coronary suture sagittal suture DOI: 10.1038/ncb3139 a b c e parietal bone suture mesenchyme parietal bone bone marrow ura ura ura f parietal bone ura suture mesenchyme bone g ura osteogenic front suture mesenchyme osteogenic front ura Supplementary Figure 1 Anatomy an histology of craniofacial sutures. (a-c) MicroCT images of one-month-ol wil type mice. Each craniofacial bone is labele with a ifferent color. (-f) HE staining of the sagittal suture (), parietal bone (e) an coronal suture (f). Dotte lines outline the calvarial bones. (g) Schematic rawing of suture organization. Scale bars in panels a-c, 1mm; other scale bars, 100µm. WWW.NATURE.COM/NATURECELLBIOLOGY 1 2015 Macmillan Publishers Limite. All rights reserve
occipital a parietal b c interparietal parietal interparietal parietal zygomatic e f g h zygomatic pre Gli1-LacZ i j k l m pre n β-gal Sp7 o β-gal Runx2 basosphenoi basosphenoi p P8 p-a q P30 p-a p-b p-b Posterior Frontal Supplementary Figure 2 Gli1+ cells are etectable in the mesenchyme of most craniofacial sutures. (a-m) LacZ staining of craniofacial sutures of one-monthol Gli1-LacZ mice. Gli1+ cells are etectable in the mi-suture mesenchyme of the lamboi (a), interparietal-occipital (b), parietal- (c), zygomatic (), -zygomatic (e), -pre (f), - (g), - (h), -pre (i), nasal- (j), inter (k), basosphenoi- (l) an basosphenoi- (m) sutures. (n-o) Immunohistochemical staining of osteogenic ifferentiation markers Sp7 or Runx2 an lacz staining (βgal) of craniofacial sutures of one-month-ol Gli1- LacZ mice. Arrows inicate positive Sp7 or Runx2 signal. (p) Whole mount LacZ staining of the posterior suture of 8-ay-ol (P8) Gli1-LacZ pups. The two panels on the right (p-a, p-b ) are sections of the posterior an sagittal sutures an their positions are shown with the arrow an arrowhea. Asterisks inicate the suture mesenchyme. Dotte lines outline the bone surface. (q) LacZ staining of the posterior suture of one-month-ol Gli1- LacZ mice. Scale bar in panel p, 1mm; other scale bars, 100µm. 2 WWW.NATURE.COM/NATURECELLBIOLOGY 2015 Macmillan Publishers Limite. All rights reserve
a b c 1 week post-inuction e f g h i j k l Gli1-Cre ERT2 ;R26Ttomato flox 1 month post-inuction m n Sp7 a b c e f g h i j k l m n Sp7 o Runx2 Supplementary Figure 3 Lineage tracing of Gli1+ cells in ault craniofacial sutures. Fluorescence imaging of sutures in Gli1- Cre ERT2 ;R26Ttomato fl mice one week (a-n) an one month (a -o ) after inuction at 1 month of age. Sutures visualize inclue the lamboi (a, a ), interparietal-occipital (b, b ), parietal- (c, c ), zygomatic (, ), -zygomatic (e, e ), -pre (f, f ), - (g, g ), - (h, h ), -pre (i, i ), nasal- (j, j ), inter (k, k ), basosphenoi- (l, l ) an basosphenoi- (m, m ). (n, n, o ) Immunostaining of Sp7 or Runx2 in the osteogenic front of Gli1-Cre ERT2 ;R26Ttomato fl mice. Arrowheas inicate Sp7+ or Runx2+ cells in the osteogenic front. Dotte lines outline the bone surfaces. Scale bars, 100µm. WWW.NATURE.COM/NATURECELLBIOLOGY 3 2015 Macmillan Publishers Limite. All rights reserve
Gli1-LacZ a 120% 100% b bone marrow Gli1+ cells suture Gli1+ cells 80% 60% 40% 98% 90% 90% 85% 85% 90% 90% 20% Gli1-Cre ERT2 ; R26Ttomato flox c 0% parietal occipital palatal basosphenoi Supplementary Figure 4 Gli1+ cells in the marrow also contribute to bone formation. (a) LacZ staining of the parietal bone of one-month-ol Gli1-LacZ mice. Arrows inicate positive signal. (b) Percentage of suture Gli1+ cells in the parietal,, occipital, ry, palatal, basosphenoi an bones of one-monthol Gli1-LacZ mice. Values are plotte as mean, n=5 samples. (c-) Visualization of Gli1+ cells in Gli1-Cre ERT2 ;R26Ttomato fl mice inuce at 1 month of age. Gli1+ cells are etectable in the marrow space of the basosphenoi bone (arrows in c). One month after inuction, osteocytes close to the bone marrow space are also labele (arrows in ), although bloo cells in the bone marrow are not. Scale bars, 100µm. 4 WWW.NATURE.COM/NATURECELLBIOLOGY 2015 Macmillan Publishers Limite. All rights reserve
