Purchase this article with an account.
A Phil Oel, Michele DuVal, W Ted Allison; Transcriptional changes in photoreceptor development genes during regeneration after cone ablation in zebrafish. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):452.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Genes controlling differentiation of developing photoreceptors have been identified in mouse and zebrafish; however, regulation of the teleost ability to robustly regenerate lost photoreceptors remains uncharacterized. We hypothesize that this regeneration recapitulates development; here, we test whether developmentally important genes are transcriptionally active during the regeneration period of post-injury neural retinas isolated from adult zebrafish.
We induced regeneration in adult zebrafish in two ways: intense light exposure causing widespread photoreceptor death, and conditional genetic ablation of either UV or blue light-sensitive cones. Conditional genetic ablation of cones occurs when the pro-drug metronidazole (MTZ) is added to tank water of transgenic fish expressing bacterial nitroreductase (NTR) in either UV or blue cones; cells expressing NTR convert MTZ into a DNA-crosslinking agent, causing cell-autonomous apoptosis. After injury by light exposure or MTZ treatment, we isolated the total RNA from neural retinas, and examined the transcriptional activity of several photoreceptor differentiation genes over a time-course by quantitative PCR analysis.
Through light-based ablation we have found changes in expression among rx1, nr2e3, nrl, and thrβ2. Compared to uninjured controls, light blasted retinas show a 2-fold reduction in rx1 transcripts, starting 12hours post ablation (hpa) and lasting until the end of the experiment (96hpa). Expression of nr2e3 is reduced 4-fold and nrl nearly 2-fold over the same period, whereas thrβ2 trends toward increased expression. Transgenic fish exposed to MTZ respond as predicted, showing a loss of NTR-expressing photoreceptors within 15 hours and regenerating new NTR-expressing photoreceptors within a week of recovery after transient treatment with MTZ, with qPCR studies pending.
We examined gene activity during regeneration after either widespread or targeted photoreceptor death. This allows us to evaluate how closely regeneration recapitulates development; to identify genes critical to the regeneration of cone photoreceptors; and finally, to use transgenic tools to modify the endogenous retinal stem cell population, to modulate the outcome of regeneration. This has implications for the design of stem cell-based treatments for blindness, where the restoration of cone-mediated sight is a paramount goal.
This PDF is available to Subscribers Only