Synaptic target selection in Drosophila

描述(申请人提供)：果蝇的遗传筛选鉴定了许多细胞表面和分泌(Css)蛋白，这些蛋白目前在脊椎动物和无脊椎动物系统中都被作为轴突引导的调节器进行了深入的研究。这项建议描述了一种在胚胎/幼虫神经肌肉系统中作为突触靶标的css蛋白的遗传筛选。该系统是研究靶标记机制的理想系统，因为它只包含36个运动神经元和30个肌肉靶，并且具有不变的神经模式。每个已识别的运动神经元支配一种特定的肌肉纤维。虽然在这个系统中已经发现了许多调控轴突引导的基因，但我们对单个肌肉纤维如何被运动轴突识别为靶标知之甚少。为了解决这个问题，我们首先定义了当CSS蛋白在所有肌肉纤维上过度表达时会导致轴突错误定位的蛋白质。我们通过构建果蝇中编码可能参与细胞识别事件的CSS蛋白的所有基因的数据库来做到这一点。然后，我们搜索了所有现有的含有UAS(GAL4结合位点)(‘EP-like’)的元件线的集合，以寻找这些CSS基因的上游插入片段，这些插入片段可以通过将它们与GAL4“驱动”线杂交来提供组织特异性的高水平表达。我们获得了EP类插入片段，可以驱动数据库中979个基因中的410个，或者说超过40%的假定细胞识别库。我们将每个品系与一只泛肌肉GAL4司机杂交，并通过抗体染色和共聚焦显微镜检查F1后代幼虫。我们发现了30个基因，它们在所有肌肉上的表达导致高外显性轴突错误定位表型，但不会扰乱肌肉结构。其中六个基因属于编码蛋白质的特定家族，其胞外结构域包含富含亮氨酸的重复序列(LRR)，这是蛋白质相互作用的模块。这项建议描述了评估四种在肌肉中表达的、似乎具有突触靶标功能的LRR蛋白的功能的实验，并确定LRR家族是否编码额外的靶标。第一个特定目标涉及Tartan(Trn)和Caps(Caps)蛋白。TRN和CAPS的功能丧失表型表明，它们在胚胎中以部分冗余的方式发挥作用。在幼虫中，Trn或Caps在肌肉12上的选择性表达只会导致靶向性的改变。我们将确定由于Trn和Caps在单个肌肉或所有肌肉中被击倒而产生的功能丧失(LOF)幼虫表型。我们还将尝试开发一种标记幼虫单个运动轴突的方法，以便我们可以观察遗传扰动如何影响单个识别的轴突的靶向。具体目标2和3涉及两个“新基因”，即CG14351/HAF和CG8561。我们利用遗传和RNAi分析表明，这些基因编码的蛋白质是腹外侧肌正常神经支配所必需的。我们将在这些基因中进行零突变，并进行遗传相互作用筛选，以寻找CG14351/HAF信号通路的组成部分。我们还将确定CG8561(哺乳动物IGF-1结合蛋白的同源基因)是否是胰岛素/IGF-1信号通路的组成部分。最后一个具体目标描述了检查整个LRR家族以确定它是否编码其他肌肉靶标的实验。为此，我们将构建UAS-cDNA结构，获得或制作41个LRR基因的RNAi系，并评估它们在幼虫中的表型。对于所有产生表型的基因，我们将绘制它们在轴突生长期间在肌肉纤维中的表达模式图。这些信息将使我们能够开始结合LRR蛋白的扰动，敲除特定肌肉上的多个基因，以便检查肌肉纤维是否被标记为通过表达特定的LRR蛋白来靶向。与公共健康相关：这是一个基础研究项目，旨在发现在发育过程中参与创建神经元回路的机制。虽然这项工作是在果蝇身上进行的，但我们正在研究的大多数基因都与人类的基因相对应。我们希望揭示一些一般性的原理，这将有助于理解人类大脑连接在出生前后是如何被控制的。有关神经连接机制的知识可能有助于研究人员了解神经元连接模式发生变化的疾病。这些疾病包括精神分裂症和自闭症。

DESCRIPTION (provided by applicant): Genetic screens in Drosophila identified many of the cell-surface and secreted (CSS) proteins that are intensively studied today as regulators of axon guidance in both vertebrate and invertebrate systems. This proposal describes a genetic screen for CSS proteins that function as synaptic target labels in the embryonic/larval neuromuscular system. This system is ideal for examination of target labeling mechanisms, because it contains only 36 motor neurons and 30 muscle targets and has an invariant innervation pattern. Each identified motor neuron innervates a specific muscle fiber. Although many genes that regulate axon guidance in this system have been identified, we know very little about how individual muscle fibers are recognized as targets by motor axons. To address this problem, we first defined CSS proteins that cause axonal mistargeting when they are overexpressed on all muscle fibers. We did this by constructing a database of all genes in Drosophila that encode CSS proteins likely to be involved in cell recognition events. We then searched through all the existing collections of UAS (GAL4 binding site)-containing ('EP-like') element lines to find insertions immediately upstream of these CSS genes that could be used to confer tissue-specific, high-level expression by crossing them to GAL4 "driver" lines. We obtained EP-like insertions that can drive 410 of the 979 genes in the database, or over 40% of the putative cell recognition repertoire. We crossed