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Nanog-dependent feedback loops regulate murine embryonic stem cell heterogeneity

Nature Cell Biology volume 14, pages 1139–1147 (2012)Cite this article

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Abstract

A number of key regulators of mouse embryonic stem (ES) cell identity, including the transcription factor Nanog, show strong expression fluctuations at the single-cell level. The molecular basis for these fluctuations is unknown. Here we used a genetic complementation strategy to investigate expression changes during transient periods of Nanog downregulation. Employing an integrated approach that includes high-throughput single-cell transcriptional profiling and mathematical modelling, we found that early molecular changes subsequent to Nanog loss are stochastic and reversible. However, analysis also revealed that Nanog loss severely compromises the self-sustaining feedback structure of the ES cell regulatory network. Consequently, these nascent changes soon become consolidated to committed fate decisions in the prolonged absence of Nanog. Consistent with this, we found that exogenous regulation of Nanog-dependent feedback control mechanisms produced a more homogeneous ES cell population. Taken together our results indicate that Nanog-dependent feedback loops have a role in controlling both ES cell fate decisions and population variability.

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Figure 1: Quantifying the molecular effects of Nanog fluctuations.
Figure 2: Transcriptome changes during periods of transient Nanog depletion.
Figure 3: Expression changes and promoter occupancy by Nanog.
Figure 4: Gene expression changes are regulated in a highly combinatorial manner.
Figure 5: Single-cell gene expression patterns.
Figure 6: Feedback in the ES cell TRN
Figure 7: Cell–cell variability in wild-type and NanogR populations.

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Acknowledgements

We thank K. Hochedlinger for the Nanog–GFP mouse ES cells53. We gratefully acknowledge funding support by the NIH (GM078465) and NYSTEM (C024410) to I.R.L. and by the NIH (GM095942) and NYSTEM (C026420) to J.W. This work was also supported by an EPSRC Doctoral Training Centre grant (EP/G03690X/1) and an EPSRC 2011/12 Institutional Sponsorship Award (EP/J501530/1).

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  1. Ben D. MacArthur and Ana Sevilla: These authors contributed equally to this work

Authors and Affiliations

  1. Centre for Human Development, Stem Cells and Regeneration, Institute of Developmental Sciences, University of Southampton, Southampton SO17 1BJ, UK

    Ben D. MacArthur & Patrick S. Stumpf

  2. School of Mathematics, University of Southampton, Southampton SO17 1BJ, UK

    Ben D. MacArthur & Sonya J. Ridden

  3. Institute for Life Sciences, University of Southampton, Southampton SO17 1BJ, UK

    Ben D. MacArthur

  4. Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, New York 10029, USA

    Ana Sevilla, Miguel Fidalgo, Jianlong Wang & Ihor R. Lemischka

  5. Black Family Stem Cell Institute, Mount Sinai School of Medicine, New York, New York 10029, USA

    Ana Sevilla, Miguel Fidalgo, Jianlong Wang & Ihor R. Lemischka

  6. Aachen Institute for Advanced Study in Computational Engineering Science, Rheinisch-Westfälische Technische Hochschule Aachen, Aachen 52062, Germany

    Michel Lenz, Berhard M. Schuldt & Andreas A. Schuppert

  7. Zentrum für Integrative Psychiatrie, Kiel 24105, Germany

    Franz-Josef Müller

  8. Institute for Complex Systems Simulation, University of Southampton, SO17 1BJ, UK

    Sonya J. Ridden

  9. Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, New York 10029, USA

    Avi Ma’ayan & Ihor R. Lemischka

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Contributions

B.D.M., A.S. and I.R.L. designed the project and prepared the manuscript. A.S., M.F. and J.W. performed the experiments. B.D.M., M.L., F.J.M., B.M.S., A.A.S., S.J.R., P.S.S. and A.M. performed the bioinformatic analyses and mathematical modelling. All authors reviewed and approved the manuscript.

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Correspondence to Ben D. MacArthur, Ana Sevilla or Ihor R. Lemischka.

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MacArthur, B., Sevilla, A., Lenz, M. et al. Nanog-dependent feedback loops regulate murine embryonic stem cell heterogeneity. Nat Cell Biol 14, 1139–1147 (2012). https://doi.org/10.1038/ncb2603

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