Author ORCID Identifier

https://orcid.org/0000-0001-5652-1348

Year of Publication

2020

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department

Biology

First Advisor

Dr. Ann C. Morris

Abstract

The formation of a healthy and functioning eye requires coordinated interactions between numerous signaling pathways and gene regulatory networks within the developing neural retina. Tight regulation of gene expression is required for cell specification and differentiation in this multilayered, light sensitive tissue. The photoreceptors are the light detecting cells of the retina, capable of functioning in both intense sunlight and dim light at night. When pigment cells of the photoreceptor outer segment are activated by light, a complex chain of events called phototransduction leads to the electrical signal cascade that is transmitted through the retina and ultimately to the brain to be interpreted as a visual image. Defects in expression of the genes involved in retinal and photoreceptor development have been implicated in many eye diseases, often leading to vision loss. The development, maintenance and function of photoreceptors has been studied in a wide range of model organisms, including the zebrafish. Uniquely, the adult zebrafish is capable of regenerating the photoreceptors and other retinal cells types in response to injury. While immense work has been done to understand the function of genes involved in retinal development, regeneration, and blinding eye diseases, our understanding about many of these genes is still incomplete.

The two genes studied in this dissertation have been either implicated in retinal development, degeneration, or regeneration. Calpain-5 (capn5) is a member of a family of calcium-dependent, non-lysosomal cysteine proteases. A mutation in CAPN5 has been shown to cause autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV). ADNIV is a devastating inherited autoimmune disease of the eye that displays features commonly seen in other eye diseases, such as retinitis pigmentosa and diabetic retinopathy, and ultimately results in retinal degeneration and blindness. When activated by influxes of calcium, calpains can either degrade their protein substrates or modify the activity of their targets through proteolytic processing. Although capn5 has been extensively studied in the brain, very little is known about its role during retinal development or in the adult eye. The second gene of interest is Hairyrelated (Her) factor Her9, a member of the Hairy/Enhancer of Split (Hes) superfamily of genes. Her9 is a basic-helix-loop-helix-orange (bHLH-O) transcription factor. Her factors have been previously shown to play roles in neural tube closure, floor plate development, and development of various components of the central nervous system as well as the cranial sensory placodes. However, the role of her9 in retinal development and regeneration is still poorly understood.

Chapter 1 of this dissertation is an overview of retinal development, with an emphasis on photoreceptor development, retinal degenerative diseases, regeneration, and the known roles of capn5 and her9 in other tissues. Chapter 2 characterizes the expression of capn5 during development and the adult retina. Using acute light damage and a zebrafish model for chronic rod degeneration and regeneration, this study provides evidence that capn5 has a role in photoreceptor maintenance, survival, and regeneration. Chapter 3 characterizes the expression of her9 during retinal development and uses a combination of molecular and behavioral experiments to characterize the retinal phenotypes present in a her9 CRISPR/Cas9 knockout. The data presented in this chapter demonstrate that Her9 plays a role in photoreceptor differentiation, maintenance and survival. Chapter 4 examines other phenotypes present in the her9 knockout. Craniofacial, pigment, and gut defects provide supporting evidence that Her9 is required for the differentiation and survival of neural crest cell lineages. Chapter 5 is a discussion of the conclusions we can draw from these studies, potential future directions for this work, and how these results impact our broader understanding of retinal development and regeneration.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2020.477

Funding Information

University of Kentucky Lyman T. Johnson fellowship (Cagney E. Coomer, 2015-2018)

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