Myomaker is required for the fusion of fast-twitch myocytes in the zebrafish embryo

Abstract

During skeletal muscle development, myocytes aggregate and fuse to form multinucleated muscle fibers. Inhibition of myocyte fusion is thought to significantly derail the differentiation of functional muscle fibers. Despite the purported importance of fusion in myogenesis, in vivo studies of this process in vertebrates are rather limited. Myomaker, a multipass transmembrane protein, has been shown to be the first muscle-specific fusion protein essential for myocyte fusion in the mouse. We have generated loss-of-function alleles in zebrafish myomaker, and found that fusion of myocytes into syncytial fast-twitch muscles was significantly compromised. However, mutant myocytes could be recruited to fuse with wild-type myocytes in chimeric embryos, albeit rather inefficiently. Conversely, overexpression of Myomaker was sufficient to induce hyperfusion among fast-twitch myocytes, and it also induced fusion among slow-twitch myocytes that are normally fusion-incompetent. In line with this, Myomaker overexpression also triggered fusion in another myocyte fusion mutant compromised in the function of the junctional cell adhesion molecule, Jam2a. We also provide evidence that Rac, a regulator of actin cytoskeleton, requires Myomaker activity to induce fusion, and that an approximately 3kb of myomaker promoter sequence, with multiple E-box motifs, is sufficient to direct expression within the fast-twitch muscle lineage. Taken together, our findings underscore a conserved role for Myomaker in vertebrate myocyte fusion. Strikingly, and in contrast to the mouse, homozygous myomaker mutants are viable and do not exhibit discernible locomotory defects. Thus, in the zebrafish, myocyte fusion is not an absolute requirement for skeletal muscle morphogenesis and function.