Molecular mechanisms of axon guidance receptor regulation and signaling

项目摘要 确定神经元是如何被正确指定并组装成功能电路的， 深入了解神经系统发育障碍，并可能提出治疗方法， 促进神经再生。Slit和Netrin，以及它们的Robo和Fra/DCC受体， 在调节神经元发育中发挥重要作用的信号分子保守家族; 然而，了解这些受体是如何调节的，以及它们是如何发出信号引导轴突的， 成长和指导是不完整的。这些都是重要的问题，因为这些扰动 信号通路与神经系统发育疾病有关。狭缝、Netrin及其 受体也在神经系统外发挥重要作用， 与几种癌症有关。我们的研究计划侧重于三个广泛的领域， 这些分子在神经元发育中的作用和调节， 以果蝇胚胎神经系统为模型。首先，我们正在努力定义功能和 赋予神经元亚型身份的保守转录调节因子之间的分子联系， Robo和Fra/DCC受体协调轴突导向和树突形态发生， 对Slit和Netrin的回应。在这里，我们将使用遗传和分子筛选方法，包括 运动神经元的确定子集的荧光激活细胞分选（FACs），连同 在野生型和突变体背景中的转录谱分析，以系统地鉴定 这些转录程序。其次，我们正在描述一种新发现的机制， 其中Frazzled/DCC受体胞内结构域（ICD）本身可以在细胞核中作为一种免疫调节剂起作用。 转录激活因子调节无连合表达，以确保连合轴突避免 对中线驱避剂Slit的过早反应在这里，我们将使用遗传和分子方法， 鉴定与Fra ICD合作调节转录的因子和转录物分析方法 以确定联邦储备委员会ICD的其他目标。此外，我们将探讨是否从信号 核是轴突导向受体的共同属性，通过合作，我们将测试这一点， 是导向受体的保守特性第三，我们正在确定 使用分子，遗传， 生物化学和细胞生物学方法。具体来说，我们使用体内基因操作， 与荧光标记的受体和报告信号分子活性在体外，以确定细胞 受体激活的生物输出与亚细胞分辨率。我们的研究计划将定义 轴突引导分子生物学中的新概念，为相关蛋白质的研究提供了信息， 哺乳动物系统，并可能会增强我们对神经发育障碍的了解。

PROJECT SUMMARY Determining how neurons are correctly specified and assembled into functional circuits will provide insight into developmental disorders of the nervous system and may suggest therapeutic approaches to promote nerve regeneration. Slit and Netrin, and their Robo and Fra/DCC receptors, are evolutionary conserved families of signaling molecules that play important roles in regulating neuronal development; however, the understanding of how these receptors are regulated and how they signal to direct axon growth and guidance is incomplete. These are important questions because perturbations of these signaling pathways are implicated in diseases of nervous system development. Slits, Netrins and their receptors also play essential roles outside of the nervous system and disruptions of these pathways are associated with several kinds of cancer. Our research program focuses on three broad areas related to the roles and regulation of these molecules in neuronal development using the genetically tractable Drosophila embryonic nervous system as a model. First, we are working to define functional and molecular links between conserved transcriptional regulators that impart neuronal subtype identity and the Robo and Fra/DCC receptors that coordinate axon guidance and dendrite morphogenesis in response to Slit and Netrin. Here, we will use genetic and molecular screening approaches, including Fluorescence Activated Cell sorting (FACs) of defined subsets of motor neurons, together with transcript profiling in wild type and mutant backgrounds, to systematically identify additional effectors of these transcriptional programs. Second, we are characterizing a newly discovered mechanism through which the Frazzled/DCC receptor intracellular domain (ICD) itself can act in the nucleus as a transcriptional activator to regulate commissureless expression to ensure that commissural axons avoid premature responses to midline repellent Slit. Here, we will use genetic and molecular approaches to identify factors that cooperate with the Fra ICD to regulate transcription and transcript profiling methods to identify additional targets of the Fra ICD. In addition, we will explore whether signaling from the nucleus is a common property of axon guidance receptors and through collaboration we will test if this is a conserved property of guidance receptors. Third, we are determining the molecular mechanisms underlying Robo and Fra/DCC receptor signaling during axon guidance using molecular, genetic, biochemical and cell biological approaches. Specifically, we use in vivo genetic manipulation, together with fluorescently tagged receptors and reporters of signaling molecule activity in vitro, to define the cell biological outputs of receptor activation with sub-cellular resolution. Our research program will define new concepts in the molecular biology of axon guidance, inform studies of related proteins in mammalian systems and will likely enhance our understanding of neural developmental disorders.