PROXIMODISTAL AXIS AND TISSUE SPECIFIC GENE REGULATION

本提案中概述的实验目的是解决发育中的两个基本问题：近端远端（P/D）轴是如何在发育的肢体中形成的，以及如何区分不同类型的肢体。我们的工作集中在果蝇远端基因编码的同源结构域转录因子的遗传和生化相互作用。Dll是已知最早在昆虫四肢发育中特异性表达的基因，在异位表达时可启动P/D轴的形成，在触角、腿和肢体衍生的颌部结构中P/D模式元件的精细化是必需的，在区分果蝇触角和腿方面起着至关重要的作用。D11的不同发育作用可能是通过不同的靶基因组介导的。然而，D11本身几乎没有DNA结合特异性，我们假设D11与不同辅助因子的组织和结构域特异性相互作用对于实现调节不同靶点所需的特异性是必不可少的。因此，如果我们想要了解D11促进肢体发育的机制，我们必须确定D11辅助因子和D11靶基因。利用遗传学方法，我们已经确定了spalt （sal）是D11在天线中的调控靶点，而Homothorax （Hth）和Exd （Exd）是D11调控sal的推定辅助因子。在这里，我们建议扩展我们的遗传学研究，以确定其他可能的靶点和辅助因子，并使用分子和生化方法来确定靶基因增强子（如sal的天线1增强子）中的D11响应元件，并测试D11， Hth和Exd之间的物理相互作用。由于D11同源基因（Dlx基因）在包括脊椎动物在内的至少6种体腔动物门的附属物发育中表达，我们预计，拟议的研究将为脊椎动物以及节肢动物的肢体发育提供见解。此外，由于哺乳动物的Dlx基因在正常大脑和鳃弓发育的各个方面都是必需的，我们期望从这些实验中获得的机制见解也将增加我们对Dll和D1x在肢体以外的组织和器官中的功能的理解。

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.