Localization and Identification of Cytoplasmic Determinants

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Early embryonic development is thought to be mediated by determinants present in the egg cytoplasm, but these substances have yet to be characterized. In this proposal we continue our studies on cytoplasmic determinants involved in endodermal alkaline phosphatase (AP), muscle and embryonic axis development in the ascidian Styela. The possibility that AP determinants consist of maternal AP mRNAs, which are localized in the egg and translationally activated after segregation to the endodermal cells will be examined by in situ hybridization with a cloned AP probe. The spatial distribution of muscle and axial determinants will be examined by UV microbeam irradiation of the egg and microsurgical deletion of cells from the early embryo. The general class of molecules to which the muscle and axial determinants belong will be investigated using a combination of UV action spectrum analysis and a bioassay involving microinjection of fractionated cytoplasmic components of normal eggs into eggs whose ability to develop axis or muscle has been impaired by localized UV irradiation. The potential role in axis determination of a maternal mRNA encoding a 25 kd protein, a component that is absent in axis deficient embryos, will also be investigated by UV irradiation and in vitro translation. It is anticipated that new information will be obtained concerning the localization and identity of determinants involved in three different developmental decisions. %%% One of the most important mechanisms regulating the control of early embryogenesis is the segregation of developmental information, "determinants" or "morphogens," to various regions of the embryo during early development. It is believed that these morphogens determine the fate of specific cell lineages and guide them to differentiate into their final tissue such as muscle, nerve and gut. The molecular nature of these determinants has not yet been determined. Dr. Jeffery has made important contributions to this area and is now on the verge of identifying those molecules necessary for muscle and gut development as well as for the establishment of the dorsal- ventral axis itself.