Grhl2 deficiency impairs otic development and hearing ability in a zebrafish model of the progressive dominant hearing loss DFNA28

Congenital and progressive hearing impairment is a common distressing disease. The progressive dominant hearing loss DFNA28 in human is associated with a frameshift mutation of Grainyhead-like 2 (GRHL2) but its etiology and mechanism remain unknown. Here we report a zebrafish grhl2b(T086) mutant line in which grhl2b expression is interrupted by an insertion of a Tol2 transposon element. The mutants exhibit enlarged otocysts, smaller or eliminated otoliths, malformed semicircular canals, insensitiveness to sound stimulation and imbalanced swimming motion. Since grainyhead-like family members can regulate epithelial adhesion, we examined the expression of some genes encoding junction proteins in mutants. We show that the expression of claudin b (cldnb) and epcam is abolished or dramatically reduced and apical junctional complexes are abnormal in otic epithelial cells of mutant embryos. Co-injection of cldnb and epcam mRNA could largely rescue the mutant phenotype. Injection of human wild-type GRHL2 mRNA but not the mutant GRHL2 mRNA derived from DFNA28 patients into grhl2b(T086) mutant embryos could rescue the inner-ear defects. Furthermore, we demonstrate that Grhl2b directly binds to the enhancers and promotes the expression of cldnb and epcam. Thus, this work reveals an evolutionarily conserved function of Grhl2 in otic development and provides a fish model for further studying mechanisms of Grhl2-related hearing loss.