Wnt/calcium signaling mediates axon growth and guidance in the developing corpus callosum

Abstract

It has been shown in vivo that Wnt5a gradients surround the corpus callosum and guide callosal axons after the midline (postcrossing) by Wnt5a-induced repulsion via Ryk receptors. In dissociated cortical cultures we showed that Wnt5a simultaneously promotes axon outgrowth and repulsion by calcium signaling. Here to test the role of Wnt5a/calcium signaling in a complex in vivo environment we used sensorimotor cortical slices containing the developing corpus callosum. Plasmids encoding the cytoplasmic marker DsRed and the genetically encoded calcium indicator GCaMP2 were electroporated into one cortical hemisphere. Postcrossing callosal axons grew 50% faster than pre-crossing axons and higher frequencies of calcium transients in axons and growth cones correlated well with outgrowth. Application of pharmacological inhibitors to the slices showed that signaling pathways involving calcium release through IP3 receptors and calcium entry through TRP channels regulate post-crossing axon outgrowth and guidance. Co-electroporation of Ryk siRNA and DsRed revealed that knock down of the Ryk receptor reduced outgrowth rates of postcrossing but not precrossing axons by 50% and caused axon misrouting. Guidance errors in axons with Ryk knockdown resulted from reduced calcium activity. In the corpus callosum CaMKII inhibition reduced the outgrowth rate of postcrossing (but not precrossing) axons and caused severe guidance errors which resulted from reduced CaMKII-dependent repulsion downstream of Wnt/calcium. We show for the first time that Wnt/Ryk calcium signaling mechanisms regulating axon outgrowth and repulsion in cortical cultures are also essential for the proper growth and guidance of postcrossing callosal axons which involve axon repulsion through CaMKII. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 71: 269–283, 2011.

Calcium signaling in axon guidance

“…Although the Wnt5a/Ca 2+ pathways were first described in vitro, similar results have since been observed in vivo [82]. Wnt5a has been implicated in repulsive guidance post corpus callosum crossing [66].…”

“…In particular Li et al [49] demonstrated both Ryk and Fz receptors are necessary for repulsive axon guidance. In contrast to other common repulsive guidance cues (such as MAG and Sema3A), Wnt5a does not decrease axon outgrowth but instead enhances it [49,82]. Increased axon outgrowth is observed both in bath application of Wnt5a and during repulsive turning induced by a Wnt5a gradient.…”

“…Wnt5a has been implicated in repulsive guidance post corpus callosum crossing [66]. Postcrossing axons have a higher rate of outgrowth than precrossing axons [82], which these authors attributed to the presence of Wnt5a after corpus callosum crossing. Both of the Wnt5a effects in growth cones are influenced by microtubule reorganization [83].…”

The Yin and Yang of Wnt/Ryk axon guidance in development and regeneration

“…Although both canonical (e.g., Wnt1, Wnt3) and noncanonical Wnts (e.g., Wnt5a, Wnt11) have been identified as Ryk ligands [9,13,36], Wnt5a/Ryk interactions have been most intensely studied [9,11,12,37]. Axon guidance is dependent on establishing correct polarity within the growth cone by the assembly and disassembly of the actin cytoskeleton.…”

Dishevelled attenuates the repelling activity of Wnt signaling during neurite outgrowth in Caenorhabditis elegans

“…Although the canonical pathway (7,15) and the Wnt/calcium pathway (16) regulate axon guidance in a few instances, Wnt-mediated axon pathfinding mainly uses the PCP pathway, which activates the small GTPases Rho, Rac, and Cdc42, which regulate cytoskeletal dynamics (17,18). Studies from mice, Drosophila, and Caenorhabditis elegans collectively suggest that the core components of PCP signaling involved in axon guidance are the Wnt receptor Fzd, the phosphoprotein Dishevelled (Dsh, Dvl), the transmembrane protein Van Gogh/ Strabismus (Vang/Stbm, Vangl) and its adaptor Prickle (Prkl, Pk), and the atypical cadherin Flamingo/starry night (Fmi/Stan, Celsrs), although the requirement for each component may depend on the specific neuron type (17,19).…”