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First published online January 13, 2009
doi: 10.1242/10.1242/dev.027706

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Glucocorticoid-dependent Wnt signaling by mature osteoblasts is a key regulator of cranial skeletal development in mice

Hong Zhou^*,, Wendy Mak^*, Robert Kalak, Janine Street, Colette Fong-Yee, Yu Zheng, Colin R. Dunstan and Markus J. Seibel

Bone Research Program, ANZAC Research Institute, The University of Sydney, Sydney, NSW 2139, Australia.

View larger version (92K): [in this window] [in a new window]   Fig. 1. Disrupted GC signaling in mature osteoblasts leads to delayed cranial bone formation. (A-F) Micro-CT images of the calvaria of wild-type and Col2.3-11βHSD2 transgenic mice. All images are of the same magnification, showing growth retardation in transgenic mice relative to wild-type animals. Scale bar: 1 mm. (A) Neonatal wild type (P1). (B) Neonatal Col2.3-11βHSD2 transgenic mouse (littermate of A). (C) Three-day-old wild type (P3). (D) Col2.3-11βHSD2 transgenic mouse (littermate of C). (E) Seven-day-old wild type (P7). (F) Col2.3-11βHSD2 transgenic mouse (littermate of E). (G-L) Embryonic skeletal preparations at E16.5. (G) Wild type. (H) Col2.3-11βHSD2 transgenic (littermate of G). (I) Enlarged head region of G. (K) Enlarged head region of H. (J) Enlarged head region of G, dorsal view. (L) Enlarged head region of H, dorsal view. Cartilage was stained using Alcian Blue and bone was stained using Alizarin Red. Areas of reduced bone formation are indicated by arrows.

View larger version (93K): [in this window] [in a new window]   Fig. 2. Ectopic cartilage formed in Col2.3-HSD2-transgenic mice. (A,B) Skeletal preparations with cartilage stained by Alcian Blue. (A) Wild-type P1, lateral and dorsal views. (B) Col2.3-11βHSD2 transgenic littermate of A. The cartilage remnants are indicated by arrows. P, parietal bone; IP, interparietal bone. (C,D) Parietal bones, apical segment. (C) Wild type; (D) Col2.3-11βHSD2 transgenic littermates. Osteoblasts are denoted by arrows. Toluidine Blue staining. Scale bars: 25 µm. (E,F) Parietal bones, base segment. (E) Wild type; (F) Col2.3-11βHSD2 transgenic littermates. Toluidine Blue staining. Scale bars: 100 µm. (G,H) Parietal bones, base segment. (G) Wild type; (H) Col2.3-11βHSD2 transgenic littermates. Alkaline phosphatase (ALP) staining. Scale bars: 100 µm. (I) The boxed region in G is enlarged to show ALP+ve pre-osteoblastic cells (arrows). Ca, cartilage. Scale bar: 25 µm. (J) The boxed region in H is enlarged to show ALP+ve osteoblastic cells (arrows) but lack of spindle shaped ALP+ pre-osteoblastic cells. Ca, cartilage. Scale bar: 25 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 3. Cranial skull defects in P7 Col2.3-11βHSD2 transgenic mice. (A,B) Skeletal preparations, cartilage stain (Alcian blue). (A) Wild type; (B) Col2.3-11βHSD2 transgenic littermate of A. Arrows denote ectopic cartilage under the sutures. P, Parietal bone; IP, interparietal bone. (C,D) Parietal bone, top segments. (C) Wild type; (D) Col2.3-11βHSD2 transgenic littermate of (C). Red arrowheads in C and D indicate calvarial bone fronts of the sagittal sutures. The black arrow in D denotes ectopic cartilage under the sagittal suture. Toluidine Blue staining. Scale bars: 100 µm. (E,F) Parietal bone, base segment. (E) Wild type; (F) Col2.3-11βHSD2 transgenic littermate of E. Toluidine Blue staining. Scale bars: 100 µm. (G,H) Parietal bone, base segment at higher magnification. (G) Wild type; (H) Col2.3-11βHSD2 transgenic littermate. Toluidine Blue staining. Scale bars: 25 µm.

View larger version (82K): [in this window] [in a new window]   Fig. 4. Mmp14 expression is reduced in neonatal (P0) transgenic parietal bone and cartilage. (A,B) Immunohistochemistry for Mmp14. Scale bars: 50 µm. (C,D) Mmp14 mRNA detected by in situ hybridization. Scale bars: 50 µm. (E,F) Areas of cartilage removal in wild-type (E) and transgenic animals (F). The arrows in E indicate areas of proteoglycan depletion and cartilage matrix degradation. Arrows in F denote the corresponding position in transgenic mice. Toluidine Blue staining. Scale bars: 50 µm. (G,H) TUNEL staining. Arrows indicate apoptotic cells. Scale bars: 50 µm. (I,J) Immunohistochemistry staining for HSD2. Scale bars: 100 µm. (K) mRNA expression for Col2a1 and Mmp14 in parietal bone. Parietal bones were dissected by cutting skull bones along coronal and lambdoid sutures. RNA was isolated from the parietal bones of wild-type and transgenic mice. Real-time PCR quantitation of relative mRNA expression levels for Col2a1 and Mmp14 after normalization by 18S expression. Data are represented as mean±s.e.m., n=6. (L) Quantitation of apoptotic chondrocytes in the parietal cartilages of wild type (WT) and Col2.3-11βHSD2 transgenic (tg) mice. Data are represented as mean±s.e.m., n=5.

