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The frog, Xenopus laevis, possesses a high capacity to regenerate various larval tissues, including the lens, which is capable of complete regeneration from the cornea epithelium. However, the molecular signaling mechanisms of cornea-lens regeneration are not fully understood. Previous work has implicated the involvement of the Wnt signaling pathway, but molecular studies have been very limited. Iris-derived lens regeneration in the newt (Wolffian lens regeneration) has shown a necessity for active Wnt signaling in order to regenerate a new lens. Here we provide evidence that the Wnt signaling pathway plays a different role in the context of cornea-lens regeneration in Xenopus. We examined the expression of frizzled receptors and wnt ligands in the frog cornea epithelium. Numerous frizzled receptors (fzd1, fzd2, fzd3, fzd4, fzd6, fzd7, fzd8, and fzd10) and wnt ligands (wnt2b.a, wnt3a, wnt4, wnt5a, wnt5b, wnt6, wnt7b, wnt10a, wnt11, and wnt11b) are expressed in the cornea epithelium, demonstrating that this tissue is transcribing many of the ligands and receptors of the Wnt signaling pathway. When compared to flank epithelium, which is lens regeneration incompetent, only wnt11 and wnt11b are different (present only in the cornea epithelium), identifying them as potential regulators of cornea-lens regeneration. To detect changes in canonical Wnt/β-catenin signaling occurring within the cornea epithelium, axin2 expression was measured over the course of regeneration. axin2 is a well-established reporter of active Wnt/β-catenin signaling, and its expression shows a significant decrease at 24 h post-lentectomy. This decrease recovers to normal endogenous levels by 48 h. To test whether this signaling decrease was necessary for lens regeneration to occur, regenerating eyes were treated with either 6-bromoindirubin-3'-oxime (BIO) or 1-azakenpaullone - both activators of Wnt signaling - resulting in a significant reduction in the percentage of cases with successful regeneration. In contrast, inhibition of Wnt signaling using either the small molecule IWR-1, treatment with recombinant human Dickkopf-1 (rhDKK1) protein, or transgenic expression of Xenopus DKK1, did not significantly affect the percentage of successful regeneration. Together, these results suggest a model where Wnt/β-catenin signaling is active in the cornea epithelium and needs to be suppressed during early lens regeneration in order for these cornea cells to give rise to a new lentoid. While this finding differs from what has been described in the newt, it closely resembles the role of Wnt signaling during the initial formation of the lens placode from the surface ectoderm during early embryogenesis.
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???displayArticle.pmcLink???PMC4842149 ???displayArticle.link???Exp Eye Res ???displayArticle.grants???[+]
Fig. 1.
Expression of wnts and frizzled receptors in larval cornea and flank epithelium. RT-PCR was conducted on total RNA isolated from larval corneaepithelium (“C”) or flank epithelium (“F”). “+” denotes a positive control from pooled embryonic total RNA. “C-” and “F-” are reverse transcriptase negative controls. Primers and corresponding product sizes are listed in Supplemental Table 1.
Fig. 2.
axin2 expression decreases early during lens regeneration. axin2 transcriptional levels in corneaepithelium regenerating for either 24, 48, 72, or 96 h. Control expression is from corneaepithelium that is not wounded and is not undergoing regeneration. axin2 expression was normalized to beta-actin (actb). Asterisk denotes a statistically significant decrease (p value = 0.0006). Error bars indicate standard error.
Fig. 3.
Activation of Wnt signaling reduces successful lens regeneration. Lens regeneration percentages for treatment with Wnt signaling activators, including 10 μM 1-azakenpaullone (A) or 1 μM BIO (B), compared to DMSO treated controls. “Percent Regenerative Success” refers to the percentage of cases that successfully regenerated based on the presence of morphologically distinct lentoids that were positively stained by an anti-lens antibody. (C) Relative expression levels of axin2 in corneas treated with 10 μM 1-azakenpaullone or 1 μM BIO. Asterisks indicate statistical significance (p < 0.05) and error bars indicate standard error. axin2 expression was normalized to ornithine decarboxylase (odc). (D–K) Representative examples of positive cases of lens regeneration for BIO or 1-azakenpaullone treated eyes. D, F, H, and J show histological eye sections labeled using an anti-lens antibody (red fluorescence). Arrowheads indicate regenerated lenses. Sometimes higher background signal is observed in retinal tissue (H and J), but lenses are easily identified due to their enhanced signaling and distinct morphology. E, G, I, and K show an overlay of anti-lens staining with the corresponding DIC images. Dark tissue is retinal pigment epithelium. All images are at the same scale, scale bar in K is 200 μm.
Fig. 4.
Wnt inhibition has no effect on lens regeneration. Lens regeneration percentages for ex vivo eyes treated with either 10 μM IWR-1 (A) or rhDKK1 protein at either 200 ng/ml or 500 ng/ml (B). “Percent Regenerative Success” refers to the percentage of cases that successfully regenerated based on the presence of morphologically distinct lentoids that were positively stained by an anti-lens antibody. Neither of these experiments showed statistically significant differences. (C) Both IWR-1 and rhDKK1 treatment significantly decreased axin2 transcriptional levels in treated corneatissue, verifying the Wnt inhibitors had the desired effect on Wnt signaling. Asterisks indicate statistical significance (p < 0.05), and error bars indicate standard error. axin2 expression was normalized to ornithine decarboxylase (odc). (D–O) Representative examples of positive cases of lens regeneration for IWR-1 or rhDKK1 treated eyes. D, F, H, J, L, and N show fluorescent (red) labeling of histological eye sections using an anti-lens antibody. Arrowheads indicate regenerated lenses. Sometimes higher background signal is observed in retinal tissue (H and J), but lenses are easily identified due to their enhanced signal and distinct morphology. E, G, I, K, M, and O show anti-lens staining overlaid with the corresponding DIC images. Dark tissue is retinal pigment epithelium. All images are at the same scale, scale bar in O is 200 μm.
Fig. 5.
Transgenic expression of DKK1 does not inhibit lens regeneration. (A) A diagram of the HGEM-DKK1 bi-cistronic transgene. The heat-shock inducible hsp70 promoter drives expression of Xenopus DKK1, and a gamma-crystallin promoter drives GFP expression specific to the lens. (B–C) HGEM-DKK1 transgenic eye showing constitutive expression of GFP in the lens (C). (D-G) Anti-lens antibody staining of regenerated lenses in a sectioned control eye kept at room temperature (“Cont.”, D and E) and a heat-shocked eye (“HS”, F and G). Arrowheads indicate regenerated lenses. Dark tissue in DIC images is retinal pigment epithelium (D and F). All images are at the same scale, scale bar in G is 200 μm. (H) Regeneration percentages of HGEM-DKK1 eyes are not significantly different (p = 0.3706). “Percent Regenerative Success” refers to the percentage of cases that successfully regenerated based on the presence of morphologically distinct lentoids that were positively stained by an anti-lens antibody. Error bars represent standard error. (I) Depletion of β-catenin (CTNNB1; 94 kDa) in western blots of HGEM-DKK1 transgenic tissue. β-tubulin (TUBB; 50 kDa) is the loading control.
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