IN SITU LOCALIZATION OF INFORMATIONAL EMBRYO RNAS

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A set of messenger RNAs has been identified which are coordinately expressed in a temporally and spatially restricted pattern during development of the sea urchin (Strongylocentrotus purpuratus) embryo. The messages accumulate in the very early blastula (VEB) and then decay rapidly. The have a non-uniform concentration along the maternally specified animal-vegetal axis, being absent from cells at the vegetal pole, but are uniformly distributed along the oral-aboral axis that is specified after fertilization. Several lines of evidence suggest that the genes encoding these messages (VEB genes) are regulated by maternal factors asymmetrically arrayed along the animal-vegetal axis. The VEB genes are activated as early as 4-cell stage, are first transcribed in specific blastomeres of the 16-cell embryo, and accumulate autonomously in cells separated beginning at 2-cell stage. The promoters of two of the VEB genes will be analyzed to identify sites of interaction with regulatory proteins, especially those factors responsible for spatial regulation. These analyses will use standard in vitro biochemical assays of protein-DNA interaction, and in vivo tests of reporter gene constructs driven by intact and altered promoters. Recombinant clones representing the mRNAs encoding the factors responsible for spatial regulation of the VEB genes will be identified, and antibodies will be raised that are specific for the corresponding proteins. These probes will be used to distinguish among possible mechanisms of localized activity of the spatial regulators-localized maternal mRNA, localized maternal protein, or localized activation of a uniformly distributed protein. Antisense methods and/or antibody inhibition will be tested for their efficacy in blocking the function of the VEB regulators, in order to investigate their role in determination and differentiation along the animal-vegetal axis.