Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Dissecting self-renewal in stem cells with RNA interference

Abstract

We present an integrated approach to identify genetic mechanisms that control self-renewal in mouse embryonic stem cells. We use short hairpin RNA (shRNA) loss-of-function techniques to downregulate a set of gene products whose expression patterns suggest self-renewal regulatory functions. We focus on transcriptional regulators and identify seven genes for which shRNA-mediated depletion negatively affects self-renewal, including four genes with previously unrecognized roles in self-renewal. Perturbations of these gene products are combined with dynamic, global analyses of gene expression. Our studies suggest specific biological roles for these molecules and reveal the complexity of cell fate regulation in embryonic stem cells.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Identification of self-renewal regulators in ES cells.
Figure 2: Genetic complementation to rescue shRNA-induced self-renewal defects.
Figure 3: Global gene expression changes after downregulation of individual self-renewal regulators.
Figure 4: Nanog rescue of phenotypes caused by shRNA directed against other self-renewal regulators.
Figure 5: Provisional model of cell fate regulatory interactions in ES cells in vitro.

Similar content being viewed by others

References

  1. Smith, A. G. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435–462 (2001)

    Article  CAS  Google Scholar 

  2. Nichols, J. et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95, 379–391 (1998)

    Article  CAS  Google Scholar 

  3. Niwa, H., Miyazaki, J. & Smith, A. G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genet. 24, 372–376 (2000)

    Article  CAS  Google Scholar 

  4. Chambers, I. et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113, 643–655 (2003)

    Article  CAS  Google Scholar 

  5. Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003)

    Article  CAS  Google Scholar 

  6. Niwa, H., Burdon, T., Chambers, I. & Smith, A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048–2060 (1998)

    Article  CAS  Google Scholar 

  7. Ying, Q. L., Nichols, J., Chambers, I. & Smith, A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281–292 (2003)

    Article  CAS  Google Scholar 

  8. Burdon, T., Stracey, C., Chambers, I., Nichols, J. & Smith, A. Suppression of SHP-2 and ERK signalling promotes self-renewal of mouse embryonic stem cells. Dev. Biol. 210, 30–43 (1999)

    Article  CAS  Google Scholar 

  9. Sato, N., Meijer, L., Skaltsounis, L., Greengard, P. & Brivanlou, A. H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nature Med. 10, 55–63 (2004)

    Article  CAS  Google Scholar 

  10. Loebel, D. A., Watson, C. M., De Young, R. A. & Tam, P. P. Lineage choice and differentiation in mouse embryos and embryonic stem cells. Dev. Biol. 264, 1–14 (2003)

    Article  CAS  Google Scholar 

  11. Bortvin, A. et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 130, 1673–1680 (2003)

    Article  CAS  Google Scholar 

  12. Tighe, A. P. & Gudas, L. J. Retinoic acid inhibits leukemia inhibitory factor signaling pathways in mouse embryonic stem cells. J. Cell. Physiol. 198, 223–229 (2004)

    Article  CAS  Google Scholar 

  13. Ivanova, N. B. et al. A stem cell molecular signature. Science 298, 601–604 (2002)

    Article  ADS  CAS  Google Scholar 

  14. Burdon, T., Smith, A. & Savatier, P. Signalling, cell cycle and pluripotency in embryonic stem cells. Trends Cell Biol. 12, 432–438 (2002)

    Article  CAS  Google Scholar 

  15. Avilion, A. A. et al. Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev. 17, 126–140 (2003)

    Article  CAS  Google Scholar 

  16. Bamshad, M. et al. Mutations in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nature Genet. 16, 311–315 (1997)

    Article  CAS  Google Scholar 

  17. Mitsunaga, K. et al. Loss of PGC-specific expression of the orphan nuclear receptor ERR-β results in reduction of germ cell number in mouse embryos. Mech. Dev. 121, 237–246 (2004)

