Regulation of Embryonic Eye Regrowth in Xenopus Laevis

Regrowth of lost organs and tissues is an amazing ability that some animals possess. For regrowth to be considered full, the organism must regrow the organ or tissue to a state that is structurally and functionally similar to that which was lost. The ability of an organism to regrow lost or damaged tissue varies among animals, both in the mechanisms utilized to achieve regrowth, and in what body parts can be regrown. Potentially even closely related species can vary wildly in regenerative ability. Therefore, regenerative research can look to other tissues and organisms for direction in elucidating the mechanisms through which regrowth is achieved, we must individually determine these mechanisms for each unique organism and tissue .The Tseng laboratory has determined that the African clawed frog, Xenopus laevis can regrow its embryonic eye. This regrowth is achieved in a period of five days following ablation of the developing optic cup and lens placode at developmental stage 27, and the regrown eye is physiologically indistinguishable from the normally developed eye and is functional. This ability is lost as the animal ages, with mature eyes being incapable of full regrowth. We are interested in determining how the embryonic eye achieves regrowth. What are the signals regulating regrowth and does regrowth recapitulate development of the eye? This dissertation helps to elucidate some of the answers to these larger questions. Within our model of regrowth, eye development and regrowth are taking place concurrently, creating interesting questions about timings and order of development/regrowth of the ablated eye. Xenopus eye development is well studied, which allows us to compare our regenerative mechanisms to well-known developmental mechanisms and timings. During Xenopus eye development, the retinal cells differentiate in a particular, overlapping order: ganglion cells, horizontal cells, cones, rods, amacrine, bipolar, and finally Müller glial cells. We determined that during eye regrowth, the retinal cells differentiate in this order as well. Additionally, the regrowing eye has a delay in eye formation compared to the normal developing eye (as determined by morphology and molecular markers) for two days, but by the third day post-surgery, it has caught up to the contralateral eye. We have determined the overall morphology of the eye during regrowth. However we do not know many of the mechanisms regulating regrowth. Previous work in our laboratory determined that inhibition of apoptosis and Pax6 both independently inhibit eye regrowth, but there are likely other regulators at work. My work determined that the Notch signaling pathway is required for regrowth of the Xenopus embryonic eye. The Notch signaling pathway is a conserved developmental pathway that regulates proliferation and differentiation. The pathway is activated through cell-to-cell signaling, with ligand binding triggering a cleavage of the receptor then acts as a transcription factor in the nucleus. I determined that Notch signaling during eye regrowth is required during the first day of the five-day period. Previous work demonstrated an increased amount of proliferation during that period, and by inhibiting Notch1 signaling during the regrowth period, a significant reduction in mitotic cells observed. It does not seem however that inhibition affects differentiation in the regrowing eye, with Notch1 inhibited eyes showing the general retinal morphology of uninhibited eyes, as well as mature rod and ganglion cells in their appropriate places. To determine the interaction of Notch with other known regenerative mechanisms, I overexpressed Notch1 during eye regrowth, while blocking V-ATPase activity. The vacuolar V-ATPase is a transmembrane enzyme that is integral to maintaining cell voltage. Membrane voltage is another regulator of regeneration, and V-ATPase is a regulator of Notch signaling in other systems. I demonstrated that overexpression of Notch1 signaling during regrowth is sufficient to rescue V-ATPase inhibition of regrowth, indicating a link between the two pathways in Xenopus laevis embryonic eye regrowth. I have helped determine the general physiology of the eye during regrowth, as well as determining the necessity and function of Notch signaling. However, there are many questions still remaining regarding embryonic eye regrowth in Xenopus laevis. It is my hope that future generations of scientists utilize this research as a foundation to discover novel mechanisms governing eye regrowth, as well as regrowth of other organs in other systems.