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In situ identification of bipotent stem cells in the mammary gland

Abstract

The mammary epithelium undergoes profound morphogenetic changes during development. Architecturally, it comprises two primary lineages, the inner luminal and outer myoepithelial cell layers. Two opposing concepts on the nature of mammary stem cells (MaSCs) in the postnatal gland have emerged. One model, based on classical transplantation assays, postulates that bipotent MaSCs have a key role in coordinating ductal epithelial expansion and maintenance in the adult gland, whereas the second model proposes that only unipotent MaSCs identified by lineage tracing contribute to these processes. Through clonal cell-fate mapping studies using a stochastic multicolour cre reporter combined with a new three-dimensional imaging strategy, we provide evidence for the existence of bipotent MaSCs as well as distinct long-lived progenitor cells. The cellular dynamics at different developmental stages support a model in which both stem and progenitor cells drive morphogenesis during puberty, whereas bipotent MaSCs coordinate ductal homeostasis and remodelling of the mouse adult gland.

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Figure 1: A novel 3D imaging strategy for validation of lineage-specific reporter strains and cell-fate mapping.
Figure 2: Elf5-expressing luminal progenitor cells contribute to morphogenesis in puberty and maintenance in adulthood.
Figure 3: K5-expressing cells labelled at the onset of puberty contribute to expansion of both epithelial lineages.
Figure 4: K5-expressing bipotent stem cells ensure expansion of both lineages in the adult gland.
Figure 5: K5 targets long-lived stem cells that contribute to maintenance and pregnancy.

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Acknowledgements

We are grateful to F. Jackling and K. Liu for genotyping, F. Vaillant for performing transplants, C. Nowell and K. Roger in the WEHI Imaging Facility for expert support, S. Firth at Monash MicroImaging, Leica and Zeiss for imaging support, D. Sieiro Mosti for help with quantification, J. Stanley for generation of transgenic strains, and the Animal, FACS and Histology facilities at WEHI. We also thank J. Adams for review of the manuscript, and H. Clevers, P. Chambon, M. Furtado, B. Hogan and M. Shen for the provision of mouse strains. This work was supported by the Australian National Health and Medical Research Council (NHMRC); the Victorian State Government through VCA funding of the Victorian Breast Cancer Research Consortium and Operational Infrastructure Support; the Australian Cancer Research Foundation; and the Qualtrough Family Bequest. A.C.R. and N.Y.F. were supported by a National Breast Cancer Foundation/Cure Cancer Australia Fellowship, G.J.L. by a NHMRC Research Fellowship and J.E.V. by an Australia Fellowship.

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Authors

Contributions

A.C.R. and N.Y.F. designed and performed all experiments, and carried out data analysis. J.E.V. and G.J.L. conceived the study and designed experiments. J.E.V., N.Y.F., A.C.R. and G.J.L. wrote the manuscript.

Corresponding author

Correspondence to Jane E. Visvader.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 FACS analysis of GFP expression in K5- and Elf5-rtTA-IRES-GFP mammary glands, and tdTomato-labelled cells in the K5-driven doxycycline-inducible model.

a, General gating strategy for flow cytometric analysis of mammary epithelial cells. Representative FACS plot showing the basal (LinCD29hiCD24+) and luminal (LinCD29loCD24+) subsets. b, c, Representative FACS histograms of GFP expression in the luminal (LinCD29loCD24+) and basal (LinCD29hiCD24+) populations isolated from the mammary glands of 4-week-old Elf5-rtTA-IRES-GFP (b) and K5-rtTA-IRES-GFP mice (c) (on a FVB/N background). dh, FACS plots of tdTomato expression in the luminal and basal populations isolated from the mammary glands of control mice (d, e) and K5-rtTA/TetO-cre/R26R-tdTomato mice injected with doxycycline (Dox) at 4 weeks of age and then analysed 2 days (f), 2 weeks (g) or 8 weeks (h) post-induction. Control mice included K5-rtTA/TetO-cre/R26R-tdTomato not injected with Dox (e) and TetO-cre/R26R-tdTomato mice injected with Dox (d). No leakiness in mammary epithelial cells was observed. No labelled tdTomato+ cells could be detected at 1 day post-induction by either FACS analysis or imaging.

