Upon Solving the Secrets for Lens Transdifferentiation - Abstract Urodele newts are the unrivaled champions of vertebrate tissue regeneration. A greater understanding of the processes which enable their regenerative capabilities could inspire novel therapeutic approaches to combating blinding ailments. After injury, the Spanish ribbed newt Pleurodeles waltl can regenerate its lens through the transdifferentiation of iris pigment epithelium cells (IPECs) into lens progenitor cells. Interestingly, this regenerative competence is restricted to IPECs within the dorsal compartment of the iris. Ventral IPECs are nominally regeneration-incompetent, despite being morphologically indistinguishable from their dorsal counterparts. Past efforts to identify molecular distinctions between dorsal and ventral IPECs have been unable to pinpoint the precise determining factors for regeneration competence. These limitations were in part owing to the large genomes of amphibians, which have precluded the robust delineation of gene structures. More recently, our group has begun to address these limitations in Pleurodeles waltl, which now has an available reference genome. Our preliminary observations suggest that the molecular foundations of IPEC regenerative competence are determined by a highly conserved signaling axis relating to vertebrate eye development, including BMP and Ephrin signaling cues. Upon surgical removal of the lens, we demonstrate that the newt iris undergoes distinct shifts in the localization of BMP signaling effectors, serving as a molecular “switch” for regeneration competence. Subsequently, the local environment of the dorsal iris is transformed into a pro-regenerative niche, whereas the ventral iris is characterized by an acute injury-response program. Furthermore, we demonstrate that pharmacologic perturbation of BMP or Ephrin signaling is sufficient to confer regenerative competence to ventral IPECs. These results collectively lead us to hypothesize that newt lens regeneration is directed by a unique gene regulatory paradigm that evolved downstream of the conserved BMP and Ephrin signaling axes. To investigate this conjecture, we propose to comprehensively interrogate the role of these signaling axes in directing dorsal and ventral IPEC injury responses. Gene regulatory mechanisms, including chromatin accessibility profiling, will be performed on dorsal and ventral irises at landmark time points during lens regeneration (Aim 1). Moreover, we will construct a spatiotemporal atlas of cell types associated with the regenerative dorsal iris and non-regenerative ventral iris by leveraging cutting-edge techniques, including single- nucleus RNA sequencing and spatial transcriptomics (Aim 2). Finally, we will systematically perturb the BMP and Ephrin pathways during lens regeneration by using in vivo screens for druggable nodes within these signaling cascades as well as transgenic newt lines (Aim 3). Collectively, these findings will greatly expand our knowledge of the cellular and molecular determinants for regeneration competence. We are hopeful that the proposed experimentation has the potential to uncover novel routes for the induction of tissue regeneration as a method to combat eye diseases.
