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• W2151001008 abstract "Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/− mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2−/− mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2−/− roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context. Signaling pathways that underlie postnatal dental and periodontal physiopathology are less studied than those of early tooth development. Members of the muscle segment homeobox gene (Msx) family encode homeoproteins that show functional redundancy during development and are known to be involved in epithelial-mesenchymal interactions that lead to crown morphogenesis and ameloblast cell differentiation. This study analyzed the MSX2 protein during mouse postnatal growth as well as in the adult. The analysis focused on enamel and periodontal defects and enamel proteins in Msx2-null mutant mice. In the epithelial lifecycle, the levels of MSX2 expression and enamel protein secretion were inversely related. Msx2+/− mice showed increased amelogenin expression, enamel thickness, and rod size. Msx2−/− mice displayed compound phenotypic characteristics of enamel defects, related to both enamel-specific gene mutations (amelogenin and enamelin) in isolated amelogenesis imperfecta, and cell-cell junction elements (laminin 5 and cytokeratin 5) in other syndromes. These effects were also related to ameloblast disappearance, which differed between incisors and molars. In Msx2−/− roots, Malassez cells formed giant islands that overexpressed amelogenin and ameloblastin that grew over months. Aberrant expression of enamel proteins is proposed to underlie the regional osteopetrosis and hyperproduction of cellular cementum. These enamel and periodontal phenotypes of Msx2 mutants constitute the first case report of structural and signaling defects associated with enamel protein overexpression in a postnatal context. Msx2 is a member of the Muscle segment homeobox gene (Msx) family, orthologous to the Msh gene of Drosophila.1Alappat S Zhang ZY Chen YP Msx homeobox gene family and craniofacial development.Cell Res. 2003; 13: 429-442Crossref PubMed Scopus (194) Google Scholar The mammalian Msx1 and Msx2 genes share highly conserved sequences.2Catron KM Wang H Hu G Shen MM Abate-Shen C Comparison of MSX-1 and MSX-2 suggests a molecular basis for functional redundancy.Mech Dev. 1996; 55: 185-199Crossref PubMed Scopus (113) Google Scholar Their encoded homeoproteins show functional redundancy during development2Catron KM Wang H Hu G Shen MM Abate-Shen C Comparison of MSX-1 and MSX-2 suggests a molecular basis for functional redundancy.Mech Dev. 1996; 55: 185-199Crossref PubMed Scopus (113) Google Scholar, 3Berdal A Molla M Hotton D Aioub M Lezot F Nefussi JR Goubin G Differential impact of MSX1 and MSX2 homeogenes on mouse maxillofacial skeleton.Cells Tissues Organs. 2009; 189: 126-132Crossref PubMed Scopus (18) Google Scholar. They act as transcriptional repressors which bind specific DNA sequences.4Catron KM Iler N Abate C Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.Mol Cell Biol. 1993; 13: 2354-2365Crossref PubMed Scopus (177) Google Scholar In several early developmental processes, MSX2 signaling pathways have been elucidated. These-ones include: cranial suture closing, epithelial–mesenchymal interactions leading to crown morphogenesis,5Satokata I Ma L Ohshima H Bei M Woo I Nishizawa K Maeda T Takano Y Uchiyama M Heaney S Peters H Tang Z Maxson R Maas R Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.Nat Genet. 