Assuntos
Fusão de Membrana/genética , Proteínas de Membrana/genética , Síndrome de Möbius/genética , Proteínas Musculares/genética , Doenças Musculares/genética , Mutação de Sentido Incorreto , Mioblastos Esqueléticos/metabolismo , Síndrome de Pierre Robin/genética , Animais , Diferenciação Celular , Fusão Celular , Modelos Animais de Doenças , Humanos , Proteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Síndrome de Möbius/metabolismo , Síndrome de Möbius/patologia , Proteínas Musculares/deficiência , Doenças Musculares/metabolismo , Doenças Musculares/patologia , Mioblastos Esqueléticos/citologia , Síndrome de Pierre Robin/metabolismo , Síndrome de Pierre Robin/patologia , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Peixe-ZebraRESUMO
The fusion of myoblasts into multinucleated muscle fibers is vital to skeletal muscle development, maintenance and regeneration. Genetic mutations in the Myomaker (mymk) gene cause Carey-Fineman-Ziter syndrome (CFZS) in human populations. To study the regulation of mymk gene expression and function, we generated three mymk mutant alleles in zebrafish using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology and analyzed the effects of mymk knockout on muscle development and growth. Our studies demonstrated that knockout of mymk resulted in defective myoblast fusion in zebrafish embryos and increased mortality at larval stage around 35-45 days post-fertilization. The viable homozygous mutants were smaller in size and weighed approximately one-third the weight of the wild type (WT) sibling at 3 months old. The homozygous mutants showed craniofacial deformities, resembling the facial defect observed in human populations with CFZS. Histological analysis revealed that skeletal muscles of mymk mutants contained mainly small-size fibers and substantial intramuscular adipocyte infiltration. Single fiber analysis revealed that myofibers in mymk mutant were predominantly single-nucleated fibers. However, myofibers with multiple myonuclei were observed, although the number of nuclei per fiber was much less compared with that in WT fibers. Overexpression of sonic Hedgehog inhibited mymk expression in zebrafish embryos and blocked myoblast fusion. Collectively, these studies demonstrated that mymk is essential for myoblast fusion during muscle development and growth.
Assuntos
Proteínas de Membrana/fisiologia , Síndrome de Möbius/fisiopatologia , Desenvolvimento Muscular , Proteínas Musculares/fisiologia , Músculo Esquelético/fisiopatologia , Doenças Musculares/fisiopatologia , Mioblastos/metabolismo , Síndrome de Pierre Robin/fisiopatologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/fisiologia , Adipócitos/fisiologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Larva/fisiologia , Proteínas de Membrana/genética , Síndrome de Möbius/metabolismo , Morfogênese , Proteínas Musculares/genética , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Doenças Musculares/metabolismo , Mioblastos/fisiologia , Síndrome de Pierre Robin/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
Multinucleate cellular syncytial formation is a hallmark of skeletal muscle differentiation. Myomaker, encoded by Mymk (Tmem8c), is a well-conserved plasma membrane protein required for myoblast fusion to form multinucleated myotubes in mouse, chick, and zebrafish. Here, we report that autosomal recessive mutations in MYMK (OMIM 615345) cause Carey-Fineman-Ziter syndrome in humans (CFZS; OMIM 254940) by reducing but not eliminating MYMK function. We characterize MYMK-CFZS as a congenital myopathy with marked facial weakness and additional clinical and pathologic features that distinguish it from other congenital neuromuscular syndromes. We show that a heterologous cell fusion assay in vitro and allelic complementation experiments in mymk knockdown and mymkinsT/insT zebrafish in vivo can differentiate between MYMK wild type, hypomorphic and null alleles. Collectively, these data establish that MYMK activity is necessary for normal muscle development and maintenance in humans, and expand the spectrum of congenital myopathies to include cell-cell fusion deficits.