Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Snail/Slug binding interactions with YAP/TAZ control skeletal stem cell self-renewal and differentiation

Nature Cell Biology volume 18, pages 917–929 (2016)Cite this article

Subjects

Abstract

Bone-marrow-derived skeletal stem/stromal cell (SSC) self-renewal and function are critical to skeletal development, homeostasis and repair. Nevertheless, the mechanisms controlling SSC behaviour, particularly bone formation, remain ill-defined. Using knockout mouse models that target the zinc-finger transcription factors Snail or Slug, or Snail and Slug combined, a regulatory axis has been uncovered wherein Snail and Slug cooperatively control SSC self-renewal, osteoblastogenesis and bone formation. Mechanistically, Snail/Slug regulate SSC function by forming complexes with the transcriptional co-activators YAP and TAZ in tandem with the inhibition of the Hippo-pathway-dependent regulation of YAP/TAZ signalling cascades. In turn, the Snail/Slug–YAP/TAZ axis activates a series of YAP/TAZ/TEAD and Runx2 downstream targets that control SSC homeostasis and osteogenesis. Together, these results demonstrate that SSCs mobilize Snail/Slug–YAP/TAZ complexes to control stem cell function.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Snail/Slug regulate SSC proliferation and differentiation.
Figure 2: Skeletal and SSC-associated defects in Snail/Slug-targeted mice.
Figure 3: Osteogenic defects in Osterix–Cre-targeted conditional knockouts of Snail/Slug.
Figure 4: Snail/Slug regulate YAP/TAZ levels in SSCs.
Figure 5: Snail/Slug-dependent regulation of YAP/TAZ stability.
Figure 6: Mapping of Snail/Slug–YAP/TAZ binding interactions and regulation of transcriptional activity.
Figure 7: Snail/Slug–YAP/TAZ complexes regulate gene expression.
Figure 8: A Snail/Slug–YAP/TAZ axis regulates SSC function.

Similar content being viewed by others

References

  1. Kfoury, Y. & Scadden, D. T. Mesenchymal cell contributions to the stem cell niche. Cell Stem Cell 16, 239–253 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. Bianco, P. & Robey, P. G. Skeletal stem cells. Development 142, 1023–1027 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Batlle, R. et al. Snail1 controls TGF-β responsiveness and differentiation of mesenchymal stem cells. Oncogene 32, 3381–3389 (2013).

    Article  CAS  PubMed  Google Scholar 

  4. Barrallo-Gimeno, A. & Nieto, M. A. Evolutionary history of the Snail/Scratch superfamily. Trends Genet. 25, 248–252 (2009).

    Article  CAS  PubMed  Google Scholar 

  5. Nieto, M. A. Epithelial plasticity: a common theme in embryonic and cancer cells. Science 342, 1234850 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Puisieux, A., Brabletz, T. & Caramel, J. Oncogenic roles of EMT-inducing transcription factors. Nat. Cell Biol. 16, 488–494 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Desgrosellier, J. S. et al. Integrin alphavbeta3 drives slug activation and stemness in the pregnant and neoplastic mammary gland. Dev. Cell 30, 295–308 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Guo, W. et al. Slug and Sox9 cooperatively determine the mammary stem cell state. Cell 148, 1015–1028 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Horvay, K. et al. Snai1 regulates cell lineage allocation and stem cell maintenance in the mouse intestinal epithelium. EMBO J. 34, 1319–1335 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Hwang, W. L. et al. MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells. Nat. Cell Biol. 16, 268–280 (2014).

    Article  CAS  PubMed  Google Scholar 

  11. Lin, Y. et al. Snail1-dependent control of embryonic stem cell pluripotency and lineage commitment. Nat. Commun. 5, 3070 (2014).

    Article  CAS  PubMed  Google Scholar 

  12. Ye, X. et al. Distinct EMT programs control normal mammary stem cells and tumour-initiating cells. Nature 525, 256–260 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Oram, K. F., Carver, E. A. & Gridley, T. Slug expression during organogenesis in mice. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 271, 189–191 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Chen, Y. & Gridley, T. Compensatory regulation of the Snai1 and Snai2 genes during chondrogenesis. J. Bone Miner. Res. 28, 1412–1421 (2013).

