Transcriptional mechanisms coordinating midline and motor axon guidance

描述（由申请人提供）：确定神经元如何正确指定和组装成功能回路将提供对神经系统发育障碍的重要见解，并可能提出促进神经再生的治疗方法。 为了实现这一目标，重要的是要了解如何轴突反应的保守家族的轴突导向线索进行调节。 Slit和Netrin以及它们的Robo和Fra/DCC受体是高度保守的信号分子，它们调节回路发育的多个方面。 在这里，我们建议调查如何响应Slit和Netrin的调节，通过定义保守的转录调节因子之间的功能和分子联系，赋予神经元亚型的身份和细胞表面轴突指导受体Slit和Netrin协调电机和中线轴突指导。 此外，我们建议探索一种新发现的机制，通过这种机制，Frazzled/DCC受体胞内结构域（ICD）本身可以调节转录，以负调节对中线排斥性Slit的反应。发育中的果蝇胚胎中枢神经系统是探索调控轴突导向的转录机制的理想系统，因为强大的遗传方法的可用性和协调电路组装的转录因子和细胞表面受体的进化保守性。 该提案的目的是确定Slit受体Robo 2和Netrin受体Frazzled（Fra）如何受转录因子（包括Hb 9，Nkx 6和Islet）的调节，以及这种调节如何反过来指导运动神经元和中间神经元的特定群体中的通路选择。 我们还将使用运动神经元的定义子集的FACs分选，以及野生型和突变背景中的转录谱分析，以系统地鉴定 这些转录程序的额外效应子。 最后，使用鲁棒的 通过体外和体内遗传和生物化学策略，我们将评估以下假设：为了促进中线交叉，Fra受体经历γ-分泌酶依赖性蛋白水解以释放胞内结构域（ICD）片段，该片段易位到细胞核以无接合地调节其靶基因（中线排斥的关键负调节因子）。 我们提出的研究将为哺乳动物系统中同源蛋白质的研究提供信息，并可以提供药理学和遗传学策略来操纵神经元亚型和受体信号传导的规范。 此外，我们的研究结果可能为各种神经系统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.