Regulation of Nervous System Wiring by the Robo3 Axon Guidance Receptor and its Ligand NELL2

项目总结 轴突对其正确靶点的引导是神经系统连接的重要一步，但分子和细胞 轴突引导的机制仍不完全清楚。这项拟议研究的目标是阐明 一种新的轴突引导信号NELL2及其受体Robo3在神经发育中的作用及其分子定义 NELL2-Robo3信号传导机制。而受体Robo1和Robo2通过 狭缝家族的引导分子，发散的Robo家族成员Robo3不结合狭缝，而是沉默 Sit-Robo1/2信令。此外，Robo3通过Netrin-1介导的Netrin-1间接增强轴突吸引 受体DCC。我们最近发现了一种新的引导信号，NELL2，作为先前孤儿的排斥配体 受体Robo3。我们产生了NELL2基因敲除小鼠，并发现NELL2/Robo3介导的排斥引导 胚胎脊髓中的连合轴突。我们的发现为轴突定义了一条新的NELL2-Robo3信号通路 引导并提高它连接整个神经系统的神经回路的可能性。为了测试这一想法，我们将 包括基因报告在内的多种手段分析NELL2和Robo3在神经发育过程中的表达 台词。将检查在NELL2表达部位附近表达Robo3或投射轴突的神经元群体 NELL2体外反应。在这些轴突引导分析中，将使用Robo3的神经元来测试对Robo3的依赖 突变的小鼠。为了确定NELL2和Robo3在连接感兴趣神经元群体、轴突中的体内作用 NELL2和Robo3缺陷小鼠的轨迹将通过胚胎神经系统的整体成像进行分析 并通过几种互补的方法。基于初步的表达数据，初步研究将集中在视网膜上 神经节细胞轴突引导。我们来自连合神经元的结果表明，Robo3在轴突引导中的功能是三- 折叠，因为它通过Robo1/2抑制缝隙诱导的排斥力，通过DCC增强Netrin-1的吸引力，并介导 来自自身配体NELL2的排斥力。目前尚不清楚哪些细胞内信号通路参与了下游的 NELL2以及Robo3如何能够同时执行其多种功能。为了解决这些问题，我们将测试 在Robo3体外结构-功能分析中，如果狭缝沉默、Netrin-1吸引和NELL2排斥可以分开 在活体内。使用候选和无偏见的生化方法，我们将识别新的NELL2受体和 Robo3信号的下游介体及其在体外轴突引导和神经回路形成中的作用 在活体内。我们的工作有望为NELL2和Robo3在大脑连接中的功能提供重要的见解。会的 也有助于阐明不同的轴突引导信号通路如何在同一神经元内相互作用，这是一个基本的 神经发育中的一个问题，目前仍知之甚少。通过定义轴突寻路的机制，提出了 这项工作有可能为旨在恢复物理损伤的轴突连接的治疗方法提供信息 神经退行性疾病的损伤或发作。此外，它还可以帮助确定本病的发病机制。 神经系统连接错误导致的疾病。

PROJECT SUMMARY The guidance of axons to their correct targets is an important step in nervous system wiring, but the molecular and cellular mechanisms of axon guidance are still not completely understood. The goal of the proposed research is to elucidate the functions of a new axon guidance cue, NELL2, and its receptor Robo3 in neural development and to define the molecular mechanisms mediating NELL2-Robo3 signaling. While the receptors Robo1 and Robo2 mediate axon repulsion by guidance molecules of the Slit family, the divergent Robo family member Robo3 does not bind Slits but instead silences Slit-Robo1/2 signaling. Furthermore, Robo3 indirectly potentiates axonal attraction by Netrin-1, mediated by the Netrin receptor DCC. We recently identified a novel guidance cue, NELL2, as a repulsive ligand for the previously orphan receptor Robo3. We generated NELL2 knockout mice and found that NELL2/Robo3-mediated repulsion guides commissural axons in the embryonic spinal cord. Our findings define a novel NELL2-Robo3 signaling pathway for axon guidance and raise the possibility that it wires neural circuits throughout the nervous system. To test this idea, we will analyze the expression of NELL2 and Robo3 during neural development by several means, including genetic reporter lines. Neuronal populations that express Robo3 or project axons close to sites of NELL2 expression will be examined for NELL2 responses in vitro. In these axon guidance assays, Robo3-dependence will be tested using neurons from Robo3 mutant mice. To determine the in vivo roles of NELL2 and Robo3 in wiring neuronal populations of interest, axonal trajectories in NELL2- and Robo3-deficient mice will be analyzed by in toto imaging of the embryonic nervous system and by several complementary approaches. Based on preliminary expression data, initial studies will focus on retinal ganglion cell axon guidance. Our results from commissural neurons show that Robo3 function in axon guidance is three- fold, as it inhibits Slit-induced repulsion through Robo1/2, potentiates Netrin-1 attraction through DCC, and mediates repulsion from its own ligand NELL2. It is unclear which intracellular signaling pathways are engaged downstream of NELL2 and how Robo3 is able to perform its multiple functions simultaneously. To address these questions, we will test if Slit silencing, Netrin-1 attraction, and NELL2 repulsion can be separated in Robo3 structure-function analyses in vitro and in vivo. Using candidate and unbiased biochemical approaches, we will identify novel NELL2 receptors and downstream mediators of Robo3 signaling and determine their roles in axon guidance in vitro and neural circuit formation in vivo. Our work is expected to provide important insights into the functions of NELL2 and Robo3 in brain wiring. It will also help elucidate how different axon guidance signaling pathways interact within the same neuron, a fundamental question in neural development that is still poorly understood. By defining mechanisms of axon pathfinding, the proposed work has the potential to inform therapeutic approaches aimed at restoring damaged axonal connections after physical injury or onset of neurodegenerative disease. Furthermore, it can help define the mechanisms underlying the etiology of diseases that result from nervous system mis-wiring.