DNA甲基化转移酶1调控斑马鱼听觉器官发育的机制研究

The morphogenesis of the auditory sensory organ is a complex and orderly process. A number of signaling pathways and factors have been identified playing an important role in regulating auditory sensory organ formation. DNA methyltransferase 1 (Dnmt1) regulates expression of many critical genes and is essential for embryonic development as well as diverse biological processes. However, the precise molecular mechanism of how Dnmt1 is involved in auditory sensory organ development remains unexplored. Our data showed that Dnmt1 was expressed in developing otic vesicles and lateral line system of zebrafish via whole-mount in situ hybridization assay. Knockdown of Dnmt1 using antisense morpholino oligonucleotides induced smaller otic vesicles, malformed semicircular canals, as well as posterior lateral line defects. These results indicate that Dnmt1 plays an important role in auditory sensory organ formation. In the current project, we propose to investigate the molecular mechanisms of Dnmt1 regulating otic and lateral line system development in zebrafish by loss- and gain-of-function analysis. Moreover, we plan to identify the genes and downstream signaling pathway of Dnmt1, which is responsible for the auditory phenotype using MeDIP-Seq and RNA sequencing. The outcome of this project will provide the importance of DNA methylation for proper control of auditory organ development, open new avenues for the potential control of auditory organ development and call attention to the long-term effects of Dnmt therapy in the treatment of hearing loss.

听觉器官发育是一个受到严格调控的复杂而有序的过程。由多种信号通路、转录因子和表观遗传相互作用参与调控前体细胞的增殖、分化和迁移。目前DNA甲基化转移酶1（Dnmt1）在听觉器官发育中的功能研究还未见报道。我们前期工作中利用斑马鱼为模式生物克隆了斑马鱼Dnmt1基因，发现其表达在发育中的内耳及侧线器，且其表达时间窗对应了斑马鱼胚胎听觉器官形成的多个关键阶段。采用斑马鱼胚胎内注射寡核苷酸Morpholino的方法抑制Dnmt1的表达，可导致斑马鱼内耳及侧线发育异常。这些前期结果提示Dnmt1是一个重要的调控听觉器官发育的因子。本项目拟进一步分析Dnmt1基因表达改变后斑马鱼听觉器官表型产生的细胞生物学机制；结合全基因组甲基化检测及转录组测序等手段鉴定Dnmt1下游信号通路或关键基因，阐述其调控听觉器官发育的分子机制。本项目将为表观遗传调控听觉器官发育提供新的理论依据和潜在的药物靶点。

耳聋不仅严重影响患者的身体健康和生活质量，对病人家庭和社会也造成了极大的经济和心理负担，但是目前耳聋的相关机制还不清楚。我们前期研究提示Dnmt1 在斑马鱼的毛细胞发育和分化过程中可能扮演着重要角色，通过聚焦Dnmt1在斑马鱼听觉器官和毛细胞发育中的作用，应用分子生物学技术、细胞生物学、转录组学、甲基化组学等多学科交叉的手段从表观遗传学的全新角度探讨听觉器官的发育机制。.我们的研究发现，Dnmt1下调诱导斑马鱼发育中的后侧线系统中神经丘数量减少，原基细胞增殖受到抑制。通过原位杂交发现，抑制Dnmt1后，斑马鱼原基Fgf信号通路受到抑制，趋化因子成员cxcr4b、cxcr7b和cxcl12a的表达受到干扰，但是lef1表达增加。 此外，Dnmt1敲降会导致耳石畸形和半规管发育迟缓，椭圆囊和球囊中毛细胞分化受到严重抑制。当Dnmt1下调时，斑马鱼内耳上pax2/5和fgf 3/8/10的RNA表达降低。WGBS分析表明，全基因组甲基化状态显著下调，细胞周期基因在Dnmt1敲降组和对照组之间表达差异最大。进一步的原位杂交证实了RNA-seq和WGBS测定的结果，即cdkn1a和tp53在敲低Dnmt1后均上调。.总之，我们的研究证实，Dnmt1通过调控细胞周期基因以及Wnt和Fgf信号通路，对斑马鱼听觉器官的发育至关重要，使其成为听觉器官和听力损失研究的重要候选基因。