The question of how a single cell, the egg, becomes a multicellular organism with differing tissue types and form is a question basic to embryology and developmental biology. It has been speculated that localized cytoplasmic components (cytoplasmic determinants) may have a regulatory role in the initial stages of embryogenesis and it has also been speculated that RNA and/or protein are likely candidates for these regulatory molecules. However, few experiments exist which demonstrate this point. The existence of regionally localized molecules (RNA, protein, or other components) in the oocyte or embryo does not necessarily mean that the component is a cytoplasmic determinant, however it is suggestive that the component may have a biologically important role (otherwise a uniform distribution of the component would be expected). In this proposal oocytes and embryos of Xenopus laevis will be used to study localized RNA and protein because: a) spatial maps of localized mRNAs exist in these oocytes (of all existing systems the Xenopus oocytes are best defined for poly(A)+ RNA and mRNA); b) the synthesis of RNA and protein are relatively well understood during early development; c) oocytes and embryos are easily obtainable and their large size allows for easy manipulation. Their large size allows the spatial maps to be used as a guide for spatial fraction (division of the single-celled oocytes and embryos into regions) and then biochemical analysis of the RNA and protein in each region. The spatial fractionation will be performed by freezing, and sectioning the eggs in a cryotome, dehydration of eggs and """"""""putting-out"""""""" of the desired region, and removal of regions of the egg cortex. In the mature oocyte the amount and type of RNA for protein) in each region will be determined. The in vitro translation products or the extracted regional RNA will be compared with: 1) the steady-state proteins in the region; 2) the distribution of newly-synthesized proteins in each region. This will determine if a mechanism exists for the sorting-out of proteins. This analysis will be performed during all of oogenesis, meiotic maturation, and early embryogenesis. Antibody probes will be made to region-specific proteins to a) analyze the spatial distribution of the proteins, 3) obtain polyribosomes for the proteins by precipitation of nascent chains in order to single out specific RNAs for future analysis. In addition, the mechanisms involved in maintaining the localized mRNA in Xenopus oocytes will be examined.
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