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Identity and dynamics of mammary stem cells during branching morphogenesis

Abstract

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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Figure 1: Pubertal mammary development follows a stochastic growth pattern leading to heterogeneity in the ductal network.
Figure 2: Number, localization and molecular characterization of pubertal MaSCs.
Figure 3: Border (but not tip) MaSCs contribute to ductal extension in the short term.
Figure 4: Pubertal mammary gland development is driven by equipotent pools of MaSCs.
Figure 5: Model of pubertal mammary ductal growth dynamics.

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Acknowledgements

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

Authors and Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Benjamin D. Simons or Jacco van Rheenen.

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Competing interests

The authors declare no competing financial interests.

Additional information

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.

Extended data figures and tables

Extended Data Figure 1 Pubertal mammary gland development is driven by elongation and bifurcation of proliferative TEBs.

a, Confocal image of whole-mount mammary glands at the onset of puberty (3.5 weeks of age) stained for DAPI (blue) and EdU (magenta, 4-h chase). Ducts are outlined in yellow. b, Magnified image of part of the fifth mammary gland outlined with a red box in a. Confocal image shows a single Z-plane of a proliferative TEB in the fifth gland at 3.5 weeks of age. c, Left, magnified image of the fifth mammary gland outlined with a yellow box in a. The top panel shows the maximum projection of the fifth gland at 3.5 weeks of age; the bottom panel shows a magnification of a representative TEB (single Z-plane, outlined with a white box). Middle, maximum projection of a fifth mammary gland at 5 weeks of age. The bottom panel shows a magnification of a representative TEB (outlined with a white box). Right, maximum projection of the fifth mammary gland at 8 weeks of age. The bottom panel shows a magnified image of a representative TEB (single Z-plane, outlined with a white box). EdU+ cells shown in red, nuclei are stained with DAPI (in blue), yellow outlines mark the ducts. d, Schematic representation of the methods used to count the branch levels in the mammary gland. In pubertal developing mammary glands, younger ducts (light grey) that were formed more recently have undergone more bifurcation events than older ducts (dark grey) that were formed earlier. e, Cumulative distribution of branch length at 5 weeks and 8 weeks, for branches formed after the beginning of puberty (level > 6) (n = 10 glands from 10 different mice). f, Bar graph showing the fraction of EdU+ cells in proliferative end buds during the course of pubertal development (three mice were used for each time point; we quantified n = 12 TEBs for the 3.5-week time point, n = 10 TEBs for the 5-week time point, and n = 10 TEBs for the 8-week time point). g, The fraction of EdU+ TEBs were measured at 3.5, 5, and 8 weeks (three mice were used for each time point; n = 12 glands for the 3.5-week time point, n = 8 glands for the 5-week time point, and n = 8 glands for the 8-week time point). The graph shows the fraction of EdU+ TEB plotted against the highest branch level. The black line represents the prediction from the stochastic model of equipotent TEBs described in the main text. h, Probability q of a TEB to terminally differentiate, shown as a function of branch level and read from the whole-gland reconstructions (n = 10 glands from 5 different mice, same data as Fig. 1e), overlaid with an linear piecewise fit (dashed green lines) which we use as an input to predict the subtree heterogeneity in Fig. 1f. Dotted lines indicate a constant value of 0.5 (horizontal line), indicative of balance between TEB bifurcation and termination and the branch level at the start of puberty (vertical line). i, Normalized average branch width as a function of branch level. Dotted lines indicate a constant value of 1 (horizontal line) and the branch level at the start of puberty (vertical line) (n = 10 glands from 5 different mice). j, Normalized average branch length as a function of branch level. Dotted lines indicate a constant value of 1 (horizontal line), and the branch level at the start of puberty (vertical line) (n = 10 glands from 5 different mice). k, Fate correlation between related TEBs, as a function of branch level. Correlation between sibling TEBs is in purple, whereas correlation between first-cousin TEBs is in green. A value of 1 indicates that the fate of two related TEBs is perfectly correlated, whereas a value of −1 indicates perfect anti-correlation. The dotted line indicates 0 (that is, no correlation) (n = 10 glands from 5 different mice). For all graphs in this figure, data are mean ± s.e.m. and data from the fourth and fifth mammary gland were combined. Scale bars, 5 mm (a); 50 μm (b); 300 μm (c).

