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Author ORCID Identifier

0000-0001-8104-4387

Date Available

7-31-2026

Year of Publication

2026

Document Type

Doctoral Dissertation

Degree Name

Doctor of Philosophy (PhD)

College

Arts and Sciences

Department/School/Program

Biology

Faculty

Jakub K Famulski

Abstract

Inherited retinal diseases (IRDs) affect millions of people worldwide. Majority of IRDs are caused by degeneration of rod and cone photoreceptor cells (PRCs) due to gene mutations. The overarching goal of my dissertation is to model and evaluate the molecular role of various gene candidates involved in IRDs such as cone rod dystrophy (CRD) and retinitis pigmentosa (RP).

Mutations in CDHR1, a photoreceptor specific cadherin have been associated with CRD and recapitulated in mouse CDHR1 knockouts. However, the molecular function of CDHR1 remains unknown. CDHR1 has been shown to localize at the leading edge of murine rod nascent outer segment (OS) making junctions to an unknown partner in the inner segment. Using Structured Illumination Microscopy (SIM), we observed that the localization of zebrafish cdhr1a extends from basal nascent OS discs above the periciliary ridge of the inner segment to a considerable length along the OS, akin to calyceal process (CPs). When labeling the CPs using pcdh15b, a CP specific cadherin, we observed that cdhr1a at the leading edge of OS juxtaposes with pcdh15b in the CP. Similar localization patterns were detected in human, macaque, xenopus, ducks, and various rodent PRCs indicating conservation. Importantly, using immunoprecipitation and K562 cell aggregation assays we demonstrate that pcdh15b and cdhr1a can interact and potentially link the OS and CP. To analyze the consequences of OS-CP interactions in CRD, we established a zebrafish cdhr1a mutant line that exhibits severe cone OS disruption and cell loss by 3 months. Rod OS defects were delayed until 3-6 months. Furthermore, we show that loss of cdhr1a function leads to disorganization and shortening of CPs coinciding with cone OS defects which is significantly exacerbated when combined with the loss of pcdh15b. In conclusion, we propose that cdhr1a and pcdh15b function to link cone OSs with CPs to maintain proper OS homeostasis thus revealing a potential novel mechanism for CRD.

Additionally, a significant number of RP patients exhibit mutations in the gene PRPH2, a vertebrate conserved tetraspanin responsible for formation and maintenance of OS morphology. Beyond RP, mutations in PRPH2 have been shown to cause diverse types of IRDs including but not limited to Leber Congenital Amaurosis and CRD. Majority of studies examining PRPH2 function have been performed in rod dominant rodent models and as such these studies have detailed pathogenesis to study PRPH2 based RP. To better understand cone PRC pathophysiology due to loss of PRPH2, we used the cone-rich zebrafish model to characterize the loss of PRPH2 function. Of the four PRPH2 zebrafish orthologs only prph2a and prph2b were found to be expressed in PRCs. CRISPR-mediated single mutants of prph2a and prph2b did not yield striking rod or cone phenotypes. Double prph2a/2b mutants exhibited early loss of all cone cells, preceded by outer segment whorls akin to the phenotypes observed in PRPH2+/- mice. Surprisingly rod photoreceptor cells were not affected and in fact exhibited a striking lengthening of rod OSs with normal OS disc formation. Overgrowth of rod OSs proceeded up to 1 year, but no degeneration was observed. To determine how rod OS can persist without prph2a/b we targeted rom1a and rom1b CRISPR. Injection of rom1a/b CRISPR constructs resulted in complete loss of both rod and cone OSs in the prph2a/b double mutants, but not in wildtype. These findings suggest that in zebrafish rom1a/b can compensate for the loss of prph2 in rod cells. This drastic difference in phenotypes across species sheds light on the potential differential role of prph2 in cone and rods PRCs of zebrafish and provides the first model to examine prph2 function in a cone rich retina.

Taken together, my approach of using the zebrafish model allows us to model and characterize macular degenerative diseases and shed light on the molecular function of causative candidates such as CDHR1 and PRPH2.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2026.366

Archival?

Archival

Funding Information

Gertrude Ribble Pilot Grant (Department of Biology at the University of Kentucky: 2021, 2022, 2023, 2024, and 2025

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