Role of Sox2 in postimplantation epiblast pluripotency
View/ Open
Date
26/11/2015Author
Wong, Ching Kwan Frederick
Metadata
Abstract
Pluripotency is defined as the capacity to differentiate into cells from each of the
three primary germ layers, the ectoderm, mesoderm and endoderm. This is a
property of cells located in the inner cell mass (ICM) of preimplantation blastocysts
and in the epiblast layer of postimplantation, presomite embryos. Preimplantation
and postimplantation pluripotency can be captured indefinitely in cultured
embryonic stem (ES) cells and epiblast stem cells (EpiSCs) respectively.
Preimplantation pluripotency in ES cells is regulated by a network of genes centred
on three transcription factors (TFs) Oct4, Sox2 and Nanog. Oct4 and Sox2 form a
mutually-reinforcing circuit and cooperatively stimulate transcription of
downstream genes, including Nanog. All three TFs are expressed in EpiSCs and in
the postimplantation epiblast. Functional studies established a role for Oct4 and
Nanog in the specification of ICM cell identity, and a role for Oct4 in the
maintenance of postimplantation pluripotency. Although the role of Sox2 in
preimplantation ICM cells is unclear, it is critical for the establishment of egg
cylinder following implantation and indispensable for ES cell pluripotency.
However, despite the presence of Sox2 in postimplantation pluripotent cells the role
of Sox2 in postimplantation pluripotency is unknown.
In this thesis the role of Sox2 in the regulation of postimplantation pluripotency was
examined. In contrast to the situation in the preimplantation ICM, Sox2 and Nanog
are expressed in opposing gradients in the gastrulation-stage postimplantation
epiblast, with Sox2 highest anteriorly and Nanog highest posteriorly. Interestingly
the posterior epiblast of neural-plate (NP)-staged embryos was shown not to be
pluripotent. Furthermore, forced expression of Sox2 but not Oct4 in this region
rescued pluripotency. The ability of Oct4 to reinstate pluripotency in the
somitogenesis-stage embryo is limited to Sox2-positive tissues. This strongly
suggests that coexpression of Sox2 and Oct4 is important for establishing
postimplantation pluripotent identity. Sox2HIGH cultured EpiSCs were not positively
correlated with NanogHIGH cells. This reciprocal relationship emerged during the
transition from ES cells to EpiSCs in culture. Using mutant cells with reduced levels
of Sox2 or Nanog, Sox2 positively influences Nanog but Nanog negatively
influences Sox2 expression post-transcriptionally. The negative influence of Nanog
on Sox2 protein level was confirmed using doxycycline-inducible Nanog
overexpressing EpiSCs. This negative relationship indicates that the regulation of
Sox2 expression is different in postimplantation pluripotency and that Nanog may
negatively regulate Sox2 on the protein level in the posterior epiblast.
Sox2 is expressed at a lower level in EpiSCs than ES cells and the significance of this
was further investigated by microarray transcription profiling using cells in which a
fluorescent reporter (tdTomato) was knocked in to the Sox2 gene. Sox2-
tdTomatoHIGH cells cultured in LIF/FCS/GMEMβ correlate with an undifferentiated
cell identity and Sox2-tdTomatoLOW cells are associated with non-neural
differentiation. Interestingly the global profile of ES cells and EpiSCs that share
similar Sox2-tdTomato signal are non-identical. This suggests that Sox2 has different
roles in different pluripotent states. ES cells with enforced Sox2 expression were
unable to enter the EpiSC state, while ES cells with lowered Sox2 levels were
inefficient in neural differentiation. Therefore, levels of Sox2 are critical for cell fate
decisions. Strikingly, given the apparent requirement for Sox2 during Oct4-induced
reinstatement of post-implantation pluripotency, deletion of Sox2 had no effect on
the maintenance of EpiSC pluripotency. This is likely due to the presence of
redundant Sox factors and indeed Sox3 is able to rescue the Sox2-null phenotype in
ES cells. Taken together, these results suggest the hypothesis that postimplantation
pluripotency is maintained by multiple Sox factors, while Nanog negatively
regulates Sox2 post-transcriptionally to repress neural specification in the posterior
epbilast. The positive influence of Sox2 on Nanog protein level suggests a possible
negative feedback loop to balance the proneural and pluripotent properties of Sox2
in postimplantation pluripotency.