Transciption Factor Codes, Guidance Receptors and Motor Axon Guidance

描述(由申请人提供)：了解不同类别的运动神经元是如何被指定并引导到适当的肌肉领域，对于设计神经再生和运动神经元疾病的治疗方法是至关重要的。众所周知，在脊椎动物和无脊椎动物的神经系统中，转录因子的组合密码决定了运动轴突路由的选择；然而，控制轴突引导的这些转录因子密码的下游靶点却没有明确的定义。这项提议试图定义运动轴突导引转录调节因子和控制路径发现的特定轴突导引受体之间的功能联系。果蝇胚胎神经系统是解决这些问题的一个有吸引力的模型，因为它有能力标记和遗传操作单个和唯一识别的运动神经元。此外，许多关键的转录调节因子以及指导信号和受体在进化上是保守的；因此，在果蝇中的发现很可能与高等脊椎动物神经系统直接相关。该建议的主要目的是：1)确定偶数跳过的同源盒转录因子是否直接或间接调节UNC-5轴突导向受体的表达，从而引导运动轴突背向；2)确定缝隙受体Robo2‘S对投射到腹肌靶点的运动轴是否反映出与腹侧投射的转录调控有关的功能联系；以及3)利用互补的遗传和分子筛选手段寻找背侧运动轴突路选择转录调控的额外下游靶点。经典的遗传和生化技术，包括遗传相互作用测试、突变分析和错误表达实验，将被用来研究Sit和Netrin受体在帮助读出运动轴突导引的转录密码中的作用。为了确定背运动轴突投射的其他决定因素，我们将利用错误表达偶数跳过的剂量依赖效应来1)对这种错误表达表型的显性增强子和抑制子进行遗传筛选，以及2)使用候选基因和cDNA微阵列进行mRNA表达筛选，以寻找在纯化的背部投射运动神经元群体中受偶跳过差异调控的基因。公共卫生相关性：拟议的研究有可能对了解神经系统发育障碍做出重要贡献，并可能提出促进脑和脊髓损伤后再生的新策略。此外，更完整地了解神经元是如何发展和形成特定连接的，对于开发干细胞疗法用于神经元替代治疗从阿尔茨海默病到肌萎缩侧索硬化症等神经疾病将是非常宝贵的。

DESCRIPTION (provided by applicant): Understanding how distinct classes of motor neurons are specified and guided to appropriate muscle domains is of fundamental importance to the design of therapeutic approaches to nerve regeneration and motor neuron disease. It is well established that combinatorial codes of transcription factors dictate motor axon pathway selection in both vertebrate and invertebrate nervous systems; however, the downstream targets of these transcription factor codes that control axon guidance are poorly defined. This proposal seeks to define functional links between transcriptional regulators of motor axon guidance and specific axon guidance receptors that control path finding. The Drosophila embryonic nervous system is an attractive model in which to address these questions because of the ability to label and genetically manipulate individual and uniquely identified motor neurons. In addition, many of the key transcriptional regulators as well as the guidance cues and receptors are evolutionarily conserved; thus, findings in Drosophila are very likely to be directly relevant to higher vertebrate nervous systems. The major aims of this proposal are: 1) to determine whether the even-skipped homeobox transcription factor directly or indirectly regulates the expression of the Unc-5 axon guidance receptor to guide motor axons dorsally, 2) to determine whether the Slit receptor Robo2's influence on motor axons that project to ventral muscle targets reflects a functional link with the transcriptional regulation of ventral projection, and 3) to identify additional downstream targets of the transcriptional regulators of dorsal motor axon pathway selection using complementary genetic and molecular screening approaches. Classical genetic and biochemical techniques, including genetic interaction tests, mutant analysis, and mis-expression experiments will be used to investigate the role of Slit and Netrin receptors in contributing to the readout of the transcriptional code for motor axon guidance. To identify additional determinants of dorsal motor axon projection, we will take advantage of dose-dependent effects of mis-expressing even-skipped to 1) perform genetic screens for dominant enhancers and suppressors of this mis-expression phenotypes and 2) perform mRNA expression screens using candidate genes and cDNA microarrays for genes that are differentially regulated by even-skipped in purified populations of dorsally projecting motor neurons. PUBLIC HEALTH RELEVANCE: The proposed research has the potential to make important contributions to the understanding of developmental disorders of the nervous system and may suggest new strategies to promote regeneration after brain and spinal cord injury. In addition, a more complete understanding of how neurons develop and form specific connections will be invaluable for developing stem cell therapies for neuronal replacement to treat neurological disorders ranging from Alzheimer's disease to Amyotrophic Lateral Sclerosis.