Deformed (Dfd) is a homeobox-containing homeotic selector gene that determines head-specific fates in the Drosophila embryo. In order to understand how the Dfd protein accomplishes its developmental programming role, we wish to understand how Dfd regulates specific downstream genes at specific times and in specific cells, and we also wish to understand the biochemical and biological functions of the downstream genes that are regulated in the Drosophila embryo. Since Dfd has mouse and human homologs that also putatively assign anterior-specific fates in the developing vertebrate body plan, many of the lessons we learn about Dfd-specific regulatory elements promise to increase our insight into mechanisms of vertebrate as well as fly development. We initially concentrate our efforts on the further characterization of the regulatory element of a recently identified candidate downstream gene, Distalless (Dll, also known as Brista). We plan to mutagenize the Dfd-dependent enhancer at Dll to define the size of the enhancer, and to test whether Dfd protein binding to the 3' element is required to mediate the Dfd-dependent regulatory effect. In addition, we plan to identify other regulatory factors that may be required for the Dfd-specificity of the Dll enhancer, and which may also restrict the activity of the Dll 3' enhancer to a subset of Dfd expressing cells in the maxillary segment. Finally, we plan to test whether other homeotic genes of the Antennapedia and Bithorax complexes might regulate patterns of Dll expression through the same element, and how this regulation is integrated with Dfd's regulation of the same gene. To identify and study new Dfd downstream genes, we intend to use both genetic and biochemical methods. One approach involves the identification of genes whose mutation results in phenotypic reversion of the homeotic transformations caused by ectopic expression of Dfd in the embryo. In addition, we plan to use subtractive methods to isolate genes that are expressed at higher levels in embryos in which Dfd is ectopically expressed. Finally, we have developed very stringent DNA binding assays that identify DNA fragments that bind Dfd protein very specifically, which should allow us to directly isolate some of the downstream regulatory regions targeted by Dfd.
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