PROJECT SUMMARY: Age-related macular degeneration and several other diseases can lead to the death of rod and cone photoreceptors. This causes permanent vision loss because the human retina lacks the capacity to regenerate itself. One possible way to reverse vision loss is by replacing dead photoreceptors. Audacious strategies to accomplish this goal include directly replacing photoreceptors from stem cell sources and reprogramming the diseased retina to endogenously regenerate its own photoreceptors. Realizing these strategies requires efficient methods to program cells to become photoreceptors. However, the mechanisms that control how cells normally become photoreceptors are not fully understood, hindering the feasibility of such therapeutic strategies. A subset of retinal progenitor cells will turn on the transcription factor Otx2 and become competent to form photoreceptors and bipolar cells. These Otx2+ cells then choose between photoreceptor and bipolar cell fates. This is controlled in part by the transcription factors Prdm1 and Vsx2. Loss of Prdm1 profoundly increases bipolars at the expense of photoreceptors and the loss of Vsx2 has the opposite effect. Photoreceptors and bipolar cells still form when both genes are deleted simultaneously. Thus, other factors must specify these fate choices in Otx2+ cells. It is unknown how these fate choices are made and how Otx2 expression is controlled. The first objective of this project is to determine how Otx2 expression is regulated in the retina. We have identified two non-coding DNA regulatory elements (enhancers) that are necessary for Otx2 expression. We will dissect each enhancer to identify binding factors and test whether they are necessary for enhancer activity and Otx2 expression. The second objective is to determine how photoreceptor and bipolar fate specification occurs. Prdm1 and Vsx2 suppress the formation of bipolars and photoreceptors, respectively. We will bypass the masking effects of Prdm1 and Vsx2 to more sensitively determine how cell fate choices occur. For this, we will use Prdm1, Vsx2, and double mutant cells to generate a single-cell transcriptomics dataset. Analysis of this dataset will reveal how photoreceptor and bipolar cell fate choices are made. Factors whose expression correlates with these fate choice events will then be tested to determine whether they are necessary and sufficient for photoreceptor and bipolar cell formation. The number of photoreceptors in the retina depends on how many cells make Otx2 and what fraction of them decide upon photoreceptor instead of bipolar fate. Our experiments will uncover how both of these events are controlled. Understanding how to generate photoreceptors may directly benefit cell and regenerative medicine strategies that aim to replace lost photoreceptors, potentially reversing vision loss in millions of people.