Fig 1.
Neural crest cell-specific Brca1 deletion causes craniofacial bone abnormalities in mice.
(A) Lateral views of control and Brca1:Wnt1-Cre mice at birth (NB). Scale bar = 5mm. (B) Alcian blue- and alizarin red-stained skulls of control and Brca1:Wnt1-Cre mice at birth. Yellow broken lines indicate osteogenic fronts. Note that the frontal bones of Brca1:Wnt1-Cre 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.
Fig 2.
Brca1 is indispensable for osteoblast proliferation and survival at mid-gestation.
(A) Coronal sections of control and Brca1:Wnt1-Cre 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 Brca1:Wnt1-Cre 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 Brca1:Wnt1-Cre 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.
Fig 3.
The Brca1-p53 interaction is critical for craniofacial bone morphogenesis.
(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, Brca1-/-:Wnt1-Cre, Brca1-/-:p53+/-:Wnt1-Cre and Brca1-/-:p53-/-:Wnt1-Cre 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.
Fig 4.
Deletion of p53 partially rescues the skull defects by preventing cell death.
(A) Coronal sections of control, Brca1-/-:Wnt1-Cre and Brca1-/-:p53-/-:Wnt1-Cre 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, Brca1-/-:Wnt1-Cre and Brca1-/-:p53-/-:Wnt1-Cre 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, Brca1-/-:Wnt1-Cre and Brca1-/-:p53-/-:Wnt1-Cre 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.
Fig 5.
Neural crest cell-specific Brca2 deletion results in a craniofacial bone phenotype similar to that of the neural crest cell-specific Brca1 deletion in mice.
(A) Alcian blue- and alizarin red-stained skulls of control and Brca2:Wnt1-Cre 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 Brca2:Wnt1-Cre 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 Brca2:Wnt1-Cre 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.
Table 1.
Primer sequences for Quantitative RT-PCR analysis.