HDAC1在斑马鱼神经丘毛细胞再生中调控机制的研究

Regulation of gene expression in development and regeneration of hair cells is a hot topic for a long time. Histone deacetylase (HDAC) is an important epigenetic regulator, and regulates neurogenesis through inhibiting Notch signaling. However its regulatory mechanism in development and regeneration of hair cells is unknown. We have found that the expression of Hdac1 is up-regulated during hair cells regeneration, and the regeneration of hair cells and expression of Atoh1a gene are inhibited by HDAC inhibitor VPA. To illustrate the regulatory mechanism of HDAC1 in hair cells regeneration, we intend to knockdown Hdac1 gene in regenerating neuromast by in vivo electroporating antisence morpholino, and analyze the regeneration of hair cells, cell proliferation and apoptosis in neuromast. Through in situ hybridization and rescue assay, we will determine the regulatory role of Hdac1 on the expression of cip/kip, Atoh1a and Notch signaling members. Furthermore, we will identify the histone modification of HDAC1 on promoters of target genes by immunohistochemistry and ChIP-qPCR. This project will reveal the role of histone modification in hair cells regeneration, thus providing a theoretical basis for directional induction of hair cells and therapy of sensorineural hearing loss.

毛细胞发育和再生过程中的基因表达调控一直是一个热点问题。组蛋白去乙酰化酶（HDAC）是一种重要的表观遗传调节子，可通过抑制Notch信号调节神经分化，但HDAC对毛细胞发育和再生的调控机制尚不清楚。近来我们发现HDAC1在再生的斑马鱼神经丘中表达上调，且其抑制剂会抑制毛细胞再生和Atoh1a基因的表达。为阐明HDAC1在神经丘毛细胞再生中的调控机制，我们将通过活体电转吗啉环反义寡核苷酸敲低再生神经丘中的Hdac1基因，分析毛细胞的再生及神经丘内细胞增殖和凋亡；然后利用原位杂交和营救实验确定Hdac1对cip/kip，Atoh1a和Notch信号成员表达的调控；最后利用免疫组化和ChIP-qPCR鉴定HDAC1对靶基因启动子区组蛋白的修饰。本研究有望揭示组蛋白乙酰化修饰在毛细胞再生调控中的作用，可为指导干细胞定向诱导和感音神经性耳聋的治疗提供理论基础。

毛细胞是脊椎动物维持听力和平衡的重要结构。与低等脊椎动物不同，哺乳动物内耳中的毛细胞受损后无法再生。利用低等模式动物阐明毛细胞再生的细胞和分子机制，对治疗人类耳聋具有重大意义。鱼类的侧线与哺乳动物内耳在结构和功能上非常相似，由于它们位于体表，易于处理和观察，成为研究毛细胞再生的理想模式。本研究旨在阐释表观遗传调控子HDAC1调控毛细胞发育和再生的细胞和分子机制。我们发现（1）利用小分子，反义morpholino和突变体造成的HDAC1失活显著抑制神经丘毛细胞的发育和再生；（2）HDAC1的失活抑制分化毛细胞染色质凝缩，同时使细胞增殖高于对照组；（3）hdac1突变和VPA处理下调了six1b, sox2, atoh1a的表达，而促进了NFkB和JAK/STAT信号通路成员的表达；（4）hdac1下游tnfrsfa的突变体可造成神经丘和毛细胞的数量无法正常维持；（5）与神经丘毛细胞类似，西伯利亚鲟的壶腹感觉毛细胞也可被抗生素损伤，且能够再生。这些发现增进了我们对毛细胞再生机理的理解。