Knockdown of TGFBI/BIGH3 Corneal Dystrophy-Linked Gene Leads to Developmental Impairment in Zebrafish

Purpose: Over 25 specific mutations in the Transforming Growth Factor beta-induced gene (TGFBI, BIGH3) are so far known to be associated with 13 different inherited clinically and histopathologically distinct phenotypes of corneal dystrophies. Despite the ubiquitous expression of the encoded keratoepithelin (KE), no other specific disease has been described in tissues other than the cornea. KE plays an important role in the extra cellular matrix, where it is mainly found and interfere with cell adhesion processes by interacting with specific integrins. Until now, no loss-of-function mutation has been observed and it would be useful to know the exact function of BIGH3.In mouse embryo, KE is expressed mainly in tissues derived from the mesoderm, including developing bones and cartilages. Still the exact function of KE remains elusive. Our aim is to investigate the role of KE during development, using the zebrafish as model. Methods: We injected the mRNA inhibitor KE-morpholino into 1 to 2-cell zebrafish embryos. Standard control-morpholino was separately injected as negative control and chordin-morpholino as positive control. At 6 days post-fertilization, larvae were fixed in 4% paraformaldehyde and embedded either in Technovit and sectioned into 5 um slices for the morphology determination, or in sucrose for cryosection and immunohistochemistry. Results: Hundreds of zebrafish embryos were microinjected using different concentrations of the various morpholinos. Injection efficacy was monitored according to the chordin morphological change. The appropriate non lethal dose for the KE-morpholino showed broad development retardation in KE morphants. The eye morphology appeared to be normal but smaller in size. Dorsal and caudal fins were underdeveloped. The notochord was smaller in size and the pericardium was enlarged. Conclusions: The effects observed in zebrafish were in agreement with expression studies in the mouse embryos. Silencing of the TGFBI/BIGH3 gene in this model lead to widespread development retardation, suggesting a primary role for KE. Fine analysis of the treated zebrafish will help elucidate the physiological role of TGFBI/BIGH3.