Elucidating the cis-regulatory landscape of retinal development and regeneration

Thesis (Ph.D.)--University of Washington, 2016-04

The mammalian retina is a heterogeneous mix of neurons and one glial cell type that mediate photo-sensation. The mechanisms of its development and the generation of cell type diversity is an area of on-going investigation. Unlike some lower vertebrates, mammalian retinas are largely incapable of regenerating lost neurons after retinal injury. In this work, I describe my efforts to map the cis-regulatory landscape of retinal development by employing DNase-hypersensitivity sequencing of retina at several important stages. The resulting data was categorized based on behavior to generate lists of putative cis-regulatory elements and transcription factors that regulate specific stages of retinal development. In addition, several putative enhancers were discovered for two key transcription factors, Otx2 and Ascl1. Next, we used this strategy to characterize the differences in cis-regulatory elements between retinal progenitors and cultured Muller glial cells. We found that the pro-neural transcription factor Ascl1, which is critical for retinal regeneration in the zebrafish, is able to partially reprogram mouse Muller glia into retinal neurons. Specifically, many retinal progenitor and neuronal genes were activated and the local chromatin environment was remodeled at ASCL1 binding sites. However, we found that ASCL1 binding within Muller glia only partially recapitulates the developmentally appropriate binding pattern found in retinal progenitors. Further, ASCL1 is able to bind to non-hypersensitive regions of chromatin in Muller glia. By comparing the accessible DNA of retinal progenitors and Muller glia, we were able to identify another factor, Zic1, which augments reprogramming. Finally, we show that perturbation of the epigenome during reprogramming, through the use of histone de-acetylase inhibitors, enhances reprogramming towards the photoreceptor cell fate. These results indicate that understanding the epigenetic state of cells can lead to insights into development and reprogramming with implications for regenerative medicine.