Extended Data Figure 2 Characterization and clonal analysis of Elf5-labelled cells in the adult mammary gland.

ad, Luminal progenitor cells express Elf5–GFP. a, Quantitative RT–PCR of Elf5 mRNA expression in sorted populations from Elf5-rtTA-IRES-GFP glands at 8 weeks of age, showing an approximately 20-fold increase in Elf5 levels in GFP+ relative to GFP cells. b, Confocal optical section from an Elf5-rtTA-IRES-GFP mammary gland stained for endogenous Elf5 (red). All Elf5+ cells also express GFP (green) from the transgene promoter, showing strong concordance between endogenous Elf5 and transgene expression. Scale bar, 20 μm. c, The vast majority of CD61+ luminal progenitors express Elf5–GFP. Mammary cell suspensions from 8-week-old mice were analysed for the expression of CD61, CD24 and CD29, resolving the luminal progenitor (CD61+CD29loCD24+) and mature luminal (CD61CD29loCD24+) subsets. d, Only Elf5–GFP+ cells in the luminal population display colony-forming activity in Matrigel. The Elf5–GFP+ and GFP luminal cells in the mammary glands of Elf5-rtTA-IRES-GFP mice were sorted by flow cytometry and 1,200 Elf5–GFP+ and GFP luminal cells were cultured in Matrigel for 7 days. e, Whole-mount 3D confocal image of an Elf5-rtTA/TetO-cre/R26R-Confetti mammary duct at 1 week post-induction in adulthood. f, g, Optical sections of the white rectangular areas illustrate small clones, defined as isolated clusters of one or more cells contacting each other; for example, f comprises one YFP+ clone (two cells), three RFP+ clones (one with two contacting cells) and one CFP+ clone (three cells). F-actin shown in blue. Scale bars, 100 μm (e, whole-mount) and 5 μm (f, g, optical sections). h, Quantification of the number of cells per clone in 3D confocal whole-mounts of Elf5-rtTA/TetO-cre/R26R-Confetti mammary glands at 1 or 8 weeks after induction with doxycycline in adult mice. Red shading indicates the relative frequency of labelled cell numbers per clone. 100% is red and 0% is white. i, Bar chart showing the percentage of labelled clones and the number of cells in each. Data represent the mean ± s.e.m. for n = 3 (1-week chase) and n = 4 mice (8-week chase).

Extended Data Figure 3 Schematic representation of lineage-tracing strategy and colour distribution of proteins expressed from the Confetti locus in the Elf5-, K5- and K14-transgenic models.

a, K5-rtTA-IRES-GFP and Elf5-rtTA-IRES-GFP mice were crossed with TetO-cre mice and a reporter strain, either R26R-Confetti or R26R-tdTomato, to generate doxycycline-inducible triple-transgenic models. b, K14-creERT2, K5-creERT2 and Lgr5- creERT2 mice were crossed, respectively, with R26R-Confetti and R26R-YFP, R26R-YFP or R26R-tdTomato to generate tamoxifen-regulatable models. c, Representation of the lineage-tracing strategy for induction in puberty or adulthood, with the various chase periods indicated. df, CFP, GFP, YFP and RFP colour representation for Elf5-rtTA/TetO-cre/R26R-Confetti (d), K5-rtTA/TetO-cre/R26R-Confetti (e) and K14-creERT2/ R26R-Confetti (f) mice at 2 weeks post-induction in puberty (4 weeks). Scale bars, 100 μm. g, h, Colour analysis of the contribution of Elf5- and K5-labelled cells to ductal morphogenesis during puberty. Bar charts showing the percentage of ducts displaying 1, 2, 3 or 4 colours in the mammary glands of Elf5-rtTA/TetO-cre/R26R-Confetti (g) and K5-rtTA/TetO-cre/R26R-Confetti mice (h), at either 2 or 8 weeks post-induction in puberty. Note the segregation of colour apparent in the K5 model after an 8-week chase, compared to the Elf5 model in which there is little change. A minimum of 50 ducts per mammary gland from three independent mice were counted. Data represents the mean ± s.e.m.

