【作者】 高燕；

【导师】 曹萤；

【作者基本信息】 南京大学 ， 生物学， 2015， 博士

【摘要】 在脊椎动物早期胚胎发育的过程中,其背腹体轴、前后体轴、左右体轴的建立是整个生物个体发育进程中的重大事件,涉及到很多信号转导通路和相应的信号转导因子的精密调控,例如Wnt信号通路、BMP信号通路、Nodal信号通路、FGF信号通路等等。其中,Wnt信号的活性在胚胎生成过程中所形成的活性梯度对于整个机体体轴的建立起到了至关重要的作用：母源的Wnt信号会特化胚胎背前部的形成；合子的Wnt信号主要负责胚胎后部腹部化的形成。Wnt/β-Catenin信号通路在调节胚胎的形成,肿瘤形成,机体内环境的稳态,机体再生以及胚胎干细胞的多能性等方面发挥了重要的作用。许多类型的癌症和Wnt/β-Catenin信号活性的错误调控都是相互关联的,同样在在非洲爪蛙胚胎中母源的P-Catenin的缺失会使organizer和背部体轴都不能形成。β-Catenin稳定性的调节是调控Wnt/β-Catenin信号活性的一种主要机制,同时核内高水平的β-Catenin也作为一些肿瘤的不良病状预断。在没有Wnt蛋白激活的情况下,胞质内会形成由APC,GSK3,CK1和β-TrCP形成的降解复合体,其中CK1和GSK3会使β-Catenin的N末端的丝氨酸和苏氨酸残基磷酸化,随后β-Catenin会被泛素化,然后被蛋白酶体降解。在有Wnt蛋白激活的情况下,胞质内不能形成降解复合体,β-Catenin也不会被磷酸化,非磷酸化的即活化的β-Catenin随后会进入细胞核内和TCF/LEF转录因子形成转录激活复合体激活靶基因的转录。之前对于Wnt信号通路中β-Catenin在胞质内调节机制的研究已经很清楚了,但是对于其在核内的调节机制还不是很清楚。赖氨酸去甲基化酶(Kdm2a/Kdm2b)作为一类对组蛋白产生特异位点去甲基化的修饰酶,目前已有数据显示有一些酶会对非组蛋白产生修饰作用,调节它所作用的底物活性和稳定性,Kdm2a/Kdm2b所作用的底物范围可能远远不止于组蛋白。之前的研究已经说明Kdm2a/Kdm2b是在核内表达的蛋白,目前为止,人们对于Kdm2a和Kdm2b在机体内的生理学作用和它们是否参与调节早期胚胎体轴的形成,以及Kdm2a/Kdm2b调节早期胚胎体轴形成的作用机制尚未有明确的结论。在此,我们利用非洲爪蛙(Xenopus leavis)为动物模型,来研究这些问题。在本篇论文中,我们利用经典的模式动物非洲爪蛙重点阐述了赖氨酸去甲基化酶Kdm2a/Kdm2b通过调节Wnt信号通路中P-Catenin的稳定性来调控非洲爪蛙胚胎形成过程中前后体轴的形成。我们在非洲爪蛙胚胎里敲降Kdm2a/Kdm2b发现胚胎体轴严重缩短,头部和尾部发育缺陷,这些表型和已知研究报道的P-Catenin过度激活表型类似。敲降Kdm2a/Kdm2b同时注射Kdm2a/Kdm2b mRNA也可以拯救这些表型。另外,母源和合子的Wnt/β-Catenin靶基因的转录被上调了。进一步地,我们的研究揭示了在敲降Kdm2a/Kdm2b之后,非洲爪蛙早期胚胎的前部标志基因被上调,后部的标志基因被下调。除此之外,Kdm2a和Kdm2b还具有功能冗余和功能互补的作用。相应地,我们还发现在敲降Kdm2a/Kdm2b之后,胚胎内活化的β-Catenin的蛋白水平是上调的,同样利用荧光素酶报告基因分析出在胚胎中和HEK293T细胞中Wnt信号的活性也是增加的。另外检测活化的β-Catenin总甲基化水平也是被上调的。同时,在RKO细胞系中Kdm2a/Kdm2b在细胞核内抑制活化的β-Catenin活性且敲降Kdm2a/Kdm2b会增加原肠胚期P-Catenin的甲基化水平。所以,Kdm2a/Kdm2b可能通过去甲基化作用诱导细胞核里非磷酸化的P-Catenin的降解,从而影响了母源和合子的Wnt/β-Catenin信号,导致了Wnt/β-Catenin信号下游靶基因不同程度的改变,这无疑影响了非洲爪蛙早期胚胎体轴的形成。综上所述,我们的研究结果表明在非洲爪蛙胚胎里,赖氨酸去甲基化酶Kdm2a/Kdm2b对于体轴的形成起着至关重要的作用。这种作用可能是通过Kdm2a/Kdm2b的去甲基化作用诱导细胞核里非磷酸化的P-Catenin的降解,从而激活胚胎里母源和合子的Wnt/β-Catenin信号,导致Wnt/β-Catenin信号下游靶基因不同程度的改变,进而影响了非洲爪蛙早期胚胎体轴的形成。更多还原

