The <i>Brca1-p53</i> interaction is critical for craniofacial bone morphogenesis.

<p>(A) Immunoblot analysis of facial region tissue from E13.5 embryos. Each sample is from individual embryos. Right chart shows quantification of p53 relative production level. (B) Alcian blue- and alizarin red-stained skulls of control, <i>Brca1</i>^<i>-/-</i>:<i>Wnt1-Cre</i>, <i>Brca1</i>^<i>-/-</i>:<i>p53</i>^<i>+/-</i>:<i>Wnt1-Cre</i> and <i>Brca1</i>^<i>-/-</i>:<i>p53</i>^<i>-/-</i>:<i>Wnt1-Cre</i> mice at birth. Yellow broken lines indicate osteogenic fronts. Scale bar = 2mm. (C) Quantification of the area ratio of frontal foramen in the frontal bone area. White box represents the mean of each genotype. (D) Quantification of sagittal length. (E) Quantification of skull width. (F) Measurement schema of C, D and E. The shaded area surrounded by the red line is the measured frontal area in C. Sagittal length was divided into anterior and posterior part at the estimated anterior fontanelle (yellow). Data in A, D and E are represented as mean ±SD, n = 3 in A and n = 10 in C, D and E in each group. *P<0.05. N.S., not significant.</p

Neural crest cell-specific <i>Brca1</i> deletion causes craniofacial bone abnormalities in mice.

<p>(A) Lateral views of control and <i>Brca1</i>:<i>Wnt1-Cre</i> mice at birth (NB). Scale bar = 5mm. (B) Alcian blue- and alizarin red-stained skulls of control and <i>Brca1</i>:<i>Wnt1-Cre</i> mice at birth. Yellow broken lines indicate osteogenic fronts. Note that the frontal bones of <i>Brca1</i>:<i>Wnt1-Cre</i> mice are separated by a large open space. Scale bar = 2mm. as, alisphenoid; bo, basioccipital; bs, basisphenoid; eo, exoccipital; fb, frontal bone; ib, interparietal bone; jb, jugal bone; md, mandible; mx, maxilla; nb, nasal bone; p, palatine; pb, parietal bone; pmx, premaxilla; ppmx, palatal process of maxilla; ppp, palatal process of palatine; ptg, pterygoid; tr, tympanic ring.</p

Neural crest cell-specific <i>Brca2</i> deletion results in a craniofacial bone phenotype similar to that of the neural crest cell-specific <i>Brca1</i> deletion in mice.

<p>(A) Alcian blue- and alizarin red-stained skulls of control and <i>Brca2</i>:<i>Wnt1-Cre</i> mice at birth. Yellow broken lines indicate osteogenic fronts. Scale bar = 2mm. (B) Quantification of the area ratio of frontal foramen in the frontal bone area. White box represents the mean of each genotype. (C) Quantification of sagittal length. (D) Coronal sections of control and <i>Brca2</i>:<i>Wnt1-Cre</i> frontal bone primordia at E12.5 and E13.5 were double-labeled with RUNX2 (red) and BrdU (green). Broken line describes the osteogenic lineage cell population. Right charts show quantification of the ratio of BrdU-positive cells over RUNX2-positive cells. Scale bar = 50μm. (E) Immunostaining for RUNX2 and TUNEL assay of sections from control and <i>Brca2</i>:<i>Wnt1-Cre</i> embryos at E12.5. Broken line describes the osteogenic lineage cell population. White arrows indicate TUNEL-positive cells. Yellow color represents the non-specific signal from red blood cells. Scale bar = 100μm. as, alisphenoid; bo, basioccipital; bs, basisphenoid; eo, exoccipital; fb, frontal bone; ib, interparietal bone; jb, jugal bone; md, mandible; mx, maxilla; nb, nasal bone; p, palatine; pb, parietal bone; pmx, premaxilla; ppmx, palatal process of maxilla; ppp, palatal process of palatine; ptg, pterygoid; tr, tympanic ring.</p

Deletion of <i>p53</i> partially rescues the skull defects by preventing cell death.

<p>(A) Coronal sections of control, <i>Brca1</i>^<i>-/-</i>:<i>Wnt1-Cre</i> and <i>Brca1</i>^<i>-/-</i>:<i>p53</i>^<i>-/-</i>:<i>Wnt1-Cre</i> frontal bone primordia at E12.5 were double-labeled with RUNX2 (red) and BrdU (green). Broken line describes the osteogenic lineage cell population. Right charts show quantification of the ratio of BrdU-positive cells over RUNX2-positive cells. Scale bar = 50μm. (B) Immunostaining for RUNX2 and TUNEL assay of sections from control, <i>Brca1</i>^<i>-/-</i>:<i>Wnt1-Cre</i> and <i>Brca1</i>^<i>-/-</i>:<i>p53</i>^<i>-/-</i>:<i>Wnt1-Cre</i> embryos at E12.5. Broken line describes the osteogenic lineage cell population. White arrows indicate TUNEL-positive cells. Yellow color represents the non-specific signal from red blood cells. Right chart shows quantification of the percentage of TUNEL-positive cells in the frontal bone primordium. (C) Immunostaining for γ-H2AX and Cleaved Caspase-3 and/or p-Chk2 of sections from control, <i>Brca1</i>^<i>-/-</i>:<i>Wnt1-Cre</i> and <i>Brca1</i>^<i>-/-</i>:<i>p53</i>^<i>-/-</i>:<i>Wnt1-Cre</i> embryos at E12.5. Broken line describes the osteogenic lineage cell population. White arrows indicate double-positive cells for γ-H2AX/Cleaved Caspase-3 and/or γ-H2AX/p-Chk2. Right chart shows quantification of the percentage of γ-H2AX, Cleaved Caspase-3 and p-Chk2 positive cells in the frontal bone primordium. Scale bar = 100μm. Data in A, B and C are represented as mean ±SD, n = 3 in each group. *P<0.05. N.S., not significant.</p

Primer sequences for Quantitative RT-PCR analysis.