Extended Data Figure 4 Contribution of Elf5-labelled cells to the alveolar lineage during pregnancy.

a, b, 3D confocal image of a whole-mounted gland (a) and enlargement (b) of an Elf5-rtTA/TetO-cre/R26R-Confetti mammary gland at 14.5 days of pregnancy stained for F-actin (white). The enlargement shows both unlabelled and labelled alveoli, with YFP+ GFP+, RFP+-only and GFP+YFP+RFP+ alveoli indicated by arrows. Scale bars, 150 μm (a, whole-mount) and 50 μm (b, enlargement). c, Bar chart showing percentage of alveoli (within large alveolar units) that exhibit either 1, 2, 3 or 4 distinct colours per alveolus for the Elf5-rtTA/TetO-cre/R26R-Confetti mice at 14.5 days of pregnancy after a doxycycline pulse during adulthood (9 weeks). Data represent the mean ± s.e.m. for 638 alveoli counted in three mice. d, 3D confocal image of a whole-mounted gland from Elf5-rtTA/TetO-cre/R26R-Confetti mice at 2 weeks post-involution and stained for F-actin (white). Scale bar, 150 μm.

Extended Data Figure 5 Confocal analysis of TEBs and ducts in glands from K5-expressing transgenic mice 2 days after induction.

a, Whole-mount 3D confocal image (left), optical section (middle) and enlargement (right) of a TEB from a 4-week-old FVB/N mammary gland immunostained for endogenous K5 (red) and labelled for EdU (green), 2 h after EdU injection. White arrows depict proliferative cap cells, one of which is dividing to yield a luminal cell. Scale bars, 40 μm (left and middle panels), 10 μm (right panel). b, Optical section (0.45 μm) of a wild-type TEB at 4 weeks of age immunostained for endogenous K5 (green), K14 (red) and Elf5 (blue). The arrow shows a triple-positive cell. Scale bar, 20 μm. c, Whole-mount 3D confocal image (left) and optical section (right) of a TEB from K5-rtTA/TetO-cre/R26R-Confetti mice in puberty at 2 days post-induction with doxycycline and 2 h post-EdU injection, immunostained for RFP (red) and EdU (green). The Confetti reporter strain was used to address clonality within the RFP+ population. Arrows depict cap cells giving rise to two luminal cells (left arrows) and one luminal cell (right arrow). Scale bars, 40 μm (whole-mount), 10 μm (optical section). d, Whole-mount 3D confocal image (top) and optical section (bottom) of a duct from a K5-rtTA/TetO-cre/R26R-tdTomato mammary gland at 2 days post-induction in adulthood (2 h after EdU injection), immunostained for EdU (green). Arrows depict EdU+ proliferative cells within the myoepithelial layer. Scale bar, 100 μm.

Extended Data Figure 6 K5-expressing cells in the K5-creERT2 model and Lgr5-expressing cells contribute to both the basal and luminal lineages.

a, b, K5-creERT2/R26R-YFP mice analysed 4 weeks post-induction by doxycycline at puberty. a, FACS plots of YFP-labelled cells in the LinCD29+ population and distribution of YFP+ cells in the luminal (LinCD29loCD24+) and basal (LinCD29hiCD24+) populations from K5-creERT2/R26R-YFP mammary glands. b, Left, 3D confocal image of a whole-mounted duct from a K5-creERT2/R26R-YFP mammary gland, immunostained for YFP using anti-GFP (green) and anti-E-cadherin (blue) antibodies. Right, optical section of the enlargement showing luminal (white arrow) and elongated myoepithelial cells. Scale bars, 150 μm (whole-mount) and 50 μm (optical section). cg, Lgr5-expressing cells contribute to both basal and luminal cells in adulthood and pregnancy. c, FACS plots of tdTomato-labelled cells in the LinCD29+ population (0.12%) and distribution of tdTomato+ cells in the luminal (LinCD29loCD24+) and basal (LinCD29hiCD24+) subsets isolated from the glands of Lgr5-GFP-IRES-creERT2/R26R-tdTomato mice chased for 8 weeks (n = 9). The same data were obtained after pulsing in puberty or adulthood. FACS analysis was performed on at least 200,000 LinCD29+ cells. d, e, 3D confocal whole-mounted duct (d) and optical section (e) from a Lgr5-creERT2/R26R-tdTomato mammary gland, immunostained for E-cadherin (blue), after induction with tamoxifen in adulthood for 8 weeks. Scale bars, 100 μm (d, whole-mount) and 50 μm (e, optical section). f, FACS plots of tdTomato-positive cells in LinCD29+ cells (0.03%) and distribution of tdTomato+ cells in the luminal (LinCD29loCD24+) and basal (LinCD29hiCD24+) populations isolated from the mammary glands of Lgr5-GFP-IRES-creERT2/R26R-tdTomato mice pulsed in adulthood and analysed in pregnancy. g, Left, 3D confocal image and right, optical section of a whole-mounted, stained for F-actin (white), at 14.5 days of pregnancy after tamoxifen injection in adulthood. Labelled lobuloalveolar units were sparsely dispersed throughout the tree but strong labelling occurred in those alveoli in which cre had been activated. Right, optical section of the rectangular area in g shows an alveolar unit composed of labelled alveolar luminal cells. Scale bars, 200 μm (whole-mount), 30 μm (optical section).

