Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation

Abstract

The retinal pigment epithelium (RPE) is a specialized monolayer of pigmented cells within the eye that is critical for maintaining visual system function. Diseases affecting the RPE have dire consequences for vision, and the most prevalent of these is atrophic (dry) age-related macular degeneration (AMD), which is thought to result from RPE dysfunction and degeneration. An intriguing possibility for treating RPE degenerative diseases like atrophic AMD is the stimulation of endogenous RPE regeneration; however, very little is known about the mechanisms driving successful RPE regeneration in vivo. Here, we developed a zebrafish transgenic model (rpe65a:nfsB-GFP) that enabled ablation of large swathes of mature RPE. RPE ablation resulted in rapid RPE degeneration, as well as degeneration of Bruch's membrane and underlying photoreceptors. Using this model, we demonstrate for the first time that larval and adult zebrafish are capable of regenerating a functional RPE monolayer after RPE ablation. Regenerated RPE cells first appear at the periphery of the RPE, and regeneration proceeds in a peripheral-to-central fashion. RPE ablation elicits a robust proliferative response in the remaining RPE. Subsequently, proliferative cells move into the injury site and differentiate into RPE. BrdU pulse-chase analyses demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological inhibition of Wnt signaling significantly reduces cell proliferation in the RPE and delays overall RPE recovery. These data demonstrate that the zebrafish RPE possesses a robust capacity for regeneration and highlight a potential mechanism through which endogenous RPE regenerate in vivo. SIGNIFICANCE STATEMENTDiseases resulting in RPE degeneration are among the leading causes of blindness worldwide, and no therapy exists that can replace RPE or restore lost vision. One intriguing possibility is the development of therapies focused on stimulating endogenous RPE regeneration. For this to be possible, we must first gain a deeper understanding of the mechanisms underlying RPE regeneration. Here, we ablate mature RPE in zebrafish and demonstrate that zebrafish regenerate RPE after widespread injury. Injury-adjacent RPE proliferate and regenerate RPE, suggesting that they are the source of regenerated tissue. Finally, we demonstrate that Wnt signaling is required for RPE regeneration. These findings establish an in vivo model through which the molecular and cellular underpinnings of RPE regeneration can be further characterized.

The immune response is a critical regulator of zebrafish retinal pigment epithelium regeneration

“…Our lab has shown that zebrafish have the capacity to regenerate RPE post-injury by utilizing a genetic ablation paradigm (25); however, the molecular components and signaling pathways involved in RPE regeneration remain unknown. This injury model utilizes a transgenic zebrafish line (rpe65a:nfsB-eGFP) wherein the RPE-specific (41) rpe65a enhancer drives expression of nitroreductase (nfsB) fused to eGFP for visualization and cell isolation.…”

“…1A). These timepoints were chosen as prior characterization identified early-(2dpi), peak-(4dpi), and late-stages (7dpi) of RPE regeneration discernible by resolution of apoptosis, peak proliferation, and recovery of RPE marker expression, respectively (25). To determine genes and pathways upregulated during RPE regeneration, differential gene expression analyses were performed and enriched Reactome pathways were identified from filtered differentially expressed genes (DEGs; fold change ≥2, false discovery rate (FDR) ≤0.05).…”

“…Previously, proliferation and pigment recovery were quantified as metrics of the extent of RPE regeneration post-ablation (25). Other studies in the retina have used a similar approach, assaying proliferation and cell recovery as a readout for repair after treatment with dexamethasone (35,67).…”

“…Thus, at present, the mechanisms driving intrinsic RPE regeneration remain elusive. Recently, our lab developed a zebrafish RPE ablation model and showed that the RPE has the intrinsic capacity to regenerate after widespread injury (25). This model uses a transgenic nitroreductase-mediated injury paradigm that enables tissue-specific ablation of the central two-thirds of the RPE.…”

“…This model uses a transgenic nitroreductase-mediated injury paradigm that enables tissue-specific ablation of the central two-thirds of the RPE. Extensive characterization showed: (i) sequential death of the RPE and photoreceptors, (ii) peripheral-to-central expansion of regenerating RPE, (iii) robust proliferation driving RPE regeneration, and (iv) recovery of visual function and RPE/photoreceptors post-injury (25).…”