Role And Regulation Of Klf4 Gene During Xenopus Early Embryogenesis

Differentiation of three germ layers is the first fundamental step during early embryogenesis, which is precisely regulated by the complex signaling network. The main pathways involved in germ layer formation and body axis patterning contain Nodal/activin, FGF, BMP, and Wnt/β-catenin signaling. Activation of these signaling pathways at right time and right place is crucial for embryo development. In Xenopus, the Nodal ligand genes, Xnrl-6, are induced by the maternal transcription factor VegT in vegetal cells. Upon ligand stimulation, Nodal/activin pathway is transmitted downstream and induces transcription of mesoderm and endoderm specific genes expression like Xbra, Mix1, Mix2, Gsc, Mixer, Milk, Soxl7, GATA4-6. Endoderm specific genes, meanwhile, inhibit mesoderm genes such that mesoderm and endoderm formation is restricted within correct locations. Maternal β-catenin signaling is enriched in dorsal-vegetal cells and induces Siamois transcription, which subsequently induces gene transcription in the Spemann’s organizer to antagonist ventral signals. Therefore, the balance of these two groups of signals establishes the correct body plan.Differentiation of early embryonic cells into germ layers is accompanied by the loss of pluripotency, which is maintained by pluripotency factors. In mammals, these factors are typically Oct4, Sox2, Nanog, cMyc and Klf4. These pluripotency genes may play important roles in early embryogenesis. However, the function and the underlying mechanism of Klf4 during early embryogenesis, especially during germ layer formation, are not well understood. Xenopus embryos provide us a utilized model animal system to study the early period of embryonic development In this thesis, we investigate the function and mechanism of Klf4 during Xenopus early embryogenesis.As a zinc finger transcription factor, Klf4 is conserved from zebrafish to human. So far Klf4 has not been reported for Xenopus laevis. In our study, we identified a Klf4 homologous protein in Xenopus. Firstly, the spatial-temporal expression patterns of Klf4 shows that Klf4 is transcribed both maternally and zygotically and the transcript exists ubiquitously and dynamically in embryos during early embryogenesis. Secondly, gain of function and loss of function analyses showed that Klf4 is critical for germ layer formation. Overexpression of Klf4 promotes neural ectoderm and endoderm formation while inhibits mesoderm formation. Knockdown of Klf4 inhibits all of the three germ layers differentiation. Thirdly, Klf4 confers the competence of early embryonic cells to the inducing signals such as Nodal/activin pathway. Fourthly, Klf4 is also involved in body axis patterning via activation of a subset of genes expressed in the Spemann’s organizer, for instance Dkkl, Noggin and Cerberus, which encode secreted antagonists for Wnt, BMP and Nodal pathways. Finally, we found that the Wnt pathway downstream transducers TCF3 and LEF1 lie upstream of Klf4 for its activation to maintain the expression level of Klf4.In summary, our study revealed the role and regulation of Klf4 gene during Xenopus early embryogenesis for the first time. Our work improved the understanding of both the molecular events during germ layer formation as well as the roles of pluripotency genes during early embryogenesis. As Klf4 plays important roles in stem cell biology, our work was instructive for the research of stem cell.