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

Abstract

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.

Midbrain-hindbrain boundary patterning and morphogenesis are regulated by diverse grainy head-like 2-dependent pathways

“…Recent studies of a mutant zebrafish line generated by Tol2 transposon-based gene trapping of an EGFP reporter into the first intron of the grhl2b gene demonstrated otic vesicle defects (Han et al, 2011). We confirmed that otolith formation was also disrupted in our ATG-blocking MO-injected morphants (supplementary material Fig.…”

“…Otic expression of both claudin b and epcam was lost in our morphants, phenocopying the expression changes observed in the grhl2b mutant line (supplementary material Fig. S3C-F) (Han et al, 2011). However, no MHB defects were noted in this line, which we attribute to persistence of tissue-specific expression of grhl2b in the MHB region or, alternatively, to the presence of an alternate spliced isoform of grhl2b in the MHB (supplementary material Fig.…”

Specification of osteoblast cell fate by canonical Wnt signaling requires Bmp2

“…A variety of human, mouse and fish studies suggest reduced expression of Grhl3, Dlx3, Dlx5 and Osx1 could explain the defects we found in calcium and phosphate metabolism (Dworkin et al, 2014;Han et al, 2011;Isaac et al, 2014;Lapunzina et al, 2010;Liu et al, 2015;Nguyen et al, 2013;Ting et al, 2003a). Mineralization of the endochondral skeleton is a complex biological process involving the formation of a calcium phosphate hydroxyapatite crystal that is deposited into an organic extracellular matrix composed primarily of type I collagen.…”

Sp8 regulates inner ear development

“…The loss of ED in the ecl mutant may lead to retention of fluid, enlargement of the OV, and its various consequences as demonstrated in this study. Grainyhead-like 2 (grhl2) mutants in zebrafish (40) showed a range of otic dysmorphogenesis very similar to the ecl mutant; however, the molecular mechanism is likely to be dissimilar. Whereas grhl2 directly regulates epithelial tissue integrity, sp8 causes more complex effects and the epithelial defects addressed in this study may be a subset of the cause of the defects.…”