G.P.9 Basement membrane pathology associated with FKRP and fukutin deficiency in zebrafish

Abstract

Deficiencies in fukutin-related protein (FKRP) and fukutin lead to aberrant glycosylation of alpha-dystroglycan, a key receptor for basement membrane proteins. There is a broad spectrum of disorders associated with FKRP and fukutin deficiency, ranging from limb-girdle muscular dystrophy to the congenital muscular dystrophy syndromes Muscle Eye Brain disease and Walker–Warburg syndrome. Here we use zebrafish to investigate the changes in several basement membranes in muscle, eye and notochord using transmission electron microscopy and immunohistochemistry in 3days post fertilisation larvae. A range of antibodies including, IIH6, laminin, zn-8 (optic nerve), zrf-1 (Müller cells) were used to study the notochord and eye phenotypes on cryosections. FKRP and fukutin were knocked down by antisense oligonucleotide morpholinos in wild type zebrafish. The morphants showed abnormal muscle fibres and disrupted vertical myosepta. Disturbances were observed in all three layers that form the peri-notochord sheath including the basement membrane. Disorganised retinal layering in both morphants was seen on toluidine blue stained sections. Dysplasia of the lens could be observed in both fukutin and FKRP morphants with a severe phenotype. The homogenous perturbation observed across the inner limiting membranes of both morphants may account for the lens dysplasia. The rod and cones in the photoreceptor cell layer were found in lower density in both morphants with the least density in fukutin knock-downs which may be the result of a disrupted external limiting membrane. We therefore conclude FKRP and fukutin are essential for the integrity of membranes in the eye, muscle and notochord of developing zebrafish larvae.