AXON GUIDANCE IN THE FRUITFLY

这项提案概述了一系列实验，其目的是 研究生长轴突被引导到的环境线索 它们的目标组织。它使用发现的感觉神经元作为其模型系统 在果蝇果蝇的翅膀上。我会攻击这件事 使用两种截然不同的方法解决问题：i)基因模式 在翅膀中的表达-因为轴突引导线索的模式在 翼已经被很好地刻画了，有强烈的限制 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陷阱技术，以及b)，检查 已知基因产物的分布，如毛发，以搜索 基因表达和细胞分化的模式对应于 我们对指导线索的期望。然后将使用突变或 利用免疫抑制技术产生和检测抗原。Ii) 体外引导的微扰--一种更有针对性的细胞方法 将使用轴突引导，这取决于固定翼的能力 片段引导分离的神经元沿着正常的轴突路径生长。 这将被用作一种生物测试，以测试先前证明的能力 用多克隆抗血清来阻断翅膀组织的正常引导。抑制性 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， 和生物化验。建议使用体外培养技术进行其他辅助项目。 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.