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.

  • Technical Report
  • Published:

Single-molecule transcript counting of stem-cell markers in the mouse intestine

Nature Cell Biology volume 14, pages 106–114 (2012)Cite this article

Abstract

Determining the molecular identities of adult stem cells requires technologies for sensitive transcript detection in tissues. In mouse intestinal crypts, lineage-tracing studies indicated that different genes uniquely mark spatially distinct stem-cell populations, residing either at crypt bases or at position +4, but a detailed analysis of their spatial co-expression has not been feasible. Here we apply three-colour single-molecule fluorescent in situ hybridization to study a comprehensive panel of intestinal stem-cell markers during homeostasis, ageing and regeneration. We find that the expression of all markers overlaps at crypt-base cells. This co-expression includes Lgr5, Bmi1 and mTert, genes previously suggested to mark distinct stem cells. Strikingly, Dcamkl1 tuft cells, distributed throughout the crypt axis, co-express Lgr5 and other stem-cell markers that are otherwise confined to crypt bases. We also detect significant changes in the expression of some of the markers following irradiation, indicating their potential role in the regeneration process. Our approach can enable the sensitive detection of putative stem cells in other tissues and in tumours, guiding complementary functional studies to evaluate their stem-cell properties.

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

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Three-colour single-molecule FISH of intestinal stem-cell markers.
Figure 2: Single-molecule FISH correlates with reporter expression in transgenic mice but provides a much broader sampling.
Figure 3: Spatial expression profiles of stem-cell markers are broad and overlap at CBC cells.
Figure 4: Single-cell correlations of stem-cell markers are validated with mutants for key regulator genes.
Figure 5: Dcamkl1 marks tuft cells occurring throughout the crypt axis that significantly co-express stem-cell markers otherwise confined to crypt bottoms.
Figure 6: 12 Gy whole-body irradiation results in significant changes in the levels and range of stem-cell markers.

Similar content being viewed by others

References

  1. Potten, C. S., Gandara, R., Mahida, Y. R., Loeffler, M. & Wright, N. A. The stem cells of small intestinal crypts: where are they? Cell Prolif. 42, 731–750 (2009).

    Article  CAS  Google Scholar 

  2. Li, L. & Clevers, H. Coexistence of quiescent and active adult stem cells in mammals. Science 327, 542–545 (2010).

    Article  CAS  Google Scholar 

  3. van der Flier, L. G. & Clevers, H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu. Rev. Physiol. 71, 241–260 (2009).

    Article  CAS  Google Scholar 

  4. Gerbe, F. et al. Distinct ATOH1 and Neurog3 requirements define tuft cells as a new secretory cell type in the intestinal epithelium. J. Cell Biol. 192, 767–780 (2011).

    Article  CAS  Google Scholar 

  5. Sato, T. et al. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415–418 (2010).

    Article  Google Scholar 

  6. Potten, C. S. Stem cells in gastrointestinal epithelium: numbers, characteristics and death. Phil. Trans. R. Soc. Lond. B 353, 821–830 (1998).

    Article  CAS  Google Scholar 

  7. Sangiorgi, E. & Capecchi, M. R. Bmi1 is expressed in vivo in intestinal stem cells. Nat. Genet. 40, 915–920 (2008).

    Article  CAS  Google Scholar 

  8. Tian, H. et al. A reserve stem cell population in small intestine renders Lgr5-positive cells dispensable. Nature 478, 255–259 (2011).

    Article  CAS  Google Scholar 

  9. Montgomery, R. K. et al. Mouse telomerase reverse transcriptase (mTert) expression marks slowly cycling intestinal stem cells. Proc. Natl Acad. Sci. USA 108, 179–184 (2011).

    Article  CAS  Google Scholar 

  10. May, R. et al. Identification of a novel putative gastrointestinal stem cell and adenoma stem cell marker, doublecortin and CaM kinase-like-1, following radiation injury and in adenomatous polyposis coli/multiple intestinal neoplasia mice. Stem. Cells 26, 630–637 (2008).

    Article  Google Scholar 

  11. Cheng, H. & Leblond, C. P. Origin, differentiation and renewal of the four main epithelial cell types in the mouse small intestine. V. Unitarian Theory of the origin of the four epithelial cell types. Am. J. Anat. 141, 537–561 (1974).

    Article  CAS  Google Scholar 

  12. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).

