Ocular phenotypes in a novel zebrafish model of albinism

Abstract

Purpose : Oculocutaneous Albinism (OCA) is a rare hypopigmentation disorder that affects an estimated one in 20,000 live births. The most common subtype of OCA, known as OCA1, is caused by variants of the TYR gene, which is involved in the melanin biosynthesis pathway.
OCA1 is characterised by reduced pigmentation in hair follicles, skin epidermis, and the retina. Consequently, affected children suffer from lifelong visual impairment, oculomotor abnormalities including infantile nystagmus and impaired horizontal optokinetic reflex (OKR).
OKR is often measured to characterise visual behaviour and oculomotor phenotypes in humans and animal models. The oculomotor phenotypes in tyr knockout zebrafish are poorly characterised. We aimed to generate a biallelic knockout of tyr in zebrafish and characterise the oculomotor phenotype to determine whether it could be a suitable animal model for human albinism.

Methods : We utilised a CRISPR-Cas9 mutagenesis method incorporating three ribonucleoproteins to generate biallelic F0 knockout zebrafish mutant larvae. Tyr gene-knockout efficiency in zebrafish mutants was visualised by assessing the penetrance of eye pigmentation as early as 2 days post-fertilisation (dpf). A novel convenient flip-phone assay was developed to measure the OKR of larvae at 5dpf. We developed a deep learning pipeline to analyse eye-movement recording and characterise oculomotor phenotypes. Optomotor (OMR) response was also assessed at 7dpf.

Results : There was a significant reduction in eliciting positive OKR of tyr mutants compared to scramble-injected controls (p<0.001). Novel nystagmus-like oscillations were identified when exposed to a stationary sinusoidal grating at 5dpf.
A high knockout efficiency (M = 91.3%) and viability of tyr mutants were achieved; comparable to scramble injected and wildtype controls. A reduced OMR response was observed at 7dpf compared to scramble injected controls.

Conclusions : High viability and efficiency of tyr mutants validates the use of the F0 biallelic CRISPR-cas9 technique. The absence of OKR and OMR at 5dpf and 7dpf recapitulates the human visual system phenotype. Moreover, it corroborates the OKR phenotype observed in mice, which takes months to develop. We demonstrate a novel rapid visual behaviour workflow from knockout to phenotypic characterisation. This substantiates the use of zebrafish as an economical and ethical animal model for future albinism research.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.