Bone tissue undergoes constant turnover supported by stem cells. Recent studies showed that perivascular mesenchymal stem cells (MSCs) contribute to the turnover of long bones. Craniofacial bones are flat bones derived from a different embryonic origin than the long bones. The identity and regulating niche for craniofacial-bone MSCs remain unknown. Here, we identify Gli1+ cells within the suture mesenchyme as the main MSC population for craniofacial bones. They are not associated with vasculature, give rise to all craniofacial bones in the adult and are activated during injury repair. Gli1+ cells are typical MSCs in vitro. Ablation of Gli1+ cells leads to craniosynostosis and arrest of skull growth, indicating that these cells are an indispensable stem cell population. Twist1+/− mice with craniosynostosis show reduced Gli1+ MSCs in sutures, suggesting that craniosynostosis may result from diminished suture stem cells. Our study indicates that craniofacial sutures provide a unique niche for MSCs for craniofacial bone homeostasis and repair.
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We thank J. Mayo and B. Samuels for critical reading of the manuscript and M. Zhang for the support. We thank R. Yang, X. Xu and S. Shi for technical support on the FACS analysis. We thank A. McMahon for providing Ihh–LacZ mice. H.Z. acknowledges training grant support from the National Institute of Dental and Craniofacial Research, NIH (R90 DE022528). This study was supported by grants from the National Institute of Dental and Craniofacial Research, NIH (DE022503, DE020065 and DE012711) to Y.C.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Anatomy and histology of craniofacial sutures.
(a–c) MicroCT images of one-month-old wild type mice. Each craniofacial bone is labelled with a different color. (d–f) HE staining of the sagittal suture (d), parietal bone (e) and coronal suture (f). Dotted lines outline the calvarial bones. (g) Schematic drawing of suture organization. Scale bars in panels a-c, 1 mm; other scale bars, 100 μm.
Supplementary Figure 2 Gli1+ cells are detectable in the mesenchyme of most craniofacial sutures.
(a–m) LacZ staining of craniofacial sutures of one-month-old Gli1-LacZ mice. Gli1+ cells are detectable in the mid-suture mesenchyme of the lambdoid (a), interparietal-occipital (b), parietal-squamous (c), maxilla-zygomatic (d), squamous-zygomatic (e), maxilla-premaxilla (f), frontal-maxilla (g), frontal-squamous (h), frontal-premaxilla (i), nasal-frontal (j), intermaxilla (k), basosphenoid-squamous (l) and basosphenoid-frontal (m) sutures. (n–o) Immunohistochemical staining of osteogenic differentiation markers Sp7 or Runx2 and lacZ staining (βGal) of craniofacial sutures of one-month-old Gli1-LacZ mice. Arrows indicate positive Sp7 or Runx2 signal. (p) Whole mount LacZ staining of the posterior frontal suture of 8-day-old (P8) Gli1-LacZ pups. The two panels on the right (p–a′, p-b′) are sections of the posterior frontal and sagittal sutures and their positions are shown with the arrow and arrowhead. Asterisks indicate the suture mesenchyme. Dotted lines outline the bone surface. (q) LacZ staining of the posterior frontal suture of one-month-old Gli1-LacZ mice. Scale bar in panel p, 1 mm; other scale bars, 100 μm.
Supplementary Figure 3 Lineage tracing of Gli1+ cells in adult craniofacial sutures.
Fluorescence imaging of sutures in Gli1-CreERT2;R26tdTomatofl mice one week (a–n) and one month (a′-o′) after induction at 1 month of age. Sutures visualized include the lambdoid (a,a′), interparietal-occipital (b,b′), parietal-squamous (c,c′), maxilla-zygomatic (d,d′), squamous-zygomatic (e,e′), maxilla-premaxilla (f,f′), frontal-maxilla (g,g′), frontal-squamous (h,h′), frontal-premaxilla (i,i′), nasal-frontal (j,j′), intermaxilla (k,k′), basosphenoid-squamous (l,l′) and basosphenoid-frontal (m,m′). (n,n′,o′) Immunostaining of Sp7 or Runx2 in the osteogenic front of Gli1-CreERT2;R26tdTomatofl mice. Arrowheads indicate Sp7+ or Runx2+ cells in the osteogenic front. Dotted lines outline the bone surfaces. Scale bars, 100 μm.