Rel. RNA level Rel. cell number Sagittal Sututre Incisor Rel. EU+ cell number Ctrl Smo flox/flox a c b c i k Gli1-Cre ERT2 ;Smo flox/flox ; Gli1-CreERT2 ; Smo R26ZsGreen flox flox/flox LacZ staining Alizarin Re e g h m n o p 3mo Ctrl 1mo Ctrl DTA f g h Gli1-Cre ERT2 ;DTA flox/flox ; R26ZsGreen flox j EU incorporation TUNEL assay Ctrl Smo flox/flox Ctrl Smo flox/flox Gli1-Cre ERT2 ;Smo flox/flox Gli1-Cre ERT2 ;Smo flox/flox EU EU TUNEL 1.2 incisor sagittal suture 0.6 EU EU TUNEL TUNEL Ctrl IHH 500ng/ml GDC0449 inhibitor 0.9 0.3 0 ctrl Mutant ctrl Mutant 1.1 0.9 0.7 0.5 4 3.5 3 2.5 2 1.5 1 0.5 0 l EU incorporation TUNEL TUNEL Ctrl IHH Inhibitor Ctrl IHH Inhibitor Ctrl IHH Inhibitor p=0.01 p=0.02 p=0.02 p=0.02 u v w x y Sagittal suture Parietal bone Palatal suture q r s 1cm z Sagittal suture Coronal suture t p=0.02 p=0.01 p=0.03 ALPase Runx2 Sp7 Osteocalcin Incisor Sagittal Sututre p=0.01 Supplementary Figure 5 Phenotypes of Smo ICKO an DTA mice. (a-h) MicroCT images of incisors of Smoothene flox/flox (control) an Gli1- Cre ERT2 ;Smoothene flox/flox (Smo ICKO) mice inuce at one month of age an analyze two months later. Arrows inicate normal calcifie tissue an arrowheas inicate isrupte calcifie tissue in sagittal (b, f) an cross (c-, g-h) sections. (i-l) HE staining of incisors in control (i, k) an Smo ICKO (j, l) mice. Normal an isrupte enamel an entin formation are inicate by the arrow an arrowhea, respectively. Asterisks inicate perioontal tissue efects. (m-o) EU incorporation an TUNEL assays in the incisors an sagittal sutures of control an Smo ICKO mice inuce at one month of age an analyze one month later. (n) Quantification of the relative numbers of EU+ cells. Values are plotte as mean ±SEM. Stuent s t-test was performe. n=4 mouse samples. (p) Lineage tracing analysis in the sagittal suture, parietal bone an palatal suture of Gli1-Cre ERT2 ;Smo fl/fl ;R26ZsGreen fl mice inuce at one month of age an analyze two months later. (q-x) LacZ an Alizarin Re staining of MSCs from the suture mesenchyme of one-month-ol Gli1-LacZ mice, either untreate (ctrl) or treate with IHH or GDC0449. (t) Quantitation of Eu incorporation an TUNEL assays of the same MSC cultures. Values are plotte as mean ±SEM an Stuent s t-test was performe. n=4 culture wells. (x) Real-time PCR of osteogenic ifferentiation markers incluing ALPase, Runx2, Sp7 an Osteocalcin of the same MSC cultures., ANOVA was performe an p values were inicate in the figure, n=4 samples. (y-z) Gli1-Cre ERT2 ;R26DTA fl/fl ;R26ZsGreen fl an control (Ctrl) mice were inuce at one month of age an analyze two months later. Fluorescently labele cells in the fuse sagittal an coronal sutures inicate cell ablation is not 100% efficient. Dotte lines outline the ental epithelium or bone. Scale bars in panels a-h, 1mm; scale bar in panel y, 1cm; other scale bars, 100µm. WWW.NATURE.COM/NATURECELLBIOLOGY 5 2015 Macmillan Publishers Limite. All rights reserve
a ura suture MSCs ura b ura IHH Gli1+ MSCs IHH osteogenic front ura Supplementary Figure 6 The suture mesenchyme provies an MSC niche for ault s. (a) Gli1+ MSCs within the suture mesenchyme give rise to the osteogenic front, an ura. These MSCs also give rise to the osteocytes either irectly in the osteogenic front region or inirectly through the or ura. (b) IHH secrete from the osteogenic front regulates the ifferentiation of Gli1+ MSCs in the suture mesenchyme. 6 WWW.NATURE.COM/NATURECELLBIOLOGY 2015 Macmillan Publishers Limite. All rights reserve