Role of Sox2 in postimplantation epiblast pluripotency
Wong, Ching Kwan Frederick
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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.