Functional Analysis of the Ascidian MyoD-family Gene

描述(申请人提供)：本项目的长期目标是了解无脊椎动物脊索动物(尾索动物亚门，海鞘亚门)的MRF基因的功能。脊椎动物的MRF基因编码一系列在骨骼肌发育中发挥重要作用的蛋白质。它们还参与受伤或停用后的肌肉修复和再生，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具有明确的影响。与脊椎动物相比，无脊椎动物的肌肉发生似乎在很大程度上独立于MRF基因的活性。这个项目中描述的实验将通过确定海鞘动物的MRF基因是否是肌肉发育所必需的，来解决我们对MRF基因功能理解上的一个重要缺口。海鞘动物是代表无脊椎动物和脊椎动物之间进化过渡的动物群体。将使用两种实验方法来解决这个问题。第一个将测试阻断MRF基因表达是否干扰肌肉发育，并涉及向胚胎显微注射反义吗啉寡核苷酸和小干扰RNA，以阻止Ciona MRF基因产生的mRNAs发挥作用。第二种方法是确定海鞘MRF基因是否可以在其正常表达的组织中诱导肌肉发育。在发育中的脊索细胞中，通过将表达由该基因编码的mRNAs的质粒电穿孔胚胎，将实现Ciona MRF的错误表达。在每种方法中，使用简单但高度特异的肌肉分化标记物将有助于分析实验结果。这些研究将提供对Ciona MRF功能的洞察，从而更好地理解这一重要的调控基因家族在肌肉发育中所扮演的角色。

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.