Genetic Control of Motor Axon Targeting

运动轴突靶向的遗传控制

• 批准号: 8442034
• 负责人: ONANONG CHIVATAKARN
• 金额: $ 5.57万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-16 至 2013-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8442034
• 关键词: Affect; Amyotrophic Lateral Sclerosis; Atrophic; Axon; Behavior; Behavioral; Biochemistry; Biological Assay; Biological Neural Networks; Candidate Disease Gene; Cell Nucleus; Cell physiology; Cells; Chick Embryo; Chromosome Mapping; Cloning; Cues; Defect; Dendrites; Development; Disease; Embryo; Embryonic Development; Ethylnitrosourea; Exhibits; Genes; Genetic; Genetic Screening; Genome; Genotype; Goals; Growth; Growth Cones; Hybrids; Imaging Techniques; Immunoblotting; Immunohistochemistry; In Situ Hybridization; In Vitro; Inbred DBA Mice; Label; Lead; Lesion; Limb structure; Locomotion; Maps; Mediating; Messenger RNA; Molecular; Molecular Genetics; Morphology; Motor; Motor Activity; Motor Neurons; Mus; Muscle; Muscular Atrophy; Mutagenesis; Mutant Strains Mice; Mutation; Nature; Neural tube; Neurodegenerative Disorders; Neurologic; Neurons; Pathology; Pathway interactions; Pattern; Peripheral; Phenotype; Physiological; Point Mutation; Positioning Attribute; Process; Property; Proteins; RNA Sequences; RNA Splicing; Recovery; Reporter; Respiration; Role; Signal Pathway; Signal Transduction; Single Nucleotide Polymorphism Map; Small Interfering RNA; Spinal; Spinal Cord; Staging; Structure; Techniques; Testing; Time; Tissue-Specific Gene Expression; Tissues; Tomatoes; Training; Transcript; Transgenic Mice; Ventral Roots; axon growth; axon guidance; cell type; design; gain of function; gain of function mutation; hindbrain; in vivo; insight; migration; motor control; motor neuron development; mutant; neuronal cell body; next generation; novel; protein distribution; protein expression; rapid technique; research study; response; spatiotemporal; synaptogenesis

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. !

描述（由申请人提供）：发育过程中神经元回路的正确连接高度依赖于精确神经连接网络的建立。这些神经网络的组装缺陷，包括细胞过程，如轴突生长、延伸和引导、细胞体迁移、树突乔木化和适当的突触形成，导致严重的神经功能障碍。本提案中描述的实验将描述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、呼吸和自主神经反应的控制，并受到发育性疾病（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中外侧、背侧和背侧轴迁移到典型位置，同时它们的轴突从中枢神经系统的特定出口点生长并导航到精确的外周目标。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精确时空活动的引导因子的机制的理解仍然是碎片化的。为了鉴定调控运动神经元发育的新基因，我们培育了具有gfp标记的运动轴突和td-番茄标记的运动细胞核的转基因小鼠。该报告小鼠用于ENU突变筛选，迄今已鉴定出三个独立的突变体（Greenlight， WrongWay和Merge），每个突变体都显示出神经管外运动轴突的缺陷。本研究的目的是利用基因定位策略和在实验室中建立的体外指导试验，对Greenlight （GrL）突变进行克隆和表征。在Aim1中，GrL将使用外杂交和SNP图谱结合最先进的高通量测序进行克隆。该基因的组织特异性表达将使用原位杂交和蛋白质免疫标记进行检查，以及突变的性质(即…Null, hypomorph, gain- function)将使用小鼠遗传学和生物化学进行测试。在Aim2中，GrL的功能表征将采用多学科方法进行，包括小鼠遗传学，生物化学，体外引导试验和先进的多维成像技术，以提供对GrL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途径将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。！