Lens regeneration from the cornea requires suppression of Wnt/β-catenin signaling

Abstract

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 hours post-lentectomy. This decrease recovers to normal endogenous levels by 48 hours. 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.

The Development, Growth, and Regeneration of the Crystalline Lens: A Review

“…Functional studies by Hamilton et al have shown that Wnt/β-catenin signalling needs to be supressed for successful lens regeneration to occur via the cornea-lens route. 116 When the Wnt pathway was held in a state of active signalling, there was a reduction in cases of successful lens regeneration in Xenopus. Conversely, when Wnt signalling was supressed, there was no reduction in regenerative ability.…”

FGF1 PromotesXenopus laevisLens Regeneration

“…These findings add to a growing base of knowledge showing various forms of lens regeneration employ different signaling mechanisms and evolved independently (e.g. retinoic acid and Wnt signaling; Hamilton et al, 2016;Thomas & Henry 2014).…”

“…Sperm nuclei for transgenesis were prepared as previously described (Sive et al, 2000). Transgenic larvae were generated using a protocol similar to the REMI method with slight modifications noted (Kroll et al, 1996;Hamilton et al, 2016). Sperm nuclei were incubated with linearized DNA without egg extract, as described in (Sparrow et al, 2000).…”

The lens regenerative competency of limbal vs. central regions of mature Xenopus cornea epithelium

“…Differences between any two groups were not statistically significant as measured by Fisher’s Exact Test (Fisher, 1922). The observed regenerative rates were higher than what Filoni et al (1997) observed; however, our experiments were carried out in an ex vivo culture system where the cornea is tucked directly into the optic cup so that it is in close contact with the neural retina, typically leading to high rates of regeneration (Fukui and Henry, 2011; Hamilton et al, 2016; Thomas and Henry, 2014). Figure 2I, shows a portion of mature cornea collected from the dorsal limbal region of a post-metamorphic froglet, tucked inside of a larval eyecup.…”

“…Larval host eyes were lentectomized and to ensure that any observed regeneration was derived from the donor tissue and not the cornea epithelium of the larval host, cornea epithelia were completely removed from larval eyes (including the point of attachment to the underlying cornea endothelium). Transplant fragments were then collected and immediately placed into the larval eyecups in an ex vivo eye culture system (see Fukui and Henry, 2011; Hamilton et al, 2016; Thomas and Henry, 2014). These eyes were then removed from the animals, and placed into culture media consisting of: 61% L-15 powder (Invitrogen, Carlsbad, CA); 100 U/ml of penicillin and 100 µg/ml of streptomycin (Mediatech, Manassas, VA); 10% fetal bovine serum (Invitrogen, Carlsbad, CA); 2.5 µg /ml of Amphotericin B (Sigma, St. Louis, IL); and 4 µg /ml of Marbofloxacin (Sigma, St. Louis, IL).…”