    Article  CAS  Google Scholar 

  18. Pekarsky, Y. et al. Tcl1 enhances Akt kinase activity and mediates its nuclear translocation. Proc. Natl Acad. Sci. USA 97, 3028–3033 (2000)

    Article  ADS  CAS  Google Scholar 

  19. Ting, D. T., Kyba, M. & Daley, G. Q. Inducible transgene expression in mouse stem cells. Methods Mol. Med. 105, 23–46 (2005)

    CAS  PubMed  Google Scholar 

  20. Yasunaga, M. et al. Induction and monitoring of definitive and visceral endoderm differentiation of mouse ES cells. Nature Biotechnol. 23, 1542–1550 (2005)

    Article  CAS  Google Scholar 

  21. Corson, L. B., Yamanaka, Y., Lai, K. M. & Rossant, J. Spatial and temporal patterns of ERK signaling during mouse embryogenesis. Development 130, 4527–4537 (2003)

    Article  CAS  Google Scholar 

  22. Saba-El-Leil, M. K. et al. An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development. EMBO Rep. 4, 964–968 (2003)

    Article  CAS  Google Scholar 

  23. Keller, G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19, 1129–1155 (2005)

    Article  CAS  Google Scholar 

  24. Ferri, A. L. et al. Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain. Development 131, 3805–3819 (2004)

    Article  CAS  Google Scholar 

  25. Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005)

    Article  CAS  Google Scholar 

  26. Kurokawa, D. et al. Regulation of Otx2 expression and its functions in mouse epiblast and anterior neuroectoderm. Development 131, 3307–3317 (2004)

    Article  CAS  Google Scholar 

  27. Essner, J. J., Branford, W. W., Zhang, J. & Yost, H. J. Mesendoderm and left-right brain, heart and gut development are differentially regulated by pitx2 isoforms. Development 127, 1081–1093 (2000)

    CAS  PubMed  Google Scholar 

  28. Evans, A. L. & Gage, P. J. Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development. Hum. Mol. Genet. 14, 3347–3359 (2005)

    Article  CAS  Google Scholar 

  29. Zhang, C., Basta, T. & Klymkowsky, M. W. SOX7 and SOX18 are essential for cardiogenesis in Xenopus. Dev. Dyn. 234, 878–891 (2005)

    Article  CAS  Google Scholar 

  30. Loh, Y. H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Genet. 38, 431–440 (2006)

    Article  CAS  Google Scholar 

  31. Luo, J. et al. Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-β. Nature 388, 778–782 (1997)

    Article  ADS  CAS  Google Scholar 

  32. Davenport, T. G., Jerome-Majewska, L. A. & Papaioannou, V. E. Mammary gland, limb and yolk sac defects in mice lacking Tbx3, the gene mutated in human ulnar mammary syndrome. Development 130, 2263–2273 (2003)

    Article  CAS  Google Scholar 

  33. Kang, S. M. et al. Impaired T- and B-cell development in Tcl1-deficient mice. Blood 105, 1288–1294 (2005)

    Article  CAS  Google Scholar 

  34. Narducci, M. G. et al. TCL1 participates in early embryonic development and is overexpressed in human seminomas. Proc. Natl Acad. Sci. USA 99, 11712–11717 (2002)

    Article  ADS  CAS  Google Scholar 

  35. Berns, K. et al. A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428, 431–437 (2004)

    Article  ADS  CAS  Google Scholar 

  36. Paddison, P. J. et al. A resource for large-scale RNA-interference-based screens in mammals. Nature 428, 427–431 (2004)

    Article  ADS  CAS  Google Scholar 

  37. Rao, M. Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells. Dev. Biol. 275, 269–286 (2004)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Smith for E14/T cells and the episomal expression system, G. Daley for Aniv15 cells, and D. Baltimore for the FUGW lentiviral expression system. We are grateful to K. A. Moore and members of the Lemischka laboratory for their assistance at various stages of these studies. We also thank A. Smith for constructive criticisms. This work was supported by funding from the NIDDK. Author Contributions N.I. and I.R.L. designed the experiments. N.I., R.L., I.K., J.L., C.D., X.S. and Y.L. performed the experiments. N.I., R.D. and I.R.L. analysed the data. N.I. and I.R.L. wrote the paper.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Natalia Ivanova or Ihor R. Lemischka.

