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Randy Igbinoba, Joshua Atkinson, Ya-Ping Lin, Gloria Orozco, Duc Nguyen, John O'Brien; Development of a Zebrafish Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2018;59(9):987. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa (RP) is an inherited blinding disorder in which rod photoreceptors degenerate and are not replaced. Current therapies aim to delay progression of the disease, but replacement of photoreceptors has been elusive. Zebrafish have robust retinal regeneration capabilities that are superior to those of mammals. When zebrafish retina is diseased or injured, it produces new progenitor cells that differentiate to form many neuron types. We hypothesized that we could develop a zebrafish model system to study regeneration of rods by expressing a rhodopsin gene containing a retinitis pigmentosa (RP) mutation in zebrafish rods.
We developed a Tol2 transgene construct containing mouse rhodopsin with a C-terminal Flag tag and a P23H mutation driven by a zebrafish rhodopsin promoter. This construct was microinjected into zebrafish embryos to create transgenic RP mutant zebrafish. We examined transient transgenic fish from 4-14 days post-fertilization (dpf), and stable transgenic fish from 4 dpf to adult by immunostaining sections of retina. The antibodies used were targeted to rhodopsin to label rods, Flag to visualize the mutant rhodopsin, Zpr-1 to visualize double cones, Brn2 to label neural progenitor cells, and PCNA to label retinal stem cells.
Immunostaining for the Flag epitope showed that the transgenic zebrafish expressed the P23H rhodopsin only in rods. At 7 dpf P23H rhodopsin was delocalized throughout the soma and terminal. Neurites extended from the rod terminals into the inner nuclear layer. Adult P23H zebrafish had rods and endogenous rhodopsin was localized to the outer segments; however, rods were morphologically compromised and mutant rhodopsin was delocalized throughout the soma and terminal. Neural progenitor cell proliferation in P23H mutant larvae at 7 dpf was significantly greater than in wild type fish.
Zebrafish expressing the P23H rhodopsin transgene experience rod photoreceptor degeneration and enhanced proliferation of retinal progenitor cells. The zebrafish model of RP that we created can be used to study photoreceptor replacement during retinal degeneration.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Mutant Rhodopsin with Flag label in 7 day old fry.
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