Investigating the role of CYP26 and retinoic acid signaling regulation in vertebrate cornea and lens regeneration

The larvae of Xenopus laevis can naturally regenerate a lost lens from the outer cornea epithelium after it is triggered to do so by signals from the neural retina. The signals have been widely studied, and FGFs are reported to play a key role in causing the cornea to transform into a lens. However, the factors that make the cornea itself competent to respond to these signals are unknown. Understanding the factors that underlie regeneration competency is the key to granting otherwise ordinary tissues the ability to regenerate, including in our own bodies. Thus, the focus of this work has been on the cornea, in order to unravel the signaling schemes that operate within it. The Retinoic Acid (RA) signaling pathway is a major cellular signaling pathway involved in development, organogenesis, and regeneration. It was strongly implicated in the regeneration of the lens in another model system, the newt, where retinal signals trigger the dorsal region of the iris to differentiate and give rise to a lens. Antagonism of RA signaling was shown to inhibit lens regeneration, demonstrating its necessity. We investigated whether the same was true in Xenopus. We inhibited RA signaling using inhibitors of RA synthesizing enzymes, and of the RAR nuclear receptors. In all cases we found there to be no effect on regeneration. We validated that the drug treatments were meaningful by observing, via qPCR, a decrease in the expression cyp26a1, a well-established marker of RA signaling. We also examined the expression of multiple RA signaling pathway members both in control corneas and in corneas harvested in the first 4 days following lentectomy. In both these normal and regenerating tissues we found the expression of cyp26 genes, which encode the RA metabolizing enzyme CYP26. In light of this finding, we assessed whether CYP26 was necessary for supporting lens regeneration. In contrast to the experiment described above, exogenous addition of an antagonist of CYP26 greatly inhibited lens regeneration. Likewise, a synthetic retinoid that activates RA signaling without being metabolized by CYP26 also inhibited regeneration, as did excess exogenous RA. In all treatments, we observed profound upregulation of the RA signaling marker cyp26a1. Taken together, we demonstrated that the action of CYP26 is necessary for lens regeneration, which implies a necessity to attenuate RA signaling by metabolism in order for lens regeneration to occur in Xenopus. This represents a species-specific difference in the signaling schemes that underlie lens regeneration, and a previously undescribed role for CYP26 in regeneration. Using immunohistochemistry and a whole-cornea mounting technique, we observed the widespread expression of RALDH and CYP26 enzymes within the corneal layers under a confocal microscope. We next investigated the possible mechanistic roles of CYP26 that could explain its necessity in lens regeneration. We assessed whether RA signaling regulated cell proliferation in the cornea by quantifying changes in cell division following various treatments. We found that CYP26 antagonism, but not exogenous retinoids, lead to a significant reduction in cell proliferation. This finding lead us to examine the possibility that CYP26 may actively generate RA-metabolites whose absence would lead to reduced corneal cell division, and therefore stunted regeneration. This would suggest a mechanistic role of CYP26 as a ligand generator, rather than simply an RA signaling attenuator. We tested this by supplementing ex vivo cultures with both Liarozole (a CYP26 antagonist), and 4-oxo-RA (a metabolic product of RA). These co-treated cultures failed to regenerate lenses, just like Liarozole-only controls. Additionally, 4-oxo-RA supplementation does not recover the loss of cell division that occurs from Liarozole. We also documented that 4-oxo- RA causes the upregulation of cyp26a1 in corneas, confirming its transcriptional effects. Taken together, it appears that CYP26 likely plays a role to simply attenuate RA signaling, and does not act to generate novel signaling ligands. We additionally examined the possibility that RA signaling modulates the expression of key corneal stem cell markers like sox2, oct60, and p63. We examined their expression under RA antagonism and stimulation, and found that CYP26 does not likely mediate its effects through such markers. However, we discovered that RA can attenuate the expression of the eye markers fgfr2 and pax6 in the cornea, and CYP26 may prevent this attenuation during regeneration. Lastly, we looked at whether CYP26 was involved in early, late, or all stages of lens regeneration by varying the timepoints at which compounds were added to ex vivo cultures. We discovered that CYP26 activity is only important during hours 12-48 post-lentectomy. This suggests a role in establishing or maintaining lens- competency in the cornea, rather than lens cell differentiation/maturation. In summary, the principal finding of these studies is that CYP26 is important for Xenopus lens regeneration, likely by acting to attenuate RA signaling during early regenerative events