Dnmt1 is required for the development of auditory organs via cell cycle arrest and Fgf signalling

Objectives: To explore the role of DNA methyltransferase 1 (DNMT1) in the development of auditory system using zebrafish as experimental model. Methods: 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. Results: 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. Conclusion: 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.