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Title:New insights into cornea-lens regeneration in Xenopus laevis: the role of Wnt/beta-catenin signaling and the regenerative capacity of the limbal region
Author(s):Hamilton, Paul William
Director of Research:Henry, Jonathan J
Doctoral Committee Chair(s):Henry, Jonathan J
Doctoral Committee Member(s):Chen, Jie; Newmark, Phillip A; Sears, Karen E; Smith-Bolton, Rachel
Department / Program:Cell & Developmental Biology
Discipline:Cell and Developmental Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Xenopus laevis
Abstract:One of the large outstanding questions in the field of developmental biology is how some tissues and organs of certain species are able to regenerate while others cannot. It is only by understanding the molecular mechanisms that drive residual cells in a damaged or diseased tissue to proliferate and differentiate to replace lost structures that we will have the knowledge to attempt to recapitulate these regenerative processes in other species, including our own. Towards this end, the focus of this work is centered on understanding the cell and molecular mechanisms of lens regeneration in the frog, Xenopus laevis, which possesses a high capacity to regenerate larval tissues, such as the complete regeneration of the lens from the cornea epithelium. To fill in a large void in our current knowledge of the cell signaling pathways necessary for regeneration, we investigated the Wnt/β-catenin signaling pathway in the context of lens regeneration (Chapter 2) as it has been shown to be important in both embryonic lens development as well as in Wolffian lens regeneration that takes place in newts and salamanders; however, it had not been functionally studied in the context of cornea-lens regeneration in Xenopus despite being implicated to be involved in the early events of lens regeneration from two independent studies. 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 components of the Wnt signaling pathway. When compared to flank epithelium, which is lens regeneration incompetent, only wnt11 and wnt11b are different (expressed 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 larval cornea epithelium and needs to be suppressed during early lens regeneration in order for these cornea cells to give rise to a new lens. 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 of the vertebrate eye. This similarity between larval lens regeneration and embryonic lens development may not be surprising when one looks at the histological structure of the larval cornea epithelium which is similar to that of the fetal cornea in humans. However, as larvae mature through metamorphosis, the cornea epithelium (and underlying layers of the cornea) matures to become structurally very similar to our own. In light of a new model suggesting that cornea-lens regeneration in the frog, Xenopus, may be driven by oligopotent stem cells and not transdifferentiation of mature cornea cells, we investigated the regenerative potential of the limbal region in post-metamorphic cornea, where the stem cells of the cornea are thought to reside. It has been reported that the mature cornea is competent to regenerate under experimental conditions, despite the fact that the in vivo capacity to regenerate is lost; however, that work did not examine the regenerative potential of different regions of the cornea. Using the thymidine-analog 5-Ethynyl-2’-deoxyuridine, we identify long-term label retaining cells in the basal cells of peripheral post-metamorphic Xenopus cornea, consistent with slow-cycling stem cells of the limbus that have been described in other vertebrates. Additionally, the pattern of label being lost from the central cornea is consistent with a model of centripetal migration of cornea epithelial cells away from the limbal region and into more superficial layers. Using this data to identify putative stem cells of the limbal region in Xenopus, we tested the regenerative capacities of the dorsal and ventral limbal regions, and compared that to the central cornea. All three regions showed a similarly high ability for the cells of the basal epithelium to express lens proteins when cultured in proximity to larval retina. This indicates that the regenerative capacity in post-metamorphic cornea is not restricted to stem cells of the limbal region, but also occurs in the transit amplifying cells located throughout the basal layer of the cornea epithelium. In contrast, there was no clear evidence that apical differentiated cells are contributing to lens regeneration. Finally, in order to more precisely monitor in vivo cell behavior during regenerative phenomena in future studies, we developed a new prolonged imaging technique (Chapter 4). While live imaging of embryonic development over long periods of time is a well-established method for embryos of the frog Xenopus laevis, once development has progressed to the swimming stages (when most regenerative phenomena that are studied currently occur), continuous live imaging becomes more challenging because the tadpoles must be immobilized. Current imaging techniques for these advanced stages generally require bringing the tadpoles in and out of anesthesia for short imaging sessions at selected time points, severely limiting the resolution of the data. Here we demonstrate that creating a constant flow of diluted tricaine methanesulfonate (MS-222) over a tadpole greatly improves their survival under anesthesia. Based on this result, we describe a new method for imaging stage 48 to 65 X. laevis (when lens regeneration occurs), by circulating the anesthetic using a peristaltic pump. This supports the animal during continuous live imaging sessions for at least 48 hr. The addition of a stable optical window allows for high quality imaging through the anesthetic solution and provides for the first time a method for continuous observations of developmental and regenerative processes in advanced stages of Xenopus over 2 days. Together this work provides new insights into the cell signaling mechanisms during larval regeneration, and sets the stage for using new imaging techniques in vivo in future studies of the regenerative process and how it may change as the cornea epithelium develops through metamorphosis.
Issue Date:2016-07-11
Rights Information:Copyright 2016 Paul Hamilton
Date Available in IDEALS:2016-11-10
Date Deposited:2016-08

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