【Abstract】 During early vertebrate embryogenesis, embryonic anterior-posterior, dorsal-ventral and left-right body axis establishment are major events in the biological process of individual development. These events refer to a mass of signaling transduction pathways and relevent signaling transducers mediating precise modulation, for example, Wnt signaling, BMP signaling, Nodal signaling, FGF signaling, ect. Especially, the activity gradient of Wnt signaling during Xenopus early embryogenesis plays a crucial role in body axis formation:maternal Wnt signaling specifies the dosal-anterior axis; zygotic Wnt signaling leads to the posterior-ventral axis formation. The Wnt signal cascade controls plenty of biological phenomena throughout development of all metazoans. The canonical Wnt/β-Catenin signaling pathway plays vital roles in multiple biological processes, such as embryogenesis, homeostasis, regeneration, and stem cell pluripotency. In parallel, aberrant Wnt signaling is correlated with a wide range of pathologies in humans, such as colorectal cancer and breast cancer. Xenopus leavis embryos will induce the deficiencies of dorsal body axis development when the Wnt/β-Catenin signaling activity is low. The regulation of P-Catenin stability is a major mechanism to control Wnt/β-Catenin signaling activity. The deletion of maternal β-Catenin in Xenopus leavis will disturb the organizer and dorsal body axis formation. Simultaneously, the high level of β-Catenin in nucleus also acts as some adverse symptoms of the tumor prognosis. In the absence of Wnt signal activation, the destruction complex consists of APC, GSK3, CK1 and β-TrCP in the cytoplasm, where CK1 and GSK3 phosphorylate the N-terminal serine and threonine residues of β-Catenin. Subsequently, the phosphorylated β-Catenin is ubiquitylated and transported into proteosome for degradation. In the presence of Wnt signal activation, the destruction components are unable to assemble in the cytoplasm and β-Catenin cannot be phosphorylated. Afterwards, the non-phosphorylated (also called active) β-Catenin transports into nucleus, where β-Catenin can interact with the transcriptional factors TCF/LEF family members to activate target gene transcription. Currently, it has been clear that the mechanism of modulating β-Catenin stability in cytoplasm. However, it is unknown how β-Catenin is modulated in nucleus in Wnt signaling pathway.Currently, data has shown that histone lysine demethylases (Kdm2a/Kdm2b) function as demethylating specific sites on histone H3. Whereas, previous data has shown that some of the KDMs play a role in non-histone modification, which affects activity and stability of the substrates. Therefore, the scope of KDM substrates might extend far beyond histones. Previous study claimed that Kdm2a and Kdm2b are located in the nucleus. Whether Kdm2a and Kdm2b affect early embryogenesis? How do Kdm2a and Kdm2b affect early embryogenesis? What is the functional mechanism? So far, these questions have not been very clear yet. In this thesis, we use Xenopus leavis as model animal to study these questions.To explore the role of histone lysine demethylases Kdm2a/Kdm2b during early embryogenesis, we carry out antisense morpholino oligos (MOs) to knockdown Kdm2a/Kdm2b in Xenopus embryos. We find some defects in body axis development in Kdm2a/Kdm2b morphants, such as shrinking of head structure, truncation in posterior structure and shorten A-P body axis. These changes in development resemble, at least in part, the phenotypes resulting from β-Catenin over-activation. The morphants could be effectively rescued when Kdm2a/Kdm2b mRNA were coinjected with the MOs. Furthermore, maternal and zygotic Wnt/β-Catenin signaling target genes were upregulated. Besides, Kdm2a and Kdm2b performed with functional redundancy and complementation. Accordingly, we revealed that active β-Catenin levels were upregulated in gastrula embryos after Kdm2a/Kdm2b knockdown. We also showed Wnt signaling activity was upregulated via detecting the luciferase reporter activity in embryos and HEK293T cells. Beyond that, knockdown of Kdm2a or Kdm2b enhanced active β-Catenin methylation in gastrula embryos. In RKO cell line, only nuclear b-catenin was downregulated in the presence of Kdm2a and Wnt3a treatment, whereas the cytoplasmic and membranous fractions were unaffected.In conclusion, our research results manifest that histone lysine demethylases Kdm2a/Kdm2b play a vital role in body axis formation during Xenopus embryogenesis. Kdm2a/Kdm2b might demethylate active P-Catenin and induce degradation of active P-Catenin in nucleus. Consequently, transcription of Wnt/β-Catenin target genes is attenuated.更多还原