Elucidating competence and fate choice dynamics during retinal development

The National Eye Institute (NEI), under the Vision Research program (CFDA 93.867), awarded $830,024 to The Regents of The University of Colorado-Denver for a two-year project (September 1, 2025 – August 31, 2027) titled "Elucidating Competence and Fate Choice Dynamics During Retinal Development." The project aims to advance understanding of retinal cell development mechanisms by investigating how Notch signaling regulates cell fate decisions in the developing mouse retina. The research employs newly identified enhancer sequences for the transcription factor OTX2 to dynamically manipulate Notch signaling in specific cell populations over time, with the ultimate objective of generating foundational knowledge applicable to therapeutic strategies for vision restoration, including endogenous neuronal regeneration and stem cell-derived retinal tissue transplantation.

The research deliverables are generated through a collaborative structure involving a sub-award to Bioinformatic Solutions LLC (a small disadvantaged business based in Sheridan, Wyoming), obligated on October 29, 2025. The sub-awardee provides bioinformatics research support services essential to the project's data analysis and computational requirements. The combined research outputs—basic science findings on retinal developmental mechanisms and supporting bioinformatic analyses—are expected to inform future regenerative medicine approaches for degenerative retinal diseases that currently result in permanent vision loss.

Abstract

Degenerative diseases that cause retinal neuronal cell death often result in permanent vision loss. This is because retinal neurons, like rod and cone photoreceptors, do not regenerate. The most promising potential therapeutic strategies for restoring lost vision include artificially stimulating endogenous neuronal regeneration or programming human stem cells into transplantable retinal tissue. However, realizing such strategies is hindered by our limited understanding of the developmental mechanisms used to build the retina. During retinal development, proliferative multipotent progenitor cells choose between seven major fate outcomes. This choice process, called fate specification, is determined by a combination of a cell's potential (i.e., competence) and instructive factors that select between competing fate choices. Retinal fate specification is a dynamic, probabilistic process that is controlled by the intersection of intrinsic gene regulatory networks and environmental cell-cell signaling mechanisms. The notch signaling pathway impacts competence and fate choice decisions in the retina. However, its mechanisms are poorly understood due to a lack of genetic tools that can dynamically manipulate signaling in specific subpopulations of competent cells over time. To overcome this barrier, we identified enhancer sequences for the key transcription factor OTX2 that drive discrete spatial and temporal activity patterns in the mouse retina. Using these narrowly tailored enhancer tools, our initial findings show that notch signaling plays multiple discrete fate choice roles throughout development. Our objective is to finely dissect how notch signaling functions to understand the probabilistic nature of retinal cell fate specification. In aim 1 of this proposal, we will investigate how notch signaling regulates multiple different fate decisions throughout retinal development. We will activate notch signaling at discrete stages of retinal development and use single cell RNA sequencing and histological approaches to determine how cells change competence and fate choices over time. These data will be used to determine whether notch signaling delays decision making or acts by specifically instructing fate choices in competent cells at different stages of development. In aim 2, we will use developmental and genetic techniques to explore how notch signaling exposure (dosage and duration) differentially impacts competence and retinal cell fate decisions. Leveraging our unique genetic tools, this project will reveal how the multifaceted notch signaling pathway impacts competence and dynamic fate choice probabilities in the developing retina. This knowledge is essential for creating regenerative and cell-based therapies to replace lost neurons, which may restore vision in millions of people suffering from retinal degenerative diseases.