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Nicole C L Noel, Gordon Hagerman, Michele G DuVal, A Phil Oel, W Ted Allison; Effects of Targeted Cone Ablation on the Integrity of Neighbouring Photoreceptor Subtypes. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2250.
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© ARVO (1962-2015); The Authors (2016-present)
Zebrafish are optimal for the genetic investigation of cone photoreceptor physiology, regeneration, and functional integration. We have engineered a pair of novel transgenic zebrafish models enabling targeted ablation of specific cone subtypes. This model allows us to determine whether specifically ablating cone subtypes results in a bystander effect on neighbouring photoreceptors, toxic or otherwise, and ultimately provides opportunity to explore other retinal responses to specific cone loss, such as regeneration, functional integration, and connectivity changes.
We engineered two transgenic zebrafish lines to express nitroreductase (NTR) in blue- or UV-sensitive cones, allowing conditional cone ablation upon the introduction of an otherwise inert prodrug, metronidazole (MTZ). A cell death assay was performed on MTZ-treated and DMSO (vehicle control)-treated larvae, and the number of apoptotic cells counted. Co-localization of apoptotic markers with a NTR-mCherry expressing blue or UV cone demonstrated the death of a target. Abundance of remaining cone types was assessed via antibody labeling.
Increased cell death was not detected amongst adjacent cells when blue (p>0.85, n=11 for MTZ, n=12 for DMSO) or UV cones were ablated (p>0.99, n=9 for MTZ, n=5 for DMSO). The predicted increase in apoptosis for target cell types upon introduction of MTZ was observed (p<0.001 for blue ablation, p<0.05 for UV ablation). Similarly, the relative abundance of non-target cone types did not change between MTZ- or DMSO-treated fish for blue (p>0.97, n=12 for both treatments) or UV ablation (p>0.78, n=5 for MTZ & n=6 for DMSO).
We investigated whether a bystander effect was elicited when specifically ablating blue- or UV-sensitive cones using the NTR method. There was no significant increase in apoptosis of non-target (non-mCherry expressing) cone cells when treated with MTZ for either cone ablation model. As expected, both lines showed an increase in the number of mCherry-expressing apoptotic cells, demonstrating that the cells expressing NTR-mCherry are ablated when exposed to MTZ. This model of conditional targeted cone ablation thus does not appear to cause off-target, deleterious effects to surrounding photoreceptors, and can be used to explore changes in retinal physiology, cone connectivity, and regenerative responses after only one cone subtype is lost.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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