The Role Of RNA Methyltransferase Mettl3 And Demethylase Fto In The Development Of Cerebral Cortex In Mice

The mammalian cerebral cortex is the most complex structure known,controlling almost all complex behavior of animals.The cortex consists of diverse cell types and forms a well-organized inside-out laminar architecture,which is achieved through tight gene expression regulations during embryonic and postnatal development.Cortical development is an extremely precise process,controlled by genetic and epigenetic factors.Moreover,epitranscriptomic regulation in brain development and function also has drawn attention,in particular N6-methyladenosine(m6A)modification.m6 A is the most abundant RNA modification in eukaryotes and plays a critical role in the post-transcriptional regulation of genes.Methyltransferase-like protein 3(Mettl3)is one of the most important components of the m6 A methyltransferase complex,responsible for the installation of methyl groups to the appropriate location on RNA.Fat mass and obesity-associated protein(Fto)is one of the two main demethylases of m6 A,responsible for removing methyl groups from RNA.The roles of Mettl3 and Fto have been extensively elucidated.However,their roles in mammalian cortical development have not been reported.In this study,Mettl3 conditional knockout mice(M-c KO)and Fto conditional knockout mice(F-c KO)were constructed,and cortical specific knockout mice were obtained by mating with Emx1-Cre mice.Observation and sequencing analysis of mice showed that:1.Deletion of Mettl3 resulted in severe postnatal dysplasia in mice,and the lactation survival rate decreased to 30%.Deletion of Mettl3 resulted in a significant increase in the number of Intermediate Progenitors(IPs)and a decrease in the generation of neurons at E15.5.Moreover,Redial Glial Cells(RGCs)and IPs demonstrated disordered stratification and folds.However,folds disappear after birth,reducing the number of neurons and leading to a thinning of the cortex.2.RNA Sequencing(RNA-seq)and Ribosome Profiling(RIbo-seq)of cerebral cortex tissues of M-c KO mice at E15.5 showed that deletion of Mettl3 resulted in large-scale transcriptional and translational disorders.At the same time,the Translational Efficiency(TE)in the cerebral cortex also showed significant changes.Genes highly expressed in RGCs and IPs also undergo a large number of transcriptional and translational changes in M-c KO,and these differential genes are mainly riched in biological processes such as cell cycle,neurogenesis,axon projection and cell differentiation.3.Deletion of Fto causes only mild symptoms.At the individual level,the lactation survival rate decreased slightly to 80%.And there were no significant differences at the tissue and cell levels.4.RNA-seq and Ribo-Seq showed that deletion of Fto also led to a large number of transcriptional and translational disorders,while the TE gene in the F-c KO cerebral cortex also showed changes.Differential genes with high expression in RGCs and IPs were mainly enriched in biological processes such as response to trauma and toxic substances.This may account for the decreased survival rate in F-c KO mice.5.A combined analysis of M-c KO and F-c KO showed that deletion of Mettl3 resulted in larger transcriptional and translational changes than deletion of Fto.Meanwhile,deletion of Mettl3 but not Fto caused significant downregulation in key genes controlling RGCs and IPs development in mouse embryonic brain.6.Several m6 A related enzymes were up-regulated in M-c KO but not in F-c KO.This may be the main cause of the difference in gene expression.Our results suggest that Mettl3 and Fto have different functions in mammalian cortical development.Mettl3 plays a more critical role in the regulation of m RNA transcription and translation,which is necessary for the normal development of cerebral cortex.