Embryonic differentiation is mediated in part by determinants asymmetrically distributed in the cytoplasm of the egg. The vegetal region of frog eggs appears to contain at least four sets of determinants, those specifying: mesoderm, endoderm, the dorsal axial structures and the germ cell lineage. Localized maternal mRNAs are a rare class of RNAs that can code for determinants. The mechanism through which the spatial distribution of RNAs is established and maintained is unknown and represents a central problem in both cell and developmental biology. Modern and classical studies have suggested that the cytoskeleton plays an important role. Recently, we have isolated two novel Xenopus cytoskeletal associated transcripts in oocytes that are localized to the vegetal pole (XCAT-2 and XCAT-3). Remarkably, XCAT-2 is homologous to manos, the posterior determinant in Drosophila. In this proposal experiments are outlined to: (1) study the spatial and temporal expression of XCAT-2 and XCAT-3 during normal development. (2) characterize the XCAT-2 and XCAT-3 protein products and examine their function in early development. (3) determine the RNA localization signal and protein binding components involved in XCAT-2 and XCAT-3 localization. The general strategy will be to establish how the XCATs are inherited during embryogenesis by RNA blot and in situ hybridization studies. Mis- expression and non-expression of XCAT-2 and XCAT-3 proteins in the oocyte, embryo, and animal caps will be used to characterize their possible functions in development. Microinjection studies of XCAT-2 and XCAT-3 mutant constructs will allow us to identify the RNA sequences required for localization and to assess the function of associated proteins. By studying localized maternal RNAs and their relationship to the cytoskeleton, we hope to understand how molecular polarity is established in the oocyte and how it directs regional specification of the early embryo.
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