Elucidation of cellular reprogramming processes that drive lens regeneration in axolotl as a basis for future therapeutic approaches

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Salamanders, like newts and axolotls, can regenerate ocular tissues when injured. This is possible due to the ability of their pigmented epithelial cells to reprogram to retina and lens. The goal of this research is to unravel the reprogramming mechanism and leverage it for regenerative medicine applications. The main focus of the study is the ability of iris pigmented epithelial (IPE) cells to get activated, proliferate, reprogram, and ultimately regenerate the lens upon its complete removal. In newts, this process is restricted to IPE cells residing in the dorsal iris while ventral IPE cells are thought to not participate at the cellular level. In contrast, juvenile axolotls cannot regenerate their lens. These regenerative discrepancies enable the use of experimental paradigms that could provide important insights into the molecular mechanism of IPE reprogramming. In Specific Aim 1, a combination of comparative transcriptomics, flow cytometry sorting and lineage tracing will be used to identify and characterize IPE subpopulations capable of triggering a regenerative response. These approaches will also be paired with a novel transgenic newt capable of tracking changes in eye transcriptional factor dynamics during reprogramming. This will allow us to better understand the association between molecular and cellular changes during regeneration. In Specific Aim 2, functional characterization of lens regeneration will be performed. To successfully accomplish that, the avian RCAS/TVA system will be implemented to provide spatial and temporal control of target gene expression in the pigmented epithelium. The avian TVA receptor will be ubiquitously expressed in eye tissues, in dorsal IPE cells, or in ventral IPE cells making them susceptible to the RCAS retrovirus. Using this system, the effects of genes and pathways will be tested for their ability to induce regeneration from the ventral, or inhibit regeneration from the dorsal IPE cells, respectively. This approach provides a functional readout by performing necessity and sufficiency experiments. In Specific Aim 3, the relationship between aging, DNA damage, and regeneration potential will be explored. Newts can regenerate their lens from IPE cells throughout life and the ability appears unaffected by aging and repeated injury. First, a repeated injury model will be developed and coupled with molecular hallmarks of aging and DNA damage. Then, pharmacological and genetic methods will be used to study the effects of elevated genotoxic stress on the ability of the pigmented epithelium to reprogram to eye tissues.