Transciption Factor Codes, Guidance Receptors and Motor Axon Guidance

描述（由申请人提供）：了解不同类型的运动神经元如何被指定并引导到适当的肌肉区域，对于设计神经再生和运动神经元疾病的治疗方法至关重要。在脊椎动物和无脊椎动物的神经系统中，转录因子的组合编码决定了运动轴突通路的选择。然而，这些控制轴突引导的转录因子编码的下游靶点尚未明确。本研究旨在确定运动轴突引导的转录调节因子与控制寻径的特定轴突引导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，因为它能够标记和基因操纵个体和独特识别的运动神经元。此外，许多关键的转录调控因子以及引导线索和受体都是进化保守的；因此，在果蝇身上的发现很可能与高等脊椎动物的神经系统直接相关。这项建议的主要目的是：1)确定均匀跳过的同源盒转录因子是否直接或间接调节Unc-5轴突引导受体的表达，从而引导运动轴突背向；2)确定Slit受体Robo2对运动轴突投射到腹侧肌肉靶点的影响是否与腹侧投射的转录调节存在功能联系。3)利用互补的遗传和分子筛选方法确定背运动轴突通路选择转录调控因子的其他下游靶点。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，将用于研究Slit和Netrin受体在运动轴突引导的转录密码读取中的作用。为了确定背侧运动轴突投射的其他决定因素，我们将利用错误表达even-跳过的剂量依赖性效应，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，2)使用候选基因和cDNA微阵列对背侧运动神经元纯化群体中由even-跳过差异调节的基因进行mRNA表达筛选。公共卫生相关性：拟议的研究有可能对理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，更全面地了解神经元是如何发育和形成特定连接的，对于开发用于神经元替代的干细胞疗法，以治疗从阿尔茨海默病到肌萎缩侧索硬化症等神经系统疾病，将是无价的。

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.