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

Zhenhang Xu,Jian Zuo,Vikrant Rai

doi:10.3389/fncel.2021.759223

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.

Highlights

Hearing loss is one of the most common impairments affecting approximately 1.57 billion people worldwide, and there are 403.3 million people who still have moderate or severe hearing loss after adjusting for hearing aid use (Haile et al, 2021)
Compared to ectopic expression of Atoh1 alone, studies have shown that co-activation of Atoh1 together with other transcription factors (Pou4f3, Gfi1, Gata3, Nmyc, etc.) induces more Supporting cells (SCs) transdifferentiate to Hair cell (HC)-like cells, and the newly generated cells are more mature in neonatal and mature murine cochleae (Liu et al, 2012; Walters et al, 2017; Chen et al, 2021), indicating the significance of co-activation of multiple factors relating to the HC regeneration
greater epithelial ridge (GER) cells are a group of transient cells that will be replaced with inner sulcus cells during the postnatal maturation of the organ of Corti; at neonatal ages, the lateral GER are abundant of Sox2+ cells which have more plasticity and capacity for transdifferentiation (Kubota et al, 2021), regenerated HCs were acquired by ectopically overexpressing Atoh1 in GER cells in cultured cochlear explants through electroporation (Zheng and Gao, 2000)

Summary

INTRODUCTION

Hearing loss is one of the most common impairments affecting approximately 1.57 billion people worldwide, and there are 403.3 million people who still have moderate or severe hearing loss after adjusting for hearing aid use (Haile et al, 2021). GER cells are a group of transient cells that will be replaced with inner sulcus cells during the postnatal maturation of the organ of Corti; at neonatal ages, the lateral GER are abundant of Sox2+ cells which have more plasticity and capacity for transdifferentiation (Kubota et al, 2021), regenerated HCs were acquired by ectopically overexpressing Atoh in GER cells in cultured cochlear explants through electroporation (Zheng and Gao, 2000) All of these indicate that the lateral GER cells can serve as progenitor sources for HC regeneration at neonatal ages. We optimized the cochlear explant electroporation procedure and electroporated these transcription factors alone or together with Atoh to see if anyone of these transcription factors or the combination could induce more regenerated HCs in the GER cells

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ETHICS STATEMENT