| 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. |
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