Extended Data Figure 7 Analysis of different clonal regions labelled by K5-expressing cells at 1 and 8 weeks post-induction in the adult gland.

a, 3D reconstruction of a whole-mount ductal tree at 1 week post-induction in adulthood immunostained for E-cadherin (blue). Scale bar, 100 μm. Independent clones can clearly be visualized. Enlargements show four representative clones. The CFP+ clone (1) comprises several luminal cells and one myoepithelial cell. The left YFP+ clone (2) comprises myoepithelial and luminal cells, whereas the right YFP+ clone (3) contains myoepithelial cells only, and the RFP+ clone (4) comprises a single myoepithelial cell. Quantitation of >1,000 clones at 1 week and >200 clones at 8 weeks post-induction was performed (see Extended Data Fig. 8). b, c, 3D confocal image of a ductal tree at 1 week post-induction in adulthood immunostained for F-actin (blue), showing two examples of CFP+ myoepithelial cells attached to luminal cells in the inner layer. Enlargements shown in Fig. 4e and panel c. Arrow depicts a luminal cell tethered to a myoepithelial cell. Scale bars, 50 μm (whole-mount) and 10 μm (optical sections). d, Luminal-rich clonal region derived from K5-expressing cells at 8 weeks post-induction in adulthood: 3D confocal image of whole-mount ducts from K5-rtTA/TetO-cre/R26R-Confetti mice and optical sections of the white rectangular area showing a YFP+ cell-enriched patch with many luminal cells and two myoepithelial cells (bottom panels). Optical section of the same area stained for F-actin (white) to indicate cell morphology (bottom right). Scale bars, 50 μm (whole-mount) and 20 μm (optical sections). e, 3D whole-mount confocal image of a ductal tree and optical sections of the white rectangular area showing a YFP and RFP-enriched myoepithelial patch. The bottom right panel depicts an optical section of the same area immunostained for E-cadherin (blue), revealing no luminal cells. Scale bars, 150 μm (whole-mount) and 15 μm (optical sections).

Extended Data Figure 8 Quantitative clonal analysis of K5-labelled cells in adult mammary glands at 1 and 8 weeks post-induction.

a, b, The number of luminal and myoepithelial cells were scored per clone via scanning of 3D confocal whole-mounts of K5-rtTA/TetO-cre/R26R-Confetti mammary glands after 1 (a) or 8 (b) weeks post-induction with doxycycline in adult mice (9 weeks). Luminal cells are shown across the x axis and myoepithelial cells are quantified along the y axis. Note the change in scale along the x axis in b, to account for the presence of very large luminal-cell-enriched clones (and a few myoepithelial cells). Red shading indicates the relative frequency of labelled cell numbers per clone. 100% is red and 0% is white. n = 5 mice for the 1-week chase and n = 3 for the 8-week chase.

Extended Data Figure 9 High doses of tamoxifen profoundly affect mammary gland development.

a, C57BL/6 mice at puberty (4 weeks old) were injected with vehicle (sunflower seed oil) or the indicated amount of tamoxifen. Mammary glands were collected and stained with carmine alum for whole-mount analysis at 10 weeks after the last tamoxifen injection. Representative images are shown. Scale bar, 2 mm. b, C57BL/6 mice (4 weeks old) were injected with vehicle or tamoxifen (as indicated), and mammary glands collected 4 days after the last injection. Immunohistochemical staining of glands from tamoxifen-treated mice for BrdU incorporation. Scale bar, 50 μm. c, d, Whole-mount analysis and BrdU immunohistochemical staining of glands from doxycycline-treated mice (2 mg) shows no toxic effects. n = 3 mice for each treatment (ad). Scale bars, 2 mm (c) and 50 μm (d). e, Adult C57BL/6 mice (8 weeks old) were injected with vehicle or tamoxifen and subjected to pregnancy 6 weeks later. Mammary glands were collected at 14.5 days of pregnancy. n = 2 for tamoxifen treatment; n = 3 for vehicle. Scale bar, 1 mm.