    Article  CAS  PubMed  Google Scholar 

  15. Chen, Y. & Gridley, T. The SNAI1 and SNAI2 proteins occupy their own and each other’s promoter during chondrogenesis. Biochem. Biophys. Res. Commun. 435, 356–360 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Vega, S. et al. Snail blocks the cell cycle and confers resistance to cell death. Genes Dev. 18, 1131–1143 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Perez-Mancera, P. A. et al. Adipose tissue mass is modulated by SLUG (SNAI2). Hum. Mol. Genet. 16, 2972–2986 (2007).

    Article  CAS  PubMed  Google Scholar 

  18. Worthley, D. L. et al. Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential. Cell 160, 269–284 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhou, B. O., Yue, R., Murphy, M. M., Peyer, J. G. & Morrison, S. J. Leptin-receptor-expressing mesenchymal stromal cells represent the main source of bone formed by adult bone marrow. Cell Stem Cell 15, 154–168 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Day, T. F., Guo, X., Garrett-Beal, L. & Yang, Y. Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev. Cell 8, 739–750 (2005).

    Article  CAS  PubMed  Google Scholar 

  21. Liu, Y. et al. Isolation of murine bone marrow derived mesenchymal stem cells using Twist2 Cre transgenic mice. Bone 47, 916–925 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tang, Y. et al. MT1-MMP-dependent control of skeletal stem cell commitment via a beta1-integrin/YAP/TAZ signaling axis. Dev. Cell 25, 402–416 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Murray, S. A., Oram, K. F. & Gridley, T. Multiple functions of Snail family genes during palate development in mice. Development 134, 1789–1797 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Karsenty, G., Kronenberg, H. M. & Settembre, C. Genetic control of bone formation. Annu. Rev. Cell Dev. Biol. 25, 629–648 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Zhao, H. et al. The suture provides a niche for mesenchymal stem cells of craniofacial bones. Nat. Cell Biol. 17, 386–396 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Tang, Y. et al. TGF-beta1-induced migration of bone mesenchymal stem cells couples bone resorption with formation. Nat. Med. 15, 757–765 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chen, J. et al. Osx-Cre targets multiple cell types besides osteoblast lineage in postnatal mice. PLoS ONE 9, e85161 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mizoguchi, T. et al. Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev. Cell 29, 340–349 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zhou, X. et al. Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. Proc. Natl Acad. Sci. USA 107, 12919–12924 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Liu, Y. et al. Osterix-cre labeled progenitor cells contribute to the formation and maintenance of the bone marrow stroma. PLoS ONE 8, e71318 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Rodda, S. J. & McMahon, A. P. Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 133, 3231–3244 (2006).

    Article  CAS  PubMed  Google Scholar 

  32. Cordenonsi, M. et al. The Hippo transducer TAZ confers cancer stem cell-related traits on breast cancer cells. Cell 147, 759–772 (2011).

    Article  CAS  PubMed  Google Scholar 

  33. Hong, J. H. et al. TAZ, a transcriptional modulator of mesenchymal stem cell differentiation. Science 309, 1074–1078 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Mo, J. S., Park, H. W. & Guan, K. L. The Hippo signaling pathway in stem cell biology and cancer. EMBO Rep. 15, 642–656 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Varelas, X. The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease. Development 141, 1614–1626 (2014).

    Article  CAS  PubMed  Google Scholar 

  36. Pan, D. The hippo signaling pathway in development and cancer. Dev. Cell 19, 491–505 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Piccolo, S., Dupont, S. & Cordenonsi, M. The biology of YAP/TAZ: hippo signaling and beyond. Phys. Rev. 94, 1287–1312 (2014).

    CAS  Google Scholar 

  38. Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011).