View larger version (121K): [in this window] [in a new window]   Fig. 5. Wnt/β-catenin signaling is reduced in neonatal (P0) transgenic parietal bone and cartilage. (A,B) Immunohistochemistry for β-catenin at regions of overlapping bone and cartilage in P0 parietal bones. Arrows indicate osteoblasts. b, bone; ca, cartilage; br, brain. Scale bars: 50 µm. (C,D) Immunohistochemistry for β-catenin (β-cat) at the sagittal sutures of P0 parietal bones. Red arrowheads indicate bone growth fronts in the sagittal sutures. The black arrows indicate mesenchymal progenitors in the non-ossified suture area. b, bone. Scale bars: 50 µm. (E,F) Parietal bones. In situ hybridization for Wnt9a at an area of overlapping bone and cartilage. b, bone; ca, cartilage. Scale bars: 50 µm. (G) mRNA expression for Wnt9a and Wnt10b in P0 parietal bones. Col2.3-11βHSD2-transgenic mice were bred with Col2.3-GFP mice to generate Col2.3-11βHSD2-GFP-transgenic and Col2.3-GFP littermates. Parietal bones were dissected from P0 Col2.3-11βHSD2-GFP-transgenic and Col2.3-GFP littermates and RNA was isolated. Real-time PCR quantitation of relative mRNA expression levels for Wnt 10b and Wnt9a after normalization by GFP expression. Data are represented as mean±s.e.m. (*P<0.05 versus wild type, n=6).

View larger version (118K): [in this window] [in a new window]   Fig. 6. Wnt3a treatment rescues the phenotype of Col2.3-11βHSD2 transgenic mice. (A-C) Representative Micro-CT images of the calvaria of 3-day-old (p3) wild-type and Col2.3-11βHSD2 transgenic mice. (A) Wild-type vehicle control; (B) Col2.3-11βHSD2 transgenic littermate, vehicle control; (C): Wnt3a-treated Col2.3-11βHSD2 transgenic mouse (littermate of A,B). (D) Quantitation of unmineralized suture areas. Results are expressed as percent of total skull area. Micro-CT images were analysed using Image J image analysis software. Data are represented as mean±s.e.m. (*P<0.05; **P<0.01 versus transgenic, n=8). tg, transgenic mice; tg Wnt3a, transgenic mice receiving recombinant Wnt3a treatment. (E-M) Representative coronal sections of parietal bones. (E,H,K) Wild-type vehicle control; (F,I,L) Col2.3-11βHSD2 transgenic, vehicle control (CTR) littermate; (G,J,M) Col2.3-11βHSD2 transgenic, Wnt3a-treated littermate. Toluidine Blue stain. (E-G) Parietal bones, base segment. Arrows in F indicate areas of remnant cartilage, whereas arrows in E and G indicate the corresponding positions in wild-type vehicle control and Wnt3a-treated Col2.3-11βHSD2 transgenic littermates. Scale bars: 400 µm. (H-J) Parietal bone, sagittal suture area. Red arrowheads indicate the position of active bone expansion (`growing bone fronts'). b, bone. Ectopic cartilage is present in the sagittal sutures with greater separation of parietal bones in Col2.3-11βHSD2 transgenic vehicle controls (I) compared with the wild-type vehicle control mice (H). In Wnt3a-treated transgenic mice, this phenotype is rescued with only a small cartilage remnant remaining (J). Scale bars: 100 µm. (K-M) Enlarged suture area of H-J. Red arrowheads indicate the position of growing bone fronts. b, bone. Parietal bones are well formed in Wnt3a-treated transgenic mice (M), comparable with what is seen in wild-type animals (K). By contrast, bone is thin and osteoblasts are disorganized in transgenic vehicle control mice (L). Scale bars: 100 µm.

View larger version (23K): [in this window] [in a new window]   Fig. 7. The role of endogenous glucocorticoids in cranial bone development. Proposed model of relevant molecular mechanisms and signaling pathways. Glucocorticoids (GC) stimulate differentiated osteoblasts to produce Wnt proteins, which activate the Wnt/β-catenin signaling cascade in: (A) cranial mesenchymal progenitor cells, promoting osteoblastogenesis and inhibiting chondrogenesis through up-regulation of Runx2 and downregulation of Sox9 expression; (B) cranial osteoblasts, through upregulation of Mmp14 to initiate the remodeling of the collagenous matrix surrounding the osteoblast; and (C) cranial chondrocytes, through up-regulation of Mmp14 to initiate the cranial cartilage degradation.

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