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

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The goal of the project is to harness the intrinsic ability of axolotl salamanders to regenerate ocular tissues and unravel natural ways to improve human stem cell function for use in regenerative medicine. Axolotl’s ability to regenerate the retina and the lens stems from the pigmented epithelium, which reprograms its cellular state upon injury. To understand the mechanism of regeneration I will focus on iris pigmented epithelial (IPE) cells and their capacity to reprogram to lens epithelial cells. This process is age-dependent in axolotls because regeneration can only occur for a short time window of two weeks after hatching and is lost thereafter. This allows me to both identify and functionally characterize the potency of candidate molecules to induce regenerative responses in the same context. Studying axolotl IPE reprogramming will provide important insight into how cellular plasticity has naturally evolved in organisms as a mean to regenerate lost organs. Modern regenerative medicine can learn from this intrinsic regenerative mechanism and apply it to coax reprogramming of any adult cell in a safer and more efficient way. In addition, its application could lead to groundbreaking treatments for blind patients by inducing ocular regeneration. In Specific Aim 1, I will study the role of Eya2 during IPE reprogramming. My preliminary data indicate that Eya2 is required for progenitor cell survival upon genotoxic stress during regeneration. I will also study Eya2’s ability to facilitate IPE reprogramming by regulating the activity of transcription factors required to maintain specific eye-tissue fates. In Specific Aim 2, I will generate a transgenic axolotl model that will allow tracking of lens regeneration by fluorescence in vivo. This transgenic animal will enable me to screen thousands of compounds in a high- throughput manner for their ability to either inhibit or induce regeneration from the IPE. In Specific Aim 3, I will study the cellular heterogeneity of the IPE with single cell RNA-seq. Pseudotime lineage trace analysis will be used to identify whether an IPE-residing progenitor population is present before injury and if it is responsible for regeneration. The in silico identification of a regeneration-poised IPE subpopulation will be further validated and characterized using the gold-standard CreERT2/loxP system in which the recombinase will be targeted in progenitor cell-specific genetic loci. My long-term career goal is to lead a research laboratory with central aim to bridge the regenerative ability gap between salamanders and mammals. My experience working with newts, axolotls and mice, in addition to the data I will acquire during the award training period in the Whited laboratory at Brigham and Women’s Hospital and Harvard Medical School, will enable me to build a foundation for pilot translational experiments in mammals in the near future. My access to and participation in a plethora of meetings, journal clubs, seminars and workshops provide me with invaluable opportunities to communicate my research in academic and clinical settings, receive constructive feedback as well as enhance my professional and leadership skills on my path to becoming a successfully independent scientist.