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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.

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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.

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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.

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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

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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.

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Correspondence to Alexander van Oudenaarden.

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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

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