Mechanisms of Target-Specific Axon Regeneration

Project Summary Nerve damage is a common affliction that causes sensory and/or motor deficits. Recovery involves a regenerative process in which damaged axons within a nerve fiber must re-extend to the appropriate target tissues, in a process known as target-specific regeneration. This process often fails in humans, leaving patients with chronic health problems. Improving clinical outcomes requires a better understanding of how target-specific regeneration is regulated. We know that components of the nerve support scaffold can guide axon re-extension along simple paths. However, when axons reach nerve branch points, they require more specific guidance mechanisms to differentiate between multiple paths and select the correct one. We have little understanding of what environmental cues guide these decisions, and how they are appropriately interpreted by regrowing axons. The objective of this proposal is to identify cellular and molecular mechanisms that regulate axon targeting decisions to promote target-specific regeneration. I have established the zebrafish vagus nerve as a model to elucidate mechanisms of target-specific axon regeneration. Regenerating vagus axons select between five nerve branches to robustly re-innervate the correct target tissue, although how they do so is not known. I hypothesize that two non-mutually-exclusive mechanisms regulate target-specific regeneration: 1) chemosensation, in which a regenerating axon can interpret spatially patterned chemical guidance cues in the environment that direct its growth; 2) fasciculation, in which a regenerating axon can recognize undamaged axons that are innervating its intended target and use them as a substrate for directed growth. The three aims of this proposal will comprehensively identify how growing axons interact with their environment at the cell biological and molecular levels during target-specific regeneration. In Aim 1, I will combine a novel single-cell chimera regeneration assay with live imaging and genetic and pharmacological manipulations to establish a conceptual understanding of how in vivo axon-environment interactions guide targeting decisions. In Aim 2, I will combine a novel method to label and isolate live neurons based on their innervation target with in vivo and in vitro techniques to precisely measure how axons of each of the five innervation target groups interact with other axons, and with chemical signals, in the environment. In Aim 3, I will combine innervation target-specific neuron isolation with RNAseq and mutant analysis for unbiased identification of molecules that regulate target selection in each of the five innervation target groups. This study will greatly enhance our fundamental understanding of how axons reinnervate their target tissues during regeneration, and provide an important knowledge base to develop improved treatments for nerve damage.