Ethics declarations

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

This file contains Supplementary Materials, Supplementary Tables 1–4, Supplementary Figures 1–18 and additional references. (PDF 2301 kb)

Supplementary Data 1

MAS5.0 normalized microarray data (ZIP 16494 kb)

Supplementary Data 2

MIAME description of microarray data (DOC 55 kb)

Supplementary Data 3

Description of microarray files (XLS 27 kb)

Supplementary Data 4

Non-redundant probes used in microarray analysis (XLS 3771 kb)

Supplementary Data 5

Probes affected at least in one shRNA time-course (XLS 503 kb)

Supplementary Data 6

901 probes down-regulated in RA differentiation time-course (XLS 732 kb)

Supplementary Data 7

Information for genes used in the over-expression studies (XLS 43 kb)

Supplementary Data 8

List of genes affected by Nanog shRNA (XLS 252 kb)

Supplementary Data 9

List of genes affected by Oct4 shRNA (XLS 189 kb)

Supplementary Data 10

List of genes affected by Sox2 shRNA (XLS 228 kb)

Supplementary Data 11

List of genes affected by Esrrb shRNA (XLS 149 kb)

Supplementary Data 12

List of genes affected by Tbx3 shRNA (XLS 136 kb)

Supplementary Data 13

List of genes affected by Tcl1 shRNA (XLS 100 kb)

Supplementary Data 14

List of genes affected by Mm.343880 shRNA (XLS 111 kb)

Supplementary Data 15

List of genes affected by Dppa4 shRNA (XLS 74 kb)

Supplementary Data 16

List of genes down-regulated during the Dppa4 shRNA time-course only. (XLS 20 kb)

Supplementary Data 17

List of genes down-regulated during the Mm.343880 shRNA time-course only. (XLS 17 kb)

Supplementary Data 18

List of genes up-regulated during the Mm.343880 shRNA time-course only. (XLS 11 kb)

Supplementary Data 19

List of genes down-regulated during the Nanog and Oct4 shRNA time-courses only. (XLS 13 kb)

Supplementary Data 20

List of genes down-regulated during the Nanog shRNA time-course only. (XLS 36 kb)

Supplementary Data 21

List of genes up-regulated during the Nanog shRNA time-course only. (XLS 25 kb)

Supplementary Data 22

List of genes down-regulated during the Oct4 shRNA time-course only. (XLS 21 kb)

Supplementary Data 23

List of genes up-regulated during the Oct4 shRNA time-course only. (XLS 20 kb)

Supplementary Data 24

List of genes up-regulated during the Oct4 and Sox2 shRNA time-courses only. (XLS 14 kb)

Supplementary Data 25

List of genes down-regulated in Pattern1 (XLS 27 kb)

Supplementary Data 26

List of genes up-regulated in Pattern1 (XLS 116 kb)

Supplementary Data 27

List of genes down-regulated in Pattern2 (XLS 48 kb)

Supplementary Data 28

List of genes up-regulated in Pattern2 (XLS 48 kb)

Supplementary Data 29

List of genes down-regulated in Pattern3 (XLS 15 kb)

Supplementary Data 30

List of genes up-regulated in Pattern3 (XLS 49 kb)

Supplementary Data 31

List of genes down-regulated during the Sox2 shRNA time-course only (XLS 16 kb)

Supplementary Data 32

List of genes up-regulated during the Sox2 shRNA time-course only (XLS 19 kb)

Supplementary Data 33

List of genes down-regulated during the Tbx3 shRNA time-course only (XLS 18 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ivanova, N., Dobrin, R., Lu, R. et al. Dissecting self-renewal in stem cells with RNA interference. Nature 442, 533–538 (2006). https://doi.org/10.1038/nature04915

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04915

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing