Synaptic target selection in Drosophila

果蝇的突触靶选择 摘要： 果蝇的遗传筛选鉴定出许多细胞表面和分泌（CSS）蛋白 作为脊椎动物和哺乳动物轴突导向的调节器， 无脊椎动物系统该提案描述了一种CSS蛋白的遗传筛选， 作为胚胎/幼虫神经肌肉系统中的突触靶标记。该系统是理想的 检查目标标记机制，因为它只包含36个运动神经元和30个 肌肉的目标，并有一个不变的神经支配模式。每个被识别的运动神经元 支配特定的肌肉纤维。尽管在这个过程中，许多调节轴突导向的基因 虽然我们已经确定了肌肉系统，但我们对单个肌肉纤维是如何工作的知之甚少。 被运动轴突识别为目标。 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蛋白质 当它们在所有肌肉纤维上过度表达时。我们建立了一个数据库， 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包含UAS（GAL 4结合位点）的集合， （'EP样'）元素行，以找到这些CSS基因的上游插入， 通过将它们与GAL 4“驱动”系杂交，可用于赋予组织特异性的高水平表达。 我们获得了类似EP的插入，可以驱动数据库中979个基因中的410个，或者更多。 40%的细胞识别系统。我们穿过每条线，得到泛肌肉GAL 4 通过抗体染色和共聚焦显微镜检查F1代幼虫。我们 发现了30个基因，它们在所有肌肉上的表达都会导致高频率轴突错误启动 表型，但不扰乱肌肉结构。其中六个基因在一个特定的家族中 编码含有富含亮氨酸重复序列（LRR）的胞外结构域的蛋白质， 蛋白质相互作用模块该提案描述了评估以下功能的实验： 在肌肉中表达的四种LRR蛋白，似乎起着突触靶点的作用 标签，并确定LRR家族是否编码额外的靶标签。 第一个具体目标涉及Tartan（Trn）和Caps（Caps）蛋白。损失- trn和caps的功能表型表明它们以部分冗余的方式发挥功能 在胚胎中。在幼虫中，在肌肉12上选择性表达Trn或Caps仅产生 靶向特异性的改变。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敲低Trn和Caps两者产生的表型。 我们还将尝试开发一种标记幼虫单个运动轴突的方法， 可以观察遗传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CG 14351/haf和CG 8561。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， 腹外侧肌的正常神经支配我们将在这些基因中制造无效突变 并进行遗传相互作用筛选以找到CG 14351/Haf信号传导的组分 通路我们还将确定是否CG 8561，哺乳动物IGF-1结合的直系同源物， 蛋白质，是胰岛素/IGF-1信号通路的组成部分。 最后的具体目标描述了实验，以检查整个LRR家族， 确定它是否编码其他肌肉目标标签。为此，我们将UAS-cDNA 构建并获得或制备41个LRR基因的RNAi株系，并评估它们在 幼虫对于所有产生表型的基因，我们将绘制它们的表达图 在轴突生长期间肌纤维的模式。这些信息将使我们能够 开始联合收割机LRR蛋白干扰，敲低特定肌肉上的多个基因， 为了检查肌纤维是否被标记用于通过表达特异性 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.