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.
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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.).
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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.
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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).
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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
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DOI: https://doi.org/10.1038/ncb3394
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Verteporfin reverses progestin resistance through YAP/TAZ-PI3K-Akt pathway in endometrial carcinoma
Cell Death Discovery (2023)
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Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis
Nature Communications (2023)
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Context-dependent transcriptional regulations of YAP/TAZ in stem cell and differentiation
Stem Cell Research & Therapy (2022)