Slit and Robo Signaling in the Specification of Motor Neuron Position

运动神经元位置规范中的狭缝和 Robo 信号

• 批准号: 8517476
• 负责人: Neal Dilip Amin
• 金额: $ 3.81万
• 依托单位: SALK INSTITUTE FOR BIOLOGICAL STUDIES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8517476
• 关键词: Address; Adhesions; Affect; Amyotrophic Lateral Sclerosis; Antibodies; Cell Adhesion; Cell Adhesion Molecules; Cell-Cell Adhesion; Cells; Cessation of life; Childhood; Chimera organism; Confocal Microscopy; Coupled; Cues; Data; Databases; Defect; Degenerative Disorder; Development; Disease; Electroporation; Embryonic Development; Figs - dietary; Floor; Foundations; Goals; Immigration; Immunohistochemistry; In Situ Hybridization; Interneurons; Knock-out; Knockout Mice; Lateral; Maps; Medial; Mediating; Mediator of activation protein; Mental Retardation; Modeling; Motor; Motor Activity; Motor Neurons; Movement; Mus; Muscle; Neural tube; Neuraxis; Neurologic; Neurons; Output; Pathway interactions; Patients; Pattern; Plasmids; Positioning Attribute; Process; Protein Isoforms; Reflex action; Role; Seizures; Sensory; Signal Pathway; Signal Transduction; Skeletal Muscle; Source; Specific qualifier value; Spinal Cord; Spinal cord injury; Staging; Structure; Surface; Synapses; Testing; Time; To specify; Ventral Roots; Wild Type Mouse; axonal guidance; base; body position; combinatorial; lissencephaly; migration; mouse model; mutant; neural circuit; neuronal cell body; novel; receptor; relating to nervous system; segregation; therapeutic target; transcription factor

DESCRIPTION (provided by applicant): All muscle movements are controlled by the output of motor neurons whose cell bodies are located in the spinal cord. Corticospinal, sensory, and inter-neurons form synapses on motor neurons early in embryonic development, establishing neural circuits that enable voluntary muscle movements, reflexes, and locomotor activity. Recent studies demonstrate that the formation of specific circuits depends upon the settling position of motor neurons within the spinal cord topographic map. Though axonal guidance has been extensively studied as a component of neural connectivity, little is known about motor neuron migration and cell adhesion despite their importance in determining cell body position. Furthermore, neuronal migration defects underlie many rare but devastating neurological conditions such as Lissencephaly that end in childhood death. The combinatorial expression of transcription factors can be used to identify motor neurons based upon subtype identity. By performing immunohistochemistry using transcription factor antibody markers, the position of motor neuron subtypes was identified in spinal cords of wild type and Robo1-/-2-/- (DKO) mice. Preliminary results demonstrate multiple interneuron and motor neuron positioning defects in Robo DKO mice and, additionally, in chicks electroporated with Slit2N-expressing plasmids compared to wild-type. These results demonstrate, for the first time, that the repulsive guidance cue Slit and its receptor Robo influence motor neuron and interneuron settling position during critical stages of motor circuit development. In-situ hybridization data identifies Slit2 and Robo co-expression by motor neurons, and it is hypothesized that these molecules are engaged in a novel cell-autonomous signaling pathway that potentially silences motor neurons from a Slit2 repulsive gradient established by the floor plate. Conditional knockout mice will be used to genetically ablate Slit2 expression in motor neurons and in the floor plate, and defects in motor neuron position will be identified to determine if cell-autonomous signaling is occurring (Aim1). Since Slit and Robo are known mediators of cell adhesion and adhesion molecules regulate motor neuron subtype clustering, it is hypothesized that mis-positioning defects in Robo DKO mice are due to disrupted cell-cell adhesion. This possibility will be investigated by performing an adhesion molecule screen to visualize changes in their expression and localization in motor neurons (Aim2). Finally, preliminary results demonstrate motor column- specific expression patterns of Robo isoforms and motor column-specific mis-positioning defects in Robo DKO mutants. From these findings, it is hypothesized that Robo isoforms mediate motor column-specific actions. By studying neuronal mis-positioning defects in Robo1-/- and Robo2-/- single knockout mice, it will be determined if Robo isoforms have differential signaling capacities (Aim3). Taken together, these proposed studies will help uncover fundamental mechanisms by which Slit and Robo are able to specify motor neuron settling position, a critical determinant of synaptic connectivity in the spinal cord.

描述（由申请人提供）：所有肌肉运动均由其细胞体位于脊髓中的运动神经元的输出控制。皮质脊髓、感觉和中间神经元在胚胎发育早期在运动神经元上形成突触，建立神经回路，实现随意肌肉运动、反射和运动活动。最近的研究表明，特定回路的形成取决于运动神经元在脊髓地形图中的固定位置。尽管轴突引导作为神经连接的一个组成部分已被广泛研究，但人们对运动神经元迁移和细胞粘附知之甚少，尽管它们在确定细胞体位置方面很重要。此外，神经元迁移缺陷是许多罕见但具有破坏性的神经系统疾病的基础，例如导致儿童死亡的无脑畸形。转录因子的组合表达可用于根据亚型身份识别运动神经元。通过使用转录因子抗体标记物进行免疫组织化学，确定了野生型和 Robo1-/-2-/- (DKO) 小鼠脊髓中运动神经元亚型的位置。初步结果表明，与野生型相比，Robo DKO 小鼠以及电穿孔 Slit2N 表达质粒的雏鸡存在多个中间神经元和运动神经元定位缺陷。这些结果首次证明，排斥引导信号 Slit 及其受体 Robo 在运动回路发育的关键阶段影响运动神经元和中间神经元的稳定位置。原位杂交数据识别了运动神经元的 Slit2 和 Robo 共表达，并且假设这些分子参与了一种新型细胞自主信号传导途径，该途径可能使运动神经元免受底板建立的 Slit2 排斥梯度的影响。条件性基因敲除小鼠将用于从基因上消除运动神经元和底板中的 Slit2 表达，并且将鉴定运动神经元位置的缺陷以确定细胞自主信号传导是否正在发生（目标 1）。由于 Slit 和 Robo 是已知的细胞粘附介质，并且粘附分子调节运动神经元亚型聚类，因此推测 Robo DKO 小鼠的错误定位缺陷是由于细胞间粘附破坏所致。将通过进行粘附分子筛选来研究这种可能性，以观察其在运动神经元中的表达和定位的变化（Aim2）。最后，初步结果证明了 Robo 同工型的运动柱特异性表达模式和 Robo DKO 突变体中运动柱特异性的错位缺陷。根据这些发现，推测 Robo 同种型介导运动柱特异性动作。通过研究 Robo1-/- 和 Robo2-/- 单基因敲除小鼠的神经元错位缺陷，将确定 Robo 亚型是否具有差异信号传导能力 (Aim3)。总而言之，这些拟议的研究将有助于揭示 Slit 和 Robo 能够指定运动神经元稳定位置的基本机制，这是脊髓突触连接的关键决定因素。