    Article  CAS  Google Scholar 

  13. Furuyama, K. et al. Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas and intestine. Nat. Genet. 43, 34–41 (2011).

    Article  CAS  Google Scholar 

  14. Zhu, L. et al. Prominin 1 marks intestinal stem cells that are susceptible to neoplastic transformation. Nature 457, 603–607 (2009).

    Article  CAS  Google Scholar 

  15. Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  CAS  Google Scholar 

  16. van der Flier, L. G. et al. Transcription factor achaete scute-like 2 controls intestinal stem cell fate. Cell 136, 903–912 (2009).

    Article  CAS  Google Scholar 

  17. Giannakis, M. et al. Molecular properties of adult mouse gastric and intestinal epithelial progenitors in their niches. J. Biol. Chem. 281, 11292–11300 (2006).

    Article  CAS  Google Scholar 

  18. Raj, A. & van Oudenaarden, A. Single-molecule approaches to stochastic gene expression. Annu. Rev. Biophys. 38, 255–270 (2009).

    Article  CAS  Google Scholar 

  19. Gregorieff, A. & Clevers, H. Wnt signaling in the intestinal epithelium: from endoderm to cancer. Genes Dev. 19, 877–890 (2005).

    Article  CAS  Google Scholar 

  20. Femino, A. M., Fay, F. S., Fogarty, K. & Singer, R. H. Visualization of single RNA transcripts in situ. Science 280, 585–590 (1998).

    Article  CAS  Google Scholar 

  21. Levsky, J. M., Shenoy, S. M., Pezo, R. C. & Singer, R. H. Single-cell gene expression profiling. Science 297, 836–840 (2002).

    Article  CAS  Google Scholar 

  22. Zenklusen, D., Larson, D. R. & Singer, R. H. Single-RNA counting reveals alternative modes of gene expression in yeast. Nat. Struct. Mol. Biol. 15, 1263–1271 (2008).

    Article  CAS  Google Scholar 

  23. Raj, A., Peskin, C. S., Tranchina, D., Vargas, D. Y. & Tyagi, S. Stochastic mRNA synthesis in mammalian cells. PLoS Biol. 4, 1707–1719 (2006).

    Article  CAS  Google Scholar 

  24. Capodieci, P. et al. Gene expression profiling in single cells within tissue. Nat. Methods 2, 663–665 (2005).

    Article  CAS  Google Scholar 

  25. Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  Google Scholar 

  26. Raj, A., Rifkin, S. A., Andersen, E. & van Oudenaarden, A. Variability in gene expression underlies incomplete penetrance. Nature 463, 913–918 (2010).

    Article  CAS  Google Scholar 

  27. Carmon, K. S., Gong, X., Lin, Q., Thomas, A. & Liu, Q. R-spondins function as ligands of the orphan receptors LGR4 and LGR5 to regulate Wnt/{β}-catenin signaling. Proc. Natl Acad. Sci. USA 108, 11452–11457 (2011).

    Article  CAS  Google Scholar 

  28. de Lau, W. et al. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476, 293–297 (2011).

    Article  CAS  Google Scholar 

  29. Zeilstra, J. et al. Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis. Cancer Res. 68, 3655–3661 (2008).

    Article  CAS  Google Scholar 

  30. Sinner, D. et al. Sox17 and Sox4 differentially regulate β-catenin/T-cell factor activity and proliferation of colon carcinoma cells. Mol. Cell Biol. 27, 7802–7815 (2007).

    Article  CAS  Google Scholar 

  31. Gregorieff, A. et al. Expression pattern of Wnt signaling components in the adult intestine. Gastroenterology 129, 626–638 (2005).

    Article  CAS  Google Scholar 

  32. Kayahara, T. et al. Candidate markers for stem and early progenitor cells, Musashi-1 and Hes1, are expressed in crypt base columnar cells of mouse small intestine. FEBS Lett. 535, 131–135 (2003).

    Article  CAS  Google Scholar 

  33. Potten, C. S. et al. Identification of a putative intestinal stem cell and early lineage marker; musashi-1. Differentiation 71, 28–41 (2003).

    Article  CAS  Google Scholar 

  34. He, X. C. et al. BMP signaling inhibits intestinal stem cell self-renewal through suppression of Wnt- β-catenin signaling. Nat. Genet. 36, 1117–1121 (2004).

