A Zic2-regulated switch in a noncanonical Wnt/βcatenin pathway is essential for the formation of bilateral circuits

Abstract

The Wnt pathway is involved in a wide array of biological processes during development and is deregulated in many pathological scenarios. In neurons, Wnt proteins promote both axon extension and repulsion, but the molecular mechanisms underlying these opposing axonal responses are unknown. Here, we show that Wnt5a is expressed at the optic chiasm midline and promotes the crossing of retinal axons by triggering an alternative Wnt pathway that depends on the accumulation of βcatenin but does not activate the canonical pathway. In ipsilateral neurons, the transcription factor Zic2 switches this alternative Wnt pathway by regulating the expression of a set of Wnt receptors and intracellular proteins. In combination with this alternative Wnt pathway, the asymmetric activation of EphB1 receptors at the midline phosphorylates βcatenin and elicits a repulsive response. This alternative Wnt pathway and its Zic2-triggered switch may operate in other contexts that require a two-way response to Wnt ligands.

Fig. 1. Wnt5A is expressed at the midline and enhances axonal growth of contralateral axons.

(A) In situ hybridization for Wnt5a in coronal sections from E13.5, E15.5, and E17.5 embryos. Scale bar, 200 μm. (B) Color-inverted Tuj1 staining on retinal explants from E14.5 embryos cultured for 12 hours. Retinal explants from the central retina treated with recombinant Wnt5a protein display longer neurites compared with vehicle-treated explants. Scale bar, 100 μm. (C) Quantification of average axon length in explants treated with vehicle or Wnt5a (n = mean length of axons/explant). Data from three biological replicates. Two-tailed Mann-Whitney U test, **P < 0.01. (D) Relative cumulative frequency histogram showing axonal length in explants growing in the presence of Wnt5a (gray; n = 927) or the vehicle (black; n = 861). Each dot represents an individual neurite. n = number of axons from three biological replicates. (E) Color-inverted immunohistochemistry for βcatenin in the growth cone of ganglion cells from contralateral axons treated for 1 hour with Wnt5a or vehicle. Note that Wnt5a-treated cones occupy a larger area and show higher levels of βcatenin. Scale bar, 5 μm. (F and G) Quantification of growth cone area and βcatenin fluorescence intensity (FI) in retinal explants. n = mean from at least four growth cones/explant. Results from three independent experiments. βCatenin levels (two-tailed Mann-Whitney U test). Growth cone area (two-tailed unpaired t test, ***P < 0.001). Results show means ± SEM. a.u., arbitrary units.

Fig. 2. βCatenin is necessary for midline crossing.

(A) Plasmids encoding EGFP and control shRNA or shRNA against βcatenin (ctnnb1 shRNAs) were electroporated into one eye of E13.5 embryos, and EGFP axons into the optic chiasm were analyzed at E18.5. Right panels are representative images of optic chiasms from embryos electroporated with ctnnb1 shRNA or scramble shRNA plasmids. Down-regulation of βcatenin in contralaterally projecting neurons inhibits midline crossing. Scale bar, 100 μm. (B) Graph represents the percent of embryos showing midline crossing defects after electroporation with scramble shRNAs [n = 10; no phenotype (NP) = 87.5%; ectopic ipsilateral projection (Ipsi) = 12.5%] or ctnnb1 shRNAs (n = 42; NP = 9.52%; Ipsi = 42.86%; and stalled = 47.62%) plasmids. (C) Color-inverted images of retinal explants from E14 embryos electroporated with ctnnb1 shRNAs plus EGFP plasmids or with control shRNA plus EGFP plasmids cultured for 12 hours with Wnt5a or vehicle. Scale bar, 100 μm. (D) Axon length quantification of control and ctnnb1 shRNAs cultured with Wnt5a or vehicle, as in (C). n = mean length of axons/explant. Results from three independent experiments. Two-tailed unpaired t test (*P < 0.05 and ***P < 0.001). Results show means ± SEM. n.s., not significant. (E) Representative immunohistochemistry for RFP and EGFP in whole-mount E16.5 retinas that electroporated at E13.5 with Top-RFP– and EGFP-encoding or Δ90-βCatenin/Top-RFP– and EGFP-encoding plasmids shows that canonical Wnt signaling is not activated in cRGCs at the time that visual axons transverse the midline. The experiment was repeated at least three times for each condition with similar results. Scale bar, 100 μm.

Fig. 3. Ipsilateral RGC axons collapse in response to Wnt5a.

