TUB and ZNF532 Promote the Atoh1-Mediated Hair Cell Regeneration in Mouse Cochleae

Abstract

Hair cell (HC) regeneration is a promising therapy for permanent sensorineural hearing loss caused by HC loss in mammals. Atoh1 has been shown to convert supporting cells (SCs) to HCs in neonatal cochleae; its combinations with other factors can improve the efficiency of HC regeneration. To identify additional transcription factors for efficient Atoh1-mediated HC regeneration, here we optimized the electroporation procedure for explant culture of neonatal mouse organs of Corti and tested multiple transcription factors, Six2, Ikzf2, Lbh, Arid3b, Hmg20 a, Tub, Sall1, and Znf532, for their potential to promote Atoh1-mediated conversion of SCs to HCs. These transcription factors are expressed highly in HCs but differentially compared to the converted HCs based on previous studies, and are also potential co-reprograming factors for Atoh1-mediated SC-to-HC conversion by literature review. P0.5 cochlear explants were electroporated with these transcription factors alone or jointly with Atoh1. We found that Sox2+ progenitors concentrated within the lateral greater epithelial ridge (GER) can be electroporated efficiently with minimal HC damage. Atoh1 ectopic expression promoted HC regeneration in Sox2+ lateral GER cells. Transcription factors Tub and Znf532, but not the other six tested, promoted the HC regeneration mediated by Atoh1, consistent with previous studies that Isl1 promotes Atoh1-mediated HC conversionex vivo and in vivo and that both Tub and Znf532 are downstream targets of Isl1. Thus, our studies revealed an optimized electroporation method that can transfect the Sox2+ lateral GER cells efficiently with minimal damage to the endogenous HCs. Our results also demonstrate the importance of the Isl1/Tub/Znf532 pathway in promoting Atoh1-mediated HC regeneration.