Source data

Extended Data Figure 2 Unbiased lineage-tracing strategy during pubertal development.

a, Cartoon depicting the gene system used for the lineage-tracing strategy. b, Timeline of the lineage-tracing strategy. c, Labelled cell fraction (product of clonal persistence and the surviving mean subclone size) as a function of segment level, for the 3–8 weeks tracing. The labelled cell fraction hovers around its initial value (reference taken for segment 6), indicative of a representative labelling. This also signifies that the persistence decays inversely to the linear increase in clone size, as expected from the model of long-term equipotent MaSCs. d, Labelled cell fraction as a function of segment level, for the 5–8 weeks trace. e, Labelled cell fraction as a function of segment level, for the 3–5 weeks trace. In both d and e, the labelled cell fraction hovers around its initial value, confirming the results of the 3–8 weeks of age trace. f, Normalized mean subclone size (basal in purple and luminal in green) as a function of branch level for the 3–5 weeks trace, showing a linear increase as branching morphogenesis proceeds due to cell segregation upon bifurcation. Subclone size is normalized by the area of the segment they are located in to correct for the segment length variability. Subclone size was also normalized to its initial value (at segment level 6), and display a similar trend to the 3–8 weeks tracing. g, Clone size distributions for luminal (left) and basal (right) clones at 5 weeks of age (lineage tracing from 3–5 weeks of age), at successive branch levels, plotted logarithmically. The clone size was rescaled by the average clone size at each time point. In each case, the data display scaling, and collapse on a single exponential (black line), as expected for an equipotent cell population. h, Quantification of the number of basal and luminal cells per micrometre in the mammary ducts of the adult mouse mammary gland (n = 17 randomly picked ducts from three different mice). For all graphs in this figure, data are mean ± s.e.m., and data from the fourth and fifth mammary glands were combined.

Source data

Extended Data Figure 3 Comparisons of branch patterns at different doses of tamoxifen and of fourth and fifth mammary glands.

a, Confocal images of whole-mount fourth mammary glands at 8 weeks of age, after injection of, from top to bottom, 3 mg tamoxifen per 25 g body weight, 1.5 mg tamoxifen per 25 g body weight or no tamoxifen, respectively, at the onset of puberty (3 weeks of age). Injection of 3 mg tamoxifen per 25 g body weight clearly interferes with branching morphogenesis, as the mammary gland did not fully invade the fat pad at the end of puberty. The mammary gland treated with 1.5 mg tamoxifen per 25 g body weight does not show any delay in mammary gland development, and shows the same branching pattern as the control gland. b, Average branch width and length as a function of branch level. No differences were observed in branch length or branch width for the different concentrations of tamoxifen. c, Probability q for a TEB to terminally differentiate as a function of branch level, read from the whole-gland reconstructions. In b and c, we analysed n = 1,480 segments from two glands (no tamoxifen), n = 1,229 segments from two glands (0.2 mg tamoxifen per 25 g body weight), and n = 3,347 segments from six glands (1.5 mg tamoxifen per 25 g body weight). No differences were observed between tamoxifen-induced glands and control glands. For graphs b and c, data from the fourth and fifth mammary glands were combined. d, Average branch widths and lengths as a function of branch level. e, Probabilities q of a TEB terminally differentiating, shown as a function of branch level and read from the whole-gland reconstructions. f, Subtree persistence, defined as the distribution of subtrees (starting at level 6) having a given maximal branch level i. g, Cumulative subtree size distributions, where size is defined as the total number of branches in a given subtree (starting at level 6). In dg, we considered fourth glands (n = 5) and fifth glands (n = 5), and plot comparisons respectively in purple and in green. Data are mean ± s.e.m. (be) or s.d. (f, g). Scale bars, 5 mm (a).

Source data

Extended Data Figure 4 Confetti+ mammary glands reveal the contribution of MaSCs during pubertal development.

a, Confocal image of a whole-mount confetti+ fourth mammary gland at 5 weeks of age, traced from 3 weeks of age. The ducts were manually reconstructed by outlining the ducts based on the K14 staining (white) and all individual confetti-labelled cells were outlined with their respective confetti colour (blue symbols: CFP+ cells; green symbols: GFP+ cells; yellow symbols: YFP+ cells; red symbols: RPF+ cells). b, Image shows a duct in the rudimentary gland containing single confetti-labelled cells (outlined with the red box in a). c, d, Confocal images showing ducts that were formed after the induction of confetti-tracing containing clonal outgrowths. Note that only one confetti colour is present in the ducts close to the border of the fat pad. Scale bars, 100 μm. e, f, n = 606 subclones analysed in four glands from two mice (same data as Fig. 4 a, b). e, Distribution of intermittency length (purple dots), defined as the number of consecutive unlabelled branches existing between branches labelled in the same colour and cell type. The black line is an exponential fit. Data are mean ± s.e.m. f, Probability of a clone being lost from the subsequent subtree, expressed as a function of its last segment level. Data are mean ± s.e.m. Scale bars, 5 mm (a), 100 μm (bd).

