Embryonic development is initiated by the activity of ooplasmic determinants. Some of these determinants are localized mRNA molecules, yet few such mRNAs have been identified and the mechanisms involved in their localization and function are poorly understood. The long term objective of this study is to understand the role of maternal mRNAs in the initiation of embryonic development. Eggs and embryos of ascidians, which contain cytoplasmic regions with distinct developmental fates, are used as a model system. This proposal exploits unique attributes of ascidians, including the ability to isolate an egg cytoplasmic region containing localized mRNAs, the autonomous and precocious differentiation of muscle cells during embryogenesis, and the existence of sibling species with radically different modes of development, to identify and characterize developmentallysignificant maternal mRNAs. I have already cloned several mRNAs that are localized in the myoplasm, a egg cytoplasmic region that specifies embryonic axis and muscle cell development, and one mRNA that is localized in the ectoplasm, a region inherited by ectodermal cells. At least one of the mRNAs localized in the myoplasm is associated with the egg cytoskeletal matrix. This proposal of six specific aims is designed to continue and extend this work. The first specific aim is to identify localized and other maternal mRNAs that are candidates for ooplasmic determinants. This will be done by screening cDNA libraries for (1) clones encoding mRNAs localized in the myoplasm, (2) clones encoding an mRNA localized in the ectoplasm, and (3) clones homologous to the vertebrate myogenic genes, MyoD and myogenin, which may be involved in muscle development; and (4) screening a subtracted cDNA library for clones with messages expressed in eggs of an ascidian species exhibiting larval development but not in eggs of a sibling species lacking larval development The second specific aim is to characterize the mRNAs by sequencing and determining their temporal and spatial expression during development. The third specific aim is to determine whether the expression of these mRNAs is changed in the species lacking larval development. The fourth specific aim is to determine whether these mRNAs are associated with the egg cytoskeletal matrix and whether this association is altered in the species lacking larval development. The fifth specific aim is to identify and characterize proteins encoded by these mRNAs with respect to their temporal and spatial expression during development and whether changes in expression patterns occur in the species lacking larval development. The sixth specific aim is to test the function of each mRNA by microinjecting synthetic transcripts into LTV irradiated eggs, which lack functional axial determinants, and eggs of the species lacking larval development, which are defective in muscle determinants.
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