[Seminar] Comparative Transcriptomic and Epigenomic Analyses of Retinal Müller Glia during Different Damage Paradigms in Zebrafish, Chick, and Mouse by Professor David R. Hyde

Comparative Transcriptomic and Epigenomic Analyses of Retinal Müller Glia during Different Damage Paradigms in Zebrafish, Chick, and Mouse

David R. Hyde

Department of Biological Sciences, Center for Zebrafish Research, and Center for Stem Cells and Regenerative Medicine, University of Notre Dame, Notre Dame, IN 46556

Retinal damage induces zebrafish Müller glia to reprogram and reenter the cell cycle to produce neuronal progenitor cells that continue to proliferate and differentiate to regenerate the lost cells. In contrast, the damaged chick retina undergoes only a limited regenerative response and the damaged mouse retina fails to regenerate, with the Müller glia undergoing gliosis. While several groups examined gene expression changes in damaged/regenerating zebrafish retinas, comparisons between different damage models and cross-species analyses have not been performed. We tackled these knowledge gaps by comparing light-damaged zebrafish retinas, which lose rod and cone photoreceptors, NMDA-damaged zebrafish retinas, in which amacrine and ganglion cells die, and zebrafish retinas coinjected with TNFa and a gamma secretase inhibitor, which stimulates Müller glia to reprogramming and proliferation without any significant cell death in Tg[gfap:EGFP] transgenic zebrafish. We FAC-sorted EGFP-positive Müller glia and GFP-negative neurons for RNA-seq and ATAC-seq analyses of gene expression and chromatin compaction, respectively, at multiple timepoints prior to Müller glia proliferation in each treatment. We also performed single cell RNA-seq (scRNA-seq) in all three treatments at the same timepoints to obtain transcriptomic data for each retinal cell type. A comparative analysis of RNA-seq, scRNA-seq, and ATAC-seq was performed across all three treatments to reveal candidate genes required for zebrafish Müller glia reprogramming and proliferation.  

Analogous treatments and analyses were also performed in chick and mouse retinas and a comparative analysis of the RNA-seq, scRNA-seq and ATAC-seq datasets from all three model organisms identified candidate genes that regulate Müller glia reprogramming and proliferation, including several transcription factors and chromatin remodeling proteins. We conditionally knocked down the expression of several candidate proteins to test their role in Müller glia programming and proliferation. These tests involved quantitative real-time expression of mRNAs to assess gene expression, immunohistochemistry to assess Müller glia proliferation, and Western blots to assess protein expression. We will discuss the roles of these transcription factors and chromatin remodeling candidates in zebrafish Müller glia reprogramming and proliferation.

Biography:

David Hyde received his B.S. degree in Biochemistry from Michigan State University in 1980. He earned his Ph.D. in Biochemistry and Molecular Biology from the Pennsylvania State University in 1985, where he studied the mechanism and regulation of a bacterial transposable element. He did his postdoctoral research at the California Institute of Technology with Dr. Seymour Benzer. His research involved one of the first transcriptomic experiments performed, where they identified thousands of genes that were expressed in the Drosophila head and using a variety of fly mutants and in situ hybridization, determined the spatial expression patterns of these genes. He took a faculty position at Notre Dame University in 1988. As an Assistant Professor, His lab cloned the elusive retinal degeneration B gene in Drosophila, which causes a light-enhanced retinal degeneration, and determined that it encodes a novel transmembrane phosphatidylinositol transfer protein. His lab also identified and cloned the G protein alpha subunit (Gqa), that is required for Drosophila phototransduction. He then decided to pursue genetic models of retinal disease in a vertebrate model and selected zebrafish. While initially studying the effects of light damage on the zebrafish retina, his lab was the first to describe the ability of the zebrafish retina to regenerate from the endogenous Muller glia. His lab subsequently made a number of key findings about Muller glia-dependent neuronal regeneration such as: TNFa is expressed by dying retinal neurons and is necessary to stimulate Muller glia reprogramming and cell cycle reentry, Notch signaling maintains Muller glia in a quiescent state and must be downregulated for Muller glia reprogramming and cell cycle reentry, and Pax6 is necessary for continued proliferation of the Muller glia-derived neuronal progenitor cells. His lab recently initiated a large scale collaboration with Johns Hopkins Medical School, Ohio State Medical School, and the University of Florida Medical School to do a comparative transcriptomics and epigenomics analysis of Muller glia in response to different damage models in zebrafish, chick, and mouse.

David is currently a Professor of Biological Sciences at the University of Notre Dame, the Kenna Director of the Center for Zebrafish Research, and the Director of the Center for Stem Cells and Regenerative Medicine. His research is funded by two different grants from the National Institutes of Health.