Molecular and cellular mechanisms of target-selective axon regeneration through a plexus

臂丛位于脊髓外，是支配前肢肌肉的周围神经网络。神经丛的损伤可能发生在接触运动或出生期间，对于轴突的再生提出了一个复杂的挑战，轴突必须重新支配其原始突触目标以实现功能恢复。引导再生轴突通过神经丛的机制以及胶质细胞在这一过程中扮演的角色尚不清楚。神经丛是不同的神经汇聚在一起，将轴突分类成靶标特异性束的区域。除了神经丛之外，引导再生轴突通过一系列循序渐进的选择点来瞄准适当肌肉的分子线索也是未知的。尽管临床意义和近一个世纪的研究表明轴突再生是不精确的，但调节轴突通过神经丛和靶点特异性轴突再生的分子机制仍未得到充分研究。为了应对这一挑战，我开发了斑马鱼幼体的胸鳍，相当于四足动物的前肢，作为脊椎动物的模型系统，在其中可视化再生轴突，同时它们导航逐步选择点。四条神经，每一条都包含几十个运动轴突，在鳍丛上分类，支配胸鳍的外展肌或内收肌。在特定的选择点，单个运动轴突会从主神经干分离出来，以固定的模式支配鳍上的肌肉纤维，这取决于它们的细胞体在脊髓中的位置。在用激光横断神经后，我观察到轴突在损伤后两天内强健、有功能和特异性地再生回其原始肌肉纤维，表明存在尚未确定的局部指导线索。因此，该系统允许实时在遗传易驯化的脊椎动物的单一轴突、单一肌肉纤维水平上对轴突再生进行整体观察。在宾夕法尼亚大学Michael Granato博士的实验室里，我将使用实时成像和细胞消融来确定雪旺细胞和神经周围神经胶质细胞在再生轴突导航其第一个主要选择点-鳍丛-选择适当肌肉时所扮演的角色(目标1)。此外，我在再生过程中对失神经的鳍进行了RNA测序，以确定局部损伤相关的指导线索，并对轴突在鳍内主动导航时上调的10个候选基因进行了优先排序。我将使用原位杂交来确定候选基因的表达模式是否具有区域特异性，并通过CRISPR/Cas9突变来确定这些候选基因是否在介导靶向特异性轴突再生方面发挥功能(目标2)。总而言之，这些努力将提供一个细胞学和机械学的切入点，以检验局部线索的协调如何在再生脊椎动物中调节精确的轴突引导。通过分子生物学技术培训和我的博士后顾问委员会的指导，这项提案中的工作将建立一个完全独立的研究利基市场，我将在其中启动我自己的实验室。

The brachial plexus, located outside the spinal cord, is a network of peripheral nerves that innervate the forelimb muscles. Injury to the plexus, which can occur during contact sports or birth, presents a complex challenge for regenerating axons which must reinnervate their original synaptic targets for functional recovery. The mechanisms that guide regenerating axons through a plexus, a region where different nerves converge to sort axons into target-specific bundles, and the role that glia play in this process, are poorly understood. Beyond the plexus, the molecular cues that guide regenerating axons through a series of stepwise choice points to target the appropriate muscle are also unknown. Despite the clinical relevance and nearly a century of studies demonstrating that axon regeneration is imprecise, the molecular mechanisms that mediate axon navigation through a plexus and target-specific axon regeneration are understudied. To address this challenge, I developed the larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, as a vertebrate model system in which to visualize regenerating axons as they navigate stepwise choice points. Four nerves, each of which contains dozens of motor axons, sort at the fin plexus to innervate either the abductor or the adductor muscles of the pectoral fin. At defined choice points, individual motor axons diverge from the main nerve trunk to innervate muscle fibers on the fin in a stereotyped pattern depending on where their cell bodies are located in the spinal cord. Following transection of nerves with a laser, I observe robust, functional, and specific regeneration of axons back to their original muscle fibers within two days after injury, indicating the existence of as yet unidentified local guidance cues. Thus, this system allows for holistic observation of axon regeneration at the single-axon, single-muscle fiber level in real time in a genetically tractable vertebrate. In the lab of Dr. Michael Granato at the University of Pennsylvania, I will use live imaging and cell ablation to determine the role of Schwann cells and perineurial glia as regenerating axons navigate their first major choice point, the fin plexus, to choose the appropriate muscle (Aim 1). Additionally, I have performed RNA sequencing on denervated fins during the regeneration process to identify local injury-dependent guidance cues and have prioritized ten candidate genes that are upregulated while axons are actively navigating within the fin. I will employ in situ hybridization to determine if there is regional specificity to the expression pattern of candidate genes and CRISPR/Cas9 mutagenesis to determine if these candidate genes play a functional role to mediate target-specific axon regeneration (Aim 2). Together, these efforts will provide a cellular and mechanistic entry point to examine how coordination of local cues mediates precise axon guidance in a regenerating vertebrate. Through training in molecular biology techniques and mentorship from my postdoctoral advisory committee, the work in this proposal will establish an entirely independent research niche from which I will launch my own laboratory.