A Critical E-box in Barhl1 3' Enhancer Is Essential for Auditory Hair Cell Differentiation.

Kun Hou,Zhouwen Xu,Hui Jiang,Jianzhong Shao,Lin Liu,Chao Zhong,Xiao Huang,Md Rezaul Karim,Minxin Guan

doi:10.3390/cells8050458

Kun Hou, Zhouwen Xu + Show 7 more

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https://doi.org/10.3390/cells8050458

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Journal: Cells	Publication Date: May 15, 2019
Citations: 11	License type: CC BY 4.0

Affiliation: Zhejiang University, Islamic University

Abstract

Barhl1, a mouse homologous gene of Drosophila BarH class homeobox genes, is highly expressed within the inner ear and crucial for the long-term maintenance of auditory hair cells that mediate hearing and balance, yet little is known about the molecular events underlying Barhl1 regulation and function in hair cells. In this study, through data mining and in vitro report assay, we firstly identified Barhl1 as a direct target gene of Atoh1 and one E-box (E3) in Barhl1 3’ enhancer is crucial for Atoh1-mediated Barhl1 activation. Then we generated a mouse embryonic stem cell (mESC) line carrying disruptions on this E3 site E-box (CAGCTG) using CRISPR/Cas9 technology and this E3 mutated mESC line is further subjected to an efficient stepwise hair cell differentiation strategy in vitro. Disruptions on this E3 site caused dramatic loss of Barhl1 expression and significantly reduced the number of induced hair cell-like cells, while no affections on the differentiation toward early primitive ectoderm-like cells and otic progenitors. Finally, through RNA-seq profiling and gene ontology (GO) enrichment analysis, we found that this E3 box was indispensable for Barhl1 expression to maintain hair cell development and normal functions. We also compared the transcriptional profiles of induced cells from CDS mutated and E3 mutated mESCs, respectively, and got very consistent results except the Barhl1 transcript itself. These observations indicated that Atoh1-mediated Barhl1 expression could have important roles during auditory hair cell development. In brief, our findings delineate the detail molecular mechanism of Barhl1 expression regulation in auditory hair cell differentiation.

Highlights

The auditory sensory epithelium within the mammalian cochlea, the organ of Corti, is elaborately organized with sensory hair cells and non-sensory supporting cells
We recently reported that Barhl1 is indispensable for hair cell differentiation in the in vitro induction system from mouse embryonic stem cell (mESC), where the mutation of Barhl1 was created on the coding sequence (CDS) region by Crispr/Cas9 technology [26]
Our results clearly suggest that disruption of the essential E-box has no impact on the inductions toward EPL cells and otic progenitors from mESCs

Summary

Introduction

The auditory sensory epithelium within the mammalian cochlea, the organ of Corti, is elaborately organized with sensory hair cells and non-sensory supporting cells. The hair cells embedded in this sensory organ serve as mechanoreceptor cells for detecting sound and transducing it into electrical signals, and their development and maintenance are critical for auditory function. Any lesion causing degeneration of these sensory cells will lead to hearing loss, which represents the main type of deafness [1]. The regeneration capacity of mammalian auditory hair cells is extremely limited comparing that of the non-mammalian vertebrates, such as birds and fishes. The regeneration of impaired hair cells remains a major challenge in the treatment of deafness by gene and cell therapy

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