Neuronal guidance landscape across development and regeneration

Dartmouth College, Hanover NH

Investigators

Linked publications & trials

Abstract

Functional neural circuits require accurate wiring by axons. Axonal targeting is mediated by a unique repertoire of guidance receptors differentially expressed on the axons of given neuronal populations. These receptors recognize spatially distributed guidance cues across an axon’s trajectory to guide directional axonal growth and ultimately the formation of appropriate synaptic connections. This complex cell to cell signaling is dynamic, spatially-regulated, and differs between neuronal populations and their targets. In a nervous system vulnerable to injury, regeneration of damaged motor axons back to their original muscle targets is essential for recovery of coordinated movement. 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 the cell signaling underlying circuit formation differ between developing and regenerating tissues? Understanding the cellular and molecular mechanisms that mediate regenerative axon guidance represents an important and unaddressed goal that is relevant to recovery from injury or disease. To fill the existing knowledge gaps, I developed a unique model using the motor innervation of the larval zebrafish pectoral fin, which is homologous to tetrapod forelimbs. Importantly, this model allows for the in vivo observation of individual axons and their synaptic targets from development through regeneration. Monitoring distinct sub-populations of motor neurons reveals that they innervate topographically defined domains in the fin musculature. To find their targets, motor neuron axons must first navigate to the fin, sort between the abductor and adductor muscles, and grow to their appropriate muscle domain where they will form functional synapses. I have previously demonstrated that after laser-mediated axon transection, individual regenerating motor axons precisely return to their originally-specified muscle and target domains in the fin via unknown mechanisms. The long-term goal of my research program is to determine the molecular and cellular signaling that enables neuronal populations to re-establish such precise targeting. Given that each of the stepwise choice points along an axon’s route requires distinct guidance signaling mechanisms, a candidate-based approach to test the role of individual guidance cues is not efficient and will not provide a comprehensive understanding of the complex landscape through which an axon is guided. Thus, a critical foundation to support my research is the generation of a spatially-resolved single cell transcriptome of the motor neuron sub-populations as well as the cells of the fin that they target. The Center for Quantitative Biology (CQB) COBRE will advance my research career goals by providing me with the necessary scientific expertise, technical support, and dedicated mentorship to accomplish the following aims: Aim 1: Determine the molecular signature that defines distinct motor neuron populations. The pectoral fin is innervated by six different motor neuron populations that target distinct domains in the musculature. I hypothesize that each neuronal sub-population expresses a molecular signature that includes guidance receptors. Using 10x Chromium single cell RNA sequencing analysis, I will identify RNA transcripts that are unique to each neuronal population in order to develop transgenic tools to label them selectively. I will also identify candidate guidance receptors that may drive the unique topographic targeting of each neuronal population. Aim 2: Define the spatial organization of guidance cues across development and regeneration. The specific guidance cues and their spatial organization in the fin are unknown. Moreover, the pectoral fin is comprised of many cell types, including muscle, perineural glia, Schwann cells, vasculature, and endoskeletal cells, which could all contribute to the spatial organization of guidance cues that drive axon targeting. First, I will employ single cell sequencing on pectoral fins to establish the molecular signature of different cell types that comprise the fin. Then, I will use 10x Xenium spatial transcriptomics to visualize the anatomical location of RNA transcripts of specific guidance and growth cues at single cell resolution across development and regeneration of fin innervation. I will combine data from these approaches to resolve the identity of guidance cues, their spatial organization, and the cell types that express them to fuel hypothesis generation and mechanistic studies. The CQB COBRE cores and staff will directly support the technical and analytical execution of these aims. Both the 10x Chromium and Xenium analyzers that perform single cell transcriptomic profiling are housed in the Single Cell Genomics Core. In collaboration with the Genome Data Science Core, I will establish an analysis pipeline to integrate information about the neuronal guidance receptors expressed in neuronal subpopulations (Aim 1) with the guidance cues expressed specifically in the territory they innervate (Aim 2). Future work that will be competitive for NIH R01 or equivalent funding 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. Insights gained from this work will have broader implications for deploying cell signaling mechanisms that build, maintain, and repair circuits across the nervous system.