Modelling FKRP and fukutin deficiency in zebrafish

Abstract

Deficiency in fukutin-related protein (FKRP) or fukutin results in aberrant glycosylation of -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 congenital disorders such as muscle eye brain disease and Walker-Warburg syndrome (WWS). fkrp and fukutin were knocked down in the zebrafish with antisense morpholino oligonucleotides (MO). The fkrp, fukutin and dystroglycan MOs each produced a spectrum of comparable phenotypes. With each MO producing a comparable morphant phenotype on morphological examination, it was hypothesised that inferences could be made about similarities and differences in the fkrp, fukutin dystroglycan axis during zebrafish development. The morphants had abnormal muscle fibres, including disruptions of the vertical myosepta and sarcolemma. Disorganised retinal layering in the eyes was found in both fukutin and fkrp morphants. Dysplasia of the lens was observed in most fukutin morphants and some of the fkrp morphants with a severe phenotype. Structural changes in basement membranes at 1-3 days post fertilisation (dpf) were investigated. The perturbation observed across the inner limiting membranes may account for the lens dysplasia. Cell density of the granular epithelium in the photoreceptor cell layer was found to be lower in both morphants with the least density in fukutin knock-downs, which may result from a disruption of the external limiting membrane. This leads to the conclusion that fkrp and fukutin are essential for membrane integrity in the eye and muscle of developing zebrafish. A transgenic zebrafish line expressing enhanced green fluorescent protein (EGFP) in vascular endothelium from the fli-1 promoter was used to investigate early vascularisation. In all morphants, including dystroglycan knock downs, the intersegmental vessels failed to reach the dorsal longitudinal anastomotic vessel at 1dpf. Additionally, in the fkrp and fukutin morphant the eye vasculature was abnormal. Interestingly, no change was observed in the eye vasculature of the dystroglycan morphants suggesting that fkrp and fukutin may modify proteins other than α-dystroglycan in the eye.