2000; 24: 391-395Crossref PubMed Scopus (623) Google Scholar and ameloblast cell differentiation.6Aioub M Lezot F Molla M Castaneda B Robert B Goubin G Nefussi JR Berdal A Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodontal osteopetrosis.Bone. 2007; 41: 851-859Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 7Bei M Stowell S Maas R Msx2 controls ameloblast terminal differentiation.Dev Dyn. 2004; 231: 758-765Crossref PubMed Scopus (52) Google Scholar A gain-of-function mutation in the Msx2 homeobox-gene sequence induces premature suture fusion (Boston-type 2 craniosynostosis; OMIM 604757).8Jabs EW Muller U Li X Ma L Luo W Haworth IS Klisak I Sparkes R Warman ML Mulliken JB et al.A mutation in the homeodomain of the human MSX2 gene in a family affected with autosomal dominant craniosynostosis.Cell. 1993; 75: 443-450Abstract Full Text PDF PubMed Scopus (580) Google Scholar Conversely, a mutation leading to reduced MSX2 DNA-binding is associated with impaired suture closing in the parietal foramina (OMIM 168500).9Wuyts W Reardon W Preis S Homfray T Rasore-Quartino A Christians H Willems PJ Van Hul W Identification of mutations in the MSX2 homeobox gene in families affected with foramina parietalia permagna.Hum Mol Genet. 2000; 9: 1251-1255Crossref PubMed Scopus (65) Google Scholar Another mutation, in the homeodomain-coding region, has been described in a family with amelogenesis imperfecta and impaired tooth eruption.10Suda N Kitahara Y Ohyama K A case of amelogenesis imperfecta, cleft lip and palate and polycystic kidney disease.Orthod Craniofac Res. 2006; 9: 52-56Crossref PubMed Scopus (21) Google Scholar Different mouse models (null mutant mice,5Satokata I Ma L Ohshima H Bei M Woo I Nishizawa K Maeda T Takano Y Uchiyama M Heaney S Peters H Tang Z Maxson R Maas R Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.Nat Genet. 2000; 24: 391-395Crossref PubMed Scopus (623) Google Scholar transgenic mice bearing overexpression constructs11Wu LY Li M Hinton DR Guo L Jiang S Wang JT Zeng A Xie JB Snead M Shuler C Maxson Jr, RE Liu YH Microphthalmia resulting from MSX2-induced apoptosis in the optic vesicle.Invest Ophthalmol Vis Sci. 2003; 44: 2404-2412Crossref PubMed Scopus (44) Google Scholar and gene mutations12Liu YH Kundu R Wu L Luo W Ignelzi Jr, MA Snead ML Maxson Jr, RE Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.Proc Natl Acad Sci USA. 1995; 92: 6137-6141Crossref PubMed Scopus (203) Google Scholar, 13Winograd J Reilly MP Roe R Lutz J Laughner E Xu X Hu L Asakura T vander Kolk C Strandberg JD Semenza GL Perinatal lethality and multiple craniofacial malformations in MSX2 transgenic mice.Hum Mol Genet. 1997; 6: 369-379Crossref PubMed Scopus (99) Google Scholar) mimic the multiple craniofacial anomalies: precocious fusion of cranial bones, development of ectopic cranial bone,12Liu YH Kundu R Wu L Luo W Ignelzi Jr, MA Snead ML Maxson Jr, RE Premature suture closure and ectopic cranial bone in mice expressing Msx2 transgenes in the developing skull.Proc Natl Acad Sci USA. 1995; 92: 6137-6141Crossref PubMed Scopus (203) Google Scholar or contrarily, persistent calvaria foramina and dental defects,5Satokata I Ma L Ohshima H Bei M Woo I Nishizawa K Maeda T Takano Y Uchiyama M Heaney S Peters H Tang Z Maxson R Maas R Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation.Nat Genet. 2000; 24: 391-395Crossref PubMed Scopus (623) Google Scholar and reduction of bone ossification13Winograd J Reilly MP Roe R Lutz J Laughner E Xu X Hu L Asakura T vander Kolk C Strandberg JD Semenza GL Perinatal lethality and multiple craniofacial malformations in MSX2 transgenic mice.Hum Mol Genet. 1997; 6: 369-379Crossref PubMed Scopus (99) Google Scholar and resorption.6Aioub M Lezot F Molla M Castaneda B Robert B Goubin G Nefussi JR Berdal A Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodontal osteopetrosis.Bone. 2007; 41: 851-859Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar The present investigation focuses on the physiopathology of enamel and periodontal tissues in a knockin transgenic mouse model.6Aioub M Lezot F Molla M Castaneda B Robert B Goubin G Nefussi JR Berdal A Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodontal osteopetrosis.Bone. 2007; 41: 851-859Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar MSX2 target-genes were first identified in osteoblasts.14Cheng SL Shao JS Cai J Sierra OL Towler DA Msx2 exerts bone anabolism via canonical Wnt signaling.J Biol Chem. 2008; 283: 20505-20522Crossref PubMed Scopus (79) Google ScholarIn vitro, the transcriptional repressor activity of MSX2 was shown to be mediated through interactions with distinct transcriptional factors such as DLX homeoproteins for osteocalcin15Newberry EP Latifi T Towler DA Reciprocal regulation of osteocalcin transcription by the homeodomain proteins Msx2 and Dlx5.Biochemistry. 