Source data

Extended Data Figure 5 MaSCs are localized in the TEBs and contribute during oestrous-driven proliferation and alveoli formation during pregnancy.

a, Cartoon depicting the gene system used for the lineage-tracing strategy and timeline of the lineage-tracing strategy. b, Confocal images of a duct and a lobuloalveolar structure in the fourth mammary gland containing lineage-traced cells after 18 months of tracing contributing to ductal turnover and the formation of lobuloalveolar structures driven by the oestrous cycle. c, Cartoon depicting the gene system and timeline used for the lineage-tracing strategy. d, Maximum projection of a part of a whole-mount fourth mammary gland after 4 days of lactation, showing the contribution of YFP+ confetti cells to alveoli formation. e, Whole-mount image of a fifth mammary gland at 5.5 weeks of age, traced from 3 weeks of age. Ducts are stained for K14 (in red). f, Maximum projection of a part of the fifth mammary gland depicted in e showing a luminal GFP clone contributing to the subtree depicted here. g, h, Maximum projection of TEBs and their adjacent ducts outlined by the yellow and green boxes in f. The most distal GFP cells of the clone are situated in the TEBs and are actively dividing, as is indicated by the most recent progeny located in the adjacent ducts. The localization of the most distal cells of a clone was determined as shown in Fig. 2e. Scale bars, 100 μm (b, d, g, h); 5 mm (e); 1 mm (f).

Extended Data Figure 6 Clonal labelling enables quantification of the number of functional MaSCs based on labelling density.

a, Cartoon depicting the method used to determine the number of MaSCs. Clonal labelling at the onset of puberty results in the confetti labelling of 1 MaSC out of N MaSCs in a TEB (red cell). Clonal analysis in the resulting subtree at the end of puberty enables us to determine the contribution of one labelled MaSC to the entire subtree that is formed after the induction of confetti tracing. The labelled fraction is calculated by dividing the clone size (red cells) by the total number of cells in the subtree (1/N). By taking the inverse, the number N of functional MaSCs can be calculated. b, Representative images of a part of a whole-mount fourth mammary gland, stained for K14 (basal marker) used to determine the identity of the labelled clones. Magnified images show a single Z-plane with basal cells (red outline), which overlap with the staining, or luminal cells (yellow outline), which do not overlap with the basal staining. Scale bars, 100 μm. c, Maximum projection of part of a whole-mount fifth mammary gland, stained for E-cadherin (luminal marker) used to determine the identity of the labelled clones. Magnified images show a single Z-plane with basal cells (yellow outline), which do not overlap with the staining, or luminal cells (red outline), which do overlap with the luminal staining. Scale bars, 1 mm (left) and 100 μm (right). d, Quantification of number of basal and luminal cells in the TEB (n = 10 TEBs, scored over three mice). Data are mean ± s.e.m. e, Representative images of TEBs at 5 weeks of age, showing a single Z-plane of the TEB stained for DAPI and K14 or E-cadherin to determine the number of luminal and basal cells per TEB. Z-stacks of complete TEBs (Z-step size, 5 μm) were used to count the total number of basal cells per TEB. Scale bars, 100 μm.

Source data

Extended Data Figure 7 StemID identifies basal and luminal cell clusters in a mixture of mammary epithelial cells.

a, Heat map of cell-to-cell transcriptome distances measured by 1 − the Pearson’s correlation coefficient. Basal and luminal cell clusters identified by StemID are colour coded along the axes. b, Cumulative transcript counts (colour legend) of cell-cycle genes expressed during G1 phase, S phase and M phase are indicated in the t-SNE maps. None of the cell cycle gene groups show a clear correlation with any of the clusters. c, Transcript counts (colour legend) of basal cell markers Acta2, Krt5 and Krt14 are indicated in the t-SNE maps. Expression of these genes is restricted to clusters 6, 7, 8, and 9. d, Transcript counts (colour legend) of luminal cell markers Krt8, Krt18 and Krt19 are indicated in the t-SNE maps. Expression of these genes is restricted to clusters 1, 2, 3, and 5. e, Transcript counts (colour legend) of previously reported MaSC markers Axin2 and Lgr5 are indicated in the t-SNE maps. The top graph shows that expression of Axin2 is assigned to the basal cells (both ductal and TEB cells), but is not restricted to one identified StemID cluster. The bottom graph shows that expression of Lgr5 is assigned to both luminal and basal cell clusters and is not restricted to one identified StemID cluster. Note that the transcript counts for both genes are very low. f, g, Heat map of differentially expressed genes comparing luminal cell cluster 1 with luminal cell clusters 2 and 3 (f) and comparing basal cell clusters 7 and 8 with basal cell clusters 6 and 9 (g). Rows are genes and columns are cells ordered based on cluster number. Genes are grouped on the basis of hierarchical clustering and the log10 expression of transcript counts is plotted.

