ENT Institute and Otorhinolaryngology Department, Eye and ENT Hospital, NHC Key Laboratory of Hearing Medicine Research, Fudan University, Shanghai, China.
Department of Otolaryngology-Head and Neck Surgery, Yijishan Hospital of Wannan Medical College, Wuhu, China.
Cell Prolif. 2022 May;55(5):e13225. doi: 10.1111/cpr.13225. Epub 2022 Mar 29.
To explore the role of DNA methyltransferase 1 (DNMT1) in the development of auditory system using zebrafish as experimental model.
Morpholino oligonucleotide was used to induce Dnmt1 deficiency. RNA sequencing, in situ hybridization (ISH), whole genomic bisulfide sequencing (WGBS) and immunostaining were used to investigate the morphologic alterations and mechanisms.
We found that downregulation of Dnmt1 induced decreased number of neuromasts and repressed cell proliferation of primordium in the developing posterior lateral line system of zebrafish. The ISH data uncovered that Fgf signalling pathway was inhibited and the expression of chemokine members cxcr4b, cxcr7b and cxcl12a were interfered, while lef1 expression was increased after inhibiting Dnmt1. Additionally, Dnmt1 downregulation led to malformed otoliths and deformed semicircular canals, and hair cell differentiation in utricle and saccule was inhibited severely. The in situ staining of otic placode markers pax2/5 and fgf 3/8/10 was decreased when Dnmt1 downregulated. The WGBS analysis demonstrated that the global methylation status was markedly downregulated, and cell cycle genes were among those most differently expressed between Dnmt1 morphants and the controls. Further ISH analysis confirmed the findings by RNA-seq and WGBS assay that cdkn1a and tp53 were both upregulated after knockdown of Dnmt1.
Our results revealed that Dnmt1 is essential for the development of zebrafish auditory organ through regulating cell cycle genes together with Wnt and Fgf signalling pathways.
以斑马鱼为实验模型，探讨 DNA 甲基转移酶 1（DNMT1）在听觉系统发育中的作用。
使用 morpholino 寡核苷酸诱导 Dnmt1 缺陷。采用 RNA 测序、原位杂交（ISH）、全基因组亚硫酸氢盐测序（WGBS）和免疫染色技术研究形态改变和作用机制。
我们发现下调 Dnmt1 会导致斑马鱼后外侧线系统发育过程中的神经丘数量减少，并抑制原基细胞增殖。ISH 数据显示，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，而 lef1 的表达在抑制 Dnmt1 后增加。此外，Dnmt1 下调导致耳石畸形和半规管变形，并且内耳囊中毛细胞的分化受到严重抑制。下调 Dnmt1 时，耳胚标记物 pax2/5 和 fgf 3/8/10 的原位染色减少。WGBS 分析表明，全局甲基化状态显著下调，细胞周期基因是 Dnmt1 缺陷型和对照组之间差异表达最显著的基因之一。进一步的 ISH 分析证实了 RNA-seq 和 WGBS 分析的结果，即 Dnmt1 敲低后 cdkn1a 和 tp53 均上调。
我们的结果表明，DNMT1 通过调节细胞周期基因以及 Wnt 和 Fgf 信号通路，对斑马鱼听觉器官的发育至关重要。
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