Peripheral nerve injury, most often caused by motor vehicle accidents, affects approximately 67,000 Americans each year and 20 million Americans suffer from peripheral neuropathies. In a vulnerable nervous system, regenerating damaged motor circuitry is essential for recovery of coordinated movement. This wiring, mediated by special processes called axons, can regenerate. However, mammals often experience targeting errors during regeneration, leading to incomplete functional recovery despite axon regrowth. Growing evidence demonstrates that axon regeneration is not simply a recapitulation of development, but that it requires unknown injury-dependent cues.
Yet, what are the guidance receptors and cues that mediate topographically correct axon targeting and what is their spatial distribution? How does this cell signaling underlying circuit formation differ between developing and regenerating tissues? The larval zebrafish pectoral fin, equivalent to tetrapod forelimbs, is an ideal vertebrate model system in which to visualize regenerating axons as they navigate stepwise choice points. After laser-mediated axon transection, individual regenerating motor axons precisely regenerate to their original specific muscle and target domains in the fin via unknown mechanisms.
To identify guidance cues that mediate axon targeting, this project will generate a spatially-resolved single cell transcriptome of motor neuron sub-populations and the cells of the fin that they target with support from the Center for Quantitative Biology (CQB) COBRE at Dartmouth College. Using 10x Chromium single cell RNA sequencing analysis, this project will identify RNA transcripts that are unique to each neuronal population to develop transgenic tools to label neuron sub-populations selectively (Aim 1).
In addition, this project will identify candidate guidance receptors that may drive the unique topographic targeting of each neuronal population. The project will then use 10x Xenium spatial transcriptomics to visualize the spatial organization of guidance cues at single cell resolution in the fin across development and regeneration of pectoral fin innervation (Aim 2).
The CQB Single Cell Genomics Core houses the 10x Chromium and Xenium analyzers and will provide essential technical support. In collaboration with the Genome Data Science Core, an analysis pipeline will be established to integrate information about the neuronal guidance receptors expressed in neuronal sub-populations with the guidance cues expressed specifically in the territory they innervate. Future work will leverage this rich dataset and the extensive toolkit in the genetically-tractable zebrafish to establish a functional role for the molecular drivers of correct axon targeting across development and regeneration.