    Article  CAS  PubMed  Google Scholar 

  39. Zaidi, S. K. et al. Tyrosine phosphorylation controls Runx2-mediated subnuclear targeting of YAP to repress transcription. EMBO J. 23, 790–799 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ducy, P. & Karsenty, G. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol. Cell Biol. 15, 1858–1869 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Zanconato, F. et al. Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth. Nat. Cell Biol. 17, 1218–1227 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Stein, C. et al. YAP1 exerts its transcriptional control via TEAD-mediated activation of enhancers. PLoS Genet. 11, e1005465 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Galli, G. G. et al. YAP drives growth by controlling transcriptional pause release from dynamic enhancers. Mol. Cell 60, 328–337 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Nishio, Y. et al. Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene. Gene 372, 62–70 (2006).

    Article  CAS  PubMed  Google Scholar 

  45. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L. & Karsenty, G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, 747–754 (1997).

    Article  CAS  PubMed  Google Scholar 

  46. Harada, H. et al. Cbfa1 isoforms exert functional differences in osteoblast differentiation. J. Biol. Chem. 274, 6972–6978 (1999).

    Article  CAS  PubMed  Google Scholar 

  47. Mingot, J. M., Vega, S., Maestro, B., Sanz, J. M. & Nieto, M. A. Characterization of Snail nuclear import pathways as representatives of C2H2 zinc finger transcription factors. J. Cell Sci. 122, 1452–1460 (2009).

    Article  CAS  PubMed  Google Scholar 

  48. Choi, S. et al. Structural basis for the selective nuclear import of the C2H2 zinc-finger protein Snail by importin beta. Acta Crystallogr. D Biol. Crystallogr. 70, 1050–1060 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Chan, C. K. et al. Identification and specification of the mouse skeletal stem cell. Cell 160, 285–298 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Isern, J. et al. The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function. eLife 3, e03696 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ono, N., Ono, W., Nagasawa, T. & Kronenberg, H. M. A subset of chondrogenic cells provides early mesenchymal progenitors in growing bones. Nat. Cell Biol. 16, 1157–1167 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. de Frutos, C. A. et al. Snail1 controls bone mass by regulating Runx2 and VDR expression during osteoblast differentiation. EMBO J. 28, 686–696 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Park, S. J. et al. The transcription factor snail regulates osteogenic differentiation by repressing Runx2 expression. Bone 46, 1498–1507 (2010).

    Article  CAS  PubMed  Google Scholar 

  54. Wang, W. et al. Defining the protein-protein interaction network of the human hippo pathway. Mol. Cell Proteomics 13, 119–131 (2014).

    Article  CAS  PubMed  Google Scholar 

  55. Ivkovic, S. et al. Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development 130, 2779–2791 (2003).

    Article  CAS  PubMed  Google Scholar 

  56. MacDonald, B. T. et al. Bone mass is inversely proportional to Dkk1 levels in mice. Bone 41, 331–339 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Nakashima, K. et al. The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108, 17–29 (2002).

    Article  CAS  PubMed  Google Scholar 

  58. Rowe, R. G. et al. Mesenchymal cells reactivate Snail1 expression to drive three-dimensional invasion programs. J. Cell Biol. 184, 399–408 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Xin, M. et al. Hippo pathway effector YAP promotes cardiac regeneration. Proc. Natl Acad. Sci. USA 110, 13839–13844 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Yu, K. et al. Conditional inactivation of FGF receptor 2 reveals an essential role for FGF signaling in the regulation of osteoblast function and bone growth. Development 130, 3063–3074 (2003).

    Article  CAS  PubMed  Google Scholar 

  61. Wu, X. et al. Inhibition of Sca-1-positive skeletal stem cell recruitment by alendronate blunts the anabolic effects of parathyroid hormone on bone remodeling. Cell Stem Cell 7, 571–580 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Anjos-Afonso, F. & Bonnet, D. Isolation, culture, and differentiation potential of mouse marrow stromal cells. Curr. Protoc. Stem Cell Biol. (2008)10.1002/9780470151808.sc02b03s7.

  63. Murdoch, A. D. et al. Chondrogenic differentiation of human bone marrow stem cells in transwell cultures: generation of scaffold-free cartilage. Stem Cells 25, 2786–2796 (2007).

    Article  CAS  PubMed  Google Scholar 

  64. McLeod, M. J. Differential staining of cartilage and bone in whole mouse fetuses by alcian blue and alizarin red S. Teratology 22, 299–301 (1980).

    Article  CAS  PubMed  Google Scholar 

  65. Tang, Y., Liu, Z., Zhao, L., Clemens, T. L. & Cao, X. Smad7 stabilizes beta-catenin binding to E-cadherin complex and promotes cell-cell adhesion. J. Biol. Chem. 283, 23956–23963 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank E. N. Olson (University of Texas Southwestern, USA) for providingYAP+/−/TAZ+/− mice. This work was supported by a grant from the Breast Cancer Research Foundation (S.J.W.). Work performed in this study was also supported by NIH Grant R01-1AR065524 (S.J.W.) and by P01-CA093900 (E.T.K.).

Author information

Authors and Affiliations

  1. Division of Molecular Medicine & Genetics, Department of Internal Medicine and the Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, USA

    Yi Tang, Tamar Feinberg, Xiao-Yan Li & Stephen J. Weiss

  2. Department of Pathology, Department of Urology and the Institute of Gerontology, University of Michigan, Ann Arbor, Michigan 48109, USA

    Evan T. Keller

Authors
  1. Yi Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tamar Feinberg
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evan T. Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xiao-Yan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stephen J. Weiss
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.T. designed and performed experiments, analysed data and wrote the paper. T.F., E.T.K. and X.-Y.L. designed and performed experiments. S.J.W. oversaw the project, designed experiments, analysed data and wrote the paper.

Corresponding authors

Correspondence to Yi Tang or Stephen J. Weiss.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Expression and Function of Snail and Slug in SSCs.

(a) LacZ expression in Snail+/LacZ 30-day old mouse skull. Scale bar: 0.5 mm. Results are representative of 3 experiments performed. (b) LacZ expression in Snail+/LacZ neonatal knee joint. Scale bar: 1 mm. Results are representative of 3 experiments performed. (c) LacZ expression in Snail+/LacZneonatal femur periosteum. Scale bar: 1 mm. Results are representative of 3 experiments performed. (d) LacZ expression in Slug+/LacZ 7-d old femur. Scale bar: 1 mm. Results are representative of 3 experiments performed. (e) LacZ expression in Slug+/LacZ 7-d old femur periosteum and on trabecular bone surface. Scale bar: 1 mm. Results are representative of 3 experiments performed. (f) Real-time PCR of Snail and Slug expression in SSCs isolated from Snailf/f/Slug+/+ or Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre (mean ± s.d., n = 3 independent experiments). ∗∗P < 0.01; one-way ANOVA. (g) Ki67 immunostaining in SSCs isolated from Snailf/f/Slug+/+ and Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre. Scale bar: 50 μm. Results are representative of 5 experiments performed. (h) Quantification of Ki67-positive cells from (g) (mean ± s.d., n = 5 independent experiments). ∗∗P < 0.01; unpaired t-test. (i) Cleaved Caspase-3 was monitored by immunohistochemistry in SSCs isolated from Snailf/f/Slug+/+ and Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre. Scale bar: 50 μm. Results are representative of 5 experiments performed. (j) SSCs isolated from Snailf/f/Slug+/+ or Snailf/f/Slug−/− mice were transduced with adeno-GFP or Cre. After culture under chondrogenic conditions for 14 d, cells were stained with Safranin O/Fast Green (upper panel) and relative mRNA expression of chondrogenic markers (Sox9, Collagen II and Aggrecan) determined by RT-PCR (mean ± s.d., n = 3 independent experiments). (k) SSCs were isolated from 3-month old Snailf/f/Slug+/+ and Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre expression vectors. Cell proliferation was determined by Ki67 immunohistochemistry (mean ± s.d., n = 3 independent experiments). P < 0.01; unpaired t-test. (l) Cells from (k) were cultured under osteogenic conditions for 14 d, relative mRNA expression of osteogenic markers (Runx2, Osterix, Alp and Bglap2) were examined by RT-PCR (mean ± s.d., n = 3 independent experiments). ∗∗P < 0.01; unpaired t-test.

Supplementary Figure 2 Phenotypic Characterization of Snailf/f/Slug+/+, Snailf/f/Slug+/+/Dermo1-Cre, Snailf/f/Slug−/− and Snailf/f/Slug−/−/Dermo1-Cre Mice.

(a) Alizarin Red/Alcian Blue staining of skulls isolated from neonatal Snailf/f/Slug+/+ and Snailf/f/Slug+/+/Dermo1-Cre mice. Scale bar: 2 mm. Results are representative of 5 experiments performed. (b) Histology of femur recovered from 6-wk old Snailf/f and Snailf/f/Dermo1-Cre mice. Scale bar: 1 mm. Results are representative of 5 experiments performed. (c) Alizarin Red/Alcian Blue staining of skulls isolated from neonatal Slug+/+ and Slug−/− mice. Scale bar: 2 mm. Results are representative of 5 experiments performed. (d) Histology of femurs recovered from 10-wk old Slug+/+ and Slug−/− mice. Scale bar: 1 mm. Results are representative of 5 experiments performed. (e) Growth plate histology of E15 Snailf/f/Slug+/+, Snailf/f/Slug+/+/Dermo1-Cre, Snailf/f/Slug−/− and Snailf/f/Slug−/−/Dermo1-Cre embryos. Scale bar: 100 μm. Results are representative of 5 experiments performed. (f) Ki67 expression in cartilage isolated from E15 Snailf/f/Slug+/+ and Snailf/f/Slug−/−/Dermo1-Cre femurs. Scale bar: 50 μm. Results are representative of 5 experiments performed. Quantification of Ki67-positive cells are presented as mean ± s.d. (n = 5 mice).∗∗P < 0.01; unpaired t-test. (g) Alizarin Red S/Fast Red staining of parietal bone isolated from E17.5 Snailf/f/Slug+/+ or Snailf/f/Slug−/−/Dermo1-Cre embryonic skulls. Scale bar: 100 μm. Results are representative of 3 experiments performed. Arrow: calcified bone. (h) Cleaved Caspase-3 expression as assessed by immunohistochemistry in the parietal mesenchymal cell layers of E15 Snailf/f/Slug+/+ and Snailf/f/ Slug−/−/Dermo1-Cre skulls. Scale bar: 50 μm. Results are representative of 5 experiments performed.

Supplementary Figure 3 Phenotypic Characterization of Snailf/f/Slug+/+, Snailf/f/Slug+/+//Osterix-Cre, Snailf/f/Slug−/− and Snailf/f/Slug−/−//Osterix-Cre mice.

(a) Cleaved Caspase-3 expression as assessed by immunohistochemistry in neonatal Snailf/f/Slug+/+ and Snailf/f/Slug−/−/Osterix-Cre femurs. Scale bar: 50 μm. Results are representative of 5 experiments performed. (b) Quantification of cleaved Caspase-3-positive cells from (a). Data are presented as mean ± s.d. (n = 5 mice).p > 0.05; unpaired t-test. (c) CFU-F generation from bone marrow cells isolated from 6-wk old Snailf/f/Slug+/+, Snailf/f/Slug+/+/Osterix-Cre, Snailf/f/Slug−/− or Snailf/f/Slug−/−/Osterix-Cre mice. Results are representative of 5 experiments performed. (d) CFU-F colony counts from (c) (mean ± s.d., n = 5 mice).∗∗P < 0.01; one-way ANOVA. (e) Osteoclast-specific TRAP staining of the proximal femur from a 12-wk old Snailf/f/Slug−/−/Osterix-Cre mouse versus a control littermate (Snailf/f/Slug+/+). Scale bar: 100 μm. Results are representative of 5 experiments performed. (f) Quantification of osteoclast number in sections captured from images shown in (e) (mean ± s.d., n = 5 mice).p > 0.05; unpaired t-test. (g) Calvarial osteoblast progenitors were isolated from Snailf/f/Slug+/+ and Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre expression vectors. Following culture under osteogenic conditions for 14 d, the cells were stained with Alizarin Red S. Results are representative of 3 experiments performed. (h) Relative mRNA expression of osteogenic markers (Runx2, Osterix, Alp and Bglap2) in cultures from (g) (mean ± s.d., n = 3 independent experiments). ∗∗P < 0.01; one-way ANOVA.

