Transcriptional mechanisms coordinating midline and motor axon guidance

描述（由申请人提供）：确定神经元如何被正确指定并组装成功能回路，将为神经系统的发育障碍提供关键的见解，并可能提出促进神经再生的治疗方法。为了实现这一目标，了解轴突对保守的轴突引导信号家族的反应是如何被调节的是很重要的。Slit和Netrin及其Robo和Fra/DCC受体是高度保守的信号分子，可调节电路发育的多个方面。在这里，我们建议通过定义赋予神经元亚型身份的保守转录调节因子与协调运动和中线轴突引导的Slit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Slit和Netrin的反应是如何被调节的。此外，我们建议探索一种新发现的机制，通过这种机制，frazzered /DCC受体胞内结构域（ICD）本身可以调节转录，从而负调控对中线排斥Slit的反应。发育中的果蝇胚胎中枢神经系统是探索调节轴突引导的转录机制的理想系统，因为强大的遗传方法的可用性以及协调电路组装的转录因子和细胞表面受体的进化守恒。本研究的目的是确定Slit受体Robo2和Netrin受体frzzled （Fra）如何受到转录因子（包括Hb9、Nkx6和Islet）的调控，以及这种调控如何反过来指导运动和中间神经元特定群体的通路选择。我们还将使用FACs对运动神经元的定义子集进行分类，并在野生型和突变背景下进行转录分析，以系统地识别

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.