1998; 37: 16360-16368Crossref PubMed Scopus (123) Google Scholar and the CCAAT/enhancer-binding protein α (C/EBPα) for amelogenin.16Xu Y Zhou YL Erickson RL Macdougald OA Snead ML Physical dissection of the CCAAT/enhancer-binding protein alpha in regulating the mouse amelogenin gene.Biochem Biophys Res Commun. 2007; 354: 56-61Crossref PubMed Scopus (17) Google Scholar In osteoblasts, MSX2 exerts either positive or negative effects on osteocalcin transcriptional activity depending on the stage of osteoblast differentiation and thus diverse MSX2 transcriptional partners.17Cheng SL Shao JS Charlton-Kachigian N Loewy AP Towler DA MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors.J Biol Chem. 2003; 278: 45969-45977Crossref PubMed Scopus (302) Google Scholar, 18Ichida F Nishimura R Hata K Matsubara T Ikeda F Hisada K Yatani H Cao X Komori T Yamaguchi A Yoneda T Reciprocal roles of MSX2 in regulation of osteoblast and adipocyte differentiation.J Biol Chem. 2004; 279: 34015-34022Crossref PubMed Scopus (163) Google Scholar, 19Lee MH Kim YJ Yoon WJ Kim JI Kim BG Hwang YS Wozney JM Chi XZ Bae SC Choi KY Cho JY Choi JY Ryoo HM Dlx5 specifically regulates Runx2 type II expression by binding to homeodomain-response elements in the Runx2 distal promoter.J Biol Chem. 2005; 280: 35579-35587Crossref PubMed Scopus (164) Google Scholar Therefore, the impact of MSX2 depends on the cellular circumstances. Recent in vivo studies addressed the role of MSX2 in late postnatal growth and adult homeostasis.3Berdal A Molla M Hotton D Aioub M Lezot F Nefussi JR Goubin G Differential impact of MSX1 and MSX2 homeogenes on mouse maxillofacial skeleton.Cells Tissues Organs. 2009; 189: 126-132Crossref PubMed Scopus (18) Google Scholar, 6Aioub M Lezot F Molla M Castaneda B Robert B Goubin G Nefussi JR Berdal A Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodontal osteopetrosis.Bone. 2007; 41: 851-859Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar These studies demonstrated additional roles for MSX2 in physiological regulation of postnatal mineralized tissue formation, modeling and homeostasis. In addition to enamel and dentin dysplasia and altered root formation, these mutant mice exhibit regional osteopetrosis resulting from site-specific down-regulation of RANKL expression in the alveolar bone6Aioub M Lezot F Molla M Castaneda B Robert B Goubin G Nefussi JR Berdal A Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodontal osteopetrosis.Bone. 2007; 41: 851-859Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar which mechanisms are not understood. In addition to osteocalcin and amelogenin, other enamel matrix proteins constitute candidate MSX2 targets. Their aberrant expression is hypothesized here to underlie the broad dento-alveolar spectrum of alterations associated with Msx2 mutation. Indeed, enamel proteins could play a dual role in mineralized tissues, acting both as a structural scaffold for amelogenesis and as signaling molecules for mineralized tissue formation and resorption. Amelogenin proteins self-assemble into nanospheres and coils which serve as an enamel scaffold.20Moradian-Oldak J Amelogenins: assembly, processing and control of crystal morphology.Matrix Biol. 2001; 20: 293-305Crossref PubMed Scopus (181) Google Scholar These transient self-assemblies run throughout the enamel layer and guide apatite crystal formation and growth.21Moradian-Oldak J Du C Falini G On the formation of amelogenin microribbons.Eur J Oral Sci. 2006; 114 (discussion 327–329, 382): 289-296Crossref PubMed Scopus (55) Google Scholar, 22Fowler CE Beniash E Yamakoshi Y Simmer JP Margolis HC Co-operative mineralization and protein self-assembly in amelogenesis: silica mineralization and assembly of recombinant amelogenins in vitro.Eur J Oral Sci. 2006; 114 (discussion 327–329, 382): 297-303Crossref PubMed Scopus (10) Google Scholar Amelogenin null mice show a significant reduction of enamel thickness which is in addition devoid of prismatic pattern.23Gibson CW Yuan ZA Hall B Longenecker G Chen E Thyagarajan T Sreenath T Wright JT Decker S Piddington R Harrison G Kulkarni AB Amelogenin-deficient mice display an amelogenesis imperfecta phenotype.J Biol Chem. 2001; 276: 31871-31875Crossref PubMed Scopus (380) Google Scholar Two other enamel proteins, enamelin and ameloblastin, also cooperate in enamel morphogenesis, as shown by their extracellular distribution in forming enamel and the phenotype of their respective null mutants.24Fukumoto S Kiba T Hall B Iehara N Nakamura T Longenecker G Krebsbach PH Nanci A Kulkarni AB Yamada Y Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts.J Cell Biol. 2004; 167: 973-983Crossref PubMed Scopus (293) Google Scholar, 25Hu JC Hu Y Smith CE McKee MD Wright JT Yamakoshi Y Papagerakis P Hunter GK Feng JQ Yamakoshi F Simmer JP Enamel defects and ameloblast-specific expression in Enam knock-out/lacz knock-in mice.J Biol Chem. 2008; 283: 10858-10871Crossref PubMed Scopus (131) Google Scholar On the other hand, dentin sialophosphoprotein (DSPP) is transiently expressed in presecretion-stage ameloblasts and initiates enamel biomineralization.26MacDougall M Nydegger J Gu TT Simmons D Luan X Cavender A D'Souza RN Developmental regulation of dentin sialophosphoprotein during ameloblast differentiation: a potential enamel matrix nucleator.Connect Tissue Res. 1998; 39 (discussion 63–67): 25-37Crossref PubMed Scopus (43) Google Scholar Therefore, these four enamel proteins (amelogenin, enamelin, amelobastin, and DSPP) cooperate in the control of biomineralization events and are essential for enamel morphogenesis. Isolated amelogenesis imperfecta, which comprises a family of hereditary diseases affecting enamel structure, including hypoplastic, hypomaturation, and hypomineralization types, is associated with mutation of the corresponding genes in humans.27Wright JT The molecular etiologies and associated phenotypes of amelogenesis imperfect.Am J Med Genet A. 2006; 140: 2547-2555Crossref PubMed Scopus (107) Google Scholar Besides their role as structural proteins, amelogenin and ameloblastin isoforms are proposed to play a role in cell signaling.28Gruenbaum-Cohen Y Tucker AS Haze A Shilo D Taylor AL Shay B Sharpe PT Mitsiadis TA Ornoy A Blumenfeld A Deutsch D Amelogenin in cranio-facial development: the tooth as a model to study the role of amelogenin during embryogenesis.J Exp Zool B Mol Dev Evol. 2009, Jul 15; 312B: 445-457Crossref PubMed Scopus (24) Google Scholar Amelogenin peptides were shown to induce bone-cell differentiation via Runx-2 pathway activation in vivo and in vitro.29Veis A Tompkins K Alvares K Wei K Wang L Wang XS Brownell AG Jengh SM Healy KE Specific amelogenin gene splice products have signaling effects on cells in culture and in implants in vivo.J Biol Chem. 2000; 275: 41263-41272Crossref PubMed Scopus (204) Google Scholar, 30Warotayanont R Zhu D Snead ML Zhou Y Leucine-rich amelogenin peptide induces osteogenesis in mouse embryonic stem cells.Biochem Biophys Res Commun. 2008; 367: 1-6Crossref PubMed Scopus (51) Google Scholar Amelogenin null mutants show regional hyperesorption mediated by activation of RANKL-RANK pathway.31Hatakeyama J Sreenath T Hatakeyama Y Thyagarajan T Shum L Gibson CW Wright JT Kulkarni AB The receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenic pathway is elevated in amelogenin-null mice.J Biol Chem. 2003; 278: 35743-35748Crossref PubMed Scopus (78) Google Scholar, 32Hatakeyama J Philp D Hatakeyama Y Haruyama N Shum L Aragon MA Yuan Z Gibson CW Sreenath T Kleinman HK Kulkarni AB Amelogenin-mediated regulation of osteoclastogenesis, and periodontal cell proliferation and migration.J Dent Res. 2006; 85: 144-149Crossref PubMed Scopus (56) Google Scholar Interestingly, the amelogenin gene was one of the first identified MSX2 target-genes for which transcriptional repression was deciphered at the molecular level.33Zhou YL Snead ML Identification of CCAAT/enhancer-binding protein alpha as a transactivator of the mouse amelogenin gene.J Biol Chem. 2000; 275: 12273-12280Crossref PubMed Scopus (71) Google Scholar While this in vitro MSX2 action has been well documented, no in vivo studies have been performed regarding the relationship between MSX2 expression and enamel proteins. Based on MSX2 transcriptional repression of the amelogenin gene established in vitro and the potential functions of enamel proteins, the purpose of this study was to scrutinize enamel proteins in vivo in Msx2 mutants mice. Postnatal phenotypes were compared in Msx2+/+ (wild type), Msx2+/− (heterozygous) and Msx2−/− (homozygous) mice. MSX2 and enamel proteins were analyzed in the dental epithelium throughout its life cycle: enamel organ during amelogenesis, Hertwig's root sheath during root formation, and Malassez remnants in the adult periodontium. A inverse relationship was observed between Msx2 gene dosage and expression levels of enamel-related genes in the dental epithelium. Our data revealed the existence of a beneficial impact of loss of a single Msx2 allele on enamel formation in Msx2+/− mice which was not previously described. In Msx2−/− mice, the data suggest a parallel relationship between epithelial amelogenin and ameloblastin levels and alveolar bone and cementum homeostasis. Knockin mice were produced by insertion of the bacterial lacZ gene within the Msx2 gene, replacing the coding sequence.6Aioub M Lezot F Molla M Castaneda B Robert B Goubin G Nefussi JR Berdal A Msx2 -/- transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodontal osteopetrosis.Bone. 2007; 41: 851-859Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar Heterozygous males and females were mated using a CD1 Swiss genetic background. Their litters were used to compare wild-type (Msx2+/+), transgenic heterozygous (Msx2+/−) and homozygous (Msx2−/−) mice. All experiments were performed in accordance with the French National Consultative Bioethics Committee for Health and Life Science, following the ethical guidelines for animal care. All experiments were performed by staff trained to conduct in vivo studies. Twenty-one 3-month-old mice were used for each group (Msx2+/+, Msx2+/−, and Msx2−/−). Half mandibles were removed, quickly frozen in liquid nitrogen and ground up. Dental epithelial cells of the continuously erupting incisor and alveolar bone were microdissected out under a stereomicroscope. The cervical loops were excised to avoid mesenchymal contamination. Three pools including three different animals per pool were generated. Total RNA was extracted using a Trizol reagent kit (Invitrogen, Cergy-Pontoise, France) according to the manufacturer's protocol. After total RNA quantification, equal amounts of RNA (1 μg) were transcribed into cDNA using SuperScript II RNase H reverse transcriptase (Invitrogen) with oligo-dT and hexanucleotide random primers. Gene expression levels were determined by real-time PCR using specific primers (Table 1). Optimal conditions and cDNA dilutions were determined for each gene. q-PCR experiments were performed with a Roche LightCycler in triplicate using FastStart DNA Master SYBR Green I (Roche, Neuilly-sur-Seine, France).Table 1Primer Sequences and PCR Conditions Used for RT-qPCR AnalysisGeneOligonucleotidesSize (bp)Tm (°C)Amelogenin P15′-GGATCAAGCATCCCTGAGTTTCAAAC-3′37060 P25′-ATCATTGGTTGCTGGGGGATCAC-3′Ameloblastin P15′-CACTTACTATCCTTCCGCAG-3′18158 P25′-GTGCTGACACTTAGACTTGC-3′Enamelin P15′-TCGGAGGGATGTTCTGAAAC-3′23258 P25′-AGGACTTTCAGTGGGTGTGG-3′MSX2 P15′-CCTGAGGAAACACAAGACCA-3′27860 P25′-AGTTGATAGGGAAGGGCAGA-3′Laminin5α3 P15′-GGGTGTGACCAAAAAGTGCT-3′20658 P25′-CATCTTCCAGGGTGACCAGT-3′Cytokeratin 5 P15′-TCAAGAAGCAGTGTGCCAAC-3′21758 P25′-TCCAGCAGCTTCCTGTAGGT-3′DSPP P15′-ACAAGAGTGGGACCCTGTTT-3′39958 P25′-TACAGTGTGGGCGTTACAAC-3′ Open table in a new tab Six mandibles from 3-month-old Msx2+/+, Msx2+/− and Msx2−/− mice were fixed for 24 hours in 4% paraformaldehyde in PBS, pH 7.4. Samples were then cut along the frontal axis into two fragments before the first molar by using a rotating diamond wheel. Previous microscanner analysis was used to validate the cutting axis (perpendicular to incisor axis). Dehydration was performed using increasing concentrations of ethanol for 24 to 48 hours each at 4°C. Each fragment was embedded in calibrated cylinders with clear polyester resin. The first fragment, corresponding to the buccal part of the incisor, was ground through bone emergence using optically controlled monitoring. The second fragment was ground until the mesial surface of the first molar appeared. Sample surfaces were polished with sandpaper of successively decreasing grits. Conditioning of the enamel surface was achieved by etching with 37% phosphoric acid for 10 seconds. Each sample was coated with palladium-gold in a vacuum evaporator and observed with a scanning electron microscope (JSM-6400, JEOL, Tokyo) at 10 kV. The morphology of incisor was used as a criteria to calibrate the section planes based on the established μCT landmarks. Msx2+/+, Msx2+/−, Msx2−/− mouse mandibles (four animals for each group, at ages 1, 2, 3, 6, 12, and 24 weeks) were dissected out, fixed by immersion in 4% paraformaldehyde for 24 hours at 4°C, and rinsed in PBS. Half of the samples were decalcified at 4°C with EDTA (4%) for 4 to 64 weeks, depending on mouse age. Right half-mandibles were rinsed, dehydrated and embedded in paraffin for sectioning. Sections of 7 μm were deparaffinized and rehydrated before being stained with hematoxylin and eosin for histological analysis or subjected to immunohistology. For the left half-mandibles, 10-μm cryostat sections without decalcification were made with a Leica CM 3050S Microtome (Leica, Rueil-Malmaison, France) and deposited onto poly-l-lysine (Sigma)-coated slides, dehydrated in a graded series of ethanol, and stored at −20°C. Amelogenin sense and antisense RNA probes were prepared from full-length cDNA (covering exons 2-3-5-6-7; see Bonass et al34Bonass WA Robinson PA Kirkham J Shore RC Robinson C Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence.Biochem Biophys Res Commun. 1994; 198: 755-763Crossref PubMed Scopus (55) Google Scholar), subcloned into a Bluescript plasmid and linearized with Xhol or Pstl endonucleases. For radioactive in situ hybridization, [35S]UTP-labeled single-stranded antisense and sense probes were synthesized using T7 and T3 RNA polymerase, respectively (Boehringer, Meylan, France).34Bonass WA Robinson PA Kirkham J Shore RC Robinson C Molecular cloning and DNA sequence of rat amelogenin and a comparative analysis of mammalian amelogenin protein sequence divergence.Biochem Biophys Res Commun. 1994; 198: 755-763Crossref PubMed Scopus (55) Google Scholar For nonradioactive in situ hybridization, the same probes were labeled with UTP-digoxigenin (Roche, France). Radioactive in situ hybridization was performed as previously described by Hotton et al35Hotton D Davideau JL Bernaudin JF Berdal A In situ hybridization of calbindin-D 28 k transcripts in undecalcified sections of the rat continuously erupting incisor.Connect Tissue Res. 1995; 32: 137-143Crossref PubMed Scopus (29) Google Scholar on non-decalcified samples. Briefly, cryostat sections were pretreated with proteinase K (Sigma), hybridized with 20 μl of labeled probes containing 60,000 cpm/μl in a humid chamber overnight at 50°C, and washed under high-stringency conditions. The slides were dipped into NTB2 autoradiographic emulsion (Kodak, Paris, France) and exposed from 2 to 4 weeks at 4°C. After developing the film, the sections were stained with hematoxylin, dehydrated, and mounted under a coverslip. The sections were examined and photographed using a