each line to a pan-muscle GAL4 driver and examined F1 progeny larvae by antibody staining and confocal microscopy. We found 30 genes whose expression on all muscles causes high-penetrance axonal mistargeting phenotypes but does not perturb muscle structure. Six of the genes are in a specific family encoding proteins with extracellular domains containing leucine-rich repeats (LRRs), which are protein interaction modules. This proposal describes experiments to assess the functions of four LRR proteins that are expressed in muscles and appear to function as synaptic target labels, and to determine if the LRR family encodes additional target labels. The first specific aim concerns the Tartan (Trn) and Capricious (Caps) proteins. Loss-of- function phenotypes for trn and caps suggest that they function in a partially redundant manner in the embryo. In larvae, selective expression of Trn or Caps on muscle 12 only produces alterations in targeting specificity. We will determine the loss-of-function (LOF) larval phenotypes generated by knockdown of both Trn and Caps in a single muscle or in all muscles. We will also attempt to develop a method for labeling single motor axons in larvae, so that we can observe how genetic perturbations affect targeting of individual identified axons. Specific aims 2 and 3 concern two "new genes", CG14351/haf and CG8561. We have used genetic and RNAi analysis to show that the proteins encoded by these genes are required for the normal innervation of ventrolateral muscles. We will make null mutations in these genes and conduct a genetic interaction screen to find components of the CG14351/Haf signaling pathway. We will also determine whether CG8561, the ortholog of a mammalian IGF-1 binding protein, is a component of the insulin/IGF-1 signaling pathway. The final specific aim describes experiments to examine the entire LRR family to determine if it encodes other muscle target labels. To do this, we will make UAS-cDNA constructs and obtain or make RNAi lines for 41 LRR genes and assess their phenotypes in larvae. For all genes producing phenotypes, we will then make a map of their expression patterns in muscle fibers during the period of axonal outgrowth. This information will allow us to begin to combine LRR protein perturbations, knocking down multiple genes on specific muscles, in order to examine whether muscle fibers are labeled for targeting by expression of specific ensembles of LRR proteins. PUBLIC HEALTH RELEVANCE: This is a basic research project to discover mechanisms involved in creation of neuronal circuits during development. Although the work is conducted in Drosophila, most of the genes we are studying have human counterparts. We hope to reveal general principles that will facilitate an understanding of how human brain wiring is controlled before and after birth. Knowledge about wiring mechanisms may help researchers to understand diseases in which neuronal connectivity patterns are altered. These include schizophrenia and autism.