Genetic Control of Motor Axon Targeting

描述(由申请人提供)：发育过程中神经元电路的正确连接高度依赖于神经连接的精确网络的建立。这些神经网络的组装缺陷，包括轴突生长、延伸和引导、细胞体迁移、树突树枝形成和正常突触形成等细胞过程，会导致严重的神经功能缺陷。这项建议中描述的实验将描述在小鼠遗传屏幕上识别的脊椎动物运动神经元连接所需的新基因的功能。运动神经元介导运动、呼吸和自主神经反应的控制，并受到发育疾病(如脊髓肌萎缩症(SMA)和神经退行性疾病(如肌萎缩侧索硬化症(ALS))的深刻影响。运动神经元在脊髓腹侧和后脑发育，它们的细胞体沿着中枢神经系统的内侧轴、肩尾轴和背腹轴迁移到刻板的位置，而它们的轴突同时从中枢神经系统的特定出口点生长并导航到精确的外周靶点。尽管已经确定了许多轴突引导分子用于肢体运动神经元的引导，但我们对最初从中枢神经系统引导运动轴突的信号以及控制引导因子精确时空活动的机制的了解仍然不完整。为了确定调控运动神经元发育的新基因，我们用GFP标记的运动轴突和TD-番茄标记的运动核构建了转基因小鼠。这只记者鼠被用于ENU的突变屏幕，到目前为止，该屏幕已经确定了三个独立的突变体(绿光、错误方式和合并)，每个突变体都显示出从神经管出来的运动轴突存在缺陷。这项研究提出的目标将集中在使用基因定位策略和实验室建立的体外指导分析来克隆和鉴定Greenlight(GRL)突变。在Aim1中，将使用异交和SNP作图结合最先进的高通量测序来克隆GRL。该基因的组织特异性表达将使用原位杂交和免疫标记蛋白质，以及突变的性质(即。零、低形态、功能获得)将使用小鼠遗传学和生化进行测试。在AIM2中，GRL的功能特征将使用多学科的方法进行，包括小鼠遗传学、生物化学、体外指导分析和先进的多维成像技术，以提供对GRL的分子和细胞特性的重要见解，并阐明其在脊髓发育中的生理作用。最终，研究介导神经元寻路和连接的分子和遗传通路将有助于我们进一步了解脊髓运动神经回路的结构及其对运动活动和运动行为反应的影响。好了！

DESCRIPTION (provided by applicant): Proper wiring of neuronal circuits during development is highly dependent on the establishment of precise networks of neural connectivity. Defects in the assembly of these neural networks, which include cellular processes such as axonal growth, elongation and guidance, cell body migration, dendrite arborization and proper synapse formation, lead to severe neurological deficits. Experiments described in this proposal will characterize the function of novel genes required for vertebrate motor neuron connectivity identified in a mouse genetic screen. Motor neurons mediate the control over locomotion, respiration and autonomic responses, and are profoundly affected by developmental diseases such as spinal muscle atrophy (SMA) and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Motor neurons develop in the ventral spinal cord and hindbrain and their cell bodies migrate to stereotypical positions along the mediolateral, rostrocaudal, and dorsoventral axes of the CNS while their axons simultaneously grow from specific exit points in the CNS and navigate to precise peripheral targets. Although numerous axon guidance molecules have been identified for motor neuron guidance in the limbs, our understanding of the signals that initially guide motor axons from the CNS and the mechanisms that control the precise spatiotemporal activity of guidance factors remain fragmentary. To identify novel genes that regulate motor neuron development, a transgenic mouse with GFP-labeled motor axons and td-tomato-labeled motor nuclei was generated. This reporter mouse was used in an ENU mutagenesis screen which has identified three independent mutants (Greenlight, WrongWay, and Merge), to date, that each display defects in motor axon exiting from the neural tube. The aims proposed in this study will focus on the cloning and characterization of the Greenlight (GrL) mutation using a gene mapping strategy and in vitro guidance assays that are well established in the lab. In Aim1, GrL will be cloned using out-crosses and SNP mapping in conjunction with state-of-the-art, high-throughput sequencing. The tissue specific expression of the gene will be examined using in situ hybridization and immunolabeling for protein, and the nature of the mutation (i.e.. null, hypomorph, gain-of-function) will be tested using mouse genetics and biochemistry. In Aim2, functional characterization of GrL will be performed using a multi-disciplinary approach, including mouse genetics, biochemistry, in vitro guidance assays, and advanced multi-dimensional imaging techniques in order to provide significant insights into the molecular and cellular properties of GrL and elucidate its physiological role during spinal cord development. Ultimately, studying the molecular and genetic pathways that mediate neuronal pathfinding and connectivity should further contribute to our understanding of the structure of the spinal motor circuit and its effects on locomotor activity and motor behavioral response. !