Fig 2: Working model.It has been widely demonstrated that when the axon grows in the absence of Wnt5a, cadherins are constantly being recycled from the plasma membrane. Our results support a model in which midline-expressed Wnt5a triggers local accumulation of ßcatenin in contralateral axons. It has been shown that ßcatenin links cadherins and actin microtubules. Thus, Wnt5a-dependent accumulation of ßcatenin would facilitate midline crossing by promoting the stabilization of the cytoskeleton at the tip of the growth cone. In contrast, our results demonstrate that Zic2 activates a different set of Wnt receptors and other intracellular Wnt proteins in ipsilaterally projecting neurons, such as Fzd1, Fzd8, Lgr5, or Apc2, to favor the accumulation of ßcatenin. We also show that EphB1 phosphorylates ßcatenin in Y654, and previous reports have demonstrated that phosphorylated Y654-ßcatenin has low affinity for cadherins. Taking all these observations together, we propose that phosphorylation of ßcatenin induced by the binding of EphB1 to ephrinB2 prevents the formation of cadherin/actin complexes facilitating axon steering.

Fig 3: Zic2 activates a program that leads to the accumulation of ßcatenin in RGC axons.(A) In situ hybridization for Fzd8 in a coronal section of an E16.5 mouse retina. Bottom panel shows higher magnification of the squared area. (B) Immunofluorescence for Apc2 and ßcatenin in the growth cone of axons growing from explants electroporated with EGFP- or Zic2-encoding plasmids. Scale bar, 10 µm. (C) Quantification of immunofluorescence intensity for Apc2 (upper graph) and ßcatenin (lower graph) (**P < 0.01 and ***P < 0.001) (n = mean of at least four growth cones/explant). (D) RFP fluorescence in whole-mount E16.5 retinas electroporated at E13.5 with the indicated plasmids. Panels at the right corner show targeted/EGFP cells in the same whole-mounted retinas. Scale bar, 100 µm. (E) Normalized quantification of RFP fluorescence intensity in whole-mount electroporated retinas. n = number of retinas (unpaired t test with Welch’s correction, **P < 0.001). (F) Optic chiasms from E16.5 embryos electroporated with plasmids encoding for ßcatenin or ?90-ßCatenin-?CT. Scale bar, 100 µm. (G) Percentage of contralaterally projecting axons at the optic chiasm normalized to the total number of targeted axons (n = number of embryos) (two-tailed unpaired t test, ***P < 0.001). Results show means ± SEM. All results come from at least three independent experiments.