Abstract
Purpose :
Mutations in PCDH15 cause severe early onset visual dysfunction, along with profound congenital deafness and loss of vestibular function. pcdh15b, previously shown by morpholino knock-down to be critical for photoreceptor function and survival with no observed effect on hearing or balance, but no zebrafish mutant studies have been published to date.
Methods :
Mutations induced by CRISPR/Cas9 injection into zygotes were verified by Sanger sequencing and propagated. We assayed hearing, balance, mechanosensory hair cells, photoreceptor morphology, and cell death with behavioral and histological methods. 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 5 day postfertilization (dpf) mutant larvae, and immunohistochemical, SEM and TEM analysis of mutant photoreceptors revealed morphologically abnormal calyceal processes and outer segments. An antibody recognizing zebrafish pcdh15 labeled the region of the calyceal processes in wild-type photoreceptors with no specific labeling in mutants. We observed elevated cell death, assayed by Caspase-3, in the outer nuclear layer. We examined levels of e1fa and CHOP by Western blot to determine whether ER stress was induced by Pcdh15b dysfunction and found no elevation in either. We observed enhanced photoreceptor death by rearing larvae in bright 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. Consistent with this finding, stereocilia of the inner ear were perturbed more in the utricle than the saccule, and expression of the ohnologous gene pcdh15a was enriched only in the saccule of 5dpf mutants compared to wild-type levels.
Conclusions :
The zebrafish model of visual and vestibular defects in USH1F exhibits severe, early defects consistent with human symptoms. Our data indicate that both pcdh15a and pcdh15b are required for hearing and balance, and that genetic compensation by pcdh15a is evident in the acoustic hair cells of pcdh15b mutants. These characterizations provide numerous measures of disease progression and an advantageous system to test potential pharmacological interventions.
This is a 2021 ARVO Annual Meeting abstract.