PROJECT SUMMARY: Age-related macular degeneration and several other diseases can cause rod and cone photoreceptors to die. This results in permanent visual impairment because the human retina does not regenerate itself. One potential way to restore vision is by replacing the lost photoreceptors. Audacious strategies to restore vision include reprogramming the diseased retina to regenerate its own photoreceptors and directly replacing the lost photoreceptors from stem cell sources. Implementing these strategies requires specific and efficient methods of programming cells to become rods and cones. However, we face a major barrier to achieving these therapeutic strategies due to our limited understanding of the mechanisms that govern how photoreceptors normally develop. Most of the undifferentiated progenitor cells of the retina will activate the transcription factor Otx2 and acquire the potential (or competence) to become photoreceptors and bipolar cell interneurons. These competent Otx2+ cells then choose between rod, cone, and bipolar cell types. This fate decision is controlled in part by the transcription factors Prdm1 and Vsx2. Loss of Prdm1 profoundly increases bipolar cells at the expense of photoreceptors. Conversely, Vsx2 mutants lack bipolar cells. Neither photoreceptors nor bipolar cells are generated when Otx2 is mutated. Therefore, the gene regulatory networks responsible for Otx2, Prdm1, and Vsx2 expression determine the cell type composition of the retina and its ability to function normally. Our goal is to decipher how this gene regulatory network functions to control cell fate decisions during retinal development. Our first objective is to determine how Otx2 expression, which is necessary for photoreceptor and bipolar cell competence, is regulated during mouse retinal development. We have identified three non-coding DNA regulatory elements (enhancers) that are essential for Otx2 expression at different times in development. Perturbing enhancer function also suggests that they dynamically interact in a tight three-dimensional structure that allows them to substitute for one another. We will test how these enhancers cooperate and substitute for each other in normal and perturbed conditions using high-resolution chromosome conformation capture and other genetic approaches. Our second objective is to understand how Otx2+ cells choose between photoreceptor and bipolar cell types. We found that deleting the bipolar-specific enhancer of Vsx2 prevented bipolar cell formation. Our results from mutating this enhancer along with Prdm1 suggested that transient Vsx2 expression permanently drives bipolar formation. Using a combination of mouse mutants, mutagenesis, misexpression, and CRISPR inhibition tools, we will determine how Vsx2 and the Vsx2 bipolar enhancer control bipolar fate choice. Our experiments will unravel the gene regulatory network that controls photoreceptor and bipolar cell competence and fate choice. Gaining this knowledg...