Differentially expressed fusogens specify myocyte states to drive myogenesis

During myogenesis, myocyte fusion leads to the formation of multinucleated muscle fibers, but how exactly this process is initiated remains poorly understood. Here, we performed single-cell RNA-sequencing on mouse somites from E9.5-E11.5 embryos, revealing multiple differentiation states during primary myogenesis. Among these, we identified two unexpected myocyte populations: one expressing both myomaker (Mymk) and myomixer (Mymx) (termed Mc1) and another expressing only Mymk (termed Mc2). Fluorescence in situ hybridization demonstrated that both populations are mononucleated and co-exist within the same somites, with only Mc1 persisting during secondary myogenesis. Lineage tracing using Mymx:Cre; RosaTdT mice demonstrated that the Mc2 cells arise from the Mc1. Mechanistically, we show that Mef2 and Rxr factors positively and negatively regulate Mymx expression, respectively. Additionally, RXRG interacts with MYOD1 and MYOG, modulating their transcriptional activity in luciferase assays. Collectively, our findings uncover two populations among the myocytes that drive primary and secondary fiber formation, challenging the traditional view of vertebrate muscle precursor homogeneity.