Generation and characterization of an adamtsl4 knockout in zebrafish: a novel animal model to study ADAMTSL4-related ocular diseases

Abstract

Purpose : Loss-of-function of human ADAMTSL4 leads to a spectrum of ocular diseases that range from autosomal recessive isolated ectopia lentis and ectopia lens et pupillae to other minor eye alterations with no displacement of the pupil and very mild displacement of the lens. Increased intraocular pressure and retinal detachment is present in some patients. The main purpose of this study was to generate a loss-of-function zebrafish mutant as an animal model to study ADAMTSL4-related ocular diseases.

Methods : Immunohistochemistry was used to evaluate ADAMTSL4 expression in human and zebrafish ocular tissues. The knockout zebrafish line was established by CRISPR/Cas9 genome editing. Adamtsl4 mRNA level was evaluated in larvae by qPCR and the phenotypes were characterized macroscopically and by histological and immunohistochemical analysis.

Results : Bioinformatic analyses revealed high evolutionary conservation between the human ADAMTSL4 gene and its zebrafish ortholog. Immunohistochemical detection of the proteins encoded by these ortholog genes showed a similar distribution in both the human and zebrafish ocular anterior segment. A zebrafish mutant line carrying the c.234-351del mutation, which presents a 75% reduction of adamtsl4 mRNA at 144 hours post fertilization (hpf), was generated. In addition, the translation of residual mutant mRNA was predicted to result in a truncated and inactive protein (p.(Gln78Hisfs*127)), supporting that the established line presents a complete adamtsl4 loss-of-function. The knockout line showed a spectrum of craniofacial defects (including microphthalmia and periocular edemas), with incomplete penetrance in 40% of the larvae (144 hpf), associated with lethality. Histological studies in these larvae showed thickening of the optic nerve, ocular anterior segment alterations consisting of corneal edema and proliferation of iris pigmented cells, neuroretinal and retinal pigment epithelium abnormalities. All adult KOs (6 months) presented bilateral posterior subluxation of the lens associated with decrease size and altered shape of the pupil. Seventy percent of these fishes also presented variable jaw alterations.

Conclusions : The adamtsl4 knockout recapitulates some of the ocular alterations observed in humans and provides a new animal model to study the function of this gene and the mechanism of disease related with its loss-of-function.

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