Synaptic target selection in Drosophila

果蝇的突触目标选择

• 批准号: 8019193
• 负责人: KAI G ZINN
• 金额: $ 9.07万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-01 至 2013-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019193
• 关键词: Address; Affect; Antibodies; Autistic Disorder; Axon; Back; Basic Science; Binding Sites; Birth; Brain; Cell surface; Cells; Collection; Complementary DNA; Complex; Confocal Microscopy; Cues; Databases; Development; Disease; Drosophila genus; Elements; Embryo; Event; Extracellular Domain; Family; Genes; Genetic; Genetic Screening; Health; Human; IGF-1 Signaling Pathway; Individual; Insulin; Insulin Signaling Pathway; Insulin-Like Growth-Factor Binding Protein 1; Insulin-Like-Growth Factor I Receptor; Invertebrates; Knowledge; Label; Larva; Leucine-Rich Repeat; Link; Maps; Methods; Motor; Motor Neurons; Muscle; Muscle Fibers; Mutation; Nature; Neuromuscular Junction; Neurons; Orthologous Gene; Paper; Pattern; Penetrance; Phenotype; Phosphotransferases; Protein Family; Proteins; RNA Interference; Research Personnel; Research Project Grants; Schizophrenia; Signal Pathway; Specificity; Staining method; Stains; Synapses; System; Tissues; Work; abstracting; axon guidance; gain of function; insulin signaling; interest; knock-down; leucine-rich repeat protein; loss of function; member; muscular structure; nerve supply; neuromuscular system; null mutation; overexpression; postsynaptic; protein function; receptor; research study; selective expression

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.

果蝇的突触目标选择 抽象的： 果蝇的遗传筛选鉴定出许多细胞表面和分泌 (CSS) 蛋白质 如今，它们作为脊椎动物和中枢神经系统轴突导向的调节剂而得到深入研究。 无脊椎动物系统。该提案描述了对具有功能的 CSS 蛋白进行遗传筛选 作为胚胎/幼虫神经肌肉系统中的突触目标标签。该系统非常适合 检查目标标记机制，因为它仅包含 36 个运动神经元和 30 个 肌肉目标并具有不变的神经支配模式。每个识别出的运动神经元 支配特定的肌肉纤维。尽管许多调节轴突引导的基因 系统已经确定，我们对单个肌肉纤维的情况知之甚少 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定位错误的 CSS 蛋白 当它们在所有肌纤维上过度表达时。我们通过构建所有数据的数据库来做到这一点 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包含 UAS（GAL4 结合位点）的现有集合 （“EP-like”）元素行查找紧邻这些 CSS 基因上游的插入，这些插入可以 通过与 GAL4“驱动”系杂交，可用于赋予组织特异性、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，或者超过 40% 的假定细胞识别库。我们跨越了每一条线，成为了全肌肉GAL4 驱动程序并通过抗体染色和共聚焦显微镜检查了 F1 后代幼虫。我们 发现了 30 个基因，其在所有肌肉上的表达会导致高外显率轴突误定位 表型但不扰乱肌肉结构。其中六个基因属于特定家族 编码具有含有富含亮氨酸重复序列（LRR）的胞外域的蛋白质，这些蛋白质是 蛋白质相互作用模块。该提案描述了评估功能的实验 四种 LRR 蛋白在肌肉中表达，似乎充当突触靶点 标签，并确定 LRR 系列是否编码其他目标标签。 第一个具体目标涉及 Tartan (Trn) 和 Capricious (Caps) 蛋白。丢失- trn 和 caps 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 12 号肌肉上的选择性表达仅产生 目标特异性的改变。我们将确定功能丧失 (LOF) 幼虫 通过敲低单个肌肉或所有肌肉中的 Trn 和 Caps 产生的表型。 我们还将尝试开发一种标记幼虫单运动轴突的方法，以便我们 可以观察遗传扰动如何影响单个已识别轴突的靶向。 具体目标2和3涉及两个“新基因”，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无效突变 并进行遗传相互作用筛选以寻找 CG14351/Haf 信号传导的成分 途径。我们还将确定 CG8561（哺乳动物 IGF-1 的直系同源物）是否与 蛋白质，是胰岛素/IGF-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 系列的实验 确定它是否编码其他肌肉目标标签。为此，我们将制作 UAS-cDNA 构建并获得或制作 41 个 LRR 基因的 RNAi 系，并评估它们的表型 幼虫。对于产生表型的所有基因，我们将绘制它们的表达图 轴突生长期间肌纤维的模式。这些信息将使我们能够 开始结合 LRR 蛋白扰动，敲低特定肌肉上的多个基因， 为了检查肌肉纤维是否被标记为通过特定表达的靶向 LRR 蛋白的集合体。