Tol2Transposon-Mediated Zebrafish Mutagenesis And Grhl2Regulation Of Inner Ear Development

Zebrafish is an excellent vertebrate model organism to explore the molecular mech-anisms of embryonic development, in which many genes involve. Generating geneticmutants is an effective approach to identify new functional genes implicated in specificdevelopmental processes. In this study, the Tol2transposon-mediated gene trap methodisutilizedtoisolatenewzebrafishmutantsandtoelucidatetherolesandmolecularmech-anisms of the trapped genes in embryonic development.This study generates219transgenic zebrafish lines, most of which express ubiq-uitous EGFP maternally with or without zygotic expression. From these lines, eightmutants are isolated. The trapped genes in three mutant lines are identified, includinggrhl2b gene from the T086line.The transgenic embryos of the T086/grhl2b line express EGFP in many organs in-cluding the inner ear and lateral line neuromasts. Mutant ears exhibit severe defectssuch as expanded otocysts, decreased or eliminated otoliths, malformed semicircularcanals, etc., resulting in hearing impairment and swimming imbalance. In this line, theTol2transposon element is inserted into the first intron of thegrhl2b locus, which causesa sharp reduction of the amount of normal transcripts. The mutant phenotype can bemimicked in wild-type embryos by knockdown of grhl2b using specific morpholinosand can be rescued by injecting grhl2b mRNA, indicating that the mutant phenotype isascribed to the loss of normal grhl2b transcripts. Further studies reveal that grhl2b isdispensable for otic induction and patterning as well as for hair cell and statoacousticganglion development. Instead, grhl2b plays an crucial role in the maintenance of theotic epithelial barrier and the endolymph homeostasis. Mechanistically, Grhl2b proteinbinds to the promoter or enhancer and regulates the transcription of cldnb and epcam,which encode junctional proteins essential for tight junction formation in the otic epithe-lial cells. Promoter assay by transgenic approach confirms that the Grhl2b binding sitein the cldnb promoter is required for the Grhl2b-dependent expression of cldnb in theinner ear.Zebrafish Grhl2b protein shares a highly conserved sequence with mouse and hu-man Grhl2proteins. Overexpression of mouse or human Grhl2mRNA can rescue theotic defects of grhl2b mutant embryos and promote the transcription of cldnb and ep- cam in wild-type embryos, indicating that Grhl2is also functionally conserved. Sincehuman GRHL2mutation leads to autosomal non-syndromic hearing loss DFNA28, thezebrafish grhl2b mutants may serve as the first animal model of DFNA28to analyze itsetiology and develop drugs that can alleviate or cure the disease.This study substantiates the application of Tol2transposon-mediated gene trap ingenerating zebrafish mutants. Meanwhile, this study provides the first animal model ofDFNA28and clarifies the essential roles and the molecular mechanisms of grhl2b ininner ear development.