Engrailed and the Control of Synaptic Circuitry in Drosophila

描述(申请人提供)：Engrailed是一种普遍存在的转录调节因子，对人类健康具有潜在的重要意义。最近，它与中脑多巴胺能神经元的命运决定和生存有关，EN基因敲除的小鼠表现出帕金森样症状，还与自闭症谱系障碍有关。因此，了解EN在调节神经元连接中所起的作用是至关重要的。这项研究的长期目标是使用简单的模型系统，即具有可识别的神经元，来研究Engraded是如何控制突触目标识别的，以及它调节的下游效应基因是什么。我之前的工作使用蟑螂谷物系统来研究这一点；然而，在这一系统的进一步发展中存在着实质性的障碍。这个试点项目的目的将使我能够在遗传上易驯化的黑腹果蝇身上开发一个类似的可识别累加表达神经元的系统。第一个目标是利用Gal4-UAS在正常表达Engraded的神经元中表达绿色荧光蛋白。然后将使用共聚焦显微镜来表征一系列感觉系统中的这些神经元，如嗅觉、听觉和触觉细胞。第二个目标是利用实验室开发的电生理和解剖学技术，以及导师罗德·默菲博士的专业知识，专注于其中一个感觉系统，以便能够测试Engraed在控制这个系统的突触连接中的作用。第三个目标将涉及异位增强与GFP的共同表达，从而能够直接测试增强控制电路中连通性的想法。在最终目标中，在罗德·默菲博士的帮助下，我将利用基因筛查，开始寻找Engraded下游控制突触专一性的其他基因。另一种策略将是测试以前由其他组织确定的EN结合靶点。 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，因为这些途径中的大多数在进化过程中都是保守的。所有动物都有积聚的蛋白质，所以在果蝇突触形成过程中受其调控的任何分子很可能在人类中也有对应的分子。这些分子在帕金森氏症或自闭症等神经系统疾病中可能具有巨大的潜在重要性。

DESCRIPTION (provided by applicant): Engrailed is a ubiquitous transcriptional regulator that is potentially of great significance to human health. It has recently been linked to fate determination and survival of midbrain dopaminergic neurons, with En knockout mice showing Parkinson-like symptoms, and has also been linked to autism spectrum disorder. It is therefore essential to understand the role En plays in regulating neuronal connectivity. The long term objectives of this research are to use simple model systems, that have identifiable neurons, to investigate how Engrailed controls synaptic target recognition, and what are the downstream effector genes that it regulates. My previous work has used the cockroach cereal system to study this; however, there are substantial barriers to further progress in this system. The aims of this pilot project will enable me to develop a similar system of identifiable Engrailed-expressing neurons in the genetically tractable Drosophila melanogaster. The first aim is to use Gal4-UAS to express green fluorescent protein in neurons that normally express Engrailed. Confocal microscopy will then be used to characterize these neurons in a range of sensory systems, such as the olfactory, auditory and touch cells. The second aim is to focus on one of these sensory systems using the electrophysiological and anatomical techniques that have been developed in the lab, and the expertise of the mentor, Dr. Rod Murphey, in order to be able to test the role of Engrailed in controlling the synaptic connectivity of this system. The third aim will involve the co-expression of ectopic Engrailed along with GFP, enabling a direct test of the idea that Engrailed controls connectivity in the circuit. In the final aim, with the help of Dr. Rod Murphey, I will use genetic screens to begin a search for other genes downstream of Engrailed that control synaptic specificity. An alternative strategy will be to test En binding targets previously identified by other groups. 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 Engrailed 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. These molecules may be of great potential importance in neurological diseases such as Parkinson's or autism.