The zebrafish retina has the same neuronal components and Mller glial cells (MG) as the mammalian retina, yet after an acute retinal injury, zebrafish MG are activated and act as tissue-specific stem cells to robustly regenerate lost neurons. Mammalian MG in vivo lack this capacity for complete regeneration. In the retinas of zebrafish carrying genetic mutations in transcriptional repressors of TGF? signaling (tgif1 and six3b), there is significantly reduced proliferation and regeneration following an acute light lesion that destroys photoreceptor cells, suggesting that these mutant fish could provide insight into the limited neural regeneration in mammals. The Objectives described in this R15 proposal will build on the fish studies to explore the effects of increased TGF? signaling (in tgif1-/-;six3b-/- fish) and decreased TGF? signaling (using small molecule inhibitors) on proliferation, cell cycle progression, and cell differentiation. This research will be performed using zebrafish as a model system due to several benefits including: 1. the large number of embryos that can be used in developmental studies, and 2. that zebrafish have a remarkable capacity for regenerating neural tissue that mammals are lacking. Developmental studies will be from 1 through 5 days post fertilization when the retina develops from a neuroepithelial layer to a functional retina; proliferation and cell cycle dynamics will be measured by examining the expression of PCNA and cyclin-dependent kinase inhibitors specifically expressed when cells are exiting the cell cycle using western blot and immunofluorescence. While previous observations showed that increased TGF? signaling led to reduced regeneration, it is unknown whether Mller glia in those studies reentered the cell cycle at a lower rate and/or if there is a reduced capacity to generate different types of neurons. Therefore, after an acute light lesion, expression of TGF? signaling pathway members will be described using immunofluorescence, and fish will be exposed to a thymidine analog to permanently label cells newly regenerated generated cells. The number Mller glia, photoreceptors, and interneurons generated during regeneration will be quantified by colocalizing cell-type specific antigens and the analog label using immunofluorescence. Finally, a novel transgenic fish will be developed that will allow researchers to distinguish between stages of the cell cycle in glial cells using fluorescence to address whether TGF? signaling prevents cell cycle re-entry or halts the cell cycle during retinal regeneration. This novel fish line will be a valuable tool for the zebrafish research community to examine questions of glial cell biology throughout the central nervous system. Because TGF? signaling is known to limit proliferation and promote scarring in the mammalian central nervous system, these experiments in fish will directly inform biomedical research in mammalian systems and provide potential therapeutic targets for the human retina to minimize scarring and promote healing or regeneration after an acute damage.
The proposed research will help define how cell to cell communication regulates stem cells in the retina, Mller glia, during development and regeneration of the eye. The results of these studies will help determine gene targets for developing therapeutic treatments for blindness and low vision in humans.
