Synaptic target selection in Drosophila

果蝇突触靶点的选择 摘要： 果蝇的遗传筛选鉴定了许多细胞表面和分泌(Css)蛋白 今天作为脊椎动物和脊椎动物轴突引导的调节者被广泛研究 无脊椎动物系统。这项建议描述了一种对具有功能的css蛋白的遗传筛选。 在胚胎/幼虫神经肌肉系统中作为突触靶标。该系统非常适合于 检查目标标记机制，因为它只包含36个运动神经元和30个 肌肉是靶子，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。尽管许多调节轴突引导的基因在这一过程中 系统已经被识别，我们对单个肌肉纤维是怎样的知之甚少 被运动神经轴突识别为目标。 为了解决这个问题，我们首先定义了导致轴突错误定位的css蛋白。 当它们在所有肌肉纤维上过度表达时。我们通过构建一个数据库来实现这一点 果蝇中编码css蛋白的基因可能参与细胞识别事件。 然后我们搜索了所有现有的UAS(GAL4结合位点)集合-包含 (‘EP-like’)元素行，找到紧邻这些css基因上游的插入片段，这些插入片段可能 通过将它们与GAL4“驱动”系杂交，用来赋予组织特异性的高水平表达。 我们获得了类似EP的插入片段，可以驱动数据库中979个基因中的410个，甚至更多 40%的假定细胞识别库。我们越过每一条线，进入了一个泛肌肉GAL4 并对F1代幼虫进行抗体染色和共聚焦显微镜检查。我们 发现了30个在所有肌肉上表达导致高外显性轴突错误定位的基因 表型，但不扰乱肌肉结构。其中六个基因属于一个特定的家族 编码含有富含亮氨酸重复序列(LRR)的胞外区的蛋白质，这些重复序列是 蛋白质相互作用模块。这项建议描述了用实验来评估 四个在肌肉中表达并似乎具有突触靶点功能的LRR蛋白 标签，并确定LRR家族是否编码额外的目标标签。 第一个特定目标涉及Tartan(Trn)和Caps(Caps)蛋白。损失-- TRN和CAP的功能表型表明，它们以部分冗余的方式发挥作用 在胚胎中。在幼虫中，Trn或Caps在肌肉12上的选择性表达只产生 靶向特异性的变化。我们将确定功能丧失(LOF)幼虫 通过在单个肌肉或所有肌肉中同时敲除Trn和Caps而产生的表型。 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 可以观察基因扰动如何影响单个已识别轴突的靶向。 具体目标2和3涉及两个“新基因”，即CG14351/HAF和CG8561。我们有 使用遗传和RNAi分析表明，这些基因编码的蛋白质是必需的 用于腹外侧肌的正常神经支配。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找到CG14351/HAF信号的组件 路径。我们还将确定CG8561，哺乳动物IGF-1结合的同源基因 蛋白质是胰岛素/胰岛素样生长因子-1信号通路的组成部分。 最终的具体目标描述了检查整个LRR家族的实验 确定它是否编码其他肌肉靶标。为了做到这一点，我们将制作UAS-cDNA 构建、获得或制作41个LRR基因的RNAi系，并对其表型进行评估 幼虫。对于所有产生表型的基因，我们将绘制它们的表达图谱 轴突生长期间肌肉纤维的模式。这些信息将使我们能够 开始结合LRR蛋白的扰动，打倒特定肌肉上的多个基因， 为了检查肌肉纤维是否被标记为靶向，通过表达特定的 LRR蛋白的集合。

Synaptic target selection in Drosophila Abstract: 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.