The establishment of polarity and provision of spatial cues are prerequisites for the development of a multicellular organism. These spatial cues can act as morphogens, evoking different responses al different concentrations and thereby instructing calls or nuclei to follow particular developmental pathways. The correct localization of such early acting morphogens within the embryo is necessarily a critical step in organizing the global body pattern. In Drosophila, the localization is accomplished by distributing early morphogens In a series of gradients which are formed early in embryogenesis. One such graded morphogen, the bicoid (bcd) protein, controls anterior pattern, and another, the caudal (cad) protein, is involved in posterior patterning. The long-term goal of the proposed work is to determine how these gradients arise and how they act. Formation of the bcd gradient depends on prelocalization of the bcd mRNA at the anterior pole. We have shown that a cis-acting element on the mRNA is required for this localization. In the proposed experiments this element will be precisely defined by systematically altering that portion of the bcd gene in vitro, reinserting it into the genome by P-element mediated transformation, and monitoring the activity of the mRNA localization element in embryos from transgenic flies. In addition, biochemical and molecular biological approaches will be used to identify and purify the trans-aging factors which recognize this element and anchor the mRNA to fixed cytoskeletal or structural components at the anterior pole of the embryo. With both the target mRNA sequence and anchoring components in hand we can ask how they interact with each other. Formation of the cad protein gradient is controlled by the bcd protein, and involves differential protein accumulation from a uniformly distributed mRNA. The signal(s) on the cad mRNA or protein which re responsible for this graded expression will be defined. As for the bcd mRNA signal, our approach is to modify the gene, reinsert it into the genome, and ask how the alterations affect cad protein distribution in embryos. Finally, we will determine how the bcd protein acts to direct formation of the cad protein gradient; because this gradient is likely to be established by translational repression, we will initially test for binding of bcd protein to cad mRNA.
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