The Regulatory Role Of M~6A Modification On Cerebellar Development In Mouse

m~6A is a dynamic and reversible methylation modification widely present in mRNA and non-coding RNAs.m~6A modification plays a regulatory role in stem cell proliferation and differentiation,neural development,and neural circuit formation.However,the current research is mainly on the m~6A methylation modification enzyme "writers" and the demethylation modification enzyme "erasers" research,and m~6A modification recognition and binding protein There are few studies on "readers".Specifically,in the study of m~6A modification to regulate cerebellar development,the functions and mechanisms of readers have not yet been reported.This subject used Nestin-cre to specifically knock out the m~6A writer METTL14 expressed in the nervous system.It was found that after METTL14 knockout,the mice became significantly smaller and the cerebellum atrophy.In view of the serious cerebellar developmental defects caused by knocking out the writer,the m~6A modification is essential for cerebellar development.We then studied m~6A modification in two major neurons of the cerebellum,namely Purkinje cells and granular cells.This topic focuses on the regulation and mechanism of m~6A modification on Purkinje cell development and function.In this project,the expression of L7-cre and Pcp2-cre specifically expressed in cerebellar Purkinje cells was determined using Rosa26 m T/m G reporter mice.We found that the expression of L7-cre was earlier than that of Pcp2-cre.Because the latter expression is too late,the use of Purkinje cell development research is relatively limited,we mainly used L7-cre mice in this research.We bred Cre mice with Mettl14fl/fl,Ythdf1fl/fl and Ythdf2fl/fl to collect c KO and littermate control ctrl mice.After perfusion fixation,dehydration embedding,cryosection and immunofluorescence observation,cerebellar mice The influence of cell development,including Purkinje cell production,dendritic development and other parameters,and also conduct some behavioral tests related to cerebellar function,including the four methods of Rota rod,Grip Strength,Open field and Footprint,observe effect of knockout on motor function controlled by mouse cerebellum.We initially found that the specific knockout of METTL14,YTHDF1 or YTHDF2 in Purkinje cells did not affect the development of cerebellar Purkinje cells,nor did it have a significant impact on the early development of the entire cerebellum.However,in the Rota rod behavioral test,the motor coordination ability of Ythdf2 c KO mice showed a significant increase,while the behavioral test of Ythdf1 c KO mice showed no significant difference.Therefore,we can initially conclude that the reader protein YTHDF2 may be an important reader regulating cerebellar development.Through further mechanistic studies,we will explain the role and mechanism of m~6A modification to regulate the function of Purkinje cells and cerebellum through its reader protein.