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Eyad Shihabeddin, Haichao Wei, Abirami Santhanam, Jiaqian Wu, John O'Brien; Identification of cells involved in photoreceptor regeneration in adult Zebrafish through single-cell analysis. Invest. Ophthalmol. Vis. Sci. 2020;61(7):2496.
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© ARVO (1962-2015); The Authors (2016-present)
Zebrafish (Zf) have a remarkable capacity to regenerate neurons following retinal injury or disease, making them a suitable model organism for regenerative studies. Current studies in Zf indicate that upon detection of retinal injury or disease, Müller glial cells (MGCs) are reprogrammed to re-enter the cell cycle, asymmetrically divide, and produce multi-potent progenitor cells. While many genes have been shown to play a role in MGC de-differentiation and differentiation of progenitor cells into other cell types, the mechanisms by which these genes work together spatially and temporally remain unknown. We hypothesize that single cell analysis of a Zf model replicating the rod degeneration pathology of a P23H rhodopsin mutation will allow us to assess the trajectory of genes driving rod photoreceptor regeneration.
Single cell 3’ RNA sequencing analysis was performed separately on dissociated WT AB and P23H mutant Zf retinas. Sequences were annotated through CellRanger’s count function and sequence quality assessed through FastQC. Cells were normalized, filtered, and scaled to generate UMAP clusters through Seurat and Monocle3. Single cell trajectory analysis was then performed in Monocle3 on each cluster map.
WT AB cluster analysis revealed MGCs clustered separately from rod photoreceptors and rod-like progenitor cells (RPCs). RPCs, rod photoreceptors, and most bipolar cells clustered together. Trajectory analysis in the WT did not show MGC de-differentiation and trajectory towards progenitor cells, however, there was weak trajectory expression from RPC into rods. Analysis of the P23H mutant model revealed MGCs clustered with rod photoreceptors. Trajectory analysis showed strong trajectory expression of potential genes involved in what appears to be MGC de-differentiation into progenitor cells as well as the re-differentiation of the progenitor cells to RPCs and RPCs into rods.
The preliminary results appear to indicate that cells from the WT AB sample may be in the quiescent state, thus showing limited trajectory expression. Results from the P23H mutant sample appears to have more cells actively de-differentiating or re-differentiating due to the constant state of disease, so cell trajectory is more clearly defined. This suggests that the P23H mutant model allows us to better illustrate the trajectory of genes driving rod photoreceptor regeneration.
This is a 2020 ARVO Annual Meeting abstract.
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