ABSTRACT: Tongue development represents a highly coordinated process orchestrated by precise transcriptional regulatory networks, however, the molecular mechanisms governing tongue muscle development remain incompletely understood. Using Six1 knockout mice and C2C12 myoblasts, we investigated the role of Six1, an important transcriptional factor for skeletal muscle development, in tongue muscle differentiation and its regulatory mechanisms. Six1 knockout mice exhibited microglossia, reduced muscle mass, and disorganized muscle architecture, accompanied by smaller fibers and downregulation of myogenic markers. RNA-seq of E14.5 tongue tissues revealed 655 differentially expressed genes in Six1−/− mice, including key myogenic factors such as Mrf4, Myod, and Actc1. Gene Ontology analysis identified strong associations with muscle system processes, muscle cell development, and muscle contraction. In vitro studies in C2C12 cells further demonstrated that Six1 knockdown impaired both cell proliferation and differentiation, as evidenced by reduced expression of myogenic genes, such as Pax7, Myog, and Myf6, as well as diminished cell viability and proliferation. Moreover, CUT&Tag analysis identified 13,454 SIX1-binding peaks in the genome, with SIX1 directly binding to the promoters of key myogenic factors, including Myf5, Myod, and Mylpf. Epigenetic modifications, such as altered H3K27Ac binding, were also observed, suggesting that Six1 regulates myogenic differentiation through chromatin remodeling. These findings establish SIX1 as a critical regulator of tongue muscle differentiation and suggest its potential involvement in congenital myopathies and craniofacial disorders. Understanding the molecular mechanisms by which Six1 regulates muscle development may provide therapeutic insights into improving muscle regeneration and function in clinical settings.
