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Identity and dynamics of mammary stem cells during branching morphogenesis

Nature volume 542, pages 313317 (16 February 2017) | Download Citation


During puberty, the mouse mammary gland develops into a highly branched epithelial network. Owing to the absence of exclusive stem cell markers, the location, multiplicity, dynamics and fate of mammary stem cells (MaSCs), which drive branching morphogenesis, are unknown. Here we show that morphogenesis is driven by proliferative terminal end buds that terminate or bifurcate with near equal probability, in a stochastic and time-invariant manner, leading to a heterogeneous epithelial network. We show that the majority of terminal end bud cells function as highly proliferative, lineage-committed MaSCs that are heterogeneous in their expression profile and short-term contribution to ductal extension. Yet, through cell rearrangements during terminal end bud bifurcation, each MaSC is able to contribute actively to long-term growth. Our study shows that the behaviour of MaSCs is not directly linked to a single expression profile. Instead, morphogenesis relies upon lineage-restricted heterogeneous MaSC populations that function as single equipotent pools in the long term.

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  1. 1.

    & Mammary ductal elongation: differentiation of myoepithelium and basal lamina during branching morphogenesis. Dev. Biol. 97, 274–290 (1983)

  2. 2.

    & Glycosaminoglycans in the basal lamina and extracellular matrix of serially aged mouse mammary ducts. Mech. Ageing Dev. 24, 151–162 (1984)

  3. 3.

    et al. Stem and progenitor cell division kinetics during postnatal mouse mammary gland development. Nat. Commun. 6, 8487 (2015)

  4. 4.

    & Mammary stem cells and the differentiation hierarchy: current status and perspectives. Genes Dev. 28, 1143–1158 (2014)

  5. 5.

    , , & Mammary gland development: cell fate specification, stem cells and the microenvironment. Development 142, 1028–1042 (2015)

  6. 6.

    et al. Purification and unique properties of mammary epithelial stem cells. Nature 439, 993–997 (2006)

  7. 7.

    et al. Generation of a functional mammary gland from a single stem cell. Nature 439, 84–88 (2006)

  8. 8.

    et al. Molecular hierarchy of mammary differentiation yields refined markers of mammary stem cells. Proc. Natl Acad. Sci. USA 110, 7123–7130 (2013)

  9. 9.

    et al. Distinct stem cells contribute to mammary gland development and maintenance. Nature 479, 189–193 (2011)

  10. 10.

    , , & In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327 (2014)

  11. 11.

    et al. Mammary stem cells have myoepithelial cell properties. Nat. Cell Biol. 16, 942–950 (2014)

  12. 12.

    & s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue. Genes Dev. 24, 1882–1892 (2010)

  13. 13.

    et al. Lgr5-expressing cells are sufficient and necessary for postnatal mammary gland organogenesis. Cell Reports 3, 70–78 (2013)

  14. 14.

    et al. Developmental stage-specific contribution of LGR5+ cells to basal and luminal epithelial lineages in the postnatal mammary gland. J. Pathol. 228, 300–309 (2012)

  15. 15.

    , & Developmental stage and time dictate the fate of Wnt/β-catenin-responsive stem cells in the mammary gland. Cell Stem Cell 11, 387–400 (2012)

  16. 16.

    et al. Identification of multipotent mammary stem cells by protein C receptor expression. Nature 517, 81–84 (2015)

  17. 17.

    & Wnt proteins are self-renewal factors for mammary stem cells and promote their long-term expansion in culture. Cell Stem Cell 6, 568–577 (2010)

  18. 18.

    et al. The Wnt receptor, Lrp5, is expressed by mouse mammary stem cells and is required to maintain the basal lineage. PLoS One 4, e6594 (2009)

  19. 19.

    , , & The branching programme of mouse lung development. Nature 453, 745–750 (2008)

  20. 20.

    , , & Hormonal and local control of mammary branching morphogenesis. Differentiation 74, 365–381 (2006)

  21. 21.

    , , , & Tissue geometry determines sites of mammary branching morphogenesis in organotypic cultures. Science 314, 298–300 (2006)

  22. 22.

    & Patterning mechanisms of branched organs. Science 322, 1506–1509 (2008)

  23. 23.

    et al. Adaptive immune regulation of mammary postnatal organogenesis. Dev. Cell 34, 493–504 (2015)

  24. 24.

    et al. Intravital imaging of cancer stem cell plasticity in mammary tumors. Stem Cells 31, 602–606 (2013)

  25. 25.

