Abstract
Purpose :
Mutations in PCDH15 cause a severe manifestation of Usher syndrome with onset of visual dysfunction in the first decade of life with profound deafness and vestibular defects. We used CRISPR/Cas9 to create a truncating mutation in a conserved 5’ region of a zebrafish orthologue, pcdh15b, previously shown by morpholino knock-down to be critical for photoreceptor function and survival.
Methods :
We induced frameshift mutations producing premature termination codons, verified by Sanger sequencing, by CRISPR/Cas9 injection into zygotes. Animals were outcrossed to isolate and maintain carriers. We assayed hearing, balance, photoreceptor morphology and cell death with behavioral and histological analyses. Light exposure experiments were performed using broad-spectrum light and petri dishes treated with tinted window film. Western blot analysis was performed on extracts from enucleated larval retinas.
Results :
Optokinetic response was detected at reduced levels in 5dpf mutant larvae, and immunohistochemical evaluation revealed presence of morphologically abnormal calyceal processes and outer segments. We observed elevated cell death, assayed by Caspase-3, in the outer nuclear layer. We examined whether ER stress was induced by Pcdh15b dysfunction and found that Elf1a and Caspase-3 levels were elevated in mutant eye extracts compared to controls, whereas CHOP levels were unchanged. We observed that photoreceptor death was enhanced by rearing larvae in elevated light, and that filtering out short wavelength while keeping Lux values constant resulted in lower rates of cell death. Although mutants exhibited normal startle responses, indicative of acoustic function, they had impaired balance incompatible with survival beyond the first week. Consistent with this finding, stereocilia organization was more perturbed in the utricle compared to the saccule.
Conclusions :
Understanding the molecular progression of USH1F retinal dysfunction is crucial for development of diagnostic and therapeutic options for affected individuals and the clinicians who care for them. The zebrafish model of visual and vestibular defects in USH1F exhibits severe, early defects consistent with human symptoms. These defects will be useful as molecular readouts of disease progression as well as providing an advantageous system to test small molecules and other potential pharmacological interventions.
This is a 2020 ARVO Annual Meeting abstract.