Requirement of the fusogenic micropeptide myomixer for muscle formation in zebrafish

Abstract

Skeletal muscle formation requires fusion of mononucleated myoblasts to form multinucleated myofibers. The muscle-specific membrane proteins myomaker and myomixer cooperate to drive mammalian myoblast fusion. Whereas myomaker is highly conserved across diverse vertebrate species, myomixer is a micropeptide that shows relatively weak cross-species conservation. To explore the functional conservation of myomixer, we investigated the expression and function of the zebrafish myomixer ortholog. Here we show that myomixer expression during zebrafish embryogenesis coincides with myoblast fusion, and genetic deletion of myomixer using CRISPR/Cas9 mutagenesis abolishes myoblast fusion in vivo. We also identify myomixer orthologs in other species of fish and reptiles, which can cooperate with myomaker and substitute for the fusogenic activity of mammalian myomixer. Sequence comparison of these diverse myomixer orthologs reveals key amino acid residues and a minimal fusogenic peptide motif that is necessary for promoting cell-cell fusion with myomaker. Our findings highlight the evolutionary conservation of the myomaker-myomixer partnership and provide insights into the molecular basis of myoblast fusion.

Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity

“…S6H-K). Interestingly, the fusion defects of fast muscle cells found in our CX-5011-treated zebrafish resemble very closely the phenotype of myomixerand myomaker-deleted zebrafish embryos (66,67). These findings support our hypothesis of a hierarchical regulation of myomixer-myomaker pair fusogenicity by CK2 catalytic activity and underline the conserved role of protein kinase CK2 in myogenesis across species.…”

“…S6H-K) somites of developing fish at both 48 and 96 hpf, which is in line with the notion that only fast muscle cells undergo fusion in zebrafish (65). The similarity between the phenotype of CX-5011-treated zebrafish and that of the recently reported myomixer-or myomaker-deficient zebrafish (66,67) confirms that CK2 activity is required for myoblast fusion in vivo by acting through the regulation of fusogenic proteins, including the myomixer-myomaker pair, in zebrafish development as well.…”

“…To investigate the role of CK2 in vertebrate muscle formation during development, zebrafish embryos were treated with CX-5011 starting from 20 hpf, a time point when embryonic muscle precursor cells are undergoing terminal differentiation and fusing to form mature somitic muscle fibers (50,65). Interestingly, 20 hpf is also the time at which the expression of the fusogenic protein pair myomixer and myomaker is maximal in developing zebrafish somites (66,67). CX-5011 treatment for 28 and 76 h strongly decreases the CK2 activity in zebrafish extracts (Fig.…”

Trout myomaker contains 14 minisatellites and two sequence extensions but retains fusogenic function

“…Recently, the muscle-specific micropeptide myomixer has been shown to be essential for myoblast fusion in mice (5)(6)(7) and zebrafish (8). Another muscle-specific transmembrane protein of 221 aa, 2 called myomaker, was found to be necessary for myocyte fusion during mouse embryonic development (9) and muscle regeneration (10).…”

DrosophilaMyoblast Fusion: Invasion and Resistance for the Ultimate Union

“…Recent studies of mouse myoblast fusion uncovered a bipartite myoblast-specific fusogen, Myomaker and Myomixer/Myomerger/Minion (14,87,97,128). Although these two proteins are conserved in vertebrate myoblast fusion (36,80,117,130), neither has a Drosophila homolog, suggesting that fusogens are likely to be species-and tissue-specific. Second, the functions of various lipids in Drosophila myoblast fusion remain unclear.…”