Defects in the zonule in loxl1 knock-out zebrafish

Purposes: To investigate the roles of lysyl oxidase-like 1 (loxl1) in zebrafish eye development and the potency of loxl1 knock-out in mimicking the ocular manifestations of Exfoliation Syndrome.Methods: CRISPR/Cas9 technology was used to generate a frameshift deletion shifting code deletion in zebrafish loxl1. Expression profiles and ocular manifestations of wildtype (WT), heterozygous mutant (loxl1+/-) and homozygous mutant (loxl1-/-) zebrafish were analyzed in a range of developmental stages from whole zebrafish larvae to dissected adult zebrafish eyes using western blot, immunofluorescence, real-time RT-PCR and transcriptome resequencing analysis (RNA-seq). Scanning electron microscopy (SEM) was used to study the zonular structure and corneal fiber organization of adult zebrafish eyes.Results: The loxl1 mutation caused zonular bundling disorders in juvenile zebrafish and accumulation of pearl-like particles adhering to the adult zebrafish zonule. SEM revealed more numerous particles, but of smaller size in loxl1-/- zebrafish compared with those in loxl1+/-, although light microscopy showed that the overall staining of the zonule in loxl1+/- was darker than that in loxl1-/-. Expression of transforming growth factor-b (tgfb) pathway-related proteins changed at the protein level, but not the mRNA level. The phosphorylated Smad3 (pSmad3) level was locally activated in the iris-side trabecular meshwork and ciliary body of loxl1-/- adults.Conclusions: Defects in the zonular bundling found in loxl1 knock-out zebrafish indicate dysplasia. Compared with adult loxl1+/- zebrafish, more numerous but smaller pearl-like particles adhered to the zonule in adult loxl1-/- zebrafish. loxl1-/- zebrafish is a promising animal model of XFS zonular pathology.