Supplementary Figure 4 Gli1+ cells in the craniofacial bone marrow also contribute to bone formation.
(a) LacZ staining of the parietal bone of one-month-old Gli1-LacZ mice. Arrows indicate positive signal. (b) Percentage of suture Gli1+ cells in the parietal, frontal, occipital, maxillary, palatal, basosphenoid and squamous bones of one-month-old Gli1-LacZ mice. Values are plotted as mean, n = 5 samples. (c–d) Visualization of Gli1+ cells in Gli1-CreERT2;R26tdTomatofl mice induced at 1 month of age. Gli1+ cells are detectable in the marrow space of the basosphenoid bone (arrows in c). One month after induction, osteocytes close to the bone marrow space are also labelled (arrows in d), although blood cells in the bone marrow are not. Scale bars, 100 μm.
Supplementary Figure 5 Phenotypes of Smo ICKO and DTA mice.
(a–h) MicroCT images of incisors of Smoothenedflox/flox (control) and Gli1-CreERT2;Smoothenedflox/flox (Smo ICKO) mice induced at one month of age and analysed two months later. Arrows indicate normal calcified tissue and arrowheads indicate disrupted calcified tissue in sagittal (b,f) and cross (c,d,g,h) sections. (i–l) HE staining of incisors in control (i,k) and Smo ICKO (j,l) mice. Normal and disrupted enamel and dentin formation are indicated by the arrow and arrowhead, respectively. Asterisks indicate periodontal tissue defects. (m–o) EdU incorporation and TUNEL assays in the incisors and sagittal sutures of control and Smo ICKO mice induced at one month of age and analysed one month later. (n) Quantification of the relative numbers of EdU+ cells. Values are plotted as mean ± s.e.m. Student’s t-test was performed. n = 4 mouse samples. (p) Lineage tracing analysis in the sagittal suture, parietal bone and palatal suture of Gli1-CreERT2;Smofl/fl;R26ZsGreenfl mice induced at one month of age and analysed two months later. (q–x) LacZ and Alizarin Red staining of MSCs from the suture mesenchyme of one-month-old Gli1-LacZ mice, either untreated (ctrl) or treated with IHH or GDC0449. (t) Quantitation of Edu incorporation and TUNEL assays of the same MSC cultures. Values are plotted as mean ± s.e.m. and Student’s t-test was performed. n = 4 culture wells. (x) Real-time PCR of osteogenic differentiation markers including ALPase, Runx2, Sp7 and Osteocalcin of the same MSC cultures. ∗, ANOVA was performed and P values were indicated in the figure, n = 4 samples. (y–z) Gli1-CreERT2;R26DTAfl/fl;R26ZsGreenfl and control (Ctrl) mice were induced at one month of age and analysed two months later. Fluorescently labelled cells in the fused sagittal and coronal sutures indicate cell ablation is not 100% efficient. Dotted lines outline the dental epithelium or bone. Scale bars in panels a-h, 1 mm; scale bar in panel y, 1 cm; other scale bars, 100 μm.
Supplementary Figure 6 The suture mesenchyme provides an MSC niche for adult craniofacial bones.
(a) Gli1+ MSCs within the suture mesenchyme give rise to the osteogenic front, periosteum and dura. These MSCs also give rise to the osteocytes either directly in the osteogenic front region or indirectly through the periosteum or dura. (b) IHH secreted from the osteogenic front regulates the differentiation of Gli1+ MSCs in the suture mesenchyme.
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Zhao, H., Feng, J., Ho, TV. et al. The suture provides a niche for mesenchymal stem cells of craniofacial bones. Nat Cell Biol 17, 386–396 (2015). https://doi.org/10.1038/ncb3139
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