Functional Analysis of the Ascidian MyoD-family Gene

描述（由申请人提供）：本项目的长期目标是了解无脊椎脊索动物Ciona ninteinalis（尾脊索亚门，海鞘科）的MRF基因的功能。脊椎动物的MRF基因编码了一个在骨骼肌发育中起重要作用的蛋白质家族。它们还参与损伤或废用后的肌肉修复和再生，并在某些肌肉异常和疾病状态中发挥重要作用。因此，他们的研究对人类健康问题有明确的影响。与脊椎动物相反，无脊椎动物的肌生成似乎在很大程度上独立于MRF基因活性。本项目中描述的实验将通过确定海鞘（一种代表无脊椎动物和脊椎动物之间进化过渡的动物群体）的MRF基因是否为肌肉发育所必需，从而解决我们对MRF基因功能理解中的一个重要空白。我们将采用两种实验方法来解决这个问题。第一项研究将测试阻断MRF基因表达是否会干扰肌肉发育，并涉及向胚胎微注射反义morpholino寡核苷酸和小干扰rna，以阻止Ciona MRF基因产生的mrna发挥功能。第二种方法是确定海鞘MRF基因是否能在正常情况下不表达的组织中引发肌肉发育。在发育中的脊索细胞中，用表达由该基因编码的mrna的质粒电穿孔胚胎可以实现Ciona MRF的错表达。在每种方法中，使用简单但高度特异性的肌肉分化标记将有助于分析实验结果。这些研究将提供对Ciona MRF功能的深入了解，从而更好地理解这一重要的调节基因家族在肌肉发育中的作用。

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.