Abstract
Purpose :
Mutations in USH2A are the most common cause of Usher Syndrome, characterised by retinitis pigmentosa and sensorineural hearing loss.
Aim: To generate and characterise an ush2a mutant zebrafish model to explore the pathophysiology of Usher syndrome.
Methods :
CRISPR/Cas9 gene editing was used to produce an ush2a mutant zebrafish line. Expression of ush2a was examined in the retina using RNAscope. Longitudinal histological evaluation and spectral domain optical coherence tomography (SD-OCT) were used to assess retinal structure and cone photoreceptor mosaic up to 12 months of age. Levels of apoptotic cell death were examined. Transmission electron microscopy was used to study the ush2a retinal ultrastructure. Immunostaining of retinal cryosections was performed using 4D2 (rhodopsin) and 1D4 (red opsin) antibodies. Wholemount hair cell staining was carried out in 6 days post-fertilisation (dpf) fish using anti-acetylated tubulin and Alexa Fluor 647-Phalloidin.
Results :
Mutant ush2a zebrafish (c.2131_2203+73delinsCGGCGG p.A711Rfs) were generated by targeting exon 11 of the gene. Histological evaluation and SD-OCT showed no gross abnormalities in retinal structure up to 12 months, with preservation of cone photoreceptor mosaic organisation. Apoptotic nuclei were detected in the outer nuclear layer of the ush2a retina from 6 months. Examination of the ush2a retinal ultrastructure at 6 months revealed regions of rod photoreceptor loss amongst preserved retinal tissue. The abnormal presence of lysosome-like structures were noted at the photoreceptor connecting cilia and synaptic regions. By 12 months, the ush2a retina showed almost complete disruption of the rod photoreceptor cell layer. Immunostaining of ush2a retinal sections revealed rhodopsin mislocalisation apparent by 3 months. Red opsin antibody staining showed preservation of red cones at all time points. Wholemount hair cell staining revealed abnormal development of the neuromasts and sensory maculae at 6 dpf.
Conclusions :
The ush2a mutant zebrafish show an adult onset photoreceptor degeneration predominantly affecting rod photoreceptors, combined with developmental abnormalities in the mechanosensory hair cells. These findings resemble the clinical phenotype observed in USH2A patients, highlighting this mutant as a valuable model in which to gain insight into the disease pathophysiology and potential therapies.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.