<p>Primer sequences for Quantitative RT-PCR analysis.</p

<i>Brca1</i> is indispensable for osteoblast proliferation and survival at mid-gestation.

<p>(A) Coronal sections of control and <i>Brca1</i>:<i>Wnt1-Cre</i> frontal bone primordia at E12.5 and E13.5 were double-labeled with RUNX2 (red) and BrdU (green) to detect osteogenic cells and proliferative cells, respectively. Broken line describes the osteogenic lineage cell population. Right charts show quantification of the ratio of BrdU-positive cells over RUNX2-positive cells. Scale bar = 50μm. (B) Immunostaining for RUNX2 and TUNEL assay of sections from control and <i>Brca1</i>:<i>Wnt1-Cre</i> embryos at E12.5. Broken line describes the osteogenic lineage cell population. White arrows indicate TUNEL-positive cells. Yellow color represents the non-specific signal from red blood cells. Scale bar = 100μm. (C) Immunostaining for γ-H2AX and Cleaved Caspase-3 and/or p-Chk2 of sections from control and <i>Brca1</i>:<i>Wnt1-Cre</i> embryos at E12.5. Broken line describes the osteogenic lineage cell population. White arrows indicate γ-H2AX- and/or Cleaved Caspase-3-positive cells. Scale bar = 100μm. (D) Quantification of the percentage of γ-H2AX-, Cleaved Caspase3- and TUNEL-positive cells in the frontal bone primordium. Data in A and D are represented as mean ±SD, n = 3 in each group. *P<0.05.</p

Morphometric analysis of femur.

Morphometric analysis of femur.</p

Effect of β-aminopropionitrile (BAPN) on matrix formation and collagen components produced by MC3T3-E1 cells.

(a) BAPN treatment did not affect the proliferation of MC cells, as demonstrated by MTS assay. (b) Expression of core-binding factor alpha 1/runt-related transcription factor 2 (Runx2/Cbfa1) and type I collagen α2 chain (Col1a2) did not change, while the levels of lysyl oxidase (Lox) were significantly elevated after BAPN treatment in a dose-dependent manner. (c) Collagen components were analyzed by electrophoresis. In the control, all chains (α, β, and γ) were clearly observable. However, BAPN treatment led to the inhibition of β- and γ-chain formation. Picrosirius Red staining was visualized under bright-field (d) and polarized light (e). Collagen quantity was not affected by BAPN, while the alignment of collagenous fibers and matrix maturation were impaired. Bar: 100 μm. (f) Collagen content was slightly increased following 0.5 mM BAPN treatment, but it decreased after the administration of 2 mM BAPN. Hyl/Hyp × 300 decreased after the treatment with 2 mM BAPN. Divalent DHLNL and HLNL levels decreased with 0.5 mM BAPN treatment and were not detectable following the treatment with 1.0 and 2.0 mM BAPN. Trivalent pyridinoline levels significantly decreased after the application of 0.5 mM BAPN, while they were undetectable following the treatment with 1.0 and 2.0 mM BAPN. *p < 0.05, compared to the control.</p

Effect of low cross-linked matrices on osteoblast and osteoclast.

(a) The ablation of cellular components by DOC was confirmed by DAPI staining. Bar: 50 μm. (b)The Lox activity of prepared matrices before and after the DOC treatment was analyzed. The Lox activity was significantly decreased in the matrices produced by BAPN-treated cells. DOC treatment did not significantly affect the Lox activity of prepared matrices, regardless of the BAPN concentration. (c) The proliferation of MC cells was significantly increased in the low cross-link density matrices. (d) Alkaline phosphatase (ALP) activity increased in cells seeded on low cross-linked matrices. (e) Gene expression of Cbfa1/Runx2, type I collagen α2 chain (Col1a2), alkaline phosphatase (Alpl), and osteocalcin (Spp1) significantly increased in these matrices at 3 and 7 days of culture. (f) Osteoclasts were cultured on differentially cross-linked matrices for 6 days under the differentiation condition. The number of multi-nuclear tartrate-resistant acid phosphatase (TRAP)-positive cells increased in the low cross-link density matrices. (g) Gene expression of cathepsin K (Ctsk), nuclear factor of activated T-cells cytoplasmic 1 (Nfatc1), and dendritic cell-specific transmembrane protein (DCstamp) increased in low cross-link density matrices. *p < 0.05, compared to the control.</p

Histological and histomorphometric analysis.

(a) Hematoxylin and eosin, and tartrate-resistant acid phosphatase (TRAP)-stained histological sections of the distal femur epiphysis at 0 week (after 8-week of the BAPN consumption). (b) No differences of osteoblast and osteoclasts activities were observed at 0 and 2 weeks after replacing to the control diet. After 4 weeks, number of osteoblasts per bone surface (N.Ob/BS) and osteoblast surface per bone surface (Ob.S/BS), representing osteoblast activity, significantly increased, while number of osteoclasts per bone surface (N.Oc/BS) and osteoclast surface per bone surface (Oc.S/BS), representing osteoclast activity did not change. *p < 0.05, compared to the control. Bar: 50 μm. (c) Picrosirius red-stained samples at 16 weeks of age (after 8-week of the BAPN consumption followed by 4-week of control diet) are analyzed under polarized light. After 4-week of control diet, immature/irregular collagen matrix still detected both in cortical and cancellous bone. Bar: 50 μm. Quantitative data also confirmed that the immature collagens, detected in green, retained high value after 4-week of control diet in cancellous bone.</p