Genetic Control of Motor Axon Targeting

描述（由申请人提供）：发育期间神经元回路的正确布线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，包括细胞过程，如轴突生长，伸长和引导，细胞体迁移，树突树枝化和适当的突触形成，导致严重的神经功能缺损。本提案中描述的实验将表征在小鼠遗传筛选中鉴定的脊椎动物运动神经元连接所需的新基因的功能。运动神经元介导对运动、呼吸和自主反应的控制，并且受到诸如脊髓性肌萎缩（SMA）的发育疾病和诸如肌萎缩性侧索硬化（ALS）的神经变性疾病的深刻影响。运动神经元在腹侧脊髓和后脑中发育，它们的细胞体沿着CNS的中外侧、吻尾侧和背腹侧轴迁移到常规位置，而它们的轴突同时从CNS中的特定出口点生长并导航到精确的外周靶点。虽然许多轴突导向分子已被确定为运动神经元的指导在四肢，我们的理解，最初引导运动轴突从中枢神经系统的信号和机制，控制精确的时空活动的指导因素仍然支离破碎。为了鉴定调节运动神经元发育的新基因，产生了具有GFP标记的运动轴突和td-番茄标记的运动核的转基因小鼠。该报告小鼠用于ENU诱变筛选，该筛选迄今已鉴定出三种独立的突变体（Greenlight、WrongWay和Merge），每种突变体在从神经管退出的运动轴突中显示缺陷。本研究中提出的目标将集中在使用基因定位策略和在实验室中建立良好的体外指导测定的绿光（GrL）突变的克隆和表征。在Aim 1中，GrL将使用异交和SNP作图结合最先进的高通量测序进行克隆。将使用原位杂交和蛋白质免疫标记检查基因的组织特异性表达，以及突变的性质（即，无效、亚型、功能获得）将使用小鼠遗传学和生物化学进行测试。在Aim 2中，GrL的功能表征将使用多学科方法进行，包括小鼠遗传学，生物化学，体外指导试验和先进的多维成像技术，以提供GrL的分子和细胞特性的重要见解，并阐明其在脊髓发育过程中的生理作用。最终，研究介导神经元寻路和连接的分子和遗传途径应进一步有助于我们理解脊髓运动回路的结构及其对运动活动和运动行为反应的影响。! 公共卫生相关性：运动神经元在发育性疾病如脊髓运动萎缩（SMA）和神经退行性疾病如肌萎缩性侧索硬化（ALS）中选择性丢失。虽然这些疾病病理学的基础机制在很大程度上是未知的，但已知有遗传成分。通过鉴定和表征调节运动神经元生长、指导和连接的基因（及其信号传导途径），治疗可以有效地靶向脊髓，促进在这种衰弱性疾病中丧失的运动功能的恢复。

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. ! PUBLIC HEALTH RELEVANCE: Motor neurons are selectively lost in developmental diseases such as spinal motor atrophy (SMA) and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Although the mechanisms that underlie these disease pathologies are largely unknown, it is known that there is a genetic component. By identifying and characterizing genes (and their signaling pathways) that regulate motor neuron growth, guidance, and connectivity, therapies may be effectively targeted within the spinal cord, facilitating recovery of motor function that is lost in such debilitating diseases.