In this proposal we will continue our recent investigations of the pattern of mRNA distribution during early development and initiate studies on the mechanism of mRNA localization and the role of localized mRNA in embryonic determination. The eggs and embryos of the ascidian Styela, a classic organism for the study of embryonic determination which exhibits colored cytoplasmic regions of known morphogenetic fate, is used as a model system.
The specific aims are as follows. 1.) Examination of the distribution of specific mRNA sequences, in particular actin mRNA and other yellow crescent mRNAs, in the egg. 2.) Determination of the oogenetic orgin and embryonic fate of specific mRNA localizations. The first two aims will be accomplished by in situ hybridization with cloned DNA probes and in vitro translation of mRNA prepared from mass isolated egg cytoplasmic regions. 3.) Investigation of the role of the cytoskeletal framework in specific mRNA localization. This will be accomplished by a combination of non-ionic detergent extraction and in situ hybridization. Finally, 4.) bioassays, based on UV irradiation and microinjection, will be developed to assess the role of localized mRNA molecules in embryonic determination. It is anticipated that these studies will provide insight into the spatial distribution of mRNA in embryos and the role of localized mRNA during embryogenesis.
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