Transcriptional mechanisms coordinating midline and motor axon guidance

描述(申请人提供)：确定神经元是如何正确指定和组装成功能回路的，将提供对神经系统发育障碍的关键洞察，并可能建议促进神经再生的治疗方法。为了实现这一目标，重要的是要了解轴突对保守的轴突引导线索家族的反应是如何调节的。Sit和Netrin以及它们的Robo和Fra/DCC受体是高度保守的信号分子，调节着电路发育的多个方面。在这里，我们建议通过定义保守的转录调节因子和Sit和Netrin的细胞表面轴突引导受体之间的功能和分子联系来研究对Sit和Netrin的反应是如何调控的，Sit和Netrin负责协调运动和中线轴突引导。此外，我们建议探索一种新发现的机制，通过该机制，Frazzleed/DCC受体胞内结构域(ICD)本身可以调节转录，从而负向调节对中线排斥剂狭缝的反应。发育中的果蝇胚胎中枢神经系统是探索调控轴突引导的转录机制的理想系统，因为强大的遗传学方法的可用，以及协调电路组装的转录因子和细胞表面受体的进化保守。这个建议的目的是确定缝隙受体Robo2和Netrin受体Frazzleed(FrA)是如何受到HB9、NKX6和Islet等转录因子的调控，以及这种调控如何指导特定的运动神经元和中间神经元群体中的路径选择。我们还将使用FACS对定义的运动神经元亚群进行分类，以及野生型和突变背景下的转录谱分析，以系统地识别 这些转录程序的其他效应器。最后，使用组合中的健壮性 在体外和体内的遗传和生化策略中，我们将评估这样的假设，即为了促进中线交叉，FrA受体经历依赖于伽马分泌酶的蛋白分解，释放一个胞内结构域(ICD)片段，该片段移位到细胞核，调节其靶基因CommIsless(中线斥力的关键负调节因子)。我们提出的研究将为哺乳动物系统中同源蛋白的研究提供信息，并可能提供药理学和遗传学策略来操纵神经元亚型和受体信号的规范。此外，我们的研究结果可能会为神经系统的各种疾病提供新的治疗靶点。

DESCRIPTION (provided by applicant): Determining how neurons are correctly specified and assembled into functional circuits will provide critical insight into developmental disorders of th nervous system and may suggest therapeutic approaches to promote nerve regeneration. To achieve this goal it is important to understand how axon responses to conserved families of axon guidance cues are regulated. Slit and Netrin, and their Robo and Fra/DCC receptors, are highly conserved signaling molecules that regulate multiple aspects of circuit development. Here, we propose to investigate how responses to Slit and Netrin are regulated by defining functional and molecular links between conserved transcriptional regulators that impart neuronal subtype identity and the cell surface axon guidance receptors for Slit and Netrin that coordinate motor and midline axon guidance. In addition, we propose to explore a newly discovered mechanism through which the Frazzled/DCC receptor intracellular domain (ICD) itself can regulate transcription to negatively regulate responses to the midline repellant Slit. Te developing Drosophila embryonic CNS is an ideal system to explore transcriptional mechanisms that regulate axon guidance because of the availability of powerful genetic approaches and the evolutionary conservation of the transcription factors and cell surface receptors that coordinate circuit assembly. The aims of this proposal are to determine how the Slit receptor Robo2 and the Netrin receptor Frazzled (Fra) are regulated by transcription factors, including Hb9, Nkx6 and Islet, and how, in turn, this regulation instructs pathway selection in defined populations of motor and interneurons. We will also use FACs sorting 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. Finally, using a combination of robust in vitro and in vivo genetic and biochemical strategies, we will evaluate the hypothesis that in order to promote midline crossing, the Fra receptor undergoes gamma-secretase dependent proteolysis to release an intracellular domain (ICD) fragment that translocates to the nucleus to regulate its target gene commissureless (a key negative regulator of midline repulsion). Our proposed research will inform studies of homologous proteins in mammalian systems and could provide pharmacologic and genetic strategies to manipulate the specification of neuronal subtypes and receptor signaling. Furthermore, the results of our research may suggest new therapeutic targets for diverse disorders of the nervous system.