Understanding the effects of guidance cues and their receptors on the growth cone during outgrowth will be critical to a comprehensive understanding of axon guidance in normal and diseased states. The Netrin family of secreted guidance cues act as both attractants and repellants in axon guidance. By analyzing the effects of mutations in Netrin signaling on the growth cone during outgrowth, we have developed a new model of Netrin mediated control of directed growth cone migration called the polarity/protrusion model. Our results using live growth cone imaging indicate that UNC-6/Netrin signaling, via the UNC-5 receptor, polarizes protrusion and F-actin in growth cones, resulting in dorsal protrusion away from the ventral UNC-6/Netrin source. We found that Netrin also inhibits protrusion ventrally via UNC-5, and stimulates protrusion dorsally via the Netrin receptor UNC-40, based on growth cone polarity (Figure 1). Thus, UNC-6/Netrin signaling both polarizes the growth cone, and regulates the extent of protrusion based on this polarity. By inhibiting protrusion ventrally and stimulating protrusion dorsally, Netrin drives directed growth dorsally away from the UNC-6/Netrin source. A key ramification of this model is that UNC-6/Netrin receptor function will be distributed asymmetrically across the growth cone, with UNC-5 inhibitory activity localized ventrally and UNC-40 stimulatory activity localized to the dorsal area of the growth cone. We will test this key aspect of the polarity/protrusion model by assaying Netrin receptor localization in growth cones. Significance: These studies will significantly advance understanding of axon guidance, as they invoke and test a fundamentally novel model of directed growth cone migration in axon guidance, the polarity/protrusion model. In this new model, UNC-6/Netrin polarizes the growth cone such that the asymmetric activity of both pro- and anti-protrusive forces, each controlled by UNC-6/Netrin in the same growth cone, lead to directed migration away from UNC-6/Netrin. Our results, combined with studies in vertebrates showing that floorplate Netrin is dispensable for commissural axon guidance, suggest a new model of axon guidance involving regulation of growth cone polarity and protrusion.
Guided axon outgrowth is critical for brain and nervous system development and function. The goal of this proposal is to understand the basic molecular mechanisms of axon outgrowth using the model organism nematode worm C. elegans. Preliminary studies have led to a fundamentally novel model of axon guidance. These studies have the potential to translate to human development, neurological disorders, and regenerative medicine.