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）：所有肌肉运动由运动神经元的输出控制，其细胞体位于脊髓。在胚胎发育早期，皮质脊髓神经元、感觉神经元和间神经元在运动神经元上形成突触，建立神经回路，使随意肌运动、反射和运动活动成为可能。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沉降位置。尽管轴突引导作为神经连通性的一个组成部分被广泛研究，但运动神经元迁移和细胞粘附在决定细胞体位置方面的重要性却鲜为人知。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，如无脑畸形，最终导致儿童死亡。转录因子的组合表达可用于基于亚型身份的运动神经元识别。利用转录因子抗体标记进行免疫组化，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，表达slit2n质粒电穿孔的Robo DKO小鼠和小鸡中存在多个中间神经元和运动神经元定位缺陷。这些结果首次证明了斥力引导线索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定位。原位杂交数据确定了运动神经元中Slit2和Robo的共表达，并假设这些分子参与了一种新的细胞自主信号通路，可能使运动神经元从地板建立的Slit2排斥梯度中沉默。条件敲除小鼠将被用于从基因上切除运动神经元和底板中的Slit2表达，并将识别运动神经元位置的缺陷，以确定是否发生细胞自主信号传导（Aim1）。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，因此假设Robo DKO小鼠的错误定位缺陷是由于细胞-细胞粘附被破坏。这种可能性将通过粘附分子筛选来观察它们在运动神经元中的表达和定位的变化（Aim2）。最后，初步结果表明，在Robo DKO突变体中，Robo异构体的运动柱特异性表达模式和运动柱特异性错误定位缺陷。根据这些发现，假设Robo同工型介导运动柱特异性动作。通过研究Robo1-/-和Robo2-/-单敲除小鼠的神经元错位缺陷，将确定Robo异构体是否具有差异信号传导能力（Aim3）。综上所述，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定位的基本机制，运动神经元定位是脊髓突触连接的关键决定因素。