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-eGFP) 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 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 incorporation assays demonstrate that the regenerated RPE is likely derived from remaining peripheral RPE cells. Pharmacological disruption using IWR-1, a Wnt signaling antagonist, significantly reduces cell proliferation in the RPE and impairs 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.

Neurodegeneration, Neuroprotection and Regeneration in the Zebrafish Retina

“…Additionally, protection of surviving neurons following degeneration or injury is long sought after. The major targets identified for retinal neuroprotection and regeneration using zebrafish include signaling pathways such as: neurotrophic [ 100 , 163 ]; other growth factors [ 91 , 158 , 161 , 167 , 174 , 202 , 203 ]; microglia/macrophage [ 159 , 182 , 191 , 207 , 216 ]; Jak/Stat [ 162 , 175 , 192 ]; Wnt [ 86 , 194 , 212 ]; Shh [ 169 , 179 ] and NMDA receptor-mediated signaling [ 160 , 180 ]. There is currently limited use of the zebrafish compared to more popular rodent models, but the trend towards utilizing this unique animal model is likely to increase over time and lead to exciting new developments in retinal neuroprotection and regeneration.…”

“…Other retinal cells, such as bipolar cells and RPE, have also been ablated and their regeneration patterns were studied. Zebrafish are capable of regenerating RPE cells, which involves the Wnt pathway, in a peripheral to central manner [ 212 ]. Bipolar cells regenerate seven days after ablation and are able to reestablish most of the connections [ 224 , 225 ].…”

Dual roles of the retinal pigment epithelium and lens in cavefish eye degeneration

“…The number and organization of the retinal layers in the AE strain is similar to the surface fish retina, and in most cases the outer layer expresses zpr2 antibody, indicative of the presence of differentiated photoreceptor cells (Qin et al, 2011;Bader et al, 2102). The coexistence of a normal retina and defective RPE in the AE strain appears to conflict with studies implicating a functional and pigmented RPE in proper retinal lamination and photoreceptor cell development in other vertebrates (Raymond & Jackson, 1995;Jeffery, 1997;Hanovice et al, 2019;Longbottom et al, 2009). As originally pointed out by Strickler et al (2007), however, this conflict would be resolved if the lens, which is present in the AE strain, normally functions alone or in concert with the RPE to regulate neuroretinal development.…”

“…Because analysis of mutant phenotypes (Gross et al, 2005;Rojas-Muñoz, Dahm, & Nüsslein-Volhard, 2005) and genetic ablation (Hanovice et al, 2019;Longbottom et al, 2009), which have been established to study RPE function in zebrafish, are unavailable in Astyanax, we developed an alternative approach using hybrid crosses and artificial selection to study the role of the RPE in eye growth.…”

“…The possibility that the RPE is defective in cavefish is supported by the absence of melanization in larval Pachón cavefish eyes (Bilandžija, Ma, Parkhurst, & Jeffery, 2013;McCauley, Hixon, & Jeffery, 2004) and less extensive eye degeneration in those cavefish populations with a hypopigmented RPE, such as Tinaja and Chica (Zilles, Tillmann, & Bennemann, 1983). Furthermore, molecular ablation experiments in other vertebrates have shown that the RPE is essential for normal eye development (Hanovice et al, 2019;Raymond & Jackson, 1995;Rymer & Wildsoet, 2005). Therefore, it is important to determine the role of the RPE in A. mexicanus eye development to fully understand the mechanisms of cavefish eye degeneration.…”