SYNAPTIC CONNECTIVITY IN CENTRAL BRAIN

本建议的总体目标是表征中枢中间神经元之间突触建立和调节的分子机制。我们将通过(1)进一步表征果蝇POU结构域转录调节因子Acj6的功能及其在中枢大脑中建立正确突触连接中的作用，(2)鉴定在突触形成过程中由Acj6调节的通路的其他组分，以及(3)表征氨基末端Acj6 POUIV盒在调节Acj6功能中的作用来实现这一广泛目标。这项建议的具体目的是：(1)通过完成Acj6突变表型增强子和抑制子的基因筛选，鉴定Acj6调控通路的其他组分；(2)通过生成Acj6神经元特异性表达转座子，作为细胞特异性标记和错误表达实验，进一步表征Acj6突变体突触连接缺陷；(3)表征氨基端POUIV盒的选择性剪接导致的Acj6异构体的神经元特异性表达；(4)完成与Acj6 POUIV box相互作用的外源蛋白酵母双杂交筛选。突触发生的过程需要精确的初始选择正确的突触伙伴和随后的末端过程和连接的细化。这种突触选择、完善、加强和/或消除的过程被认为对学习和记忆的形成很重要，可能是许多神经疾病的根源。果蝇（Drosophila melanogaster）突触功能的遗传分析对我们学习和记忆机制的认识有重要贡献。在初步研究中，我们已经发现果蝇的转录调节因子Acj6对中枢中间神经元的末端轴突分支和突触发生的调节至关重要。在过去的几年中，转录因子相互作用的显著进化守恒已经证明了在果蝇等生物中详细描述发育调节机制的价值，这可以更容易地在实验中进行操作。通过对果蝇中Acj6蛋白所起的基本发育作用的分析获得的信息可以外推到高等生物中，以更好地理解突触连通性和某些发育异常所涉及的类似过程。

The overall goal of this proposal is to characterize the molecular mechanisms by which synapses between central interneurons are established and modulated. We will work toward that broad goal by (1) further characterizing the function of the Drosophila POU domain transcriptional regulator, Acj6, and its role in establishing correct synaptic connections in the central brain, (2) identifying other components of the pathway(s) regulated by Acj6 during synapse formation and (3) characterizing the role of the amino terminal Acj6 POUIV box in regulating Acj6 function. The specific alms of this proposal are: (1) to identify other components of the pathway(s) regulated by Acj6 through completion of a genetic screen for enhancers and suppressors of the acj6 mutant phenotype, (2) to further characterize the acj6 mutant defects in synaptic connectivity by generating Acj6 neuron-specific expression transposons for use both as cell-specific markers and for misexpression experiments, (3) to characterize the neuron-specific expression of Acj6 isoforms resulting from alternative splicing of the amino terminal POUIV box; and (4) to complete a yeast two-hybrid screen for heterologous proteins interacting with the Acj6 POUIV box. The process of synaptogenesis requires precise initial selection of the correct synaptic partners and subsequent refinement of terminal processes and connections. This process of synaptic selection, refinement, strengthening and/or elimination is thought to be important for learning and memory formation and may lie at the root of a number of neuropathologies. Genetic analysis of synaptic function in the fruit fly, Drosophila melanogaster, has contributed significantly to our knowledge of learning and memory mechanisms. In preliminary studies, we have shown that the Drosophila transcriptional regulator, Acj6, is essential for regulation of terminal axon branching and synaptogenesis in a subset of central interneurons. The significant evolutionary conservation of transcription factor interactions shown over the past several years has demonstrated the value of detailed characterization of developmental regulatory mechanisms in an organism such as Drosophila which can be more readily manipulated experimentally. Information gained from an analysis of the fundamental developmental roles played by the Acj6 protein in Drosophila can be extrapolated to higher organisms to better understand analogous processes involved in synaptic connectivity and certain developmental abnormalities.