Slit and Robo Signaling in the Specification of Motor Neuron Position

描述(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。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，使肌肉能够随意运动、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安置位置。虽然轴突引导作为神经连接的一个组成部分已被广泛研究，但对运动神经元迁移和细胞黏附知之甚少，尽管它们在确定细胞体位置方面具有重要作用。此外，神经元迁移缺陷是许多罕见但毁灭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同一性识别运动神经元。采用免疫组织化学方法，对野生型和Robo1-/-2-/-(DKO)小鼠脊髓运动神经元亚型进行定位。初步结果表明，与野生型相比，在Robo DKO小鼠中存在多个中间神经元和运动神经元定位缺陷，此外，在电穿孔Slit2N表达质粒的鸡中也存在缺陷。这些结果首次证明，在运动神经回路发育的关键阶段，排斥性引导线索狭缝及其受体Robo影响运动神经元和中间神经元的稳定位置。原位杂交数据证实运动神经元共表达Slit2和Robo，并假设这些分子参与了一种新的细胞自主信号通路，该通路潜在地使运动神经元从底板建立的Slit2排斥梯度中沉默。条件基因敲除小鼠将被用来从基因上消除运动神经元和底板中Slit2的表达，并将识别运动神经元位置的缺陷，以确定是否发生了细胞自主信号(Aim1)。由于Sit和Robo是已知的细胞黏附介质，黏附分子调节运动神经元亚型聚集，推测Robo DKO小鼠的错位缺陷是由于细胞-细胞黏附中断所致。这种可能性将通过进行黏附分子筛查来可视化它们在运动神经元中表达和定位的变化(AIM2)。最后，初步结果证明了Robo亚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错位缺陷。根据这些发现，推测Robo亚型介导了运动柱特异的动作。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能力(Aim3)。综上所述，这些拟议的研究将有助于揭示Sit和Robo能够确定运动神经元稳定位置的基本机制，这是脊髓中突触连接的关键决定因素。 公共卫生相关性：我的提案将调查影响运动神经元迁移的因素，运动神经元迁移是中枢神经系统胚胎发育过程中的一个基本过程。神经元迁移的缺陷是破坏性疾病的基础，如轻脑和皮质下带状异位，这些疾病困扰着癫痫发作、精神发育迟缓和早期死亡。我对运动神经元(及其神经元间突触伙伴)迁移的研究可能会为21世纪的治疗奠定基础，例如那些可能允许靶向治疗新神经元的治疗方法，以取代那些因退行性疾病(例如肌萎缩侧索硬化症)或脊髓损伤而受损的神经元。

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. PUBLIC HEALTH RELEVANCE: My proposal will investigate the factors that influence motor neuron migration, a fundamental process occurring during the embryonic development of the central nervous system. Defects in neuronal migration underlie devastating diseases such as Lissencephaly and Subcortical Band Heterotopia that afflict patients with seizures, mental retardation, and early death. My study of migration in motor neurons (and their interneuron synaptic partners) may lay the foundations for twenty-first century treatments such as those that might permit targeting the therapeutic delivery of new neurons to replace those damaged from degenerative diseases (e.g. amyotrophic lateral sclerosis) or spinal cord injury.