Antagonistic functions of Dishevelleds regulate Frizzled3 endocytosis via filopodia tips in Wnt-mediated growth cone guidance.

How growth cones detect small concentration differences of guidance cues for correct steering remains a long-standing puzzle. Commissural axons engage planar cell polarity (PCP) signaling components to turn anteriorly in a Wnt gradient after midline crossing. We found here that Frizzled3, a Wnt receptor, undergoes endocytosis via filopodia tips. Wnt5a increases Frizzled3 endocytosis, which correlates with filopodia elongation. We discovered an unexpected antagonism between Dishevelleds, which may function as a signal amplification mechanism in filopodia where PCP signaling is activated: Dishevelled2 blocks Dishevelled1-induced Frizzled3 hyperphosphorylation and membrane accumulation. A key component of apical-basal polarity (A-BP) signaling, aPKC, also inhibits Dishevelled1-induced Frizzled3 hyperphosphorylation. Celsr3, another PCP component, is required in commissural neurons for anterior turning. Frizzled3 hyperphosphorylation is increased in Celsr3 mutant mice, where PCP signaling is impaired, suggesting Frizzled3 hyperphosphorylation does correlate with loss of PCP signaling in vivo. Furthermore, we found that the small GTPase, Arf6, which is required for Frizzled3 endocytosis, is essential for Wnt-promoted outgrowth, highlighting the importance of Frizzled3 recycling in PCP signaling in growth cone guidance. In a Wnt5a gradient, more Frizzled3 endocytosis and activation of atypical protein kinase C was observed on the side of growth cones facing higher Wnt5a concentration, suggesting that spatially controlled Frizzled3 endocytosis is part of the key mechanism for growth cone steering.