Abstract
Purpose :
Mutations in CLRN1 cause Usher syndrome type 3A (USH3A), a recessive inherited disorder leading to combined hearing and vision loss. In this study, we created and characterized a novel zebrafish model lacking clrn1 to investigate the pathophysiology of USH3A.
Methods :
CRISPR/Cas9 was used to delete 90% of the zebrafish clrn1 coding sequence. High-intensity light (3500 lux) exposure experiments were performed in 5-7dpf larvae, along with phalloidin and TUNEL staining. In 4 to 20-month adults, we assessed the retinal structure and the cone photoreceptor mosaic using spectral-domain optical coherence tomography. We also analyzed photoreceptor morphology using hematoxylin and eosin staining, and cell death and regeneration using staining for activated caspase-3, and BrdU incorporation assays.
Results :
High-intensity light treated clrn1 mutant zebrafish presented with a significant increase in the number of TUNEL positive photoreceptor nuclei and regions of absent or disrupted phalloidin staining, indicating that the absence of Clarin1 sensitizes photoreceptors to stress. In adult clrn1 mutant zebrafish raised under normal lighting, we observed progressive alterations of the photoreceptor mosaic from 4 to 20-months of age, suggesting a loss of photoreceptor integrity. Histological analysis of clrn1 mutant zebrafish retinas showed gross changes at 20 months compared to wildtype. Specifically, we observed a significant thinning of the outer nuclear layer (ONL) in the central retina, an indication of photoreceptor cell death. At the time points investigated, we did not observe a significant increase in activated-caspase 3 staining. However, there was a small but significant increase in BrdU incorporation at 4-months, suggesting regeneration and therefore loss of photoreceptors at an early age. BrdU incorporation decreased with age, potentially as a consequence of losing regenerative potential in older adults.
Conclusions :
The clrn1 mutant zebrafish represents the first model of USH3 disease that displays photoreceptor disorganization and degeneration. Collectively, our data indicates that the loss of Clarin1 function in zebrafish induces a slow, progressive retinal degeneration. We will utilize this model, along with cell-specific clrn1 re-expression and RNAseq analysis, to investigate the mechanisms promoting retinal degeneration in USH3A.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.