The Paired Homeodomain transcription factor Pax6 is an important regulator of cell fate in the human eye, and mutations cause congenital eye defects, including aniridia, leading to blindness. Recent studies in both vertebrates and in Drosophila show that Pax6 has a role both in proliferation of retinal progenitor cells, and in regulating their transition to differentiation, in part through activation of proneural genes. Accordingly, Pax6 is currently the topic of intense work aimed towards converting e.g. induced pluripotent stem cells to retinal neurons. Such work holds spectacular promise for treating neurodegenerative eye disease. However, for this type of approach to be successful as well as safe, it is imperative that we understand the mechanisms by which Pax6 regulates gene expression during the transition between proliferation and differentiation. The long- term goal of this application is to understand the mechanisms by which Pax6 regulates gene expression during proliferative phases of retinal development as well as during the transition to differentiation. Our previous work has demonstrated that Drosophila Pax6, Eyeless (Ey), is physically and functionally linked during eye development with the transcriptional regulator CtBP, which is known to link sequence-specific transcription factors to chromatin modification factors. Additional data suggests that Ey and CtBP have antagonistic functions in regulating proliferation as well as expression of the proneural gene atonal. This data provides an exceptional opportunity to probe the mechanisms by which Pax6 regulates gene expression during the transition from proliferation to differentiation. The proposed research will test the hypothesis that CtBP interacts with Drosophila Ey/Pax6 and other eye specification factors to regulate gene expression important for proliferation as well as differentiation. We will integrate the powerful genetics available in Drosophila with molecular and transcriptomic approaches to determine (1) whether CtBP antagonizes Ey's function in regulating proliferation and ato expression, (2) which ato enhancer elements respond to CtBP and other repressive factors linked to CtBP [e.g. the bHLH factor Hairy and the Notch pathway transcription factor Su(H)], (3) the mechanisms by which Ey and CtBP associate and whether the switch in target gene expression between proliferating and differentiating cells depends on the presence of different cofactors, and (4) what genes besides ato are co-regulated by Ey and CtBP, which will provide abundant future opportunities for understanding the mechanisms by which Pax6 functions. In summary, the proposed research will provide a novel mechanistic approach to the study of Pax6 function in eye specification and proliferation, and lay the foundation for development of new treatments for blindness.
The Pax6 gene is a critical regulator of cell fate in the human eye and mutations cause congenital eye defects, including aniridia, that lead to blindness. Furthermore, Pax6 is at the center of recent efforts to use stem cells to treat neurodegenerative disease. A detailed understanding of how Pax6 works is therefore important because it will facilitate the development of novel therapies for blindness.