nitroreductasemetronidazole-mediated-ablation-matlab-platform-rpegen

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent ...

University of Pittsburgh School of Medicine

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.

Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation.

Diseases that result in retinal pigment epithelium (RPE) degeneration, such as age-related macular degeneration (AMD), are among the leading causes of blindness worldwide. Atrophic (dry) AMD is the most prevalent form of AMD and there are currently no effective therapies to prevent RPE cell death or restore RPE cells lost from AMD. An intriguing approach to treat AMD and other RPE degenerative diseases is to develop therapies focused on stimulating endogenous RPE regeneration. For this to become feasible, a deeper understanding of the mechanisms underlying RPE development, injury responses and regenerative potential is needed. In mammals, RPE regeneration is extremely limited; small lesions can be repaired by the expansion of adjacent RPE cells, but large lesions cannot be repaired as remaining RPE cells are unable to functionally replace lost RPE tissue. In some injury paradigms, RPE cells proliferate but do not regenerate a morphologically normal monolayer, while in others, proliferation is pathogenic and results in further disruption to the retina. This is in contrast to non-mammalian vertebrates, which possess tremendous RPE regenerative potential. Here, we discuss what is known about RPE formation during development in mammalian and non-mammalian vertebrates, we detail the processes by which RPE cells respond to injury, and we describe examples of RPE-to-retina and RPE-to-RPE regeneration in non-mammalian vertebrates. Finally, we outline barriers to RPE-dependent regeneration in mammals that could potentially be overcome to stimulate a regenerative response from the RPE.

The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems.

Loss of the retinal pigment epithelium (RPE) because of dysfunction or disease can lead to blindness in humans. Harnessing the intrinsic ability of the RPE to self-repair is an attractive therapeutic strategy; however, mammalian RPE is limited in its regenerative capacity. Zebrafish possess tremendous intrinsic regenerative potential in ocular tissues, including the RPE, but little is known about the mechanisms driving RPE regeneration. Here, utilizing transgenic and mutant zebrafish lines, pharmacological manipulations, transcriptomics, and imaging analyses, we identified elements of the immune response as critical mediators of intrinsic RPE regeneration. After genetic ablation, the RPE express immune-related genes, including leukocyte recruitment factors such as  We demonstrate that macrophage/microglia cells are responsive to RPE damage and that their function is required for the timely progression of the regenerative response. These data identify the molecular and cellular underpinnings of RPE regeneration and hold significant potential for translational approaches aimed toward promoting a pro-regenerative environment in mammalian RPE.

Article	Year
Regeneration of the zebrafish retinal pigment epithelium after widespread genetic ablation. PLoS genetics\| PubMed ID: 30695061	2019
The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems. Progress in retinal and eye research\| PubMed ID: 33901682	2021
The immune response is a critical regulator of zebrafish retinal pigment epithelium regeneration. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 34006636	2021

Lyndsay L. Leach

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