Supplemental Data. Wnt/β-Catenin Signaling in Mesenchymal Progenitors. Controls Osteoblast and Chondrocyte

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1 Supplemental Data Wnt/β-Catenin Signaling in Mesenchymal Progenitors Controls Osteoblast and Chondrocyte Differentiation during Vertebrate Skeletogenesis Timothy F. Day, Xizhi Guo, Lisa Garrett-Beal, and Yingzi Yang Supplemental Results Delayed Chondrocyte Maturation and Blood Vessel Invasion in the Long Bones of the β-catenin Conditional Mutant Embryo As blood vessel invasion into the hypertrophic cartilaginous region is required for trabecular bone formation (Maes et al., 2002; Vu et al., 1998; Zelzer et al., 2004), we examined blood vessel invasion by performing TRAP and PECAM staining, which recognize tartrate acidic phophosphatase (TRAP) in osteoclasts and vascular endothelial cells, respectively. We found that although endochondral bone formation is severely compromised, blood vessel invasion occurred and osteoclasts were present in the developing long bones by 17.5 dpc, judged by the presence of TRAP- and PECAM-positive cells in both Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre embryos (Supplemental Figure S3I and data not shown). In addition, we examined the expression of MMP9 and MMP13. Both MMP9 and MMP13 were expressed in Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre embryos at levels comparable to those in the Catnby c/+ ;Col2a1-Cre control embryos at 17.5 dpc (Supplemental Figure S3I and data not shown). Interestingly, we also noticed the expression of MMP9 and MMP13 in the front of the wedge-shaped ectopic chondrocyte region in both Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre embryos (Supplemental Figure S3I and data not shown). In addition, cells ectopically expressing the hypertrophic chondrocyte marker ColX were not limited to the wedge-shaped region. Instead, they were detected throughout the region where blood vessel invasion had occurred (Figure 6D and Supplemental Figure S2). These results indicate that the ectopic chondrocytes that differentiated from perichondrial mesenchymal progenitors may also migrate into the hypertrophic chondrocyte region while they are maturing, just like the osteoblasts that migrated into the cartilage to form the trabecular bone in the control embryo. Nonetheless, the process of blood vessel invasion and osteoblast migration was delayed when β-catenin was inactivated. At an earlier developmental stage at 15.5 dpc, blood vessel invasion just started in the Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre mutants, whereas this process had already occurred extensively in the control embryo (Supplemental Figure S3IB, S3IB, S3ID, and S3ID ). This is supported by another observation: the hypertrophic chondrocyte region was significantly expanded in both Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre embryos (Figure 6D and Supplemental Figure S2). As the size of the hypertrophic domain results from the interplay of chondrocyte hypertrophy (a gain in hypertrophic chondrocytes) and chondrocyte apoptosis during blood vessel invasion (loss of hypertrophic chondrocytes), delayed blood vessel invasion will cause expansion of the hypertrophic region as has been observed in the MMP9 / mutant mouse (Vu et al., 1998). Consistent with the delay in blood vessel invasion, we found that in both Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre embryos at 15.5 dpc, expression of MMP9 and MMP13 were both significantly reduced (Supplemental Figure S3IE, S3IF, and S4). Expression of osteoblast markers including ColI, Runx2, Osx, and Osteocalcin was also greatly decreased and confined to the periosteum, whereas they were expressed in both periosteum and migrating osteoblasts in control embryos (Supplemental Figures S3II and S4). Because chondrocyte hypertrophy is required for blood vessel invasion, we then examined chondrocyte hypertrophy by analyzing the expression of Ihh and ColX. At 15.5 dpc, extensive chondrocyte hypertrophy, indicated by Ihh and ColX expression, could be found in both Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1- Cre embryos (Supplemental Figures S3II and S4). In addition, the initial onset of chondrocyte hypertrophy and PTHrP expression in Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre embryos was normal at 12.5 Page 1 of 8

