Characterization of ectopic cartilage in <i>Axin2^−/−</i> suture.

<p><b>A</b>, comparative qPCR analysis of <i>Col2α1</i> and <i>Col1α1</i> genes expression in <i>Axin2^−/−</i> PF<b>-</b>sutures shows overtime elevated levels of <i>Col2α1</i>, whereas <i>Col1α1</i> expression is significantly lower. <b>B,</b> immunohistochemistry of type I and type II collagen performed on coronal sections of <i>Axin2^−/−</i> PF-sutures. Chondrocyte staining is more intense for type II Collagen as compared to type I Collagen, indicating a hyaline nature of the cartilage Scale bar: 50 µm.</p

Partial inhibition of cWnt-signaling decreases ectopic cartilage size.

<p><b>A,</b> X-gal staining, representative of endogenous active cWnt signaling, performed on P8 <i>Axin2^−/−</i> PF-suture treated with the inhibitors of cWnt signaling Dkk1 (2 µg) and sFRP1 (2 µg) (n = 7) and untreated control (n = 7). X-gal staining is reduced in both cartilage areas as well as in the ectocranial layer and pericranium upon treatment with inhibitors (<b>left panel</b>). Pentachrome staining of adjacent slides reveals diminished cartilage in <i>Axin2^−/−</i> PF-suture treated with Dkk1 and sFRP1 (<b>right panel</b>). <b>B</b>, Histomorphometry of X-gal staining and cartilage surface confirmed these findings. Quantification of X-gal staining (<b>left panel</b>) showing inhibition of cWnt signaling (73.8%) in Dkk1 and sFRP1 treated PF-suture compared to untreated control. In PF-suture treated with inhibitors the cartilage size is reduced by (63.8%) compared to untreated control (<b>right panel</b>). Scale bar: 100 µm.</p

Lack of endochondral ossification and closure delay in <i>Axin2^−/−</i> PF-sutures.

<p><b>A</b>, pentachrome staining of PF-sutures from <i>Axin2^−/−</i> and wild type mice during the timing of physiological closure reveals an involuting ectopic cartilage in the suture mesenchyme of <i>Axin2^−/−</i> PF-sutures, while endochondral ossification occurs in wild type PF-sutures. Closure is delayed in <i>Axin2^−/−</i> PF-sutures compared to wild type PF-sutures. <i>Axin2^−/−</i> PF-sutures closure is observed at day P25. <b>B,</b> measurements of the distance between the approaching osteogenic fronts of endocranial and ectocranial bone plates in <i>Axin2^−/−</i> and wild type PF-sutures show significantly greater distances between the osteogenic fronts of the endocranial bone plates in <i>Axin2^−/−</i> PF-sutures from P7 to P17. Scale bar: 100 µm.</p

Comparative analysis of Mmp-9 between <i>Axin2^−/−</i> and wild type sutures.

<p><b>A,</b><b> </b><i>Mmp-9</i> gene expression analysis by qPCR at different time points reveals extremely low levels of <i>Mmp-9</i> expression in <i>Axin2^−/−</i> PF-sutures. In contrast, in wild type PF-sutures is observed upregulation of <i>Mmp-9</i> starting by day P9 with a peak at day 11. <b>B,</b> MMP-9 immunohistochemistry does not detect protein in the cartilage area of <i>Axin2^−/−</i> PF-sutures, whereas strong staining is detected in the hypertrophic cartilage of wild type PF-suture at day P11. Scale bar: 100 µm.</p

Chondrogenic profile of <i>Axin2^−/−</i> and wild type sutures.

<p><b>A,</b> qPCR analysis of chondrogenic markers at different time points coinciding with timing of physiological closure of PF-sutures through endochondral ossification. Expression profile of cartilage signatures gene is unique in <i>Axin2^−/−</i> PF-sutures compared to wild type PF-sutures. Elevated levels of <i>Sox9</i> gene expression are observed starting from day P7 and remain sustained at later time points. Expression of <i>Col2α1</i> also is higher (P13 and P17) and sustained compared to wild type PF-sutures. In contrast to wild type PF-sutures, in <i>Axin2^−/−</i> PF-sutures is not detected upregulation of <i>Col10α1</i> gene, a marker of hypertrophic chondrocytes. By day P13, expression of the osteocalcin gene <i>Bglap</i> is already upregulated in wild type PF-sutures. Conversely, in <i>Axin2^−/−</i> PF-sutures <i>Bglap</i> expression is observed at P25. The relative mRNA level in each sample is normalized to its <i>Gapdh</i> content. Values are given as relative to <i>Gapdh</i> expression.</p

Enhanced activation of endogenous Wnt signaling in FOb and exogenous activation of Wnt signaling in POb cells protects from apoptosis.

