Functional Analysis of the Ascidian MyoD-family Gene

Rhode Island College, Providence RI

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Abstract

DESCRIPTION (provided by applicant): The long-term objective this project is to understand the function of the MRF gene of the invertebrate chordate, Ciona intestinalis (subphylum Urochordata, Class Ascidiaceae). The MRF genes of vertebrates encode a family of proteins that play essential roles in skeletal muscle development. They are also involved in muscle repair and regeneration following injury or disuse, and they play significant roles in certain muscle abnormalities and disease states. Thus, their study has clear implications for issues of human health. In contrast to vertebrates, myogenesis in invertebrates appears to be largely independent of MRF gene activity. The experiments described in this project will address an important gap in our understanding of MRF gene function by determining whether the MRF gene of ascidians - an animal group that represents an evolutionary transition between invertebrates and vertebrates - is required for muscle development. Two experimental approaches will be used to address this question. The first will test whether blocking MRF gene expression interferes with muscle development, and involves microinjecting embryos with antisense morpholino oligonucleotides and small interfering RNAs that prevent the mRNAs produced by the Ciona MRF gene from functioning. The second approach will be to determine whether the ascidian MRF gene can elicit muscle development in a tissue where it is not normally expressed. Misexpression of Ciona MRF will be achieved by electroporating embryos with plasmids that express the mRNAs encoded by this gene in developing notochord cells. In each approach, the use of simple, but highly specific markers of muscle differentiation will facilitate the analysis of experimental outcomes. These studies will provide insight into Ciona MRF function, and thus lead to a better understanding of the roles this important family of regulatory genes plays in muscle development.