Extrinsic Factors and RPE Regeneration
Date
2024-01-24
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Abstract
The retinal pigment epithelium (RPE) is a monolayer of pigmented cells that closely interacts with photoreceptor outer segments of the outer vertebrate retina to maintain visual function. Damage to the RPE, for instance in a disease such as Age-Related Macular Degeneration, results in photoreceptor degeneration and subsequently, vision loss. In contrast to mammals, zebrafish can intrinsically regenerate a functional RPE layer after injury. Specific molecular pathways are known to regulate RPE proliferation in culture, but the pathways that function in vivo to promote RPE regeneration remain largely unknown. My aim is to determine potential pathways that influence RPE regeneration in zebrafish. First, I examine the importance of the secreted ligand Semaphorin 3F (SEMA3F), expressed in the RPE of both mammals and zebrafish, in RPE regeneration. I use a sema3fa homozygous mutant zebrafish on a transgenic RPE injury background (Tg(rpe65a:NTR-EGFP)) where timed application of the drug metronidazole (MTZ) to the bath results in nitroreductase-mediated RPE-specific cell death. My data suggest Sema3fa has no effect on the extent of RPE injury in this model, though RPE apoptosis may be delayed and increased in the absence of Sema3fa. Further, loss of Sema3fa may induce an initial increase in proliferation in the RPE as well as increased proliferation in the photoreceptor outer nuclear layer. Second, I provide an initial assessment of the involvement of additional pathways in zebrafish RPE regeneration. These pathways impact proliferation and/or migration of cells in culture and are expressed within the RPE. I use in situ hybridization to visualize larval RPE expression of 10 candidate genes before and after RPE injury. Genes that may show changes in expression post-injury include bmp7b, caska, foxm1, her4.1, msnb, rpe65a, trpm7, and vrk1. Future work could include using loss-of-function approaches in the RPE injury model to determine potential roles of these genes in RPE regeneration. In the long-term, this work may impact gene therapies for patients suffering from retinal degenerative diseases.
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Keywords
RPE, Retinal Pigment Epithelium, Regeneration, Semaphorin, Zebrafish, Eye, AMD, Age-related Macular Degeneraton, Ablation, Injury, in vivo, Metronidazole, in situ hybridization
Citation
Selje, S. J. (2024). Extrinsic factors and RPE regeneration (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.