Extended Data Figure 10 K14-targeted cells are bipotent and contribute to expansion and homeostasis of the adult mammary gland.

a, Quantification of YFP-labelled cells in the LinCD29+ population and distribution of YFP+ cells in the luminal (LinCD29loCD24+) and basal (LinCD29hiCD24+) populations isolated from the mammary glands of K14-creERT2/R26R-YFP mice pulsed at the onset of puberty (4 weeks) and chased for 4 weeks. b, 3D confocal image of a whole-mounted duct from a K14-creERT2/R26R-YFP mammary gland, pulsed in puberty and chased for 4 weeks, and immunostained for YFP using anti-GFP (green) and anti-E-cadherin (blue) antibodies (top panel). Optical sections (bottom panels) showing luminal (left) and myoepithelial (right) cells, indicated by the white rectangles above. Scale bars, 150 μm (whole-mount) and 50 μm (optical sections). ce, Whole-mount 3D confocal images of a TEB from a K14-creERT2/R26R-Confetti mammary gland at 2 days post-induction (c) and ductal trees from the mammary glands of K14-creERT2/R26R-Confetti mice stained for F-actin (white) and analysed 2 weeks after tamoxifen injection at the onset of puberty (d, e). Scale bars, 40 μm (TEB), 100 μm (whole-mount), 20 μm (optical section). fk, Whole-mount 3D confocal image of ducts and optical sections from K14-creERT2/R26R-Confetti glands immunostained for E-cadherin (blue) or stained for F-actin (white) at 8 weeks post-induction in adulthood. Optical sections (0.45 μm) in g of the enlargement from the whole-mounted ductal tree duct (f) showing the myoepithelial (top) and luminal (bottom) layers of a RFP+ clonal region. Scale bars, 100 μm (whole-mount) and 20 μm (optical sections). An example of a multicoloured duct is shown in h. The enlargements of the indicated areas (i, j) represent optical sections (0.45 μm) from anti-E-cadherin-immunostained ducts of YFP+ clonal regions comprising the luminal (top) and myoepithelial (bottom) layers. Scale bars, 50 μm (whole-mount), 25 μm (optical sections). k, 3D confocal image of a whole-mounted duct immunostained for E-cadherin (blue) illustrating different clonal patches in K14-creERT2/R26R-Confetti mice induced in adulthood after an 8-week chase. Thick optical sections (2 μm) of the luminal and myoepithelial layers in the area depicted by the white rectangle are shown. Scale bars, 100 μm.

Extended Data Figure 11 Bipotent stem cells drive remodelling of the mammary gland during involution.

a, Whole-mount 3D confocal image of a ductal portion from K14-creERT2/R26R-Confetti mice, stained for F-actin (white), after two cycles of pregnancy and analysed at 14 days of involution. Enlargement shows an optical section with multicoloured cells in the luminal and myoepithelial layers of the duct undergoing remodelling. White arrow indicates luminal cells. Scale bars, 200 μm (whole-mount), 100 μm (optical section). b, Whole-mount 3D confocal image of a ductal tree from K5-rtTA/TetO-cre/R26R-Confetti mice, stained for F-actin (white) at 14 days of involution after a single round of pregnancy. Enlargements show an optical section (top right) of a multicoloured region (white rectangular area, left), and optical sections of the luminal (middle right) and myoepithelial (bottom right) layers of the indicated RFP+ clone. Scale bars, 150 μm (whole-mount), 100 μm (top right panel), 10 μm (middle and bottom right panels).

Supplementary information

3D visualization of the whole-mount mammary gland presented in Fig. 1a.

The video shows 3D rendering using maximum intensity projection and then zooms in to show myoepithelial cells in the xy-plane at high resolution. The end of the movie shows a series of xy-planes (optical sections) through the 3D reconstruction data. (MOV 13226 kb)

3D visualization of a whole-mounted duct immunostained with E-cadherin, using Imaris blend mode. (MOV 10269 kb)

3D visualization of a whole-mount duct for the K5-rtTA-IRES-GFP mammary gland presented in Fig. 1d using Imaris blend mode.

The whole-mount was immunostained for GFP (green) to reveal the myoepithelial cells, E-cadherin (blue) to show luminal cells and DAPI (red) to show all nuclei including stromal cells. (MOV 10243 kb)

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Rios, A., Fu, N., Lindeman, G. et al. In situ identification of bipotent stem cells in the mammary gland. Nature 506, 322–327 (2014). https://doi.org/10.1038/nature12948

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