    Article  CAS  Google Scholar 

  35. Schepers, A. G., Vries, R., van den Born, M., van de Wetering, M. & Clevers, H. Lgr5 intestinal stem cells have high telomerase activity and randomly segregate their chromosomes. EMBO J. 30, 1104–1109 (2011).

    Article  CAS  Google Scholar 

  36. Kohler, T., Prols, F. & Brand-Saberi, B. PCNA in situ hybridization: a novel and reliable tool for detection of dynamic changes in proliferative activity. Histochem. Cell Biol. 123, 315–327 (2005).

    Article  Google Scholar 

  37. Batlle, E. et al. β-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111, 251–263 (2002).

    Article  CAS  Google Scholar 

  38. Potten, C. S., Booth, C. & Pritchard, D. M. The intestinal epithelial stem cell: the mucosal governor. Int. J. Exp. Pathol. 78, 219–243 (1997).

    Article  CAS  Google Scholar 

  39. Gerbe, F., Brulin, B., Makrini, L., Legraverend, C. & Jay, P. DCAMKL-1 expression identifies Tuft cells rather than stem cells in the adult mouse intestinal epithelium. Gastroenterology 137, 2179–2180 (2009) (author reply 2180-2171).

    Article  CAS  Google Scholar 

  40. van der Lugt, N. M. et al. Posterior transformation, neurological abnormalities, and severe hematopoietic defects in mice with a targeted deletion of the bmi-1 proto-oncogene. Genes Dev. 8, 757–769 (1994).

    Article  CAS  Google Scholar 

  41. Kirsch, D. G. et al. p53 controls radiation-induced gastrointestinal syndrome in mice independent of apoptosis. Science 327, 593–596 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank H. Youk, S. Semrau, S. Klemm and K. Hilgendorf for comments on the manuscript, and X. Wu, Z. Peng Fan and A. Yang for help with the cell segmentation software. This work was supported by the National Institutes of Health (NIH)/National Cancer Institute Physical Sciences Oncology Center at MIT (U54CA143874) and an NIH Pioneer award (1DP1OD003936) to A.v.O., and in part by Cancer Center Support (core) grant P30-CA14051 from the National Cancer Institute. S.I. acknowledges support from a European Molecular Biology Organization postdoctoral fellowship, the International Human Frontiers Science Program Organization and the Machiah Foundation. I.C.B. acknowledges support from the Howard Hughes Medical Institute Gilliam fellowship. T.J. is the D. H. Koch Professor of Biology and a D. K. Ludwig Scholar.

Author information

Authors and Affiliations

  1. Department of Physics, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    Shalev Itzkovitz, Anna Lyubimova & Alexander van Oudenaarden

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    Shalev Itzkovitz, Anna Lyubimova, Irene C. Blat, Jacqueline Lees, Tyler Jacks & Alexander van Oudenaarden

  3. Hubrecht Institute–KNAW (Royal Netherlands Academy of Arts and Sciences) and University Medical Center Utrecht, Uppsalalaan 8, 3584 CT Utrecht, Netherlands

    Anna Lyubimova, Johan van Es, Hans Clevers & Alexander van Oudenaarden

  4. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    Irene C. Blat, Mindy Maynard, Jacqueline Lees, Tyler Jacks & Alexander van Oudenaarden

  5. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA

    Tyler Jacks

Authors
  1. Shalev Itzkovitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Anna Lyubimova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Irene C. Blat
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Mindy Maynard
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Johan van Es
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jacqueline Lees
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Tyler Jacks
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Hans Clevers
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alexander van Oudenaarden
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

S.I. and A.v.O. conceived the project. S.I., I.C.B. and A.L. carried out most of the experiments. S.I. analysed the data. M.M., J.L., J.v.E., T.J. and H.C. provided mice and assisted with experiments. S.I., H.C. and A.v.O. wrote the paper.

Corresponding author

Correspondence to Alexander van Oudenaarden.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Information (PDF 1473 kb)

Supplementary Table 1

Supplementary Information (XLS 87 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Itzkovitz, S., Lyubimova, A., Blat, I. et al. Single-molecule transcript counting of stem-cell markers in the mouse intestine. Nat Cell Biol 14, 106–114 (2012). https://doi.org/10.1038/ncb2384

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb2384

This article is cited by

Search

Advanced 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