Nanog safeguards pluripotency and mediates germline development

Abstract

Nanog is a divergent homeodomain protein found in mammalian pluripotent cells and developing germ cells1,2. Deletion of Nanog causes early embryonic lethality2, whereas constitutive expression enables autonomous self-renewal of embryonic stem cells1. Nanog is accordingly considered a core element of the pluripotent transcriptional network3,4,5,6,7. 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.

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Figure 1: Nanog expression within the undifferentiated embryonic stem-cell population is reversible.
Figure 2: Nanog -/- embryonic stem cells retain expression of pluripotency markers and the capacity for in vitro self renewal.
Figure 3: Nanog -/- cells retain the potential for embryo colonization and somatic contribution to chimaeras.
Figure 4: Nanog is required for cell state transitions during germ cell development and for cell state reversions in embryonic stem-cell cultures.

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Acknowledgements

We are grateful to V. Karwacki, A. Waterhouse, R. Wilkie, R. MacLay and J. Ure for technical assistance, to C. Manson, J. Verth and colleagues for animal husbandry, and to V. Wilson for comments on the manuscript. This research was supported by the Wellcome Trust, the Juvenile Diabetes Research Foundation, the Medical Research Council and the Biotechnological and Biological Sciences Research Council of the United Kingdom, and a Human Frontier Science Program Fellowship (to L.G.).

Author Contributions I.C. designed the experimental strategy and analysed the data; J.S., J.N. and A.S. contributed to the experimental design. J.S., J.N. and K.J. performed the chimaera study, and J.S. the confocal analyses. D.C. conducted gene targeting and cell biological analysis, and together with J.V. ran the FACS experiments. M.R. and B.N. performed molecular biological analyses. L.G. produced and characterized the ROSA26-Cre-ERT2 cells. I.C. and A.S. conceived the study and wrote the paper.

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Correspondence to Ian Chambers.

Supplementary information

Supplementary Information

The file contains Supplementary Tables 1-4 and Supplementary Figures 1-12 (PDF 4603 kb)

Supplementary Video 1

This file contains Supplementary Video 1 showing bright field of GFP- TNG cells from 24-44h post-plating (MOV 32601 kb)

Supplementary Video 2

This file contains Supplementary Video 2 showing fluorescent field of GFP TNG cells from 24-44h post-plating (MOV 42173 kb)

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Chambers, I., Silva, J., Colby, D. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007). https://doi.org/10.1038/nature06403

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