Nervous system function depends on precise organization of axonal connections established during embyrogenesis. Much of this axonal organization is established along major anatomical axes, such as anterior-posterior (A–P), dorsal-ventral (D–V) (equivalent to medial-lateral) and inferior-superior. Molecular guidance cues provide directional information for navigating axons during both pathfinding and target selection (Tessier-Lavigne and Goodman, 1996
) (Dickson, 2002
) (Zou and Lyuksyutova, 2007
). Although many axon guidance molecules and their receptors have been identified, the signal transduction mechanisms leading to directed growth cone turning remain unsolved. In particular, where exactly signaling components are localized in the growth cone and how they respond to guidance cues and interact with each other to create asymmetric signaling leading to turning are unknown in virtually all axon guidance systems.
Multiple subpopulations of commissural axons first project along the dorso-ventral axis towards the ventral midline, cross the floor plate to the contralateral side of the spinal cord and turn either anteriorly or posteriorly along the longitudinal axis. Wnt-Frizzled signaling is required for anterior turning of the dorsal-most populations of commissural axons after they have crossed the midline (Lyuksyutova et al., 2003
) (Wolf et al., 2008
) (Zou and Lyuksyutova, 2007
). Wnt proteins are secreted glycoproteins, a subset of which are expressed by the ventral midline cells in the floor plate of the spinal cord, where they are expressed in an anterior-posterior gradient. Wnts attract post-crossing commissural axons and the loss of function mutation of a Wnt receptor, Frizzled3, results in the randomized turning of commissural axons along the A–P axis after midline crossing (Lyuksyutova et al., 2003
) (Wolf et al., 2008
Wnt-Frizzled signaling activates several pathways and plays multiple roles in development and function (Logan and Nusse, 2004
) (Zou, 2004
). Among the known signaling pathways that mediate Wnt functions, the planar cell polarity (PCP) pathway is an appealing candidate for Wnt-mediated axon guidance because of its ability to introduce cellular asymmetry in response to environmental cues (Zou, 2004
). PCP refers to cell and tissue polarity along the planar axis of epithelia or mesenchymal cell sheets, perpendicular to the apical-basal axis (Wang and Nathans, 2007
) (Zallen, 2007
) (Goodrich, 2008
) (Simons and Mlodzik, 2008
). PCP signaling pathway is highly conserved and regulates the polarized cellular and tissue morphology exhibited in a number of processes, including orientation of epithelial prehair in the Drosophila
wing, directed cell movement during vertebrate gastrulation and the polarized organization of mammalian stereocilia of cochlear hair cells. Furthermore, in C elegans
Wnts are instructive signals for PCP and control spindle orientation during neuroblast division (Goldstein et al., 2006
The PCP signaling pathway involves two sets of regulators, the Frizzled/Flamingo core group and the Fat/Dachsous PCP system (Simons and Mlodzik, 2008
). The Frizzled/Flamingo group of conserved core components include the seven transmembrane domain protein Frizzled (Fzd), the atypical cadherin with seven-pass transmembrane domains Flamingo/starry night (Fmi/Stan or Celsrs in vertebrates), the four-pass transmembrane protein Van Gogh or Strabismus (Vang/Stbm or Vangl), the Fzd-binding intracellular protein Dishevelled (Dsh, Dvl), the ankyrin repeat protein Diego (Dgo) and the Fzd-binding Lim domain protein Prickle (Prkl, Pk). Furthermore, it is known that PCP signaling leads to activation of c-Jun N-terminal kinase (JNK) and c-Jun by phosphorylation (Boutros et al., 1998
) (Yamanaka et al., 2002
). Until now, very little is known about the biochemical functions of the PCP signaling components and their cell biological mechanisms of action with the exception that some components directly bind to each other and in some cases their proper subcellular locations are correlated to proper PCP signaling. For example, Fzd and Dvl colocalize to the distal membrane of the epithelial cells of the Drosophila
wing epithelial cells and Vang, Pk and Dgo are localized to the proximal membrane. How their subcellular localization is regulated biochemically and what signaling effects these cellular localizations trigger or reflect are completely unknown.
We report here that the core PCP components are present in commissural axon growth cones at the time they are making anterior turns and in addition to Frizzled3
, both Celsr3
are required for proper A–P guidance of commissural axons in vivo
. PCP components, Frizzled3 and Vangl2, mediate Wnt5a-stimulated commissural axon growth in culture. Wnt5a activates JNK signaling in commissural neurons, and JNK activity is required for A–P guidance of commissural axons. We uncovered a Dvl1 mediated negative feedback loop upon Wnt-Frizzled activation. This feedback loop involves Dvl1-induced Frizzled3 hyperphosphorylation, which causes accumulation of Frizzled3 on plasma membrane. Point mutations of Frizzled3 result in prolonged and enhanced PCP signaling. Vangl2 antagonizes Dvl1 inhibition by reducing Frizzled3 phosphorylation and cell surface accumulation. It has been shown that Dvl binds to AP-2 and clathrin-mediate endocytosis of Fzd is required for PCP signaling (Sato et al.
) (Yu et al., 2007
). In commissural axon growth cones, Fzd3 and Dvl1 are localized primarily in intracellular vesicles by themselves. When co-expressed, they target each other to the plasma membrane. Vangl2, which is primarily localized on plasma membrane, again antagonizes the Dvl1-mediated Frizzled3 accumulation on the growth cone plasma membrane. Finally, Vangl2 protein is found predominantly on the growth cone plasma membrane and highly enriched on the tips of stable or growing filopodia or in patches of plasma membrane where new filopodia emerge in live growth cones. We propose that the antagonistic interaction between Vangl2 and Dvl1 on Frizzled3 phosphorylation of internalization (and thus PCP signaling) maybe a general biochemical mechanism used to create asymmetric signaling in setting up planar cell polarity. And, in neuronal growth cones, this opposing interaction makes the tip of the filopodia more sensitive to guidance cues by allowing Wnt/PCP signaling to enter the growth cones via these tips.