On the function of Xenopus Oct4 protein homologs: Molecular construction of dominant interference variants and functional analysis in early frog development

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.