The role of pluripotency gene regulatory network components in mediating transitions between pluripotent cell states

Abstract

Pluripotency is a property that early embryonic cells possess over a considerable developmental time span. Accordingly, pluripotent cell lines can be established from the pre-implantation or post-implantation mouse embryo as embryonic stem (ES) or epiblast stem (EpiSC) cell lines, respectively. Maintenance of the pluripotent phenotype depends on the function of specific transcription factors (TFs) operating within a pluripotency gene regulatory network (PGRN). As cells move from an ES cell to an EpiSC state, the PGRN changes with expression of some TFs reduced (e.g. Nanog) or eliminated (e.g. Esrrb). Re-expressing such TFs can move cells back to an earlier developmental identity and is being applied to attempt establishment of human cell lines with the properties of mouse ES cells.

Figure 1

Nanog, Oct4 and Sox2 expression dynamics during murine development. Whereas Oct4 and Sox2 mRNAs are inherited maternally, Nanog mRNA is first detected in blastomeres of the 8 cell stage embryo [62^••]. At E3.5 Nanog is expressed heterogeneously in the ICM, with segregation of Nanog positive and negative cells leading to formation of the epiblast and hypoblast at E4.5. Around implantation Nanog is downregulated before being re-expressed in the posterior epiblast. Nanog remains regionally expressed but progressively declines until being lost at the onset of somitogenesis. Oct4 and Sox2 also become regionally expressed post-implantation, with Sox2 higher in the anterior epiblast and Oct4 becoming progressively posterior. In PGCs (that retain Oct4 expression throughout development) Sox2 becomes detectable at neural plate stage, with Nanog upregulated by the 2–3s stage. Nanog and Sox2 are expressed by PGCs until E12.5 (an E12.5 genital ridge is represented) before becoming undetectable at E15.5 and E14.5 in males and females respectively. Oct4 expression is also lost at E14.5 as female germ cells enter meiosis but persists longer in male PGCs (E, embryonic day; s, number of somites).

Figure 2

Positively regulated transcriptional targets of Nanog. Expression changes of selected significantly upregulated genes identified by analysing the response to acute Nanog induction in a Nanog^−/− ES cell line (data from supplementary information in [33^••]).

Figure 3

Schematic representation comparing the role of Nanog during development and reprogramming. Nanog interacts with other pluripotency TFs, as well as chromatin remodellers, such as NuRD and Tet1. By recruiting proteins to target gene chromatin Nanog can induce or repress mediators of self-renewal and differentiation. (a) Nanog is required twice during development: for the correct specification of a pluripotent epiblast in the blastocyst and for completion of germ cell development beyond E11.5. In both contexts Nanog may operate by modulating expression of its target genes and by counteracting accumulation of 5-methyl-cytosine at regulatory elements and consequent epigenetic silencing of key pluripotency genes. Nanog downregulation appears to be required in the epiblast to reduce expression of pluripotency TFs and allow dismantling of the pre-implantation PGRN. (b) Overexpression of Nanog induces LIF independent self-renewal and blocks ES cell differentiation. Nanog^−/− EpiSC can be converted to a pre-implantation pluripotent state by complementation with Nanog or Esrrb. Conversion of wild-type EpiSC to ES cells can be achieved by overexpressing Nanog (possibly through transcriptional activation of the indicated TFs) or Esrrb. An alternative LIF-mediated conversion (which may operate through Klf4 induction) can be accelerated by additional enforced expression of the individual TFs indicated (oval). (c) Nanog expression is required for complete reprogramming of pre-iPSCs from somatic cells, potentially by inducing Esrrb expression and contributing to the reversion of 5-methyl-cytosine marks by Tet1 recruitment. Esrrb can substitute for Nanog during reprogramming of pre-iPSCs when used together with 5-azacytidine. N, Nanog; E, Esrrb; T, Tet1. Boxes associated with arrows indicate potential mechanisms of action.