Engrailed targets and the control of synaptic circuits in Drosophila

果蝇的纠缠目标和突触回路的控制

• 批准号: 8257801
• 负责人: JONATHAN M BLAGBURN
• 金额: $ 35.21万
• 依托单位: UNIVERSITY OF PUERTO RICO MED SCIENCES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8257801
• 关键词: Adult; Afferent Neurons; Animals; Auditory; Auditory system; Autistic Disorder; Automobile Driving; Axon; Binding; Biological Assay; Biological Models; Brain; Branchiostoma floridae AmphiEn protein; Cell Adhesion Molecules; Cell surface; Coupling; Culicidae; Data; Databases; Dengue; Development; Disease; Disease Vectors; Drosophila genus; Drosophila melanogaster; Electrophysiology (science); Ensure; Evolution; Fiber; Gene Targeting; Genes; Goals; Health; Human; Insecta; Knock-out; Knockout Mice; Knowledge; Label; Leucine-Rich Repeat; Link; Malaria; Mammals; Measures; Methods; Midbrain structure; Modeling; Molecular; Morphology; Neuraxis; Neurons; Parkinson Disease; Partner in relationship; Pathway interactions; Pattern; Pilot Projects; Play; Positioning Attribute; Process; Protein Overexpression; Proteins; RNA Interference; Regulation; Repression; Research; Role; Specificity; Symptoms; Synapses; System; Testing; autism spectrum disorder; axon guidance; axonal guidance; axonal pathfinding; connectin; design; disorder control; dopaminergic neuron; feeding; fly; human disease; innovation; knock-down; member; nervous system disorder; neural circuit; neurobiotin; neuroglian; neuron development; neuronal survival; overexpression; synaptogenesis; transcription factor; visual stimulus

DESCRIPTION (provided by applicant): Engrailed (En) is a transcription factor first discovered in Drosophila but later found to be present in all animals, playing an important role in controllin neuronal development. Little is known about the cell surface molecules that it regulates. The long-term goal of this research is to find out how En regulates synaptic connectivity in the CNS, with a particular focus on identifying and characterizing its target genes of cell surface effector molecules. Preliminary data show that overexpression of En in Drosophila olfactory neurons alters their axonal path finding to their targets, the olfactory glomeruli. Additionally, ectopic E expression in a normally En- negative subset of auditory neurons allows them to form synaptic connections with the Giant Fiber (GF) escape neuron. The first aim is to selectively knock out En in olfactory neurons using fly lines in which RNAi is driven by the Gal4-UAS system. I will determine the effects on axonal guidance by assaying changes in the morphology of GFP-labeled olfactory axons, and alterations in the positions of immunolabeled olfactory glomeruli. The second aim will drive En RNAi in the auditory neurons and measuring their synaptic input to the GF. The third aim is to test whether increasing or knocking down expression of the En target Connectin, a member of the conserved LRR superfamily of adhesion molecules, alters axon guidance or glomerulus positioning. I will also test whether En knockout results in overexpression of this protein, as would be expected if it is a target of En repression. In the finl aim, I will ectopically express or knock down the En-binding target gene Neuroglian, a cell surface adhesion molecule homologous to vertebrate L1-CAM, then use the auditory synapse assays of connection to the GF. I will also test whether its immunostaining is altered by overexpression or knockout of En, as would be expected if it is negatively regulated by En. Relevance: En has been shown to control the survival of midbrain dopaminergic neurons, with En knockout mice showing Parkinson-like symptoms, and it has also been linked to autism spectrum disorder. Drosophila models are particularly useful for the discovery of molecular pathways that are directly relevant to human health, because most of these pathways have been conserved during evolution. All animals have En protein, so it is very likely that any molecules that are regulated by it during the process of synapse formation in Drosophila have their counterparts in humans, playing similar roles. These molecules may be of great potential importance in neurological diseases such as Parkinson's or autism. In addition, the basic knowledge gained from this project about the development of dipteran olfactory and auditory systems could be of use in designing ways to disrupt the feeding and mating patterns of mosquitoes, which are vectors of diseases such as malaria and dengue fever. PUBLIC HEALTH RELEVANCE: This research will help our understanding of the molecular pathways involved in the formation of synaptic connections in the central nervous system, using the fruit fly, Drosophila melanogaster, as a model system. The long-term goal of this research is to find out how a protein that is present in all animals' brains regulates the accuracy of the formation of synaptic connections, by controlling the presence of other cell surface molecules. The results of this study may have relevance to diseases of humans such as Parkinson's disease and autism, and may help in the control of disease-transmitting insects such as mosquitoes.

描述（申请人提供）：Engrailed (En) 是一种转录因子，首先在果蝇中发现，后来发现它存在于所有动物中，在控制神经元发育中发挥重要作用。人们对它所调节的细胞表面分子知之甚少。这项研究的长期目标是找出 En 如何调节 CNS 中的突触连接，特别关注识别和表征其细胞表面效应器的靶基因 分子。初步数据表明，果蝇嗅觉神经元中 En 的过度表达会改变其轴突路径寻找目标（嗅觉肾小球）。此外，听觉神经元的正常 En- 负性子集中的异位 E 表达使它们能够与巨纤维（GF）逃逸神经元形成突触连接。第一个目标是使用由 Gal4-UAS 系统驱动 RNAi 的蝇系选择性敲除嗅觉神经元中的 En。我将通过分析 GFP 标记的嗅觉轴突的形态变化以及免疫标记的嗅觉肾小球位置的变化来确定对轴突引导的影响。第二个目标是驱动听觉神经元中的 En RNAi 并测量它们对 GF 的突触输入。第三个目的是测试增加或敲低 En 靶标 Connectin（保守的 LRR 粘附分子超家族成员）的表达是否会改变轴突引导或肾小球定位。我还将测试 En 敲除是否会导致该蛋白质的过度表达，如果它是 En 抑制的目标，那么正如预期的那样。在最终目标中，我将异位表达或敲除 En 结合靶基因 Neuroglian（一种与脊椎动物 L1-CAM 同源的细胞表面粘附分子），然后使用与 GF 连接的听觉突触测定。我还将测试其免疫染色是否因 En 的过度表达或敲除而改变，如果它受到 En 的负向调节，这将是预期的。相关性：En 已被证明可以控制中脑多巴胺能神经元的存活，En 敲除小鼠表现出帕金森样症状，并且它也与自闭症谱系障碍有关。果蝇模型对于发现与人类健康直接相关的分子途径特别有用，因为大多数这些途径在进化过程中都是保守的。所有动物都有En蛋白，因此果蝇突触形成过程中受其调节的任何分子很可能在人类中也有对应的分子，发挥着相似的作用。这些分子可能对帕金森病或自闭症等神经系统疾病具有巨大的潜在重要性。此外，从该项目中获得的有关双翅目嗅觉和听觉系统发育的基本知识可用于设计扰乱蚊子摄食和交配模式的方法，蚊子是疟疾和登革热等疾病的传播媒介。 公共健康相关性：这项研究将帮助我们了解中枢神经系统中突触连接形成所涉及的分子途径，以果蝇（Drosophila melanogaster）作为模型系统。这项研究的长期目标是找出所有动物大脑中存在的蛋白质如何通过控制其他细胞表面分子的存在来调节突触连接形成的准确性。这项研究的结果可能与帕金森病和自闭症等人类疾病有关，并可能有助于控制蚊子等传播疾病的昆虫。