Institute of Cell and Developmental Biology, College of Life Sciences, Zhejiang University, Hangzhou 310058, China.
Department of Biotechnology and Genetic Engineering, Faculty of Biological Sciences, Islamic University, Kushtia 7003, Bangladesh.
Cells. 2019 May 15;8(5):458. doi: 10.3390/cells8050458.
, 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 regulation and function in hair cells. In this study, through data mining and in vitro report assay, we firstly identified as a direct target gene of Atoh1 and one E-box (E3) in 3' enhancer is crucial for Atoh1-mediated 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 . Disruptions on this E3 site caused dramatic loss of 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 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 transcript itself. These observations indicated that Atoh1-mediated expression could have important roles during auditory hair cell development. In brief, our findings delineate the detail molecular mechanism of expression regulation in auditory hair cell differentiation.
，一个与果蝇 BarH 类同源盒基因同源的小鼠基因，在内耳中高度表达，对介导听觉和平衡的听觉毛细胞的长期维持至关重要，但对于其在毛细胞中的调控和功能的分子事件知之甚少。在这项研究中，通过数据挖掘和体外报告测定，我们首次鉴定出 作为 Atoh1 的直接靶基因，并且 3'增强子中的一个 E 盒（E3）对于 Atoh1 介导的 激活至关重要。然后，我们使用 CRISPR/Cas9 技术在这个 E3 位点 E 盒（CAGCTG）上产生了一个携带破坏的小鼠胚胎干细胞（mESC）系，这个 E3 突变的 mESC 系进一步进行了高效的逐步毛细胞分化策略。这个 E3 位点的破坏导致 的表达显著丢失，并显著减少了诱导的毛细胞样细胞的数量，而对早期原始外胚层样细胞和耳前体的分化没有影响。最后，通过 RNA-seq 分析和基因本体论（GO）富集分析，我们发现这个 E3 盒对于 的表达是必不可少的，以维持毛细胞的发育和正常功能。我们还分别比较了 CDS 突变和 E3 突变 mESC 诱导细胞的转录谱，除了 转录本本身外，结果非常一致。这些观察结果表明，Atoh1 介导的 表达在听觉毛细胞发育过程中可能具有重要作用。总之，我们的研究结果描绘了听觉毛细胞分化中 表达调控的详细分子机制。
