Cellular and molecular characterization of the corneal epithelium in Xenopus frogs

The vertebrate cornea is a transparent, avascular tissue that forms the front window of the eye. Specifically, the corneal epithelium is exposed to detrimental conditions including infections, solar (Ultraviolet, UV) irradiation, mechanical injuries, and undergoes constant self-renewal. Corneal epithelial stem cells (CESCs) and their progeny, the transit amplifying cells (TACs), play a prominent role in the maintenance of corneal homeostasis, transparency, and wound repair processes. Towards this end, my thesis focuses on understanding the molecular signature of the corneal epithelium using the frog, Xenopus laevis, with the goal to explore novel markers that will reliably identify populations of CESCs and TACs. In Chapter 2, I start by examining the expression of known corneal biomarkers (previously reported in literature from corneal studies of various vertebrates) in the vertebrate species, the African clawed frog. Here, I used antibody-based immunohistochemical staining to molecularly characterize the expression of nine proteins in the corneas of both Xenopus larvae and post-metamorphic adults. I found that localization of some markers changes between tadpole and juvenile adult stages. Markers such as p63, Keratin19, and beta1-integrin are restricted to basal corneal epithelial cells of the larvae. After metamorphosis their expression is found in basal and intermediate layer cells of the adult frog cornea epithelium. Another protein, Pax6 was expressed in the larval corneas, but surprisingly it was not detectable in the adult corneal epithelium. For the first time we report that Tcf7l2 can be used as a marker to differentiate cornea vs. skin in frogs. Tcf7l2 is present only in the frog skin, which differs from reports indicating that the protein is expressed in the human cornea. Furthermore, I identified the transition between the inner, and the outer surface of the adult frog eyelid as a key boundary in terms of marker expression. Although these markers are useful to identify different regions and cellular layers of the frog corneal epithelium, none was unique to CESCs or TACs. The results of this study substantiate findings from other studies in the field indicating that there may not be a single conserved, specific CESC marker in vertebrates. To overcome the limitation of candidate-based biomarker characterization, however, I undertook single-cell genomics approaches to understand the temporal cell atlas of the frog cornea (Chapter 3). Using ~22,000 corneal cells isolated from two distinct developmental time-points, this work provides key insights about the amphibian corneal transcriptome. The data also reveals several novel genes expressed in corneal cells and spatiotemporal changes in gene expression during corneal differentiation. In addition, the data helps in understanding the developmental trajectory of corneal cells during development and differentiation, and identifies key gene regulatory networks that are involved in corneal maturation. In conclusion, this work provides a detailed molecular characterization of the Xenopus corneal epithelium and establishes distinct and newly identified biomarkers of corneal cellular layers. Although a single biomarker for CESCs was not identified in this work (and may not even exist), it helps identify a range of proteins that could be tested for their functional role in regulating the population of corneal stem cells in vertebrates, including humans. Furthermore, this work will be valuable for future studies to understand the critical factors that regulate cornea epithelial cells and lens regeneration, the latter phenomenon being unique to the larval stages of Xenopus frogs.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste