- Email: [email protected]
Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis
Developmental Cell
Previews mitochondria. Because these calcium signals can be modified by neuronal activation, neuromodulator stimulation, or ROS release, they represent a separate neuroglia communication system that is independent of metabotropic receptors. This may be linked to the ability of astrocytes to respond to neuronal activity with glycogenolysis and the release of lactate or pyruvate to support the metabolic demands of neurons (Magistretti and Allaman, 2015). The clever application of new genetically encoded indicators and mitochondrial-targeted proteins has revealed a striking new layer of neuron-astrocyte communication.
REFERENCES Agarwal, A., Wu, P.-H., Hughes, E.G., Fukaya, M., Tischfield, M.A., Langseth, A.J., Wirtz, D., and Bergles, D.E. (2017). Neuron 93, 587–605.e7. Bernardi, P., Rasola, A., Forte, M., and Lippe, G. (2015). Physiol. Rev. 95, 1111–1155.
Rizzuto, R., De Stefani, D., Raffaello, A., and Mammucari, C. (2012). Nat. Rev. Mol. Cell Biol. 13, 566–578. Rungta, R.L., Bernier, L.P., Dissing-Olesen, L., Groten, C.J., LeDue, J.M., Ko, R., Drissler, S., and MacVicar, B.A. (2016). Glia 64, 2093– 2103.
Kanemaru, K., Sekiya, H., Xu, M., Satoh, K., Kitajima, N., Yoshida, K., Okubo, Y., Sasaki, T., Moritoh, S., Hasuwa, H., et al. (2014). Cell Rep. 8, 311–318.
Shigetomi, E., Bushong, E.A., Haustein, M.D., Tong, X., Jackson-Weaver, O., Kracun, S., Xu, J., Sofroniew, M.V., Ellisman, M.H., and Khakh, B.S. (2013). J. Gen. Physiol. 141, 633–647.
Magistretti, P.J., and Allaman, I. (2015). Neuron 86, 883–901.
Shigetomi, E., Patel, S., and Khakh, B.S. (2016). Trends Cell Biol. 26, 300–312.
Otsu, Y., Couchman, K., Lyons, D.G., Collot, M., Agarwal, A., Mallet, J.M., Pfrieger, F.W., Bergles, D.E., and Charpak, S. (2015). Nat. Neurosci. 18, 210–218.
Srinivasan, R., Huang, B.S., Venugopal, S., Johnston, A.D., Chai, H., Zeng, H., Golshani, P., and Khakh, B.S. (2015). Nat. Neurosci. 18, 708–717.
Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis Erin Greenwood,1,2 Emma D. Wrenn,1,2 and Kevin J. Cheung1,* 1Translational Research Program, Public Health Sciences and Human Biology Divisions, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA 2Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.02.014
The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth. Over the lifetime of mammals, from in utero development to puberty to pregnancy, the mammary gland undergoes remodeling and regeneration, resulting in large-scale transformations in tissue form and function (Visvader and Stingl, 2014). At birth, the gland is composed of a stromal fat pad with a small epithelial rudiment arising from the nipple. During puberty, in response to ovarian hormone release, this rudiment rapidly expands over a course of weeks to form a complex branched network of epithelial ducts that fills the mammary gland. It has been long speculated that a mammary stem cell (MaSC) population is needed to keep pace with proliferation in order to generate this ductal tree. Prior studies have hinted that MaSCs reside in the
tips of growing ducts (also known as terminal end buds; TEBs), potentially in the front-most cap cells (Williams and Daniel, 1983), which are enriched for the stem cell marker s-SHIP (Visvader and Stingl, 2014), and retain label in pulse chase experiments (dos Santos et al., 2013). However, the contribution of cells in the TEB to morphogenesis has not been tested directly by lineage tracing. Reporting in Nature, Scheele et al. (2017) now uncover surprising insights into the location, fate, and dynamics of MaSCs during mammary branching morphogenesis. Scheele et al. (2017) apply lineage tracing and theoretical modeling in innovative ways to study MaSCs during branching morphogenesis (Figure 1A). The authors took an approach agnostic
328 Developmental Cell 40, February 27, 2017 ª 2017 Elsevier Inc.
to specific, known stem cell markers and instead use sparse clonal lineage labeling with the tamoxifen-inducible Confetti reporter to induce randomized labeling in <1% of cells at the beginning of puberty. This enabled the authors to identify Confetti-labeled cells that contributed to multiple progeny along the duct. In conjunction, the authors build a theoretical model for branch decisions from the observed tree structure and its relationship to cell proliferation. From this model, the authors uncover two properties of the branched network essential to interpret their lineage traces. First, nearly all mammary duct branching is a result of TEB bifurcation rather than side branching. Second, proliferation occurs in the TEB, not in mature ducts. These two
Developmental Cell
Previews A
Mammary fat pad Duct
Neonatal B
TEB
Pubertal
Border stem cells expand Tip stem cells self-renew
C
TEB cells mix and change positions over time
MaSCs randomly segregate during bifurcation Figure 1. Lineage-Tracing MaSCs in Mammary Branching Morphogenesis Upon puberty, TEBs proliferate and bifurcate to generate a branched, bilayered epithelial tree. (A) The authors fluorescently labeled cells randomly before puberty. Labeled MaSCs leave behind a trail of fluorescent progeny along the duct (shown in blue in inset). These MaSCs arise from TEBs. (B) MaSC cell fate is positionally biased, such that cells near the border generate daughters that contribute to ductal elongation, while cells near the tip favor self-renewal. (C) Cells mix within the TEB. MaSCs change position from border to tip and randomly segregate into one of two new branches after bifurcation.
properties enabled the insight that distance along the ductal tree is a proxy for time. Therefore, Confetti-labeled cells found throughout the branched ductal tree can be interpreted as a ‘‘frozen record’’ of the developmental history of a MaSC. By following labeled progeny along the ductal tree, Scheele et al. (2017) establish that the majority of MaSCs are located within TEBs. By their estimates, each TEB harbors 260 MaSCs, and given the average number of cells in each TEB, this indicates that most TEB cells are MaSCs. Next, the authors examined how these MaSCs contribute to different cell types of the growing duct. Mammary ducts are hollow bilayered tubes composed of an inner layer of luminal cells surrounded by an outer layer of contractile basal myoepithelial cells. The authors found that Confettilabeled MaSCs gave rise to daughters
that were only luminal or only basal, indicating that the MaSCs are unipotent in this developmental context, a finding observed independently in another study of mammary branching morphogenesis using sparse Confetti lineage tracing (Davis et al., 2016). Because these data involve sparse labeling, rare multipotent MaSCs that escape color randomization could be missed in these analyses (Visvader and Stingl, 2014). However, these data appear to be broadly consistent with the idea that unipotent progenitors in the TEB contribute to the bulk of cell proliferation during branching morphogenesis. In addition, the authors found that the behavior of a MaSC was determined in part by its position within the TEB. MaSCs in the adjacent border region produced clones in adjacent ducts, whereas MaSCs in the tip largely did not. Because both border and tip regions have similar
proliferative activity, the authors conclude that MaSCs at the border of the TEB contribute daughters to the growing duct, while those residing in the tip of the TEB favor self-renewal (Figure 1B). Left unresolved are the precise boundaries separating tip and border regions, how these regions relate to the position of cap cells, and the effect of positional bias on basal versus luminal cell expansion. Nonetheless, these findings are conceptually interesting because the spatial regulation of stem cell fate is also observed in other developmental contexts. For example, in the intestinal crypt, stem cells stochastically compete for access to the niche, and cells at the base are biased toward self-renewal, whereas at the niche border, they are biased toward loss and replacement (Ritsma et al., 2014). Likewise, in the mouse hair follicle, stem cells located in the upper bulge remain uncommitted, while those located in the lower bulge are more likely to amplify and give rise to differentiated lineages (Rompolas et al., 2013). In each of these developmental systems, stem cell fate is linked to its position within a largely stationary niche. The current study adds to this concept by showing that positional bias also occurs in highly migratory branching organs and suggests the hypothesis that a MaSC niche migrates along with the TEB. Unexpectedly, the authors also observed significant intra-TEB cell migration, or ‘‘mixing,’’ of MaSCs during the branching process. The authors’ analysis of clonality implies that with each TEB bifurcation, MaSCs randomly segregate between the two branches; therefore, in the long-term, MaSCs act as an equipotent pool (Figure 1C). Consistent with these results, the authors performed intravital imaging demonstrating that cells in the TEB can exchange positions. This type of movement is also seen within collectively migrating buds in normal mammary organoids (Ewald et al., 2008) and in other branching organs, including by Id2+ distal tip cells during lung development, as well as in Ret-dependent ureteric bud morphogenesis (Rawlins et al., 2009; Shakya et al., 2005). These observations suggest that stem cell mixing is functionally important for mammary branching morphogenesis, but this remains to be tested. A further implication of these findings is that the
Developmental Cell 40, February 27, 2017 329
Developmental Cell
Previews rate of MaSC cell migration is linked to stem cell fate, suggesting that altering intra-epithelial cell migration behavior could shift the balance between selfrenewal and differentiation. The current work by Scheele et al. (2017) shows that MaSCs within the TEB drive branching morphogenesis and that these MaSCs are subject to dynamic positional regulation, including both positional bias in MaSCs’ shortterm contribution to duct elongation and cellular rearrangements during bifurcation that reassign this fate bias among MaSCs. An important direction for future research will be to integrate how the TEB-associated MaSC behaviors uncovered in this study are regulated by signals emanating from the microenvironment, such as TGF-b, which potently inhibits branching. Interestingly, TEB-associated MaSCs display heterogeneity not only in location and fate, but also in gene expression at
the single-cell level. When the authors performed single-cell RNA sequencing, they did not find a single transcriptional signature that identified mammary stem cells and instead uncovered a spectrum of luminal and basal mammary stem cell states. Further work is needed to understand how this diverse stem cell pool coordinates decision making in order to generate a complex tissue such as the mammary gland and will inform our broader understanding of the collective cellular mechanisms underlying tissue morphogenesis.
REFERENCES Davis, F.M., Lloyd-Lewis, B., Harris, O.B., Kozar, S., Winton, D.J., Muresan, L., and Watson, C.J. (2016). Nat. Commun. 7, 13053. dos Santos, C.O., Rebbeck, C., Rozhkova, E., Valentine, A., Samuels, A., Kadiri, L.R., Osten, P., Harris, E.Y., Uren, P.J., Smith, A.D., and Han-
330 Developmental Cell 40, February 27, 2017
non, G.J. (2013). Proc. Natl. Acad. Sci. USA 110, 7123–7130. Ewald, A.J., Brenot, A., Duong, M., Chan, B.S., and Werb, Z. (2008). Dev. Cell 14, 570–581. Rawlins, E.L., Clark, C.P., Xue, Y., and Hogan, B.L. (2009). Development 136, 3741–3745. Ritsma, L., Ellenbroek, S.I., Zomer, A., Snippert, H.J., de Sauvage, F.J., Simons, B.D., Clevers, H., and van Rheenen, J. (2014). Nature 507, 362–365. Rompolas, P., Mesa, K.R., and Greco, V. (2013). Nature 502, 513–518. Scheele, C.L.G.J., Hannezo, E., Murano, M.J., Zomer, A., Langedijk, N.S.M., van Oudenaarden, A., Simons, B.D., and van Rheenen, J. (2017). Nature. http://dx.doi.org/10.1038/nature21046. Shakya, R., Watanabe, T., and Costantini, F. (2005). Dev. Cell 8, 65–74. Visvader, J.E., and Stingl, J. (2014). Genes Dev. 28, 1143–1158. Williams, J.M., and Daniel, C.W. (1983). Dev. Biol. 97, 274–290.
Previews mitochondria. Because these calcium signals can be modified by neuronal activation, neuromodulator stimulation, or ROS release, they represent a separate neuroglia communication system that is independent of metabotropic receptors. This may be linked to the ability of astrocytes to respond to neuronal activity with glycogenolysis and the release of lactate or pyruvate to support the metabolic demands of neurons (Magistretti and Allaman, 2015). The clever application of new genetically encoded indicators and mitochondrial-targeted proteins has revealed a striking new layer of neuron-astrocyte communication.
REFERENCES Agarwal, A., Wu, P.-H., Hughes, E.G., Fukaya, M., Tischfield, M.A., Langseth, A.J., Wirtz, D., and Bergles, D.E. (2017). Neuron 93, 587–605.e7. Bernardi, P., Rasola, A., Forte, M., and Lippe, G. (2015). Physiol. Rev. 95, 1111–1155.
Rizzuto, R., De Stefani, D., Raffaello, A., and Mammucari, C. (2012). Nat. Rev. Mol. Cell Biol. 13, 566–578. Rungta, R.L., Bernier, L.P., Dissing-Olesen, L., Groten, C.J., LeDue, J.M., Ko, R., Drissler, S., and MacVicar, B.A. (2016). Glia 64, 2093– 2103.
Kanemaru, K., Sekiya, H., Xu, M., Satoh, K., Kitajima, N., Yoshida, K., Okubo, Y., Sasaki, T., Moritoh, S., Hasuwa, H., et al. (2014). Cell Rep. 8, 311–318.
Shigetomi, E., Bushong, E.A., Haustein, M.D., Tong, X., Jackson-Weaver, O., Kracun, S., Xu, J., Sofroniew, M.V., Ellisman, M.H., and Khakh, B.S. (2013). J. Gen. Physiol. 141, 633–647.
Magistretti, P.J., and Allaman, I. (2015). Neuron 86, 883–901.
Shigetomi, E., Patel, S., and Khakh, B.S. (2016). Trends Cell Biol. 26, 300–312.
Otsu, Y., Couchman, K., Lyons, D.G., Collot, M., Agarwal, A., Mallet, J.M., Pfrieger, F.W., Bergles, D.E., and Charpak, S. (2015). Nat. Neurosci. 18, 210–218.
Srinivasan, R., Huang, B.S., Venugopal, S., Johnston, A.D., Chai, H., Zeng, H., Golshani, P., and Khakh, B.S. (2015). Nat. Neurosci. 18, 708–717.
Un(MaSC)ing Stem Cell Dynamics in Mammary Branching Morphogenesis Erin Greenwood,1,2 Emma D. Wrenn,1,2 and Kevin J. Cheung1,* 1Translational Research Program, Public Health Sciences and Human Biology Divisions, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA 2Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.devcel.2017.02.014
The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth. Over the lifetime of mammals, from in utero development to puberty to pregnancy, the mammary gland undergoes remodeling and regeneration, resulting in large-scale transformations in tissue form and function (Visvader and Stingl, 2014). At birth, the gland is composed of a stromal fat pad with a small epithelial rudiment arising from the nipple. During puberty, in response to ovarian hormone release, this rudiment rapidly expands over a course of weeks to form a complex branched network of epithelial ducts that fills the mammary gland. It has been long speculated that a mammary stem cell (MaSC) population is needed to keep pace with proliferation in order to generate this ductal tree. Prior studies have hinted that MaSCs reside in the
tips of growing ducts (also known as terminal end buds; TEBs), potentially in the front-most cap cells (Williams and Daniel, 1983), which are enriched for the stem cell marker s-SHIP (Visvader and Stingl, 2014), and retain label in pulse chase experiments (dos Santos et al., 2013). However, the contribution of cells in the TEB to morphogenesis has not been tested directly by lineage tracing. Reporting in Nature, Scheele et al. (2017) now uncover surprising insights into the location, fate, and dynamics of MaSCs during mammary branching morphogenesis. Scheele et al. (2017) apply lineage tracing and theoretical modeling in innovative ways to study MaSCs during branching morphogenesis (Figure 1A). The authors took an approach agnostic
328 Developmental Cell 40, February 27, 2017 ª 2017 Elsevier Inc.
to specific, known stem cell markers and instead use sparse clonal lineage labeling with the tamoxifen-inducible Confetti reporter to induce randomized labeling in <1% of cells at the beginning of puberty. This enabled the authors to identify Confetti-labeled cells that contributed to multiple progeny along the duct. In conjunction, the authors build a theoretical model for branch decisions from the observed tree structure and its relationship to cell proliferation. From this model, the authors uncover two properties of the branched network essential to interpret their lineage traces. First, nearly all mammary duct branching is a result of TEB bifurcation rather than side branching. Second, proliferation occurs in the TEB, not in mature ducts. These two
Developmental Cell
Previews A
Mammary fat pad Duct
Neonatal B
TEB
Pubertal
Border stem cells expand Tip stem cells self-renew
C
TEB cells mix and change positions over time
MaSCs randomly segregate during bifurcation Figure 1. Lineage-Tracing MaSCs in Mammary Branching Morphogenesis Upon puberty, TEBs proliferate and bifurcate to generate a branched, bilayered epithelial tree. (A) The authors fluorescently labeled cells randomly before puberty. Labeled MaSCs leave behind a trail of fluorescent progeny along the duct (shown in blue in inset). These MaSCs arise from TEBs. (B) MaSC cell fate is positionally biased, such that cells near the border generate daughters that contribute to ductal elongation, while cells near the tip favor self-renewal. (C) Cells mix within the TEB. MaSCs change position from border to tip and randomly segregate into one of two new branches after bifurcation.
properties enabled the insight that distance along the ductal tree is a proxy for time. Therefore, Confetti-labeled cells found throughout the branched ductal tree can be interpreted as a ‘‘frozen record’’ of the developmental history of a MaSC. By following labeled progeny along the ductal tree, Scheele et al. (2017) establish that the majority of MaSCs are located within TEBs. By their estimates, each TEB harbors 260 MaSCs, and given the average number of cells in each TEB, this indicates that most TEB cells are MaSCs. Next, the authors examined how these MaSCs contribute to different cell types of the growing duct. Mammary ducts are hollow bilayered tubes composed of an inner layer of luminal cells surrounded by an outer layer of contractile basal myoepithelial cells. The authors found that Confettilabeled MaSCs gave rise to daughters
that were only luminal or only basal, indicating that the MaSCs are unipotent in this developmental context, a finding observed independently in another study of mammary branching morphogenesis using sparse Confetti lineage tracing (Davis et al., 2016). Because these data involve sparse labeling, rare multipotent MaSCs that escape color randomization could be missed in these analyses (Visvader and Stingl, 2014). However, these data appear to be broadly consistent with the idea that unipotent progenitors in the TEB contribute to the bulk of cell proliferation during branching morphogenesis. In addition, the authors found that the behavior of a MaSC was determined in part by its position within the TEB. MaSCs in the adjacent border region produced clones in adjacent ducts, whereas MaSCs in the tip largely did not. Because both border and tip regions have similar
proliferative activity, the authors conclude that MaSCs at the border of the TEB contribute daughters to the growing duct, while those residing in the tip of the TEB favor self-renewal (Figure 1B). Left unresolved are the precise boundaries separating tip and border regions, how these regions relate to the position of cap cells, and the effect of positional bias on basal versus luminal cell expansion. Nonetheless, these findings are conceptually interesting because the spatial regulation of stem cell fate is also observed in other developmental contexts. For example, in the intestinal crypt, stem cells stochastically compete for access to the niche, and cells at the base are biased toward self-renewal, whereas at the niche border, they are biased toward loss and replacement (Ritsma et al., 2014). Likewise, in the mouse hair follicle, stem cells located in the upper bulge remain uncommitted, while those located in the lower bulge are more likely to amplify and give rise to differentiated lineages (Rompolas et al., 2013). In each of these developmental systems, stem cell fate is linked to its position within a largely stationary niche. The current study adds to this concept by showing that positional bias also occurs in highly migratory branching organs and suggests the hypothesis that a MaSC niche migrates along with the TEB. Unexpectedly, the authors also observed significant intra-TEB cell migration, or ‘‘mixing,’’ of MaSCs during the branching process. The authors’ analysis of clonality implies that with each TEB bifurcation, MaSCs randomly segregate between the two branches; therefore, in the long-term, MaSCs act as an equipotent pool (Figure 1C). Consistent with these results, the authors performed intravital imaging demonstrating that cells in the TEB can exchange positions. This type of movement is also seen within collectively migrating buds in normal mammary organoids (Ewald et al., 2008) and in other branching organs, including by Id2+ distal tip cells during lung development, as well as in Ret-dependent ureteric bud morphogenesis (Rawlins et al., 2009; Shakya et al., 2005). These observations suggest that stem cell mixing is functionally important for mammary branching morphogenesis, but this remains to be tested. A further implication of these findings is that the
Developmental Cell 40, February 27, 2017 329
Developmental Cell
Previews rate of MaSC cell migration is linked to stem cell fate, suggesting that altering intra-epithelial cell migration behavior could shift the balance between selfrenewal and differentiation. The current work by Scheele et al. (2017) shows that MaSCs within the TEB drive branching morphogenesis and that these MaSCs are subject to dynamic positional regulation, including both positional bias in MaSCs’ shortterm contribution to duct elongation and cellular rearrangements during bifurcation that reassign this fate bias among MaSCs. An important direction for future research will be to integrate how the TEB-associated MaSC behaviors uncovered in this study are regulated by signals emanating from the microenvironment, such as TGF-b, which potently inhibits branching. Interestingly, TEB-associated MaSCs display heterogeneity not only in location and fate, but also in gene expression at
the single-cell level. When the authors performed single-cell RNA sequencing, they did not find a single transcriptional signature that identified mammary stem cells and instead uncovered a spectrum of luminal and basal mammary stem cell states. Further work is needed to understand how this diverse stem cell pool coordinates decision making in order to generate a complex tissue such as the mammary gland and will inform our broader understanding of the collective cellular mechanisms underlying tissue morphogenesis.
REFERENCES Davis, F.M., Lloyd-Lewis, B., Harris, O.B., Kozar, S., Winton, D.J., Muresan, L., and Watson, C.J. (2016). Nat. Commun. 7, 13053. dos Santos, C.O., Rebbeck, C., Rozhkova, E., Valentine, A., Samuels, A., Kadiri, L.R., Osten, P., Harris, E.Y., Uren, P.J., Smith, A.D., and Han-
330 Developmental Cell 40, February 27, 2017
non, G.J. (2013). Proc. Natl. Acad. Sci. USA 110, 7123–7130. Ewald, A.J., Brenot, A., Duong, M., Chan, B.S., and Werb, Z. (2008). Dev. Cell 14, 570–581. Rawlins, E.L., Clark, C.P., Xue, Y., and Hogan, B.L. (2009). Development 136, 3741–3745. Ritsma, L., Ellenbroek, S.I., Zomer, A., Snippert, H.J., de Sauvage, F.J., Simons, B.D., Clevers, H., and van Rheenen, J. (2014). Nature 507, 362–365. Rompolas, P., Mesa, K.R., and Greco, V. (2013). Nature 502, 513–518. Scheele, C.L.G.J., Hannezo, E., Murano, M.J., Zomer, A., Langedijk, N.S.M., van Oudenaarden, A., Simons, B.D., and van Rheenen, J. (2017). Nature. http://dx.doi.org/10.1038/nature21046. Shakya, R., Watanabe, T., and Costantini, F. (2005). Dev. Cell 8, 65–74. Visvader, J.E., and Stingl, J. (2014). Genes Dev. 28, 1143–1158. Williams, J.M., and Daniel, C.W. (1983). Dev. Biol. 97, 274–290.