The long term objective of the study is to understand the role of localized maternal mRNA in embryonic development. As a model system, we employ eggs and early embryos of the ascidian Styela, because they exhibit colored cytoplasmic regions which appear to delimit the distribution of localized mRNAs and cytoplasmic determinants. We have already cloned the gene corresponding to a maternal muscle actin mRNA and found that its transcripts are localized primarily in the yellow crescent of the egg and segregated into the embyronic muscle cells. Other evidence indicates that localized maternal mRNAs are bound to the egg cytoskeletal matrix. This proposal is designed to continue this work; it has four specific aims.
The first aim i s to identify and characterize further examples of localized mRNAs. This will be done by screening an egg cDNA library with probes encoding tissue-specific, cytoskeletal, and yellow crescent mRNAs. The spatial distribution of candidates for localized mRNAs in the egg will then be examined by in situ hybridization.
The second aim i s to examine the developmental expression of localized mRNAs during oogenesis and embryogenesis. This will be accomplished by Northern blot analysis and in situ hybridization of oocytes, eggs, and developing embryos.
The third aim i s to determine the mechanism of mRNA localization, specifically whether various localized mRNAs are bound to the egg cytoskeletal matrix and, if so, the nature of the binding sites. This will be accomplished by a combination of Northern blots, in situ hybridization, RNase protection analysis, and UV-crosslinking of mRNA in isolated cytoskeletal matrices.
The final aim i s to determine the developmental significance of localized mRNAs. This will be accomplished by experiments in which antisense or sense synthetic RNA corresponding to localized mRNAs are microinjected into normal eggs or used to rescue eggs that exhibit developmental defects caused by UV-irradiation. These studies should provide insight into the mechanism of mRNA localization and the function of localized mRNA in eggs and early embryos.
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