Regeneration of ultraviolet sensitive cones following light damage in tbx2b (lots-of-rods^p25bbtl) mutant zebrafish.

Abstract

Purpose : Zebrafish possess the remarkable capability to regenerate photoreceptors following injury. Most research focuses on the reprogramming of Muller glia and characterization of the mitotic progenitors; however, fewer studies investigate the factors regulating photoreceptor subtype specification. We tested the role of the transcription factor tbx2b in the specification of photoreceptors during regeneration.

Methods : Adult zebrafish (3-6 months of age) were used throughout this study. Ultraviolet-sensitive (UV) cones were visualized using the Tg(-5.5sws1:EGFP) reporter. To induce light damage (LD), wildtype (n=8) and homozygous tbx2b^p25bbtl (n=8) zebrafish were dark adapted for 7 days then exposed to 120,000 lux of light for 30 minutes. This was followed by constant illumination at 70,000 lux of white light for 96 hours. At 96 hours, half (n=4) of each treatment group was injected with EdU and euthanized 4 hours later. The remaining fish were returned to cyclic light, injected with EdU on 3 consecutive days and allowed to recover for 10 days before euthanasia. Retinas were fixed and sectioned at 10 microns. Cell death was assayed using the Apoptag detection kit. EdU uptake was detected with Click-iT technology. Sections were imaged using a Nikon confocal microscope. The number of UV cones was quantified at the site of recovery and at the retinal margin. Data were analyzed using a two-way ANOVA and Bonferroni post-hoc test.

Results : Prior to LD, tbx2b mutant zebrafish had significantly fewer UV cones in the central retina (<1 UV cone/100 µm) than wildtype zebrafish (10.1 ±5.1 UV cones/ µm, p<0.0001). We report that following LD and recovery, tbx2b mutants had significantly more UV cones (7.4 ±1.1 UV cones/ 100 µm) in the central retina compared to untreated siblings (p<0.0001). The regeneration of UV cones in LD tbx2b mutant and LD wildtype fish was not significantly different (p>0.999). TUNEL and EdU labeling confirmed that LD and recovery were restricted to the central retina.

Conclusions : Light damage provides a means to test the function of transcription factors during photoreceptor regeneration. Our results uncovered distinct differences in the impact of the tbx2b^p25bbtl allele on the number of UV cones in the developing and regenerating retinas. Future studies will explore the molecular nature of these differences, and test the role of other genes in regeneration.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.