2 dpc (Supplemental Figure S6). However, the final maturation of the ColX-expressing hypertrophic chondrocytes was significantly delayed, as shown by diminished expression of MMP13, which is normally expressed in the most mature hypertrophic chondrocytes, in both Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre (Supplemental Figures S3IF and S4). The delay in chondrocyte maturation was confirmed by delayed expression of VEGF (Supplemental Figure S4G ), which is stronger in more mature hypertrophic chondrocytes (Supplemental Figure S4G). The phenotype in the Catnby c/c ;Dermo1-Cre embryo was more severe, possibly due to the broader and earlier expression of Cre in the Dermo1-Cre mouse. Interestingly, in contrast to the control embryos in which hypertrophic chondrocytes were in direct contact with perichondrium/periosteum, ColII and Ihh expression persisted at the periphery of the hypertrophic regions that expressed ColX in both Catnby c/- ;Col2a1-Cre and Catnby c/c ;Dermo1-Cre (Supplemental Figures S3II and S4). This resulted in blockage of the direct contact of perichondrium with the hypertrophic chondrocytes that secrete signals such as VEGF required for blood vessel invasion, by a layer of nonhypertrophic chondrocytes. Again, such blockage was more severe in the Catnby c/c ;Dermo1- Cre embryo (Supplemental Figures S3II and S4). These observations suggest that impaired interaction of perichondrium and hypertrophic chondrocytes also contributed to the delay of blood vessel invasion. Furthermore, it appears that signals from cells in the perichondrium and periosteum, likely differentiating osteoblasts, is required for chondrocyte hypertrophy of the neighboring chondrocytes. All these results indicate that the Wnt/β-catenin signaling acts directly in chondrocytes to regulate the progression of chondrocyte maturation and indirectly in the perichondrium to affect chondrocyte hypertrophy. Together, these two activities regulate blood vessel invasion and osteoblast migration. Experimental Procedures TRAP and PECAM Staining For TRAP staining, sections were deparaffinized, rehydrated, and stained for TRAP (tartrate acidic phosphatase) activity using the Leukocyte kit 387 (Sigma-Aldrich Co., St. Louis, MO) according to the manufacturer's instruction and counterstained by hematoxylin. For PECAM staining, sections were deparaffinized, rehydrated, and incubated with Trypsin-EDTA (Invitrogen) for 10 min at 37 C. Slides were then rinsed in distilled water and PBS, blocked with sheep serum for 10 min, incubated with anti-mouse CD31 antibody (BD Pharmigen) at 1:500 dilution in 2% sheep serum overnight at 4 C, and rinsed twice in PBS, and signals were detected using the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA). The slides were then counterstained with Alcian blue. References Maes, C., Carmeliet, P., Moermans, K., Stockmans, I., Smets, N., Collen, D., Bouillon, R., and Carmeliet, G. (2002). Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech. Dev. 111, Vu, T.H., Shipley, J.M., Bergers, G., Berger, J.E., Helms, J.A., Hanahan, D., Shapiro, S.D., Senior, R.M., and Werb, Z. (1998). MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93, Zelzer, E., Mamluk, R., Ferrara, N., Johnson, R.S., Schipani, E., and Olsen, B.R. (2004). VEGFA is necessary for chondrocyte survival during bone development. Development 131, Page 2 of 8

Supplemental Figure S1. Incomplete Removal of β-catenin in the Catnby c/c ;Dermo1-Cre Embryo Sections of the developing frontal bone at 13.5 dpc (A and B) and humerus at 15.

Cells still containing β-catenin after Cre-mediated recombination are indicated by arrows. Cal, calvarium; PC, perichondrium; Msl, muscle. Big yellow spots are autofluorescent blood cells.

3 Supplemental Figure S1. Incomplete Removal of β-catenin in the Catnby c/c ;Dermo1-Cre Embryo Sections of the developing frontal bone at 13.5 dpc (A and B) and humerus at 15.5 dpc (C and D) were examined by fluorescent immunohistochemistry with an anti-β-catenin antibody (green). Nucleus is stained by DAPI (blue). Cells still containing β-catenin after Cre-mediated recombination are indicated by arrows. Cal, calvarium; PC, perichondrium; Msl, muscle. Big yellow spots are autofluorescent blood cells. Supplemental Figure S2. Analysis of Early Chondrocyte and Osteoblast Gene Expression during Endochondral Ossification Consecutive sections of the developing humerus at 17.5 dpc were examined by in situ hybridization with indicated 35 S-labeled riboprobes. In the Catnby c/c ;Dermo1-Cre embryo, chondrocyte markers Sox9 and ColII (A and B ) and markers for more mature chondrocytes including ColX and Ihh (C and D ) were ectopically expressed in the wedge-shaped region (arrows). The hypertrophic chondrocyte region indicated by the double-arrowheaded lines is expanded (D ). In the periosteum (arrowhead), ColI expression (E ) was reduced and early osteoblast-specific marker expression including Runx2 and Osx (F and G ) was diminished. Expression of mature osteoblast marker Osteocalcin was missing (H ). Page 3 of 8

Chondrocyte Maturation in the Absence of β-catenin

(A, A, B, and B ) High-magnification pictures of

in the developing humerus after blood vessel

5 dpc. (A ) Catnby c/- ;Col2a1-Cre embryo at 17.

4 Supplemental Figure S3. Delayed Blood Vessel Invasion and Progression of Chondrocyte Maturation in the Absence of β-catenin Function (I) Delayed blood vessel invasion. (A, A, B, and B ) High-magnification pictures of TRAP staining showing osteoclasts (purple, arrows) in the developing humerus after blood vessel invasion. (A) Catnby c/+ ;Col2a1-Cre embryo at 17.5 dpc. (A ) Catnby c/- ;Col2a1-Cre embryo at 17.5 dpc. (B) Catnby c/+ ;Col2a1-Cre embryo at 15.5 dpc. (B ) Catnby c/- ;Col2a1-Cre embryo at 15.5 dpc. Page 4 of 8

(C F ) In situ hybridization with MMP9 and MMP13 probes in the developing long bones. (C and D) Humerus of the Catnby c/+ ;Col2a1-Cre embryo at 17.5 dpc.