Supplementary Figure 4 Snail/Slug Regulate YAP/TAZ levels during SSC Differentiation and Bone Development.

(a) YAP and TAZ mRNA expression in SSCs isolated from Snailf/f/Slug+/+ or Snailf/f/Slug−/− mice that were transduced with adeno-GFP or Cre expression vectors and cultured in the absence or presence of osteogenic medium (mean ± s.d., n = 3).p > 0.05; unpaired t-test. (b) Ctgf and Ankdr1 mRNA expression in SSCs isolated from Snailf/f/Slug+/+ or Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre expression vectors (mean ± s.d., n = 3 independent experiments).∗∗P < 0.01; unpaired t-test. (c) Western blotting of Snail, Slug, YAP and TAZ in SSCs cultured under osteogenic conditions for the indicated time periods. Results are representative of 3 experiments performed. (d) Deletion of Snail/Slug in osteoblast progenitors blunts BMP2-induced osteogenesis. Calvarial osteoblast progenitors isolated from Snailf/f/Slug+/+ or Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre expression vectors were treated with 100 ng ml−1 BMP2 for 7 d. Relative mRNA expression of osteogenic markers (Runx2, Osterix, Alp and Bglap2) were assessed (mean ± s.d., n = 3 independent experiments). ∗∗P < 0.01; unpaired t-test. (e) Immunostaining of YAP/TAZ in calvaria from E15 Snailf/f/Slug+/+ or Snailf/f/Slug−/−/Dermo1-Cre mice. Scale bar: 100 μm Results are representative of 3 experiments performed. (f) Immunostaining of YAP/TAZ in femurs recovered from neonatal Snailf/f/Slug+/+ or Snailf/f/Slug−/−/Osterix-Cre mice. Scale bar: 100 μm Results are representative of 3 experiments performed. (g) Western blot of YAP and TAZ levels in SSCs isolated from YAPf/+/TAZf/+ mice and transduced with adeno- GFP or Cre expression vectors. Results are representative of 3 experiments performed. (h) Immunofluorescence staining of Slug in SSCs isolated from YAPf/+/TAZf/+ mice and transduced with adeno- GFP or Cre expression vectors. Results are representative of 3 experiments performed. (i) Western blot of Flag-tagged Snail expression in SSCs isolated from YAPf/+/TAZf/+ mice and transduced with an adeno-Cre expression vector. Results are representative of 3 experiments performed. (j) Snail increases SSC proliferation. Proliferative response in cells from (i) were assayed by XTT assay (mean ± s.d., n = 3 independent experiments). ∗∗P < 0.01; unpaired t-test. (k) Relative mRNA expression of osteogenic markers (Runx2, Osterix, Alp and Bglap2) was assessed in cells from (i) cultured under osteogenic conditions for 7 d (mean ± s.d., n = 3 independent experiments). ∗∗P < 0.01; unpaired t-test.

Supplementary Figure 5 Snail/Slug Regulate YAP/TAZ Protein Levels.

