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.
Injuries to the peripheral nervous system, most often caused by motor vehicle accidents, can leave patients with devastating motor defects that can be disabling long-term. Despite the capacity for axons to regenerate in the peripheral nervous system, adults with peripheral nerve injuries rarely fully recover largely due to axon targeting errors. To understand how regrowing axons, know where to grow, this proposal outlines an approach to determine the cellular and molecular mechanisms that enable target-selective axon guidance in vivo.
