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First published online March 24, 2005
doi: 10.1242/10.1242/dev.01786

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The role of Axin2 in calvarial morphogenesis and craniosynostosis

¹ Center for Oral Biology, Department of Biomedical Genetics, Abs Institute of Biomedical Sciences, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
² Max Delbruck Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13122 Berlin, Germany
³ Department of Orthopedics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642, USA
⁴ Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA

View larger version (80K): [in a new window]   Fig. 1. Cranial skull defects in the Axin2–/– mice. Lateral (A) and dorsal (B,C) views of 27-day-old control (+/+) and Axin2 mutant (–/–) mice show that the skull is shortened in the Axin2–/– mice. Whole-mount (D,E) and skeletal staining (F-I) analyses of the cranial skull reveal abnormalities of the Axin2–/– suture at 4 weeks. The arrowheads indicate the sutures (black, metopic suture; white, jugum limitans) that lie between the nasal (N) and frontal (F) bones. The jugum limitans has disappeared and there is unilateral fusion of the metopic suture (arrows). Scale bars: 4 mm in A-C; 2 mm in D,E; 1 mm in F-I.

View larger version (110K): [in a new window]   Fig. 2. Targeted disruption of Axin2 in mice induces craniosynostosis. Histological sections show that cranial sutures are prematurely fused in the Axin2–/– mice. The sutures of control (A,C,F) and Axin2-null (B,D,E,G) mice at postnatal day 0 (A,B), 8 (C-E) and 17 (F,G) were analyzed by histology. In the controls (A,C,F), the metopic suture remains patent (indicated by arrowheads) in the first 4 weeks of postnatal development. The Axin2–/– suture did not show fusion at birth (B). However, unilateral (D) or bilateral (E) fusion of the Axin2–/– suture (arrows) occurred at day 8. The overlaying of cranial bones and endocranial bridging (arrows) is evident by day 17 (G). Compared with controls, the Axin2–/– sutures are morphologically more advanced with increased ossification (orange). The skeletogenesis stimulated in the Axin2 mutant was characterized by expression of FGFR1, a marker for osteoblast precursor (H-K). Immunohistochemical staining reveals an increase in FGFR1-expressing osteoblast precursors (asterisks) in the Axin2–/– suture at P17 (I,K). Sections were immunostained with -FGFR1 antibody (brown) and counterstained with Hematoxylin (blue). Enlargements of the insets (H,I) are shown in J and K. Scale bars: 100 µm in A,B; 200 µm in C-G; 50 µm in H,I.

View larger version (63K): [in a new window]   Fig. 3. Axin2 deficiency stimulates expansion of osteoprogenitors. The osteoblast precursors undergoing active divisions in the Axin2+/+ (A) and Axin2–/– suture (B) were identified by the expression of Ki67. Immunohistochemical staining reveals increases in the presence of the mitotic osteoprogenitors in the Axin2–/– suture at P8. In the defined suture region (broken lines), osteoprogenitors positive (brown) and negative (blue) for Ki67 were counted to measure proliferation abnormalities. At least six different regions from the same developmental stage were analyzed to obtain the average percentage of Ki67 positive (Ki67+) cells at E16.5, P8 and P17 (C). Primary osteoblast precursors isolated from nasal and frontal bones of the Axin2+/+ (D) and Axin2–/– (E) were grown in culture media for in vitro BrdU incorporation analysis. Cells were immunostained with -BrdU antibody (brown), and counterstained with Hematoxylin (blue). The stained images were taken randomly to determine the percentage of proliferating cells by counting the BrdU-positive cells in a total of 1000 cells. The graph shows the average percentage of Axin2+/+ and Axin2–/– in three independent experiments (F). Scale bars: 50 µm in A,B; 200 µm in D,E.

View larger version (74K): [in a new window]   Fig. 4. Defects of calvarial osteoblast differentiation caused by the Axin2 mutation. Primary calvarial osteoblast precursors isolated from the Axin2+/+ and Axin2–/– littermates were cultured in differentiation media for up to 9 days. Liquid (A) and histochemical (B) assays for alkaline phosphatase were performed at different time points of osteoblast (OB) differentiation as indicated. The enzyme activity is expressed as micrograms of p-nitrophenol (Pi) released per microgram of protein per minute. A diagram summarizes neural crest contribution (blue) to the skeletal elements and sutures of the mouse skull vault [diagram modified from Jiang et al. (Jiang et al., 2002)] (C). The neural crest or mesoderm-derived osteoblasts were isolated from nasal/frontal (highlighted by a red line) or parietal (highlighted by a green line) bones, respectively. c, coronal suture; F, frontal bone; IP, interparietal bone; JL, jugum limitans; l, lambdoid suture; m, metopic suture; N, nasal bone; nc, nasal cartilage; P, parietal bone; s, sagittal suture. (D) The neural crest or mesoderm-derived primary osteoblasts of Axin2+/+ and Axin2–/– were cultured in differentiation media for up to 12 days. Liquid assays for alkaline phosphatase were performed at different time points as indicated. A and D are representative of three independent experiments. Scale bar: 200 µm in B.

