m6A修饰对小鼠小脑发育的调控作用研究

THE REGULATORY ROLE OF M6A MODIFICATION ON CEREBELLAR DEVELOPMENT IN MOUSE

林小燕

11849122

硕士

生物工程

姬生健

2020-06-03

2020-07-08

哈尔滨工业大学

深圳

m6A是一种在mRNA和非编码RNAs中广泛存在的，动态且可逆的甲基化修饰。在神经发育以及神经环路形成等过程中m6A修饰发挥着调控作用。但是，现进行的研究主要是对m6A的甲基化修饰酶“writers（刻写器）”和去甲基化修饰酶“erasers（擦除器）”的研究，而对m6A修饰的识别和结合蛋白“readers（阅读器）”的研究很少，具体在m6A修饰调控小脑发育的研究中，其readers的功能和机制目前还未见报道。本课题利用神经系统特异性表达的Nestin-cre敲除m6A的writer METTL14，发现METTL14敲除后的小鼠体型明显变小，小脑萎缩。鉴于敲除writer后导致严重的小脑发育缺陷，说明m6A修饰对于小脑发育至关重要。我们分别在小脑的两类主要神经元即浦肯野细胞和颗粒细胞中分别研究m6A修饰，本课题主要集中研究m6A修饰对浦肯野细胞发育和功能的调控作用和机制。本课题利用Rosa26mT/mG报告基因鼠确定了小脑浦肯野细胞特异表达的L7-cre和Pcp2-cre在小脑中的表达谱，我们发现L7-cre的表达要早于Pcp2-cre。因为后者表达过晚，对于浦肯野细胞发育研究的用途比较有限，我们在本课题研究中主要使用了L7-cre小鼠。我们将Cre鼠分别与Mettl14fl/fl，Ythdf1fl/fl和Ythdf2fl/fl交配收集cKO和同窝对照ctrl小鼠，经过灌流固定，脱水包埋，冷冻切片和免疫荧光观察敲除后对小鼠小脑浦肯野细胞发育的影响，包括浦肯野细胞产生，树突发育等参数，同时也进行小脑功能相关的一些行为学测验，包括Rota rod、Grip Strength、Open field和Footprint这四种方法，观察敲除后对小鼠小脑控制的运动功能的影响。我们初步发现浦肯野细胞中特异性敲除METTL14、YTHDF1或YTHDF2后，小脑浦肯野细胞的产生和早期发育没有受到明显的影响，对整个小脑的早期发育也没有产生明显的影响。但是在Rota rod行为学测验中，浦肯野细胞特异性敲除的Ythdf2 cKO小鼠的运动协调能力表现出了明显的增强，而Ythdf1 cKO小鼠的行为学测验没有明显差异。因此我们初步可以得出浦肯野细胞中的阅读器蛋白YTHDF2可能是调控小脑功能的一个重要reader。通过后续进一步的机制研究，我们将阐释m6A修饰通过其阅读器蛋白调控小脑神经环路和功能的作用和机制。

m6A is a dynamic and reversible methylation modification widely present in mRNA and non-coding RNAs. m6A 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 m6A methylation modification enzyme "writers" and the demethylation modification enzyme "erasers" research, and m6A modification recognition and binding protein There are few studies on "readers". Specifically, in the study of m6A 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 m6A 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 m6A modification is essential for cerebellar development. We then studied m6A modification in two major neurons of the cerebellum, namely Purkinje cells and granular cells. This topic focuses on the regulation and mechanism of m6A 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 Rosa26mT/mG 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 cKO 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 cKO mice showed a significant increase, while the behavioral test of Ythdf1 cKO 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 m6A modification to regulate the function of Purkinje cells and cerebellum through its reader protein.

m6A YTHDF1 YTHDF2 小脑 浦肯野细胞 行为学

m6A YTHDF1 YTHDF2 cerebellum Purkinje cells behavior

中文

联合培养

南方科技大学

林小燕. m6A修饰对小鼠小脑发育的调控作用研究[D]. 深圳. 哈尔滨工业大学,2020.