The aim of the experiments outlined in this proposal is to address two fundamental issues in development: how the proximodistal (P/D) axis is formed in developing limbs and what distinguishes different limb types from one another. Our efforts are focused on the genetic and biochemical interactions of a homeodomain transcription factor encoded by the Drosophila Distal-less (Dll) gene. Dll is the earliest known gene to be specifically expressed in developing insect limbs, can initiate P/D axis formation when expressed ectopically, is required for the elaboration of P/D pattern elements in the antenna, the legs and the limb-derived gnathal structures, and plays an essential role in distinguishing the Drosophila antenna from the leg. The distinct developmental roles of D11 are likely to be mediated via distinct suites of target genes. However, D11 has little DNA binding specificity of its own, and we hypothesize that tissue and domain specific interactions with different co-factors are essential for D11 to achieve the specificity needed to regulate distinct targets. Therefore, if we want to understand the mechanisms by which D11 contributes to limb development, we must identify both D11 co-factors and D11 target genes. Using genetic approaches, we have identified spalt (sal) as a target of D11 regulation in the antenna and Homothorax (Hth) and Extradenticle (Exd) as a putative co-factors of D11 for the regulation of sal. Here, we propose to extend our genetic studies to identify other putative targets and co-factors and to use molecular and biochemical approaches both to identify the D11 responsive elements in target gene enhancers, such as the atenna1 enhancers of sal, and to test for physical interactions among D11, Hth and Exd. Because D11 homologs (the Dlx genes) are expressed in developing appendages in at least six coelomate phyla, including vertebrates we anticipate that the proposed studies will provide insights into vertebrate, as well as arthropod, limb development. In addition, since mammalian Dlx genes are required for aspects of normal brain and branchial arch development, we expect that the mechanistic insights gained from these experiments also will increase our understanding of Dll and D1x function in tissues and organs other than limbs.