<p>(A) Time-course Caspase-3 activity performed on FOb and POb cells undergoing osteogenic differentiation with or without Dkk and sFRP (150 ng/ml of each), as illustrated by histogram this treatment upregulates the apoptotic activity in treated FOb cells (B) The same treatment reduces dramatically mineralization of the extracellular matrix in both cell types. (C) Contrary, treatment with Wnt3a (50 ng/ml) robustly decreases apoptosis in POb cells compared to untreated cells. Wnt3a has a similar effect also on FOb cells. (D) Mineralization of the extracellular matrix detected by Alizarin red staining reveals a significant increase of osteogenesis in treated POb cells. (E) and (F) Immunodetection of active/unphosphorylated β-catenin showing the effective inhibition and/or activation of Wnt signaling upon specific treatments.</p

Apoptotic profile of FOb and POb cells undergoing to osteogenic differentiation.

<p>(A) Alizarin red staining of FOb and POb cells at differentiation day 21 shows striking differences between FOb and POb, with FOb cells having a more robust mineralization and larger bone nodules as seen in the lower panel (Magnification 10X). (B) Quantification of Alizarin red staining. (C) Time course of Caspase-3 activity performed during osteogenic differentiation of FOb and POb cells harvested from different mouse stages reveals significant higher apoptotic activity in POb cells with a peak at d12 of differentiation. Abbreviations: (E), embryonic; (P), postnatal.</p

Differential activation of endogenous TGF-β and BMP signaling between FOB and POb cells.

<p>(A) Immunoblotting analysis using specific anti-phosphoSmad-2 and phoshoSmad-1/5 antibodies shows a more intense phosphorylation of Smad-2 in POb than FOb cells. In contrast, analysis with anti-phoshoSmad-1/5 antibody reveals a stronger staining in FOb. To assess for the total amount of endogenous Smad-2 and Smad-1/5 and to control for equal loading and transfer of the samples membranes were reprobed with anti-Smad-2, Smad-1/5 and anti-α-Tubulin antibodies. Histogram below represents quantification of phosphorylated Smad-2 and Smad-1/5 proteins obtained by Image J program. The relative intensity of each band was normalized to their respective α-Tubulin loading controls. (B) immunofluorescent staining using anti-phosphoSmads antibodies as above confirms the results obtained by immunoblotting analysis. Immunofluorescent staining using anti-phosphoBcl-2 antibody detects higher levels of the anti-apoptotic protein Bcl-2 in FOb compared to POb. Dapi nuclear counterstaining. (C) Immunoblotting analysis performed as above (A) showing that the differential activation of the two signaling pathways observed between FOb and POb cells is maintained throughout their osteogenic differentiation.</p

Exogenous activation of BMP signaling decreases apoptosis in POb cells.

<p>(A) Histogram illustrating a time-course of Caspase-3 activity performed on FOb and POb cells undergoing osteogenic differentiation with or without exogenous BMP-2 (200 ng/ml). BMP-2 treatment decreases apoptosis markedly in POb cells compared to untreated POb, a lower apoptotic activity is also observed in BMP-2 treated FOb as compared to untreated FOb cells. (B) Alizarin red staining and its quantification (lower panel) showing higher levels of mineralization in BMP-2 treated cells. (C) Profile of Caspase-3 activity in FOb and POb cells undergoing osteogenic differentiation with or without 200 ng/ml of noggin, inhibitor of BMP signaling, reveals that this treatment upregulated apoptosis with major effect on P7 osteoblasts (D) Noggin treatment inhibits dramatically mineralization of the extracellular matrix on both FOb and POb treated cells, as assessed by Alizarin red staining. The lower panel represents quantification of Alizarin red staining. (E) and (F) immunoblotting analysis with specific anti-phosphoSmad-1/5 antibody to validate the effective inhibition and/or activation of BMP signaling upon specific treatments. To control for equal loading and transfer of the samples membranes were reprobed with anti-Smad-1/5 and anti-α-Tubulin antibodies.</p

Development of the bony skull during metamorphosis (stages 59–61).

<p>A′–C′. Dorsal view of unstained <i>Xenopus</i> cranium demonstrates gross morphology, 0.7× magnification. D′–F′. Alizarin red (bone) and Alcian blue (cartilage) whole-mount staining of cranial skull. G′–I′. μCT scans delineate ossified portions of the cranium. Note ossification of the frontoparietal bone proceeding from the lateral aspect of the bone corresponding to the Alizarin red staining.</p