(a) Lats(1/2)-YAP/TAZ complex levels as detected following immunoprecipitation in SSCs isolated from Snailf/f/Slug+/+ and Snailf/f/Slug−/− mice and transduced with adeno-GFP or Cre. Results are representative of 3 experiments performed. (b) Calvarial-derived osteoblast progenitors were isolated from Snailf/f/Slug+/+ or Snai1f/f/Slug−/− mice and transduced with adeno- GFP or Cre expression vectors. These cells were then transfected with Flag-tagged YAP, and protein synthesis blocked by treatment of 50 μg ml−1 cycloheximide (CHX) for the indicated time. YAP protein levels were monitored by Western blot (upper panels), and the relative YAP levels were quantified by the ratio between Flag-YAP and actin, which was arbitrarily set 1.0 at time point 0 (lower panels). T1/2 is the half-life of the protein. Results are representative of 3 experiments performed. (c) Calvarial-derived osteoblast progenitors were isolated from Snai1f/f/Slug+/+ or Snai1f/f/Slug−/− mice and transduced with adeno- GFP or Cre expression vectors. The cells were then transfected with Flag-tagged TAZ, and protein synthesis blocked by treatment of 50 μg ml−1 CHX for the indicated times. TAZ protein levels were monitored by Western blot (upper panels), and the relative TAZ levels quantified as the ratio between Flag-TAZ and actin, which was arbitrarily set 1.0 at time point 0 (lower panels). T1/2 is the half-life of the protein. Results are representative of 3 experiments performed. (d) Western blot of YAP/TAZ levels in Snailf/f/Slug−/−/Cre SSCs treated with the proteasome inhibitor, MG132 (10 μM), for 4 h. Results are representative of 3 experiments performed. (e) Mesenchymal progenitor C3H10T1/2 cells were transfected with Flag-YAP, Flag-TAZ, Snail-HA or Slug-Myc, respectively. Protein-protein interactions between YAP/TAZ and Snail/Slug were assessed by proximity ligation assay. Scale bar: 10 μm. Results are representative of 3 experiments performed. (f) Western blot of SNAIL and TEADs(1–4) in human SSCs transfected with siGFP, siSnail and siTEADs(1–4). Results are representative of 3 experiments performed. (g) Western blot of SLUG in human SSCs transfected with siGFP and siSlug. Results are representative of 3 experiments performed. (h) Co-localization of endogenous SNAIL/SLUG and YAP/TAZ in human SSCs. Scale bar: 10 μm. Results are representative of 3 experiments performed.

Supplementary Figure 6 Snail/Slug-YAP/TAZ Binding Domains.

(a) Schematic of Flag-tagged YAP mutants. (b) Snail complexes with the WW domain of YAP. Cos-1 cells were co-transfected with the indicated Flag-tagged wild-type YAP or YAP mutant constructs in tandem with HA-tagged Snail. Cell lysates were subjected to anti-Flag immunoprecipitation, and the presence of Snail in the complexes assessed by anti-HA immunoblotting. Results are representative of 3 experiments performed. (c) Slug interacts with the WW domain of YAP. Cos-1 cells were co-transfected with the indicated Flag-tagged YAP or YAP mutant constructs and Myc-tagged Slug. Cell lysates were immunoprecipitated with anti-Flag antibodies, and the presence of Slug in the complexes assessed by anti-Myc immunoblotting. Results are representative of 3 experiments performed. (d) Schematic of Flag-tagged TAZ mutants. (e) Snail binding interactions with the WW domain of TAZ. Cos-1 cells were co-transfected with the indicated Flag-tagged TAZ or TAZ mutant constructs with Snail-HA. Cell lysates were subjected to anti-Flag immunoprecipitation, and the presence of Snail in the complexes determined by anti-HA immunoblotting. Results are representative of 3 experiments performed. (f) Slug interacts with the WW domain of TAZ. Cos-1 cells were co-transfected with the indicated Flag-tagged TAZ or TAZ mutant constructs along with Myc-tagged Slug. Cell lysates were immunoprecipitated with anti-Flag antibodies and the presence of Slug in the complexes assessed by anti-Myc immunoblotting. Results are representative of 3 experiments performed. (g) Schematic of Myc-tagged Slug and Slug mutants. (h) YAP interacts with the SNAG domain of Slug. Cos-1 cells were co-transfected with the indicated Myc-tagged Slug or Slug mutant constructs and Flag-tagged YAP. Cell lysates were immunoprecipitated with anti-Myc antibodies, and the presence of YAP in the complexes assessed by anti-Flag immunoblotting. Results are representative of 3 experiments performed. (i) TAZ interacts with the SNAG domain of Slug. Cos-1 cells were co-transfected with the indicated Myc-tagged Slug or Slug mutant constructs and Flag-tagged TAZ. Cell lysates were immunoprecipitated with anti-Flag antibodies and the presence of Slug or Slug mutants in the complexes assessed by anti-Myc immunoblotting. Results are representative of 3 experiments performed.

