The Role And Mechanism Of M6A Methylase METTL16 In Mouse Molar Development

Background:Dental abnormalities in humans include abnormal number of teeth,abnormal morphology and delayed eruption,which are common craniofacial developmental disorders and can cause common clinical oral diseases such as dental defects,malocclusion and edentulism.It is known that tooth development begins with the sinking of the oral epidermis and occurs through interactions between the epithelial mesenchyme.Molar development in mice is similar to that of human teeth and has been used as a model organism to study human tooth development because of its short growth cycle and high genetic homology.m6A methylation is a widespread internal modification of eukaryotic RNA sequences that regulates gene expression through methylation,demethylation,and methylation reading of RNA,with METTL16 being a key RNA methylating enzyme that can independently methylation-modify RNA.The role of m6A methylation in tooth development has not been elucidated.Objective:To investigate the role of METTL16-mediated RNA methylation in mouse tooth development,to identify and validate the key target molecules of METTL16 in the regulation of tooth development,and to elucidate the molecular mechanism of METTL16 regulation of tooth development.Methods:（1）To obtain Mettl16 conditional knockout mice by crossing Mettl16^fx/fx,a gene modified mouse,with K14-Cre mice,and to observe the effects of Mettl16knockout on mouse embryonic development and tooth development;（2）To study the biological causes of the defective phenotype by using molar teeth as the target,and to investigate the changes of tissue apoptosis and proliferation;（3）Transcriptome sequencing and analysis of E14.5 mouse lower molar tooth epithelium to study the modification of dental gene expression by Mettl16 knockout;murine model（4）Analysis of the m6A modification of Mettl16 knockdown epithelium,combined with transcriptome data to find the key target molecules of METTL16 regulation of dental development;（5）Validation of target molecule expression in dental embryo tissue by real-time quantitative PCR,immunohistochemistry and Western blot;（6）In vitro cellular assays using the methylation inhibitor cyclic leucine and the RNAi method to further validate m6A methylation and the regulatory role of METTL16 on target molecules;（7）In vitro organ culture experiments to verify whether restoration at the target molecule level can rescue dental development problems caused by Mettl16knockout in missing teeth.Conclusions:（1）Mettl16^K14-Cre mice have defects in ectodermal development,including defects in tooth development and skin and hair follicle development,with normal tooth morphology maintained until E15.5 and complete loss of morphology at day E18.5;（2）Abnormally reduced cell proliferation and increased apoptosis in the cervical ring site of the dental epithelium of Mettl16 knockout mice;（3）Transcriptome sequencing results show significant down-regulation of RNA levels in Edar,the causative gene for dental dysplasia;（4）m6A sequencing analysis revealed significant downregulation of Edar methylation modification in Mettl16 knockout epithelium,which,combined with transcriptome sequencing results,led to speculation that Edar may be a key target molecule in the regulation of teeth by METTL16;（5）EDAR protein level was significantly downregulated in Mettl16 knockout dental embryos,supporting that Edar was significantly downregulated in Mettl16 knockout dental embryos.（6）In vitro cellular assays revealed that inhibition of methylation using cyclic leucine and knockdown of Mettl16 resulted in significant downregulation of EDAR expression,suggesting that Edar is regulated by m6A modifications;（7）In vitro organ culture experiments revealed that overexpression of EDAR rescued the developmental defects of Mettl16 knockout tooth embryos,suggesting that EDAR is a key target molecule in the regulation of tooth development by METTL16.In summary,this study found that Mettl16 is essential for tooth development and that the key target molecule for Mettl16-mediated m6A methylation to regulate tooth development is Edar.