During neural development growing axons (nerve fibers) are guided to their targets by responses of their motile tips (growth cones) to molecular cues in the environment. The long term goal of this work is to understand the intracellular signaling mechanisms that regulate axon outgrowth and guidance. The work will be carried out on the developing rodent cortex and thus the findings will be important for strategies promoting regeneration of injured axons in the mammalian CNS. The three specific aims will address the signaling mechanisms that regulate how growth cones respond to Wnt5a, a secreted protein important for embryonic development that has been shown in vivo to guide axons in several developing cortical pathways. In dissociated cortical cultures Wnt5a repels growing axons while increasing their rate of axon outgrowth. Separate plasma membrane receptors Ryk and Fz (Frizzled) and distinct components of calcium signaling pathways (IP3 receptors and TRP calcium channels) differentially mediate cortical axon outgrowth and repulsive growth cone turning behaviors evoked by Wnt5a. In the first specific aim, the role of these Wnt5a signaling mechanisms in growth and guidance of cortical axons in vivo will be tested in living cortical slices. Confocal microscopic imaging in the corpus callosum, a pathway linking the two cerebral hemispheres, will be used to visualize cortical growth cone behaviors in a WNT5a environment. Frequencies of calcium transients in growth cones will be measured in relation to rates of axon outgrowth and selective disruption of the Wnt receptors Ryk and Fz and calcium signaling components IP3 and TRP will be carried out to test their role in cortical axon outgrowth and guidance in the callosum. In the second specific aim, calcium imaging will be carried out in the axonal growth cones of dissociated cortical neurons to test the hypothesis that Wnt5a gradients increase axon outgrowth rates by evoking global calcium transients throughout the growth cone and induce growth cone repulsion by evoking asymmetric calcium gradients across the growth cone. To further test this hypothesis, in Wnt5a gradients patterns of calcium activity will be characterized in growth cones in which specific Wnt receptors and calcium signaling components are experimentally disrupted. In the third specific aim high resolution live cell imaging of fluorescently labeled cytoskeletal elements, actin filaments and microtubules, will be carried out to understand how the cytoskeleton reorganizes in response to guidance cues to direct growth cone extension and turning behaviors. Manipulations of Wnt5a receptors and calcium signaling will be used to identify cytoskeletal changes associated with axon growth and guidance. To determine whether these cytoskeletal changes also occur in an in vivo environment, confocal imaging of the cytoskeleton will be carried out in callosal growth cones extending in living cortical slices.
The goal of the proposed research is to characterize fundamental molecular mechanisms that regulate the growth of axons (nerve fibers) from the cerebral cortex and allow them to reach appropriate targets. An understanding of these developmental mechanisms will be essential for promoting regeneration of injured nerve fibers in the mammalian central nervous system.
