Molecular mechanisms of axon guidance receptor regulation and signaling

项目总结 确定神经元如何被正确指定并组装成功能电路将提供 对神经系统发育障碍的洞察，并可能提出治疗方法 促进神经再生。Sit和Netrin以及它们的Robo和Fra/DCC受体是进化的 保守的信号分子家族，在调节神经元发育中发挥重要作用； 然而，对这些受体是如何调节的以及它们如何向直接轴突发出信号的理解 增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。缝隙、网状结构和它们 受体在神经系统之外也扮演着重要的角色，这些通路的中断 与几种癌症有关。我们的研究计划集中在与以下三个方面相关的广泛领域 这些分子在神经细胞发育中的作用和调控 以果蝇胚胎神经系统为模型。首先，我们正在努力定义函数和 传递神经元亚型特性的保守转录调控因子与 协调轴突引导和树突形态发生的Robo和Fra/DCC受体 对斯利特和内特林的回应。在这里，我们将使用遗传和分子筛选方法，包括 确定的运动神经元亚群的荧光激活细胞分类(FACS)以及 野生型和突变型背景下的转录谱分析，以系统地识别更多的效应因子 这些转录程序。第二，我们正在通过一种新发现的机制来描述 FRAMZLED/DCC受体胞内域(ICD)本身可以在细胞核中作为一种 转录激活剂调节无连合表达以确保连合轴突避免 对中线排斥缝隙的过早反应。在这里，我们将使用遗传和分子方法来 确定与Fra ICD合作调节转录和转录图谱方法的因素 以确定Fra ICD的其他目标。此外，我们还将探讨来自 细胞核是轴突引导受体的共同属性，通过合作，我们将测试这一点 是引导感受器的保守属性。第三，我们正在确定分子机制 在轴突引导过程中使用分子，遗传， 生物化学和细胞生物学方法。具体地说，我们一起使用体内的基因操作 用荧光标记的受体和体外信号分子活性的报告，来定义细胞 具有亚细胞分辨率的受体激活的生物输出。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 这可能会增强我们对神经发育障碍的理解。

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.