Axon guidance through the bifunctional cue WFIKKN2 and its receptors

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PROJECT SUMMARY/ABSTRACT Connectivity between neurons as part of neural networks governs information flow through the nervous system and therefore shapes behavioral output. Guidance of axon to their correct targets is a key step in neural circuit assembly during embryonic development, and understanding this process is critical, as neuronal miswiring can cause circuit dysfunction and disease. We report identification of a novel axon guidance cue, WFIKKN2, as a ligand for several DCC family receptors and propose to investigate the biochemical nature of these signaling complexes and their functions in neuronal wiring. DCC is a receptor for the attractive axon guidance cue Netrin- 1, and this ligand-receptor pair shapes neuronal connectivity in organisms as diverse as nematodes and humans. In vertebrates, the DCC family comprises five members, and Netrins can bind two of these – DCC and Neogenin. Three additional family members – Punc, Nope, and Protogenin – have diverged considerable over the course of evolution, do not bind Netrins, and their functions in axon guidance have remained elusive. We found that the secreted multi-domain protein WFIKKN2 binds Punc, Nope, and Protogenin, but not DCC and Neogenin. We provide evidence that developing sensory and motor neurons express these newly identified receptors for WFIKKN2, while WFIKKN2 is expressed in multiple tissues in the body periphery. Additional preliminary results show that WFIKKN2 acts as an attractant for motor axons and a repellant for sensory axons, and phenotype analysis of WFIKKN2 knockout mice strongly supports the idea that WFIKKN2-mediated repulsion helps sensory axons navigate to their peripheral targets. We propose a multidisciplinary approach to study WFIKKN2 and its receptors further and test hypotheses about their functions in neural circuit formation. We will use protein interaction assays to map the domains mediating ligand-receptor interactions and determine how ligand binding specificity arises within the DCC family. We will also combine axon guidance assays using cultured neurons with functional manipulations to dissect the contributions of Punc, Nope, and Protogenin to WFIKKN2-dependent axon attraction and repulsion, and we will use biochemical approaches to identify downstream mediators of WFIKKN2 signaling. To elucidate the axon guidance functions of WFIKKN2 and its receptors in vivo, we will employ mouse genetics and state-of-the-art neuroanatomical methods. Our work will uncover mechanisms of motor and sensory axon guidance, and it expands the known molecular toolkit for neuronal wiring by discovering a novel, bifunctional axon guidance cue and its receptors. Our studies further have the potential to identify new intracellular mediators of guidance cue signaling. In the long term, our work can help elucidate how the evolutionary diversification of axon guidance receptor gene families may allow the wiring of more complex nervous systems. Overall, by providing important insights into mechanisms of neuronal wiring, this research will contribute to our understanding of neurodevelopmental disease etiology, and it can aid in the development of novel therapeutic approaches for neural circuit repair and nerve regeneration in various disease states.