Distribution of cell adhesion molecules in developing skull. Cellular localizations of adhesion molecules were analyzed by confocal microscopy. Sections of the 8-day-old Axin2+/+(A, C, E, G) and Axin2−/− (B, D, F, H) calvaria were stained with β-catenin (A, B, C, D), OB-cadherin (E, F), or N-cadherin (G, H) in green and counterstained with PI in red. Nuclear localization of β-catenin (arrowheads) was evident in calvarial osteoprogenitors located to the Axin2-null periosteum (B), but absent in the wild type counterparts (A). In the Axin2-null calvarial bones, strong membrane staining of β-catenin (D) and OB-cadherin (F, asterisks) was detected in mature osteoblasts. Diffused cytosolic staining was shown in the Axin2+/+ calvarial bones (C, E). N-cadherin showed weak expression in the cytoplasm of both Axin2+/+ and Axin2−/− (G, H). Scale bars, 40 μm (A–H).

BMP signaling is induced in the Axin2-null mutants during calvarial osteoblast development. Immunohistochemical staining of the 8-day-old Axin2 +/+ (A) and Axin2−/− (B) sutures with the α-phospho-Smad1/5/8 antibody reveals stimulation of BMP signaling by the Axin2 deletion (brown staining and blue counterstaining). The graph shows the average percentage of the Axin2+/+ and Axin2−/− osteoblast precursors of the suture region positive for phospho-Smad1/5/8 in five independent experiments (D). (C) CNC-derived osteoblast precursors, isolated from the Axin2+/+ and Axin2−/− littermates, were cultured in differentiation media for up to 12 days. Lysates were isolated at different differentiation days as indicated. Immunoblot analysis with α-phospho-Smad1/5/8 (PSmad1/5/8) antibodies shows that BMP signaling was stimulated in the Axin2 mutants during osteoblast differentiation. The level of Actin was also analyzed as a control for protein content of the lysates. Quantitative real-time RT-PCR analyses were performed to examine expression of BMP-2 (E) and BMP-6 (F) during the course of osteoblast differentiation. The graphs represent the expression levels (in arbitrary units, Y axes) of BMP-2 and BMP-6 during the course of osteoblast differentiation. Scale bars, 50 μm (A, B).

Effects of Noggin on osteoblast development. Primary osteoblast precursors isolated from nasal and frontal bones of the Axin2+/+ and Axin2−/− were grown in culture media for in vitro BrdU incorporation assay (A). The percentage of proliferating osteoprogenitors was obtained by analyzing BrdU positive and negative cells in a total of >1000 cells. The addition of Noggin had no obvious effect on expansion of both the Axin2+/+ and Axin2−/− osteoprogenitors. (B–E) The CNC-derived osteoprogenitors of Axin2+/+ (B) and Axin2−/− (C–E) were analyzed for osteoblast differentiation using alkaline phosphatase liquid assay at different time points as indicated. Noggin (B–D) and Ionomycin (E) were present after day 5 of differentiation. Noggin inhibits the accelerated intramembranous ossification caused by Axin2 deficiency in a dosage dependent manner as determined by alkaline phosphatase activity at differentiation day 10 (D). Graphs, except for panel D, represent the average in three independent experiments.

Requirement of β-catenin in the Axin2 mediated osteoblast proliferation and differentiation. Immunostaining of Ki67 detects cells undergoing active divisions in the 8-day-old Axin2−/− (A) and Axin2−/−; β-catenin+/− (B) sutures. A reduced level of β-catenin causes a decrease in the presence of the mitotic osteoprogenitors stimulated by Axin2 deficiency. In the suture regions, osteoblast precursors that are positive (brown) and negative (blue) for Ki67 were counted to measure the proliferation effects. At least 8 different regions from four different mice were analyzed to obtain the average percentage of Ki67 positive (Ki67+) cells at postnatal day 8 (C). The skeletogenesis was characterized by expression of FGFR1 (D, E) and phospho-Smad1/5/8 (F, G). Sections were immunostained with α-FGFR1 or α-phospho-Smad1/5/8 antibody (brown) and counterstained with hematoxylin (blue). In the Axin2−/−; β-catenin+/− suture, a reduction of the FGFR1-expressing cells is evident at postnatal day 17 and a diminution in BMP signaling is detected at postnatal day 28. The graph shows in the suture region the average percentage of the Axin2−/−; β-catenin+/+ and Axin2−/−; β-catenin+/− osteoblast precursors positive for phospho-Smad1/5/8 (PSmad) and FGFR1 in five independent experiments (H). Immunoblot analyses revealed that haploid deficiency of β-catenin induced a quantitative change of the FGFR1 and phospho-Smad1/5/8 levels in the Axin2−/− calvarial osteoprogenitors (I). The level of Actin was also analyzed as a control for protein content. Scale bars, 50 μm (A, B, D, E, F, G).