Extended Data Figure 8 Successive rounds of bifurcation lead to monoclonal conversion as pubertal development proceeds, at a speed dictated by the rate of MaSC mixing in TEBs.

a, Simulated lineage tree in two opposite limits. Top, well-mixed limit, in which cells are randomly segregated upon bifurcation, showing slow linear conversion towards monoclonality. Bottom, a zero-mixing limit, in which the position of cells during two successive bifurcations remains unchanged, showing a fast exponential conversion towards monoclonality. Branches without any labelled cells are represented as black lines. Branches containing confetti-labelled cells are represented as lines of the dominant colour, with branching points showing a pie chart representing the proportion of each colour present in the branch. The size of the pie chart represents the size of the confetti clone to the total duct. b, Mean persisting confetti-clone size as a function of branch level, in the same two opposite limits, assuming a total MaSC number of n = 200. Numerical integration in the well mixed limit (top) for three initial conditions for the initial number of cells labelled (n = 1 in blue, n = 2 in yellow, n =3 in green). The red dashed line indicates the predicted linear slope. Numerical integration in the zero-mixing limit (bottom, n = 1 in blue). The red dashed line indicates the predicted exponential increase for small clone sizes. c, Top image shows confocal images of TEBs during the bifurcation process. Bottom image shows a schematic representation of the branching process as predicted by the model in the well mixed limit. d, Evolution of the cumulative rescaled subclone size distribution at different segment numbers (7, 9, 11 and 15 in purple, green, blue and orange, respectively) for a two-compartment model of TEB, consisting of a 10% population of MaSCs that are tilted towards renewal and 90% population of progenitors tilted towards loss. Contrary to the equipotent one-population model and our data in Fig. 4, the two-compartment model does not display scaling, with MaSCs forming a long tail in the distribution. e, Representative confocal images of a confetti+ mammary epithelial duct of a fourth mammary gland at 8 weeks of age. Clones of RFP+ cells (red) are outlined and blue represents E-cadherin. TEBs are located at the right side of the image. f, Quantification of the average cluster size for basal and luminal cell lineages at 8 weeks of age. P = 0.46 (Mann–Whitney test). Scale bars, 100 μm (c); 300 μm (e).

Source data

Extended Data Figure 9 Successive rounds of bifurcation lead to clonal extinction and enrichment.

a, Confocal image of a whole-mount-imaged fourth mammary gland stained for K14 (white) and DAPI (blue). The ductal network was manually reconstructed by outlining the ducts based on the K14 staining (white) and all individual confetti-labelled cells were outlined with their respective confetti colour (blue symbols, CFP+ cells; green symbols, GFP+ cells; yellow symbols, YFP+ cells; red symbols, RPF+ cells). This information was used as input for a schematic representation of the lineage tree in Fig. 4c. bd, Confocal images showing a zoom of the location of the coloured boxes indicated in a, showing a conversion of these luminal clones towards monoclonality over successive bifurcation rounds, including both extinction and enrichment of clones. A merge of these images is also shown in Fig. 4d. Scale bars, 5 mm (a); 100 μm (bd).

Extended Data Figure 10 Theory of equipotent TEBs making near-balanced stochastic fate choices between branching and termination explains quantitatively kidney branching morphogenesis.

a, Maximum projection of a whole-mount image of a murine kidney at embryonic day (E)16. Ducts are stained for E-cadherin (green) and nuclei (blue). Scale bar, 1 mm. b, Outline from our quantitative reconstruction. c, Probability q of a bud terminally differentiating, expressed as a function of branch level and read from the whole-mount reconstruction. Green line represents a constant value of 0.5, indicative of balance between TEB bifurcation and termination. d, Distribution of branch lengths (level >6), which collapses onto an exponential distribution. This is indicative of branching occurring stochastically at any time with equal constant probability. e, f, Inter-subtree heterogeneity (level >6, data in purple) can be predicted quantitatively by a model of equipotent buds making stochastic decisions. Subtree persistence (e) is defined as the distribution of subtrees having a given maximal branch level i. Subtree size distribution (f) is defined as the total number of branches in a given subtree. The theoretical curve (green line) explains the subtree heterogeneity on both measurements well. n = 48 subtrees were analysed from one embryo (cf). Data are mean ± s.e.m. (d) or mean ± s.d. (e, f).

Supplementary information

Supplementary Information

This file contains a Supplementary Theory note and additional references. (PDF 525 kb)

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. (XLSX 82 kb)

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. (MP4 2345 kb)

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Scheele, C., Hannezo, E., Muraro, M. et al. Identity and dynamics of mammary stem cells during branching morphogenesis. Nature 542, 313–317 (2017). https://doi.org/10.1038/nature21046

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