AXON GUIDANCE IN THE FRUITFLY

该提案概述了一系列实验，其目的是 调查引导生长轴突的环境线索 它们的靶组织。它使用的模型系统的感觉神经元发现 在果蝇的翅膀上，黑腹果蝇。我会攻击这个 问题使用两种不同的方法：I）基因的模式 因为轴突引导线索的模式在翅膀中， 机翼已经被很好地描述了，在机翼上有很强的限制。 候选引导分子的分布方式。我会利用这一点 通过a）使用新开发的增强子捕获技术，和B）检查 已知基因产物的分布，如多毛，以寻找 基因表达和细胞分化的模式， 我们对引导线索的期望。然后将使用突变或 产生，并使用免疫抑制技术检测抗原。 （二） 体外指导的扰动-一种更直接的细胞方法， 将使用轴突引导，这取决于固定翼的能力 引导分离的神经元沿着正常轴突通路生长的片段。 这将被用作生物测定，以测试先前证明的能力 多克隆抗血清来阻断正常的引导抑制 将产生抗血清并通过以下方法分离抑制活性： 使用免疫印迹系统进行亲和纯化的连续步骤， 和生物测定。 建议使用体外试验方法开展额外的附带项目。 系统

This proposal outlines a series of experiments whose purpose is to investigate the environmental cues by which growing axons are guided to their target tissues. It uses as its model system the sensory neurons found on the wing of the fruitfly, Drosophila melanogaster. I will attack this problem using two rather different approaches: I) Patterns of gene expression in the wing-Because the pattern of axon guidance cues in the wing has been so well characterized, there are strong constraints upon the way candidate guidance molecules could be distributed. I will exploit this by a) using the newly developed enhancer trap technique, and b), examining the distribution of known gene products, such as hairy, to search for patterns of gene expression and cell differentiation which correspond to our expectations for guidance cues. Mutations will then be used or generated, and antigens detected using immunosuppressive techniques. II) Perturbation of guidance in vitro- A more directed, cellular approach to axon guidance will be used, which depends upon the ability of fixed wing fragments to guide growing dissociated neurons along normal axon pathways. This will be used as a bioassay to test the previously demonstrated ability of polyclonal antiserum to wing tissue to block normal guidance. Inhibitory antiserum will be generated and the inhibitory activity isolated by successive steps of affinity purification using an immunoblotting system, and bioassay. Additional side projects are proposed using the in vitro system.