Isolation of Myofibres and Culture of Muscle Stem Cells from Adult Zebrafish

Skeletal muscles generate force throughout life and require maintenance and repair to ensure efficiency. The population of resident muscle stem cells (MuSCs), termed satellite cells, dwells beneath the basal lamina of adult myofibres and contributes to both muscle growth and regeneration. Upon exposure to activating signals, MuSCs proliferate to generate myoblasts that differentiate and fuse to grow or regenerate myofibres. This myogenic progression resembles aspects of muscle formation and development during embryogenesis. Therefore, the study of MuSCs and their associated myofibres permits the exploration of muscle stem cell biology, including the cellular and molecular mechanisms underlying muscle formation, maintenance and repair. As most aspects of MuSC biology have been described in rodents, their relevance to other species, including humans, is unclear and would benefit from comparison to an alternative vertebrate system. Here, we describe a procedure for the isolation and immunolabelling or culture of adult zebrafish myofibres that allows examination of both myofibre characteristics and MuSC biology ex vivo. Isolated myofibres can be analysed for morphometric characteristics such as the myofibre volume and myonuclear domain to assess the dynamics of muscle growth. Immunolabelling for canonical stemness markers or reporter transgenes identifies MuSCs on isolated myofibres for cellular/molecular studies. Furthermore, viable myofibres can be plated, allowing MuSC myogenesis and analysis of proliferative and differentiative dynamics in primary progenitor cells. In conclusion, we provide a comparative system to amniote models for the study of vertebrate myogenesis, which will reveal fundamental genetic and cellular mechanisms of MuSC biology and inform aquaculture. Graphic abstract: Schematic of Myofibre Isolation and Culture of Muscle Stem Cells from Adult Zebrafish.