    , , , & The influence of tamoxifen on normal mouse mammary gland homeostasis. Breast Cancer Res. 16, 411 (2014)

  26. 26.

    et al. De novo prediction of stem cell identity using single-cell transcriptome data. Cell Stem Cell 19, 266–277 (2016)

  27. 27.

    & Candidate regulators of mammary branching morphogenesis identified by genome-wide transcript analysis. Dev. Dyn. 235, 3404–3412 (2006)

  28. 28.

    & Integrated morphodynamic signalling of the mammary gland. Nat. Rev. Mol. Cell Biol. 12, 581–593 (2011)

  29. 29.

    et al. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 507, 362–365 (2014)

  30. 30.

    , , & Intestinal stem cell replacement follows a pattern of neutral drift. Science 330, 822–825 (2010)

  31. 31.

    , , , & Imaging windows for long-term intravital imaging. Intravital 3, e29917 (2014)

  32. 32.

    et al. A single-cell transcriptome atlas of the human pancreas. Cell Syst. 3, 385–394.e3 (2016)

  33. 33.

    et al. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq. Genome Biol. 17, 77 (2016)

  34. 34.

    & Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

  35. 35.

    & Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010)

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The authors would like to thank members of the van Rheenen group for critically reading this manuscript, and Anko de Graaff of the Hubrecht Imaging Centre and the Hubrecht Institute animal caretakers for technical support. This work was supported by the European Research Council (consolidator grant 648804 to J.v.R.), the Worldwide Cancer Research (grant 13-0297 to J.v.R), and the Wellcome Trust (grant 098357/Z/12/Z to B.D.S. and 110326/Z/15/Z to E.H.). E.H. is funded by a Junior Research Fellowship from Trinity College, Cambridge, and a Sir Henry Wellcome Fellowship from the Wellcome Trust and acknowledges the Bettencourt-Schueller Young Researcher Prize for support. C.L.G.J.S. is funded by a Boehringer Ingelheim Fonds PhD Fellowship.

Author information

Author notes

    • Colinda L. G. J. Scheele
    •  & Edouard Hannezo

    These authors contributed equally to this work.


  1. Cancer Genomics Netherlands, Hubrecht Institute-KNAW & University Medical Centre Utrecht, Utrecht, the Netherlands

    • Colinda L. G. J. Scheele
    • , Mauro J. Muraro
    • , Anoek Zomer
    • , Nathalia S. M. Langedijk
    • , Alexander van Oudenaarden
    •  & Jacco van Rheenen
  2. Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK

    • Edouard Hannezo
    •  & Benjamin D. Simons
  3. The Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, Cambridge, UK

    • Edouard Hannezo
    •  & Benjamin D. Simons
  4. The Wellcome Trust/Medical Research Council Stem Cell Institute, University of Cambridge, UK

    • Edouard Hannezo
    •  & Benjamin D. Simons


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J.v.R., B.D.S., C.L.G.J.S., and E.H. conceived the study and designed the experiments. C.L.G.J.S., with assistance from A.Z., and N.S.M.L., performed the experiments and analyses. E.H. performed all theoretical work. M.J.M. performed single-cell mRNA sequencing and analysis. C.L.G.J.S., E.H. and M.J.M. made the figures. A.v.O., J.v.R. and B.D.S. supervized the study. All authors discussed results and participated in preparation of the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Benjamin D. Simons or Jacco van Rheenen.

Reviewer Information Nature thanks M. Bentires-Alj, A. Klein and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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    Supplementary Information

    This file contains a Supplementary Theory note and additional references.

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    Supplementary Table 1

    Differentially regulated genes within each cell cluster derived from TEBs and ducts in the pubertal mammary gland. Related to Fig. 2g and Extended Data Fig. 7.


  1. 1.

    Intravital imaging of static ducts and highly dynamic TEBs in the pubertal mammary gland

    Time-lapse videos of a 4th mammary gland at 5 weeks of age imaged through a mammary imaging window, showing that TEB cells are highly dynamic and show cell migration, cell mixing, and proliferation (top panels), whereas the ductal cells do not migrate or proliferate over time (bottom panels). The stills of these videos are depicted in Fig. 2d. Time is indicated in minutes.

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