Engrailed and the Control of Synaptic Circuitry in Drosophila

描述（由申请人提供）：Engrailed是一种普遍存在的转录调节剂，对人类健康具有潜在的重要意义。它最近与中脑多巴胺能神经元的命运决定和存活有关，En基因敲除小鼠表现出帕金森样症状，也与自闭症谱系障碍有关。因此，有必要了解En在调节神经元连接中所起的作用。这项研究的长期目标是使用具有可识别神经元的简单模型系统，研究Engrailed如何控制突触目标识别，以及它调节的下游效应基因是什么。我之前的工作是用蟑螂谷物系统来研究这个问题；然而，这一制度的进一步发展存在着重大障碍。这个试点项目的目的是使我能够在遗传上易于处理的黑腹果蝇中开发一个类似的可识别的表达神经元系统。第一个目标是利用Gal4-UAS在正常表达Engrailed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描述这些神经元在一系列感觉系统中的特征，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学和解剖学技术，以及导师罗德·墨菲博士的专业知识，专注于这些感觉系统中的一个，以便能够测试Engrailed在控制该系统突触连通性方面的作用。第三个目标将涉及异位Engrailed与GFP的共表达，从而能够直接测试Engrailed控制电路连通性的观点。最后，在罗德·墨菲博士的帮助下，我将使用基因筛选开始寻找Engrailed下游控制突触特异性的其他基因。另一种策略将是测试以前由其他小组确定的En结合目标。

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.