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。在接触性运动或分娩过程中可能发生的神经丛损伤，对再生轴突提出了复杂的挑战，轴突必须重新神经支配其原始突触靶点以实现功能恢复。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所起的作用，目前还知之甚少。神经丛是不同神经汇聚的区域，可以将轴突分类成目标特定的神经束。在神经丛之外，引导再生轴突通过一系列逐步选择点以靶向适当肌肉的分子线索也是未知的。尽管临床相关性和近世纪的研究表明，轴突再生是不精确的，介导轴突导航通过神经丛和靶特异性轴突再生的分子机制研究不足。为了应对这一挑战，我开发了幼斑马鱼胸鳍，相当于四足动物的前肢，作为脊椎动物模型系统，在其中可视化再生轴突，因为他们导航逐步选择点。四个神经，其中每一个包含几十个运动轴突，排序在鳍丛，以支配胸鳍的展肌或收肌。在确定的选择点，单个运动轴突从主神经干分叉，以刻板的模式支配鳍上的肌纤维，这取决于它们的细胞体在脊髓中的位置。在用激光横切神经后，我观察到损伤后两天内轴突恢复到原始肌纤维的强大的，功能性的和特异性的再生，这表明存在尚未识别的局部指导线索。因此，该系统允许在遗传上易处理的脊椎动物中真实的时间内在单轴突、单肌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博士的实验室中，我将使用活体成像和细胞消融来确定雪旺细胞和神经束膜胶质细胞在再生轴突导航其第一个主要选择点（鳍丛）以选择适当肌肉时的作用（目标1）。此外，我在再生过程中对失神经鳍进行了RNA测序，以确定局部损伤依赖的指导线索，并优先考虑了10个候选基因，这些基因在轴突在鳍内积极导航时上调。我将采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，并采用CRISPR/Cas9诱变来确定这些候选基因是否在介导靶特异性轴突再生中发挥功能作用（目的2）。总之，这些努力将提供一个细胞和机制的切入点，以研究如何协调当地的线索介导精确的轴突指导再生脊椎动物。通过分子生物学技术的培训和我的博士后顾问委员会的指导，本提案中的工作将建立一个完全独立的研究利基，我将从那里推出自己的实验室。

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.