5 dpc. (II) In situ hybridization with the indicated probes on consecutive sections of the developing tibia at 15.5 dpc. A layer (arrows) of nonhypertrophic chondrocytes between the perichondrium and hypertrophic chondrocytes was found in the Catnby c/- ;Col2a1-Cre embryo.

Delayed Progression of Chondrocyte Maturation in the Absence of β-catenin Function In situ hybridization with the indicated probes on consecutive sections of the developing ulna at 15.

5 (C F ) In situ hybridization with MMP9 and MMP13 probes in the developing long bones. (C and D) Humerus of the Catnby c/+ ;Col2a1-Cre embryo at 17.5 dpc. (C and D ) Humerus of the Catnby c/- ;Col2a1-Cre embryo at 17.5 dpc. Leading edge of cell migration is indicated by the arrow. (E and F) Tibia of the Catnby c/+ ;Col2a1-Cre embryo at 15.5 dpc. (E and F ) Tibia of the Catnby c/- ;Col2a1-Cre embryo at 15.5 dpc. (II) In situ hybridization with the indicated probes on consecutive sections of the developing tibia at 15.5 dpc. A layer (arrows) of nonhypertrophic chondrocytes between the perichondrium and hypertrophic chondrocytes was found in the Catnby c/- ;Col2a1-Cre embryo. Supplemental Figure S4. Delayed Progression of Chondrocyte Maturation in the Absence of β-catenin Function In situ hybridization with the indicated probes on consecutive sections of the developing ulna at 15.5 dpc is shown. (A I) Sections of the Catnby c/+ ;Dermo1-Cre embryos. (A I ) Sections of the Catnby c/c ;Dermo1-Cre embryo. A layer (arrows) of nonhypertrophic chondrocytes between the perichondrium and hypertrophic chondrocytes was found in the Catnby c/- ;Dermo1-Cre embryo (A and B ). Page 5 of 8

6 Supplemental Figure S5. Analysis of Cell Proliferation and Apoptosis during Intramembranous and Endochondral Ossification Cells at the mitotic M phase were detected by fluorescent immunohistochemistry with an antiphosphohistone H3 antibody (green, A H). Chondrocytes are shown by ColII immunohistochemistry (red). Nucleus was stained by DAPI (blue). Autofluorescent blood cells are yellow or orange. At 13.5 dpc (A D) and 15.5 dpc (E H), there is a slight reduction in the number of cells that are positive for phosphohistone H3 (arrows) in the frontal bone (C and K) and perichondrium of a humerus (D) or tibia (H) in the Catnby c/c ;Dermo1-Cre embryo. Apoptosis is detected by TUNEL assay (green, I P). Nucleus is stained by DAPI (blue). Autofluorescent blood cells are yellow or orange. At 13.5 dpc (I L), more apoptotic cells (arrows), although still few, are detected in the frontal bone (G) and perichondrium of a humerus (H) in the Catnby c/c ;Dermo1-Cre embryo. At 15.5 dpc (M P), fewer apoptotic cells (arrows), are detected in the frontal bone (O) and perichondrium of a tibia (P) in the Catnby c/c ;Dermo1-Cre embryo. Page 6 of 8

Absence of β-catenin Function Chondrocyte hypertrophy indicated by ColX

Catnby c/- ;Col2a1-Cre mouse embryos at 12.

7 Supplemental Figure S6. Normal Onset of Chondrocyte Hypertrophy and PTHrP Expression in the Absence of β-catenin Function Chondrocyte hypertrophy indicated by ColX expression was normal in the humerus of both Catnby c/c ;Dermo1-Cre and Catnby c/- ;Col2a1-Cre mouse embryos at 12.5 dpc when chondrocyte hypertrophy just started. The expression of PTHrP (arrows) was also normal in Catnby c/c ;Dermo1-Cre mouse embryo at 12.5 dpc. Page 7 of 8

cells isolated from the developing calvarium of the Catnby c/c mouse embryos at 12.

Osteogenesis was induced at day 5 when cells had reached superconfluency.

phosphohistone H3 fluorescent immunostaining (green) or TUNEL assay (green), respectively.

8 Supplemental Figure S7. Analysis of Cell Proliferation and Apoptosis in Calvarial Cell Cultures Mesenchymal progenitor cells isolated from the developing calvarium of the Catnby c/c mouse embryos at 12.5 dpc were cultured in vitro with or without Cre-adenovirus infection. Osteogenesis was induced at day 5 when cells had reached superconfluency. At different time points, cell proliferation (A C) and apoptosis (D F) were examined by phosphohistone H3 fluorescent immunostaining (green) or TUNEL assay (green), respectively. Nucleus was stained by propidium iodide (red). Arrows point to positively stained cells. Percentage of M phase cells (C) or apoptotic cells (F) were counted from three independent samples and the averages with standard deviations (error bars) are shown. Page 8 of 8

Supplemental Tables and Figures. The metalloproteinase-proteoglycans ADAMTS7 and ADAMTS12 provide an innate,

Supplemental Tables and Figures The metalloproteinase-proteoglycans ADAMTS7 and ADAMTS12 provide an innate, tendon-specific protective mechanism against heterotopic ossification Timothy Mead et al Supplemental