Nanog safeguards pluripotency and mediates germline development

Abstract

Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells. Deletion of Nanog causes early embryonic lethality, whereas constitutive expression enables autonomous self-renewal of embryonic stem cells. Nanog is accordingly considered a core element of the pluripotent transcriptional network. However, here we report that Nanog fluctuates in mouse embryonic stem cells. Transient downregulation of Nanog appears to predispose cells towards differentiation but does not mark commitment. By genetic deletion we show that, although they are prone to differentiate, embryonic stem cells can self-renew indefinitely in the permanent absence of Nanog. Expanded Nanog null cells colonize embryonic germ layers and exhibit multilineage differentiation both in fetal and adult chimaeras. Although they are also recruited to the germ line, primordial germ cells lacking Nanog fail to mature on reaching the genital ridge. This defect is rescued by repair of the mutant allele. Thus Nanog is dispensible for expression of somatic pluripotency but is specifically required for formation of germ cells. Nanog therefore acts primarily in construction of inner cell mass and germ cell states rather than in the housekeeping machinery of pluripotency. We surmise that Nanog stabilizes embryonic stem cells in culture by resisting or reversing alternative gene expression states.

Esrrb extinction triggers dismantling of naïve pluripotency and marks commitment to differentiation

“…To directly assess the effect of NANOG protein on Esrrb expression in single cells, an Esrrb‐2a‐tdTomato reporter was introduced into both Nanog −/− ESΔN and Nanog +/− TNG ESCs (Chambers et al , 2007; Festuccia et al , 2012; Fig EV1, Appendix Fig S1). Both lines have GFP knocked‐in to one of the Nanog alleles, but in ESΔN ESCs, the second Nanog allele is also inactivated (Fig EV1, Appendix Fig S2).…”

“…NANOG is the pluripotency TF for which a direct quantitative correlation between levels of expression and ESC self‐renewal efficiency has been most clearly demonstrated (Chambers et al , 2003, 2007; Abranches et al , 2014). Accordingly, NANOG is considered to act as a rheostat to tune ESC self‐renewal efficiency (Mullin & Chambers, 2012).…”

“…Consistent with this, ESRRB is able to mediate several downstream effects of NANOG, both in pluripotent cells and primordial germ cells (Festuccia et al , 2012; Zhang et al , 2018). NANOG protein is expressed in the nuclei of ESCs cultured in LIF/FCS in a heterogeneous manner, a feature shared with several other pluripotency proteins, including ESRRB (Chambers et al , 2007; Torres‐Padilla & Chambers, 2014). …”

“…The activity of a relatively well‐characterized pluripotency gene regulatory network, centered on the triumvirate OCT4, SOX2 and NANOG, controls the dual abilities of ESCs to self‐renew and to differentiate (Jaenisch & Young, 2008; Chambers & Tomlinson, 2009; Ng & Surani, 2011). While populations of ESCs cultured in LIF/FCS express relatively homogeneous levels of Oct4 and Sox2, individual ESCs show varying levels of Nanog expression (Chambers et al , 2007; Kalmar et al , 2009; Abranches et al , 2014). Heterogeneous expression has also been reported for Esrrb (van den Berg et al , 2008), Klf4 (Niwa et al , 2009), Tbx3 (Niwa et al , 2009; Russell et al , 2015), Rex1 (Toyooka et al , 2008), and Stella (Hayashi et al , 2008).…”

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

“…The differential expression patterns of some pluripotency regulators in different subpopulations have been linked to fine tuning the balance between self‐renewal and cell fate commitment 7, 14, 15, 16, 17. The mechanisms underlying gene expression heterogeneity in the pluripotent state are still not well understood, and evidence suggests that gene fluctuations could arise from stochastic noise in gene expression regulatory mechanisms 18, 19, 20, 21.…”