Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal Myogenesis.
Isolation, Culture, and Analysis of Zebrafish Myofibers and Associated Muscle Stem Cells to Explore Adult Skeletal Myogenesis.
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DOI：
10.1007/978-1-0716-3036-5_3
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发表时间：
2023-01-01
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影响因子：
--
通讯作者：
Hughes, Simon M
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文献类型：
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作者：
Ganassi, Massimo;Zammit, Peter S;Hughes, Simon M
成人骨骼肌组织经历持续的物理压力，因此需要维护和修复以确保其持续有效的功能。驻留肌肉干细胞（musc）的群体，被称为卫星细胞，驻留在成人肌纤维的基底层，有助于肌肉肥大和再生。暴露于激活刺激后，MuSCs增殖产生新的成肌细胞，分化和融合以再生或生长肌纤维。此外，许多硬骨鱼一生都在持续生长，需要不断地从musc中招募核来启动和生长新的纤维，这一过程与大多数羊膜动物观察到的确定生长形成对比。在本章中，我们描述了一种分离、培养和免疫标记成年斑马鱼肌纤维的方法，该方法允许检查肌纤维在体内的特征和体外的MuSC肌生成程序。分离肌纤维的形态计量学分析适用于评估慢肌和快肌之间的差异，或研究细胞特征，如肌节和神经肌肉连接。Pax7（一种典型的干性标记物）的免疫染色鉴定分离肌纤维上的MuSCs用于研究。此外，活肌纤维的移植允许MuSC激活和扩张，并对其增殖和分化动力学进行下游分析，从而为脊椎动物肌肉发生的研究提供了一种合适的、平行的羊膜模型。
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.