Engrailed targets and the control of synaptic circuits in Drosophila

描述（由申请人提供）：Engrailed（En）是一种转录因子，首先在果蝇中发现，但后来发现存在于所有动物中，在神经元发育中起重要作用。关于它调节的细胞表面分子知之甚少。本研究的长期目标是了解En如何调节中枢神经系统的突触连接，特别是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，在果蝇嗅觉神经元中En的过表达改变了它们的轴突路径发现其目标，嗅球。此外，在正常En阴性的听觉神经元子集中的异位E表达允许它们与巨纤维（GF）逃逸神经元形成突触连接。第一个目标是使用其中RNAi由Gal 4-UAS系统驱动的飞线选择性地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和免疫标记的嗅觉小球的位置改变来确定对轴突指导的影响。第二个目标是在听觉神经元中驱动En RNAi，并测量它们对GF的突触输入。第三个目的是测试是否增加或敲低En目标连接蛋白（粘附分子的保守LRR超家族的成员）的表达，改变轴突导向或肾小球定位。我还将测试En敲除是否会导致该蛋白的过表达，如果它是En抑制的靶点，这将是预期的。最后，我们将外源性表达或敲除与脊椎动物L1-CAM同源的细胞表面粘附分子Neuroglian，并通过听觉突触实验检测其与GF的连接。我还将测试其免疫染色是否被En的过表达或敲除所改变，如预期的那样，如果它被En负调控。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，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通路特别有用，因为这些通路中的大多数在进化过程中是保守的。所有的动物都有En蛋白，所以很可能在果蝇突触形成过程中受其调节的任何分子在人类中都有对应物，发挥类似的作用。这些分子可能在神经系统疾病如帕金森氏症或自闭症中具有巨大的潜在重要性。此外，从该项目中获得的关于双翅目嗅觉和听觉系统发展的基本知识可用于设计破坏蚊子进食和交配模式的方法，蚊子是疟疾和登革热等疾病的媒介。

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.