Transcription factor heterogeneity in epiblast pluripotency
Osorno Hernandez, Carlos Rodrigo
Hernandez, Carlos Rodrigo Osorno
Pluripotency is the ability of a cell to differentiate into derivatives of all three somatic lineages and germ cells. In vivo, pluripotent cells exist transiently in the epiblast of the developing embryo and in rare tumour cells. In vitro, pluripotent cells have been isolated and propagated from teratocarcinomas (EC cells), preimplantation epiblast (ES cells) and post-implantation epiblast (EpiSCs). Pluripotency is governed by a gene regulatory network centred on the triumvirate of transcription factors Oct4, Sox2 and Nanog. Interestingly, transcription factors that are important to direct pluripotent cell identity are not all equally distributed throughout the pluripotent cell population. While Oct4 levels are relatively homogeneous, other transcription factors, such as Nanog, are more heterogeneously expressed. Additionally, an increasing body of evidence indicates that extrinsic cues also play a critical role in the establishment and maintenance of pluripotency. Using biochemical and genetic tools in mouse ES cells, the role of FGF signaling and Sox2 levels on heterogeneous Nanog expression was examined. Interference with FGF or ERK activity by genetic ablation or signal inhibition, promoted high, homogenous Nanog expression and enhanced self-renewal. This is consistent with reports showing that similar manipulations reduced the ability of ES cells to commit to differentiation. Moreover, ES cells with reduced Sox2 levels displayed greater heterogeneity for Nanog expression than wild-type ES cells. Pluripotency is lost in the mouse embryo around E8.5, however, the precise timing and mechanism involved in this process has not yet been defined. Here it is shown that pluripotency is extinguished at the onset of somitogenesis, coincident with reduced expression and chromatin accessibility of Oct4 and Nanog regulatory regions. Prior to somitogenesis, the expression of both Nanog and Oct4 is regionalized. Interestingly, pluripotency tracks the in vivo level of Oct4, this correlation does not hold true for Nanog. However, Nanog expression reports on pluripotent cells. Indeed, ectopic Oct4 expression in somitogenesis-stage tissue provokes rapid reopening of Oct4 and Nanog chromatin, Nanog re-expression and resuscitation of moribund pluripotency. Competence to re-activate the pluripotency network upon enforced Oct4 expression is gradually lost with the progression of embryonic development. ES cells and EpiSCs are two distinct pluripotent populations as they show differences in their ability to undergo clonal propagation, re-colonize embryos, growth factor responsiveness, morphology and gene regulatory networks. It is possible to harness this differential growth factor responsiveness to convert ES cells into EpiSCs. Conversely, EpiSC can be reverted back to ES cell pluripotency through the overexpression of a small number of transcription factors. The inter-conversion of ES cells and EpiSCs has been documented, but detailed analyses of the changes that occur during such transitions had not been performed. The current work shows that Nanog levels are critical for the specification of the pluripotent state of the cells. Furthermore, it is shown that orphan nuclear receptor Esrrb is a potent inducer of ES cell pluripotency in EpiSC. Interestingly, Esrrb was able to restore naïve pluripotency in cells genetically depleted of Nanog.