Axon guidance and target-derived signals control axonal behavior by regulating the cytoskeleton through poorly defined mechanisms. In particular, how these signaling molecules regulate the growth and directionality of microtubules is not well understood. Here we examine the effect of Wnts on growth cone remodeling, a process that precedes synapse formation. Time-lapse recordings reveal that Wnt3a rapidly inhibits growth cone translocation while inducing growth cone enlargement. These changes in axonal behavior are associated with changes in the organization of microtubules. Time-lapse imaging of EB3-GFP (green fluorescent protein)-labeled microtubule plus-ends demonstrates that Wnt3a regulates microtubule directionality, resulting in microtubule looping, growth cone pausing, and remodeling. Analyses of Dishevelled-1 (Dvl1) mutant neurons demonstrate that Dvl1 is required for Wnt-mediated microtubule reorganization and axon remodeling. Wnt signaling directly affects the microtubule cytoskeleton by unexpectedly inducing adenomatous polyposis coli (APC) loss from microtubule plus-ends. Consistently, short hairpin RNA knockdown of APC mimics Wnt3a function. Together, our findings define APC as a key Wnt signaling target in the regulation of microtubule growth direction.

Leading researchers working on synthetic biology and its applications gathered at the University of Edinburgh in May 2018 to discuss the latest challenges and opportunities in the field. In addition to the potential socio-economic benefits of synthetic biology, they also examined the ethics and security risks arising from the development of these technologies. Speakers from industry, academia and not-for-profit organizations presented their vision for the future of the field and provided guidance to funding and regulatory bodies to ensure that synthetic biology research is carried out responsibly and can realize its full potential. This report aims to capture the collective views and recommendations that emerged from the discussions that took place. The meeting was held under the Chatham House Rule (i.e., a private invite-only meeting where comments can be freely used but not attributed) to promote open discussion; the findings and quotes included in the report are therefore not attributed to individuals. The goal of the meeting was to identify research priorities and bottlenecks. It also provided the opportunity to discuss how best to manage risk and earn public acceptance of this emerging and disruptive technology.

Upon arrival at their synaptic targets, axons slow down their growth and extensively remodel before the assembly of presynaptic boutons. Wnt proteins are target-derived secreted factors that promote axonal remodelling and synaptic assembly. In the developing spinal cord, Wnts secreted by motor neurons promote axonal remodelling of NT-3 responsive dorsal root ganglia neurons. Axon remodelling induced by Wnts is characterised by growth cone pausing and enlargement, processes that depend on the re-organisation of microtubules. However, the contribution of the actin cytoskeleton has remained unexplored. Here, we demonstrate that Wnt3a regulates the actin cytoskeleton by rapidly inducing F-actin accumulation in growth cones from rodent DRG neurons through the scaffold protein Dishevelled-1 (Dvl1) and the serine-threonine kinase Gsk3β. Importantly, these changes in actin cytoskeleton occurs before enlargement of the growth cones is evident. Time-lapse imaging shows that Wnt3a increases lamellar protrusion and filopodia velocity. In addition, pharmacological inhibition of actin assembly demonstrates that Wnt3a increases actin dynamics. Through a yeast-two hybrid screen, we identified the actin-binding protein Eps8 as a direct interactor of Dvl1, a scaffold protein crucial for the Wnt signalling pathway. Gain of function of Eps8 mimics Wnt-mediated axon remodelling, whereas Eps8 silencing blocks the axon remodelling activity of Wnt3a. Importantly, blockade of the Dvl1-Eps8 interaction completely abolishes Wnt3a-mediated axonal remodelling. These findings demonstrate a novel role for Wnt-Dvl1 signalling through Eps8 in the regulation of axonal remodeling.

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