(A) Top panel shows the experimental approach used to assess the response of ipsilateral RGCs to Wnt5a. (Bottom) Retinal explants from Sert-RFP E15 embryos cultured for 12 hours, incubated with vehicle or Wnt5a for 1 hour, and stained with phalloidin. Scale bar, 20 μm. (B) Quantification showing the percentage of growth cones smaller than 70 μm (% collapse) in Sert-RFP explants incubated with Wnt5a or the vehicle. Results from three independent experiments. Two-tailed unpaired t test (***P < 0.001). Results show means ± SEM. (C) Top panels show the experimental approach used to assess the response of Zic2-expressing neurons to Wnt5a. Color-inverted retinal explants from E14.5 embryos electroporated at E13.5 with EGFP- or Zic2/EGFP-encoding plasmids were isolated and cultured with or without Wnt5a. The bottom images are retinal explants from electroporated embryos incubated with Wnt5a or vehicle. Scale bar, 200 μm. (D) Quantification of axonal length of RGCs expressing EGFP or Zic2/EGFP grown in the presence of Wnt5a or vehicle (n = number of explants). Axon length values were normalized to the mean value of the axons in explants treated with the vehicle. Results from three independent experiments. Two-tailed unpaired t test, P = 0.003 (EGFP) and P = 0.563 (Zic2). (E) Schema summarizing the experimental approach. Color-inverted images show axons from representative retinal explants isolated from electroporated embryos that were cultured for 12 hours and exposed to Wnt5a or vehicle for 1 hour. Green arrowheads point out axons with growth cones smaller than 70 μm. Scale bar, 50 μm. (F) Quantification showing the percentage of collapsed growth cones in Zic2-expressing explants incubated with Wnt5a or the vehicle. n = mean length of axons/explant (two-tailed unpaired t test, ***P < 0.001). Results from three independent experiments. Results show means ± SEM.

Fig. 4. Zic2 regulates many genes involved in the Wnt signaling pathway.

(A) Scheme representing the experimental design of the RNA-seq screen. FACS, fluorescence-activated cell sorting. (B) Volcano plot showing differentially expressed genes (DEGs) 36 hours after Zic2 electroporation in contralateral RGCs. The name of relevant DEGs is indicated in the amplification inset. The significance value for the change in Zic2 is above the scale. LFC, log2 fold change. (C) PANTHER pathway enrichment analysis of genes up-regulated (top graphs) and down-regulated (bottom graphs) after Zic2-induced expression in RGCs. The bar graphs present the significance of the enrichment (right) and the number of genes involved (left). (D) GSEA of DEGs after Zic2 transduction of RGCs detects a nonrandom distribution of genes involved in Wnt signaling. The normalized enrichment score (NES) and false discovery rate (FDR) values are shown in the upper right corner of the graph. (E) Examples of RNA-seq profiles from relevant Wnt signaling genes down-regulated (Apc2) or up-regulated (Fzd1, Fzd8, and Lgr5) by Zic2.

Fig. 5. Zic2 activates a program that leads to the accumulation of βcatenin 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 βcatenin 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 βcatenin (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 βcatenin or Δ90-βCatenin-ΔCT. 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.

Fig. 6. EphB1 phosphorylates βcatenin.

(A) Optic chiasms from E16.5 embryos electroporated at E13.5 with plasmids encoding EGFP plus EphB1 alone or together with plasmids bearing shRNA against βcatenin (ctnnb1 shRNA). Scale bar, 100 μm. (B) Quantification showing the percentage of ipsilaterally projecting axons in each condition normalized to the total number of EGFP axons at the chiasm (n = number of embryos) (two-tailed unpaired t test, **P < 0.01 and ***P < 0.001). Results show means ± SEM. (C) Detection of βcatenin in the immunoprecipitation (IP) of tyrosine-phosphorylated proteins from GFP-, EphB1-, and EphB1-ΔCT–transfected cells. (D) Detection of tyrosine phosphorylation in βcatenin immunoprecipitated from GFP-, EphB1-, and EphB1-ΔCT–transfected cells. (E) Detection of Y654-βcatenin in protein extracts from GFP- and EphB1-transfected cells. (F) Immunohistochemistry using antibodies to Y654-βcatenin and total βcatenin in HEK293 cells transfected with EGFP or EphB1. Scale bar, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (G) Percentage of phospho–Y654-βcatenin cells on EGFP-expressing cells. Eleven regions of interest (ROIs) were quantified from three independent experiments for each condition. Two-tailed Mann-Whitney test (***P < 0.001). Results show means ± SEM. (H) Quantification of the area occupied by individual cells transfected with EGFP alone or EphB1- and EGFP-encoding plasmids (n = number of cells). Two-tailed unpaired t test (***P < 0.001). Results show means ± SEM. (I) pY654βcatenin immunostaining in retinal explants electroporated with EGFP- or EphB1/EGFP-encoding plasmids. Scale bar, 50 μm. Scale bar in the higher magnification panels at the right, 10 μm. Representative experiments from three independent experiments are shown.

Fig. 7. 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 βcatenin in contralateral axons. It has been shown that βcatenin links cadherins and actin microtubules. Thus, Wnt5a-dependent accumulation of βcatenin 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 βcatenin. We also show that EphB1 phosphorylates βcatenin in Y654, and previous reports have demonstrated that phosphorylated Y654-βcatenin has low affinity for cadherins. Taking all these observations together, we propose that phosphorylation of βcatenin induced by the binding of EphB1 to ephrinB2 prevents the formation of cadherin/actin complexes facilitating axon steering.