耳蜗毛细胞的发育与再生研究

Cochlear sensory epithelium contains hair cell (HCs) and supporting cells (SCs). Noise, environmental toxic and genetic defects can cause damage of HCs, leading to sensorineural deafness. It is of clinical importance to investigate how to regenerate HCs. The previous studies of applicant support that ectopic expression of HC specific gene Atoh1 converts SCs into HCs. Unfortunately, the newly generated HCs are not fully differentiated, and cannot detect sound. Our project will focus on identifying the key gene families that control the entire differentiation program of HCs. Our strategies are divided into four steps: 1) With RNA-Seq approach, we will compare gene expression profiles of HCs at five different developmental ages, and choose the most dynamic genes as candidates; 2) The necessiety of the candidate genes will be tested through an in vivo high-throughput CRISPR/Cas9-mediated knockout approach recently invented by a group in our institute, which can allow us to get homozygous knockout in one month, and is like an in vivo quick screening; the sufficiency of candidate genes will be tested by overexpressing our candidate genes with Atoh1, Pou4f3 and Gfi1 in mouse embryonic cells (mES), which is recently proved to be a good in vitro screening approach. Presence of mature HCs derived from mES cells will tell us which candidate is sufficient to promote HC differentiation; 3) An in vivo complex mouse model will be generated in which HCs is specifically damaged first, followed by overexpressing Atoh1 and our candidate genes in SCs. We will focus on determining whether fully differentiated new HCs are present as well as hearing capacity is significantly improved. Taken together, our proposed study not only put new insights into the mechanisms underlying HC development and differentiation, but also provide foundations for how to use SCs to regenerate new functional HCs in clinic.

耳蜗听觉上皮有毛细胞和支持细胞两种主要细胞类型。噪音、环境和遗传等因素引起的毛细胞损伤是感音神经性耳聋的主要原因。因此研究如何再生毛细胞有重大的临床价值。申请人之前的研究表明支持细胞在给予毛细胞特异性基因Atoh1后，能跨分化为毛细胞。遗憾的是新生的毛细胞无法完全分化成熟，也没有听觉感知功能。本项目将致力于探索调控毛细胞分化成熟的核心基因家族。我们的策略是：1）利用RNA-Seq技术，对毛细胞在五个不同的发育时间点进行全基因组表达谱分析，选择显著动态变化的做为候选基因；2）通过体内、体外基因敲除和过表达来确定真正调控毛细胞分化的基因；3）建立小鼠毛细胞损伤模型, 把这些新基因和Atoh1一起过表达在支持细胞，检测是否有成熟的新生毛细胞出现和听力是否有恢复。本项目的研究不仅大大促进我们对毛细胞发育分子机制的理解，也为临床上如何利用支持细胞再生出有功能的毛细胞提供理论依据。

与非哺乳类动物鱼和鸟类相比，哺乳类动物听觉系统丧失了自我修复的能力。哺乳类动物耳蜗外毛细胞是我们感知声音的放大器，并特异表达马达蛋白Prestin，而且也是对噪音、耳毒性药物和衰老等因素最为敏感的一类细胞。因此，听觉领域内的一个重要科学问题是如何在成年哺乳动物内实现外毛细胞再生。本课题利用小鼠为模型，通过在成年小鼠耳蜗支持细胞内过表达对外毛细胞正常发育重要的两个转录因子Atoh1和Ikzf2 (转录组测序筛选出），成功转分化成年支持细胞为Prestin+的外毛细胞，但是这个转分化效率非常低。因此，我们又构建了外毛细胞特异性杀伤的耳聋模型（Prestin-P2A-DTR/+）。我们在成年Prestin-P2A-DTR/+小鼠内注射白喉毒素，可以在一周内杀伤90%以上的外毛细胞。令人鼓舞的是，Atoh1和Ikzf2转分化成年耳蜗支持细胞为外毛细胞的效率在外毛细胞损伤的模型中提高到16%-20%。这是外毛细胞再生领域内的一个重要进展，该研究成果于2021年9月发表于eLife杂志。