Simon M. Hughes

Scoliosis affects 2–3% of people, often developing during and after adolescence, and currently has a lifetime chance of surgical intervention of ~0.1% in high income countries. Understanding of causal genetic and environmental factors is improving, with mechanical feedback interactions between the neuromuscular and skeletal systems thought to be important. While examining mechanosignalling in the zebrafish musculoskeletal system, we observed transient expression of yap1 mRNA in precursor cells of muscle and notochord and wwtr1 mRNA accumulation in differentiated muscle. Yap1 and Wwtr1/Taz are transcriptional coactivators that mediate Hippo pathway signalling, often in response to mechanosignals. Loss of function mutation of either gene alone transiently altered early larval motility and reduced survival to adulthood, but mutation of yap1 specifically diminished overall growth without an obvious histological muscle defect. Yap1 mutants had a temperature-sensitive phenotype of oedema in cardiac and other tissues, which could be rescued by rearing at low temperature. Rescued yap1 mutants showed focal defects in hypochordal col8a1a mRNA expression at 1–2 days post-fertilisation (dpf), an early motility defect at 5 dpf and subsequently developed a fully penetrant vertebral dysmorphology, reflected by a decrease in posterior vertebral height. Thereafter, frank kyphoscoliosis accompanied by additional vertebral defects developed in around a third of the surviving yap1 mutants and was first detected at 11 dpf. Thus, the mild initial vertebral defect can, in a predisposing genetic or environmental background, gradually develop into full kyphoscoliosis through a positive feedback mechanism, analogous to the Hueter-Volkmann ‘Law’. Although the cell type/s of cell autonomous yap1 action remain unclear, we hypothesise that Yap1 mechanosensation mediates feedback between bone, muscle and tendon to restrain vertebral overgrowth and protect against the development of kyphoscoliosis.

How central and peripheral circadian clocks regulate protein metabolism and affect tissue mass homeostasis has been unclear. Circadian shifts in the balance between anabolism and catabolism control muscle growth rate in young zebrafish independent of behavioral cycles. Here, we show that the ubiquitin–proteasome system (UPS) and autophagy, which mediate muscle protein degradation, are each upregulated at night under the control of the muscle peripheral clock. Perturbation of the muscle transcriptional molecular clock disrupts nocturnal proteolysis, increases muscle growth measured over 12 h, and compromises muscle function. Mechanistically, the shifting circadian balance of Ror and Rev-erb regulates nocturnal UPS, autophagy, and muscle growth through altered TORC1 activity. Although environmental zeitgebers initially mitigate defects, lifelong muscle clock inhibition reduces muscle size and growth rate, accelerating aging-related loss of muscle mass and function. Circadian misalignment such as shift work, sleep deprivation, or dementia may thus unsettle muscle proteostasis, contributing to muscle wasting and sarcopenia.

Primary sarcopenia is a progressive, age-related decline of skeletal muscle strength, size, and quality, the socio-economic and health impacts of which are set to increase due to global ageing. Despite differences in the physiology of female and male skeletal muscle being well characterised, their alteration with age is less clear. Here we report a striking sexual dimorphism in arm muscle ageing in 478,438 UK Biobank participants aged 40–82 yr. Although the sex difference in age-related arm muscle strength decline is modest, muscle mass loss is considerably more pronounced in males, both in absolute and percentage terms. We also present two alternative measures of muscle quality, each of which exhibits substantially greater age-related decline in females. These trends hold across independent analyses of separate cross-sectional and longitudinal participant groups, persist after accounting for systematic size differences between the sexes, and are apparent irrespective of female menopause status and hormone replacement therapy usage history, despite an sharp reduction in female strength during the perimenopause. Our findings confirm the importance of sex to effective diagnosis and mitigation of sarcopenia, and prompt consideration of the physiological basis of this pronounced sex difference in skeletal muscle ageing.

Adult skeletal musculature experiences continuous physical stress, and hence requires maintenance and repair to ensure its continued efficient functioning. The population of resident muscle stem cells (MuSCs), termed satellite cells, resides beneath the basal lamina of adult myofibers, contributing to both muscle hypertrophy and regeneration. Upon exposure to activating stimuli, MuSCs proliferate to generate new myoblasts that differentiate and fuse to regenerate or grow myofibers. Moreover, many teleost fish undergo continuous growth throughout life, requiring continual nuclear recruitment from MuSCs to initiate and grow new fibers, a process that contrasts with the determinate growth observed in most amniotes. In this chapter, we describe a method for the isolation, culture, and immunolabeling of adult zebrafish myofibers that permits examination of both myofiber characteristics ex vivo and the MuSC myogenic program in vitro. Morphometric analysis of isolated myofibers is suitable to assess differences among slow and fast muscles or to investigate cellular features such as sarcomeres and neuromuscular junctions. Immunostaining for Pax7, a canonical stemness marker, identifies MuSCs on isolated myofibers for study. Furthermore, the plating of viable myofibers allows MuSC activation and expansion and downstream analysis of their proliferative and differentiative dynamics, thus providing a suitable, parallel alternative to amniote models for the study of vertebrate myogenesis. Key words Zebrafish Myonucleus Skeletal muscle Pax7 MuSC Stem cell Myofiber Adult

Muscle regeneration models have revealed mechanisms of inflammation, wound clearance, and stem cell–directed repair of damage, thereby informing therapy. Whereas studies of muscle repair are most advanced in rodents, the zebrafish is emerging as an additional model organism with genetic and optical advantages. Various muscle wounding protocols (both chemical and physical) have been published. Here we describe simple, cheap, precise, adaptable, and effective wounding protocols and analysis methods for two stages of a larval zebrafish skeletal muscle regeneration model. We show examples of how muscle damage, ingression of muscle stem cells, immune cells, and regeneration of fibers can be monitored over an extended timecourse in individual larvae. Such analyses have the potential to greatly enhance understanding, by reducing the need to average regeneration responses across individuals subjected to an unavoidably variable wound stimulus. Key words Muscle wounding Needlestick injury Laser injury Zebrafish muscle Muscle precursor cells Satellite cell Pax7 Confocal live imaging Spinning disk imaging Second harmonic generation Multiphoton microscopy Muscle regeneration

To address questions of stem cell diversity during skeletal myogenesis, a Brainbow-like genetic cell lineage tracing method, dubbed Musclebow2, was derived by enhancer trapping in zebrafish. It is shown that, after initial formation of the primary myotome, at least 15 muscle precursor cells (mpcs) seed each somite, where they proliferate but contribute little to muscle growth prior to hatching. Thereafter, dermomyotome-derived mpc clones rapidly expand while some progeny undergo terminal differentiation, leading to stochastic clonal drift within the mpc pool. No evidence of cell-lineage-based clonal fate diversity was obtained. Neither fibre nor mpc death was observed in uninjured animals. Individual marked muscle fibres persist across much of the lifespan indicating low rates of nuclear turnover. In adulthood, early-marked mpc clones label stable blocks of tissue comprising a significant fraction of either epaxial or hypaxial somite. Fusion of cells from separate early-marked clones occurs in regions of clone overlap. Wounds are regenerated from several local mpcs; no evidence for specialised stem mpcs was obtained. In conclusion, our data indicate that most mpcs in muscle tissue contribute to local growth and repair and suggest that cellular turnover is low in the absence of trauma.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.