Supplementary Figure 7 Snail/Slug-YAP/TAZ Complexes Regulate Gene Expression.

(a) Western blot of the re-expressed Flag-Snail in calvarial osteoblast progenitors isolated from Snail1f/f/Slug−/− mice and transduced with adeno-Cre, and then were transfected with empty vector (EV) or Flag-Snail expressing vector. The expression level of Flag-Snail was comparing to the Snail/Slug wild type cells. Results are representative of 3 experiments performed. (b) Western blot of Flag-Snail and Flag-Slug in calvarial osteoblast progenitors isolated from Snailf/f/Slug−/− mice and transduced with adeno-Cre to generate Snail−/−/Slug−/− cells followed by transfection with an empty vector (EV), Flag-Snail or Flag-Slug expressing vectors. Results are representative of 3 experiments performed. (c) Co-occupation of Runx2 and Snail/Slug in the Bglap2 promoter. ChIP experiments using anti-Runx2 antibody were in osteoblast progenitors from (K) and then re-ChIPed with anti-Flag antibody. Results are shown as the fold-enrichment relative to IgG IP controls (mean ± s.d., n = 3 independent experiments). (d) Western blot of pan-Teads(1-4) in mouse SSCs transfected with siGFP or siTeads siRNAs. Results are representative of 3 experiments performed.

Supplementary Figure 8 A Snail/Slug-YAP/TAZ Axis Directs SSC Function.

(a) Western blot of Snailf/f/Slug−/− SSCs transduced with lentiviral constructs carrying an empty vector (EV), Flag-YAP or Flag-TAZ. Results are representative of 3 experiments performed. (b) Schematic of Snail and Snail mutants. (c) Snail (N150)-YAP/TAZ complex detected by immunoprecipitation in C3H10T1/2 cells transfected with Flag-tagged mutant Snail (N150). Results are representative of 3 experiments performed. (d) Snail/Slug-YAP/TAZ complex levels detected by immunoprecipitation in C3H10T1/2 cells transfected with mutant Snail (N150) or empty vector (EV). Results are representative of 3 experiments performed. (e) 8XGTIIC luciferase activity was measured in Cos-1 cells co-transfected with YAP, Snail or the Snail (N150) mutant (mean ± s.d., n = 3 independent experiments). (f) 6XOSE luciferase activity was determined in Cos-1 cells co-transfected with Runx2, TAZ, Snail and the Snail (N150) mutant (mean ± s.d., n = 3 independent experiments). (g) Snail (N150) mutant inhibits YAP binding to Ctgf promoter. ChIP experiments using anti-YAP antibody were performed on lysates prepared from C3H10T1/2 cells transfected with mutant Snail (N150) or empty vector (EV). Results are expressed as the fold-enrichment relative to IgG IP controls (mean ± s.d., n = 3 independent experiments). (h) Snail (N150) mutant inhibits Runx2 binding to Bglap2 promoter. ChIP experiments using anti-Runx2 antibody were performed on lysates prepared from MC3T3-E1 osteoblasts transfected with mutant Snail (N150) or empty vector (EV). Results are expressed as the fold-enrichment relative to IgG IP controls (mean ± s.d., n = 3 independent experiments).

Supplementary information

Supplementary Information

Supplementary Information (PDF 4613 kb)

Supplementary Table 1

Supplementary Information (XLSX 15 kb)

Supplementary Table 2

Supplementary Information (XLSX 12 kb)

Supplementary Table 3

Supplementary Information (XLSX 64 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tang, Y., Feinberg, T., Keller, E. et al. Snail/Slug binding interactions with YAP/TAZ control skeletal stem cell self-renewal and differentiation. Nat Cell Biol 18, 917–929 (2016). https://doi.org/10.1038/ncb3394

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3394

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing