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

DataSheet1_Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress.PDF

The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.</p