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Michel, Laura (2011): On the function of Xenopus Oct4 protein homologs: Molecular construction of dominant interference variants and functional analysis in early frog development. Dissertation, LMU München: Faculty of Medicine
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Abstract

Embryonic development represents a sophisticated multistep process. Hereby, specification, patterning and differentiation of cells and tissue need to be extremely well regulated in a temporo-spatial manner. This is based on repression and activation of a vast number of cell-type specific genes, but only a small number of transcription factors seem to be responsible for their regulation. The transcription factor network of Oct4, Sox2 and Nanog are thought to play an essential role in the maintenance of pluripotency and in timing the onset of differentiation. The importance of mouse Oct4 in the regulation of pluripotency is underscored by recent findings providing evidence that Oct4 is essential for reprogramming somatic cells. Nevertheless, little is known on the molecular function of this transcription factor during normogenesis. Given the extra uterine development of the embryos, the well-studied early development and the established manipulation methods like injection of RNA or DNA, Xenopus leavis offers an ideal model organism to study the role of Oct4 homologs in early development. In Xenopus laevis three Oct4 paralogs – Oct25, Oct60 and Oct91 – are known, which are similar in size and have a high sequence homology compared to mammalian Oct4. There are strong evidences that Xenopus Oct proteins and mammalian Oct4 share similar functions. To gain further insights into the function of Oct proteins I generated dominant activating- (VP16-Oct60), dominant repressing- (EnR-Oct60) and hormone inducible (GR-Oct60) transcription factor variants for all three Xenopus Oct proteins. Protein expression was verified in vitro as well as in vivo. Oct60 shows a unique expression pattern among Xenopus Oct proteins: Oct60 is maternally transcribed and its RNA is detectable in mature oocytes. Expression is downregulated in the gastrula, when the expression of other Xenopus POU proteins begins. Therefore, it is one of the earliest genes to be expressed. I decided to concentrate first efforts on Oct60. The transactivating functions of the Oct60 G.o.F. variants were tested in a luciferase assay on two different Oct4 reporter constructs in vivo. Oct60 and VP16-Oct60 acted as strong activators whereas EnR-Oct60 repressed both reporter constructs. By overexpression of Oct60 and its G.o.F. variants, several phenotypes were observed that affected distinct parts of the body. Beside impaired head differentiation, observed by overexpression of VP16-Oct60 and Oct60, a strong hyperpigmentation was observed by injection of EnR-Oct60 and Oct60. Additionally, EnR-Oct60 injected embryos showed hyperpigmented outgrowths in the trunk region. All injected embryos possessed a shortened body axis that was specifically curved depending on the injected mRNA. In situ hybridizations were performed to investigate the molecular mechanism of the observed phenotypic changes. Experiments revealed that all examined constructs promote neuroectodermal fate while repressing mesoderm formation. These results indicate that Oct60 plays an important role in the induction and specification of germ layer formation. By cloning and testing these different G.o.F. variants I accomplished to obtain important tools for further dissecting the molecular function of Oct4 homologs in Xenopus embryos.