The Ciona intestinalis MyoD homolog is essential for myogenesis

Abstract

We used gene over expression and gene knockdown to study the functional properties of CiMDF, the single myogenic regulatory factor gene (MRF) of Ciona intestinalis. Overexpression was achieved by injecting eggs with synthetic CiMDF mRNA. RNA-injected eggs did not develop typical tadpole morphology, but instead formed flattened balls of cells. Expression of the four muscle-specific genes assayed was strongly upregulated in muscle cells of injected embryos. Notably, these muscle-specific genes were also expressed in non-muscle lineage cells of injected embryos. This was especially apparent for troponin I, which was typically detected in all vegetal hemisphere cells of mRNA-injected embryos. Animal hemisphere cells of mRNA-injected embryos did not express muscle genes. We knocked down CiMDF expression by injecting eggs with antisense morpholino oligonucleotides (MO). MO-injected eggs often developed into quite normal tadpoles with clearly recognizable heads and tails; nevertheless, muscle-specific gene expression was reduced in these embryos compared to controls. Moreover, MO-treated larvae were almost always paralyzed and never showed the frenzied tail movements of normal larvae. Electron microscopy revealed that ‘‘muscle’’ cells of MO-treated larvae lacked the myofibrils seen in normal larval muscle. We conclude that CiMDF is a bona fide MRF because it elicits ectopic myogenesis, and that it is essential for tail muscle development. Awards from the NIH (1 R15 HD047357-01) and Rhode Island College to THM, and the Association Francaise contre les Myopathies (AFM) to HY supported this work.