The objective of this program is to identify and functionally characterize neurogenic genes that are required for CNS development. Given the high conservation in basic mechanisms used by all metazoans, our search was initiated in the fruit fly (Drosophila melanogaster) where these genes are more accessible for study. Utilizing classical genetic, molecular biology and transgenic techniques, we have continued to study both the function and the regulatory mechanisms that control the expression of castor, a novel Zinc finger gene required for Drosophila CNS development and pollux, castor's close genomic neighbor. Based on its predicted primary structure and the high expression levels in CNS neuro~blasts, castor may regulate itself and other genes involved in neuroblast maturation. To test this hypothesis, we are currently mapping the cis~regulatory elements that control its in vivo expression. Located in its 5' flank, we have found a near perfect 880bp inverted repeat and are now determining if it harbors cis~elements that regulate expression. Once identified, we will assess if lack of or ectopic castor expression modulates reporters that respond to these cis~elements. We are also determining if the pollux protein functions as a membrane~associated adhesion molecule. Analysis of its primary structure reveals that pollux contains an integrin~binding tetrapeptide RGD sequence, multiple glycosaminoglycan~binding sites, and a potential glycosaminoglycan~linkage site. pollux immunostainings have shown that a portion or all of the protein is located on the plasma membrane extracellular surface. In addition, we have observed that misexpression of pollux leads to high levels of mature male homosexual activity. Protein data bank searches, have revealed that pollux shares a 70 amino acid domain with the human tre~1 oncogene and a predicted human myoblast protein (84% and 87% similarity). We have also continued our functional analysis of the murine homeobox gene Hox 1.3 by identifying genes that it regulates. We have observed that ectopic expression of Hox 1.3 in transgenic mice correlates with the apparent repression of a hepatocyte nuclear transcription factor, HNF~3beta. During development, we have also discovered that HNF~3beta is expressed in the CNS and are now assessing if in utero ectopic Hox 1.3 expression modulates its expression.
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