Short- and long-range roles of UNC-6/Netrin in dorsal-ventral axon guidance in vivo in Caenorhabditis elegans.

Recent studies in vertebrates and Caenorhabditis elegans have reshaped models of how the axon guidance cue UNC-6/Netrin functions in dorsal-ventral axon guidance, which was traditionally thought to form a ventral-to-dorsal concentration gradient that was actively sensed by growing axons. In the vertebrate spinal cord, floorplate Netrin1 was shown to be largely dispensable for ventral commissural growth. Rather, short range interactions with Netrin1 on the ventricular zone radial glial stem cells was shown to guide ventral commissural axon growth. In C. elegans, analysis of dorsally-migrating growth cones during outgrowth has shown that growth cone polarity of filopodial extension is separable from the extent of growth cone protrusion. Growth cones are first polarized by UNC-6/Netrin, and subsequent regulation of protrusion by UNC-6/Netrin is based on this earlier-established polarity (the Polarity/Protrusion model). In both cases, short-range or even haptotactic mechanisms are invoked: in vertebrate spinal cord, interactions of growth cones with radial glia expressing Netrin-1; and in C. elegans, a potential close-range interaction that polarizes the growth cone. To explore potential short-range and long-range functions of UNC-6/Netrin, a potentially membrane-anchored transmembrane UNC-6 (UNC-6(TM)) was generated by genome editing. unc-6(tm) was hypomorphic for dorsal VD/DD axon pathfinding, indicating that it retained some unc-6 function. Polarity of VD growth cone filopodial protrusion was initially established in unc-6(tm), but was lost as the growth cones migrated away from the unc-6(tm) source in the ventral nerve cord. In contrast, ventral guidance of the AVM and PVM axons was equally severe in unc-6(tm) and unc-6(null). Together, these results suggest that unc-6(tm) retains short-range functions but lacks long-range functions due to reduced secreted UNC-6. Ectopic unc-6(+) expression from non-ventral sources did not dramatically perturb dorsal VD growth cone polarity or axon outgrowth, suggesting that ectopic UNC-6 cannot redirect polarity once it is established in the VD/DD neurons. This is not what would be expected of a growth cone dynamically reading a gradient of UNC-6, but is consistent with the Polarity/protrusion model of growth cone guidance away from UNC-6/Netrin. Author summary: In the developing nervous system, neurons extend thin cellular processes called axons that make precise contacts with other neurons in elaborate networks. The structure of these axon networks underlies the function of the nervous system. Thus precise axon guidance is critical during development to produce a functional nervous system. Axons are guided by the growth cone, a dynamic structure at the distal tup of the axon that senses and responds to signals in the environment that instruct axon guidance. One such signal if UNC-6/Netrin, a conserved, laminin-like secreted molecule that guides many aspects of axon guidance. In the nematode Caenorhabditis elegans, UNC-6 controls dorsal-ventral axon guidance of motor neurons and sensory neurons. The canonical textbook model of UNC-6/Netrin function involved the formation of a gradient of secreted UNC-6/Netrin in the ventral-to-dorsal axis which growth cones dynamically sensed and responded by growing up or down the gradient. The gradient model does not explain recent studies in vertebrates and C. elegans that have shown close-range or contact-mediated events that more resemble cell polarity events than dynamic reading of a gradient. In C. elegans, the growth cone of the VD axon is polarized dorsally by UNC-6/Netrin. UNC-6/Netrin then maintains this polarity and regulates protrusion based upon this polarity, stimulating protrusion dorsally and inhibiting protrusion ventrally, resulting in net dorsal outgrowth. This Polarity/Protrusion model resembles events that take place in ventral growth of sensory axons, the Statistically-Oriented Asymmetric Localization (SOAL) model. The Polarity/Protrusion model predicts that a short-range UNC-6 signal polarizes the VD growth cone, and that secreted, diffusible UNC-6/Netrin maintains initial polarity and regulates protrusion. This model is critically tested here by the construction of a membrane-anchored UNC-6 genome edit predicted to not produce any diffusible UNC-6, thus blocking any long-range function. Data here show that VD growth cones are initially polarized corerectly in unc-6(tm), but that this polarity is lost as the growth cones move dorsally away from the UNC-6(TM) source. This is consistent with a short-range interaction that polarizes the growth cone, and a long-range function needed to maintain polarity. Furthermore, ectopic expression of diffusible UNC-6 from ectopic sources did not perturb VD growth cone polarity, suggesting that the initial polarity event cannot be altered by later diffusible UNC-6/Netrin, as would be expected with a gradient model. These results are consistent with the Polarity/Protrusion model of UNC-6 function. [ABSTRACT FROM AUTHOR]