DRMB photomicroscope (Leica, France) under bright- and dark-field illumination. For nonradioactive in situ hybridization, sections were deparaffinized, dehydrated, and hybridized overnight with 50 μl of labeled probe (1 ng/μl) in a dark, humid chamber. The reaction was revealed by immunocytochemistry using an anti-digoxigenin alkaline phosphatase-conjugated polyclonal antibody (Vector NovaRED; Abscyss, Paris, France). The sections were then stained with Mayer's hematoxylin and dehydrated before being mounted under a coverslip and photographed. Deparaffined and rehydrated sections were incubated for 30 minutes in 3% H2O2/PBS to quench endogenous peroxidase activity, and then rinsed for 10 minutes in PBS. Nonspecific protein binding was blocked by incubation for 30 minutes in 10% normal goat serum and 1% bovine serum albumin in PBS. Specimens were incubated for 1 hour at room temperature in a humidified chamber with a polyclonal rabbit anti-mouse β-galactosidase antibody (Genetex), anti-amelogenin (generous gift of Pr. S. Sasaki, Tokyo Medical and Dental University, Department of Biochemistry, diluted at 1:1000) and anti-ameloblastin (generous gift of Pr. Tilmann Wurtz, Centre de Recherche des Cordeliers, Paris, France; diluted at 1:1400). Sections were then washed extensively three times in PBS at room temperature before treatment for 30 minutes at room temperature with the secondary biotin-labeled goat anti-rabbit IgG antibody (StrAviGen Multilink kit, Biogenex, UK) for β-galactosidase, and with horseradish peroxidase goat anti-rabbit IgG antibody (DAKO) for amelogenin and ameloblastin. Subsequently, sections were incubated for 30 minutes at room temperature with peroxidase linked to avidin (Vectastain ABC kit, Vector Laboratories, Burlingame, CA) for β-galactosidase, and with a NovaRED kit (Vector Laboratories) for amelogenin and ameloblastin. After rinsing in PBS, the immunoreactivity was visualized by development for 2 to 10 minutes with 0.1% 3,3-diaminobenzidine and 0.02% H2O2 (DAB substrate kit, Vector Laboratories). For β-galactosidase analysis, sections were counterstained with Mayer's hematoxylin, mounted with permanent mounting medium (XAM, BDH Laboratory, England) and examined by light microscopy. Amelogenin and ameloblastin labeling samples were not counterstained but mounted with aqueous medium (Labonord, Lille). A positive control was performed on untreated mouse teeth and a negative control on mouse oral mucosa. A further negative control experiment that omitted primary antibody was also performed on mouse tooth sections. MSX2 expression was analyzed during the first 4 postnatal months in the mandibular first molar of Msx2+/− mice by using β-galactosidase immunostaining. Developmental stages of ameloblasts have been identified based on their cell morphology. Presecretion stage ameloblasts are short, and are adjoining differentiated mesenchymal cells forming predentin and dentin with no enamel deposition. Secretion stage ameloblast are polygonal and elongated with a large basal nucleus and distal Tomes processes. Enamel deposition is visible. Maturation stage ameloblasts do not possess Tomes process, become abruptly much shorter and reduced in size. The enamel thickness is stable. During amelogenesis (Figure 1), MSX2 expression was stronger in presecretion-stage ameloblasts (Ab PS) (Figure 1D) and in mature, postsecretion-stage ameloblasts (Ab M) (Figure 1I) than in secretion-stage ameloblasts (Ab S) (Figure 1, A, D–H). Figure 1, B and C, show controls without primary antibody. Ab S1, S2, and S3 represent secretion-stage ameloblasts adjoining enamel area with different thicknesses, in the cervical part, the middle to u" @default.
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• W2151001008 date "2010-11-01" @default.
• W2151001008 modified "2023-10-17" @default.
• W2151001008 title "Enamel Protein Regulation and Dental and Periodontal Physiopathology in Msx2 Mutant Mice" @default.
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