Investigating Conserved Mechanisms for Embryonic Eye Regrowth in Xenopus laevis

Regeneration is a complex process to replace or restore tissue after damage. However, animals vary in their ability to regenerate tissue, and humans are limited in their abilities. Understanding how regeneration occurs in highly regenerative vertebrates, such as in Xenopus laevis frogs will lead to defining common regeneration mechanisms. In recent years, eye regeneration research in Xenopus laevis tadpoles provided new promising insight into the mechanisms necessary for repair of damaged eye tissue. Much progress has been made towards understanding the regenerative capability of the complex eye since Xenopus tadpoles can regenerate mature eye tissues, including: the optic nerve, retina, and lens. Little is known, however, whether the developing eyes of young embryos can be regrown similarly to mature eyes in tadpoles. To further our understanding of the regeneration process, this current research focused on the physiological, molecular, and cellular dissection of the Xenopus embryonic eye regrowth model. First, we examined cell proliferation activity after eye removal and found that cell proliferation was a regrowth-specific activity. We then showed that apoptosis was a required mechanism for embryonic eye regrowth. Previously, we found that connection to the brain was established through the formation of an optic nerve in the regrowing eye and normal behavior was observed through a visual preference swimming assay. Here, we assessed the functionality of the apoptosis-inhibited eye due to the presence of remnant eye cells and determined that it was not sufficient to restore visual preference in tadpoles, indicating that the restoration of visual function through eye regrowth is due to regrown tissues. Next, we determined that during regrowth, cell patterning, and retinal layer formation was delayed by 1 day but restored by 3 days when compared to eye development. Furthermore, an assessment of the differentiated cells in the retinal layer indicated that retinal birth order generated during eye regrowth was consistent with that observed in eye development. Additionally, we showed that Pax6 was required for both eye development and regrowth, but apoptosis was regrowth-specific. Finally, we explored whether a mechanism involving V-ATPase was necessary. Our experiments demonstrated that inhibition of V-ATPase blocked eye regrowth, indicating that V-ATPase is required for this process. The block of eye regrowth with V-ATPase inhibition was due to decreased eye stem cell proliferation. However, the expression of a yeast H+ pump restored eye regrowth in the absence of V-ATPase activity. Finally, we found that V-ATPase and apoptosis may act in independent pathways in embryonic eye regrowth. Overall, our results showed V-ATPase is required for eye regeneration through regulating stem cell proliferation. This research will serve as a foundation to understand the mechanisms driving the regulation of stem cells to repair tissues for the continued advancements in the development of regenerative therapies