View larger version (56K): [in a new window]   Fig. 5. Alteration of osteoblast specific gene expression and increased mineralization by the Axin2 deletion. Primary calvarial osteoblast precursors isolated from nasal/frontal bones of the Axin2+/+ and Axin2–/– littermates were cultured in differentiation media for up to 9 days. Quantitative real time RT-PCR analyses were performed to examine expression of osteopontin and osteocalcin. The graphs represent the expression levels (in arbitrary units) of osteopontin (A) and osteocalcin (B) during the course of osteoblast differentiation. Primary calvarial osteoblasts from nasal/frontal bones of the Axin2+/+ (C) and Axin2–/– (D) littermates were maintained in differentiation media for 3 weeks. Von Kossa staining analyses show increased mineralization in Axin2–/– osteoblast cultures compared with wild-type cultures (C,D). Scale bars: 0.1 mm in C,D.

View larger version (73K): [in a new window]   Fig. 6. Expression of Axin2 in developing skull. The Axin2 expression pattern was visualized by ß-gal staining in whole mounts (A-H) or sections (I-L). Axin2 is present in metopic and coronal sutures at E16.5 (A,E,I). Axin2 is detected at high levels in the osteogenic fronts (arrowheads), periosteum (asterisks) and mesenchyme of the sutures, as well as in the nasal bone (arrows) and nasal cartilage. At P4, Axin2 is highly expressed in the osteogenic fronts (arrowheads), periosteum (asterisks), nasal bone (arrows) and nasal cartilage (B,F,J). At P14, high levels of Axin2 are observed in nasal and frontal bones (C,G) in addition to the osteogenic fronts (arrowhead) and periosteum (asterisks; K,L). By P31, expression of Axin2 appears to be diminished to very low levels (D,H). First and second rows, the Axin2lacZ allele shows Axin2 expression. Third row, coronal sections of the ß-gal stained metopic sutures, counterstained with nuclear Fast Red. Scale bars: 3 mm in A,H; 5 mm in B-D; 1 mm in E,F; 2 mm in G; 100 µm in I-L.

View larger version (75K): [in a new window]   Fig. 7. Activation of ß-catenin and LEF/TCF signaling in skull development. Mice carrying the TOPGAL transgene, capable of responding to ß-catenin and LEF/TCF signaling, were used to monitor activation of this pathway by ß-gal staining analyses in cranial skulls at E16.5 (C,E) and P1 (D,F). Whole-mount staining shows activation of the canonical Wnt pathway during early skull formation (C-F). Arrowheads indicate the coronal sutures and arrows indicate the activation of ß-catenin and LEF/TCF signaling in medial parts of the nasal bones. The non-transgenic mice, with no background staining, indicate the specificity of the assay (A,B). Scale bars: 2 mm in A-D; 500 µm in E,F.

View larger version (93K): [in a new window]   Fig. 8. Alteration of the canonical Wnt pathway in the Axin2 mutants during skull morphogenesis. Sections of the Axin2+/+ (A,C,E) and Axin2–/– (B,D,F) sutures were immunostained with the primary antibody (brown) and counterstained with Hematoxylin (blue). Immunohistochemical staining with an antibody that recognizes only the activated form of ß-catenin (-ABC antibody) reveals stimulation of ß-catenin signaling (arrowheads) in the Axin2–/– suture at P8 (B) and P28 (D). Expression of cyclin D1, a bona fide target of ß-catenin, is also elevated in the Axin2–/– suture (broken lines) at P8 (F). Scale bars: 50 µm in A,B,E,F; 25 µm in C,D.

View larger version (49K): [in a new window]   Fig. 9. Stimulation of ß-catenin signaling promotes osteoblast differentiation. Whole calvarial (A, left panel) or CNC-derived (A, right panel) osteoblast precursors, isolated from the Axin2+/+ and Axin2–/– littermates, were cultured in differentiation media for up to 12 days. Lysates, isolated at different differentiation days as indicated, were first analyzed by the alkaline phosphatase liquid assay (shown in Fig. 4A,D). The same lysates were then used for immunoblotting (A). Immunoblot analyses with the -ABC (activated) and -ß-catenin (total) antibodies reveal that ß-catenin signaling is significantly stimulated by inactivation of Axin2 during osteoblast differentiation. The expression of FGFR1, which is increased upon differentiation, is elevated in the Axin2–/– cells. The level of actin was also analyzed as a control for protein content of the lysates. (B,C) Quantitative real-time RT-PCR analyses were performed to examine expression of two Wnt targets FGF4 and FGF18 in the CNC-derived osteoblasts. The graphs represent the expression levels (in arbitrary units) of FGF4 (B) and FGF18 (C) during the course of differentiation in vitro. (D) The CNC-derived osteoblasts of Axin2+/+ and Axin2–/– were cultured in differentiation media for 7 days. The TOPFLASH reporter plasmid was then transfected by lipofectamine with different combinations of the ß-catenin expression (ß-cat) (Korinek et al., 1997), ß-catenin RNA interference (ß-cat-shRNA) (Cellogenetics) and pUC19 carrier plasmids, as indicated (0.5 µg for each plasmid plus the carrier to a total of 1.5 µg DNA). Relative luciferase activity (RLA) for each sample was determined after 48 hours. (E) Activation of ß-catenin signaling by BIO stimulates osteoblast differentiation. Primary osteoblast precursors isolated from nasal and frontal bones were cultured in differentiation media with or without 2 µM BIO for up to 9 days. Liquid assays for alkaline phosphatase were performed at different time points as indicated. The diagram is representative of three independent experiments.