopy showed that both fast and slow fibers formed in the control and treated embryos, but those of the treated embryos failed to form myofibrils. Other organ systems, such as the heart and the gut, appeared histologically normal throughout the course of treatment. To investigate possible nerve independent action of DMBr on muscle development we determined the effect of this compound on the growth and differentiation of the C2C12 skeletal muscle cell line. DMBr treatment of C2C12 cell cultures did not affect the growth or survival of the cells, even at a tenfold higher concentration than that used in ovo, but myosin heavy chain expression was dramatically inhibited. We conclude that DMBr has a nerve independent blocking inhibition effect on myosin heavy chain synthesis in the developing avian embryo besides the recognized role as a non-competitive post-synaptic blocker of the neuromuscular junction.