The loss of Axin2 alters cellular compartmentalization of β-catenin in osteoprogenitors and stimulates formation of adhesion complex in mature osteoblasts. Nuclear localization of β-catenin in osteoprogenitors was stimulated by BIO and the Axin2 disruption. Primary osteoprogenitors isolated from the Axin2+/+ and Axin2−/− nasal and frontal bones were analyzed for β-catenin distribution. Confocal microscopy (A–D) revealed that β-catenin was localized to cytoplasm of the CNC-derived Axin2+/+ osteoprogenitors (A). The presence of BIO (B) or deletion of Axin2 (C) induced nuclear accumulation of β-catenin in the osteoprogenitors. The addition of Noggin has no effect on β-catenin nuclear localization in the Axin2−/− osteoprogenitors (D). Inset in panels A and C shows a superimposed fluorescent image for double labeling of β-catenin in green and propidium iodide (PI) in red under a low magnification. In mature osteoblasts, β-catenin is predominantly localized to the plasma membrane. The Axin2+/+ and Axin2−/− primary osteoprogenitors were induced for maturation by culturing in differentiation media for 10 days. Confocal microscopy revealed that β-catenin (E, F) and OB-cadherin (G, H) were located to plasma membrane of the Axin2+/+ (E, G) and Axin2−/− (F, H) differentiated CNC-derived osteoblasts. Note that the staining of β-catenin and OB-cadherin intensified by the ablation of Axin2. Cells were stained with β-catenin (A–F) or OB-cadherin (G, H) in green and counter-stained with PI in red (E–H). Pictures were taken with the same exposure time for panels A–D and for panels E–H. Scale bars, 40 μm (A–D, G, H); 125 μm (E, F).

Expansion of osteoblast precursors affected by Axin2 deficiency. Immunostaining of cyclin D1 (brown staining and blue counterstaining) showed an enhanced expression in the Axin2−/− osteoprogenitors (B), compared to the Axin2+/+ cells (A). Osteoblast precursors in developing mouse skull were analyzed by immunohistochemistry (C–H). Sections were immunostained with antibodies (brown) and counterstained with hematoxylin (blue). The deletion of Axin2 resulted in stimulation of CDK4 at postnatal day 8 (C, D). The Sox9-expressing precursor population was highly increased by the Axin2 inactivation at newborn (E, F) and postnatal day 8 (G, H). The Sox9 expression in the newborn nasal cartilage of Axin2+/+(I) and Axin2−/− (J) did not show any noticeable difference. The osteoblast precursors that are positive (brown) and negative (blue) for immunostaining analyses (A–D and G–H) were counted to obtain the percentage of positive cells. The graph shows the average percentage of the positive stained cells in five independent experiments (L). Immunoblot analyses revealed quantitative alterations in the expression of cyclin D1 (CycD1), CDK4 and Sox9 in the Axin2+/+ and Axin2−/− calvarial osteoprogenitors (K). The level of Actin serves as a loading control. Scale bars, 200 μm (A, B); 50 μm (C–J).

Noggin inhibits membrane accumulations of β-catenin and OBcad caused by the Axin2 disruption in mature osteoblasts. Confocal microscopy revealed that β-catenin (A) and OBcad (D) were predominantly located to plasma membrane of the Axin2−/− differentiated CNC-derived osteoblasts. Cells were stained with β-catenin (A–C) or OBcad (D–F) in green and counterstained with propidium iodide (PI) in red. Pictures were taken under the same parameter optimized for detection in the Axin2−/− cells. Addition of ionomycin (Iono) or Noggin (NG) disrupts the membrane localization of β-catenin (B, C) and OB-cadherin (E, F) in the Axin2-null mutants. Scale bars, 125 μm (A–C); 40 μm (D–F).

Alleviation of the Axin2-null defects by haploid deficiency of β-catenin. β-catenin+/− mice were crossed into the Axin2-null background to obtain the Axin2−/−; β-catenin+/− mice. Haploid deficiency of β-catenin alleviates the Axin2−/− skull abnormalities at 4 weeks (A, B). The arrows indicate the sutures (black, metopic suture; white, jugum limitans) that lie between nasal (N) and frontal (F) bones. Theses sutures disappearing in the Axin2−/− mutants (A) are apparent in the Axin2−/−; β-catenin+/− littermates (B). μCT analyses reveal that the suture closure defects (white long and short arrows, jugum limitans; black long and short arrows, coronal suture) caused by Axin2 deficiency (C) were alleviated by haploid deficiency of β-catenin (D). Histology shows that metopic sutures, prematurely fused in the Axin2-null mutants (E, long arrows), remain patent in the Axin2−/−; β-catenin+/− mice (F, short arrows) at 4 weeks. Scale bars, 3 mm (A, B); 5 mm (C, D); 200 μm (E, F).

Model for calvarial osteoblast development mediated by Wnt signaling. β-catenin is located to the cytoplasm of resting osteoprogenitors. An active Wnt signal promotes cell cycle entry by inducing nuclear localization of β-catenin (β-Cat) and activation of cyclin D1 (CycD1). During osteoblast differentiation, membrane distribution and stimulation of β-catenin and OBcad are mediated through a mechanism involving BMP signaling. Membrane accumulations of β-catenin and OBcad induce cell–cell interaction to promote the differentiation process. Noggin has an inhibitory effect on osteoblast differentiation, but not proliferation. Both Axin2 and Noggin regulate the Wnt and BMP pathways in a negative feedback loop, respectively. β-catenin, which functions as a transcriptional co-activator and a cell adhesion molecule, plays distinct roles in osteoblast proliferation and differentiation, respectively. Axin2, whose deficiency affects dislocation of β-catenin in osteoprogenitors and formation of adherens junctions in mature osteoblasts, negatively modulates both developmental processes.