THE DROSOPHILA CNS--A MODEL FOR PCB NEUROTOXICITY

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Cells of both invertebrate and vertebrate embryos that give rise to neurons must correctly receive and interpret positional information throughout development so that terminally differentiated neurons are precisely determined. The mis-interpretation of positional cues can lead to severe developmental abnormalities and in the most extreme case death. Ventral nervous system defective (vnd), an NK-2 type homeobox gene, specifies the identity of midline proximal ventral neuroectodermal cells and neuroblasts in Drosophila. Since the vertebrate homologues of this gene are expressed in parallel domains in the vertebrate CNS, this suggests both regulatory and functional conservation. The overall goal of this proposal is to understand how the Drosophila vnd gene integrates positional information at the transcriptional and post-translational level so that it fits into the hierarchical network involved in CNS dorsal-ventral specification. Specifically, we want to localize the enhancers responsible for vnd expression in transgenic embryos, identify conserved sequence islands in the vnd regulatory domain from two related Drosophila species, perform DNA binding studies with regulators that bind vnd enhancers and confirm the functional significance of the DNA binding sites by mutating the relevant DNA binding sites and testing their activity in transgenic embryos. The epidermal growth factor (EGF) pathway also patterns the developing CNS along the dorsal-ventral axis by inducing the phosphorylation of largely unidentified target proteins. Since expression of Vnd is affected in EGF receptor mutants, we believe that Vnd is regulated by phosphorylation. We want to detemine if Vnd is phosphorylated, and to identify the amino acids that are modified, with the long term goal of determining the role of phosphorylation in Vnd's function as a transcription factor. Finally, we want to address the role of the NK-2 box, a conserved stretch of 18 amino acids downstream of the homeodomain, in Vnd's role as a transcription factor. The capacity of mutated Vnd protein, lacking the NK-2 box, to drive reporter expression will be assayed in a tissue culture model. We recently showed that over-expression of wild-type Vnd leads to a transformation in neuronal precursor identity. Transgenic embryos that over-express mutant Vnd, lacking the NK-2 box, will be assayed for alteration in neuronal fate. These studies directly address the structure and function of vnd, a critical regulator of early neuronal development, and may serve as a prototype for vertebrate vnd-like genes.