Deletion of Dentin Matrix Protein-1 Leads to a Partial Failure of Maturation of Predentin into Dentin, Hypomineralization, and Expanded Cavities of Pulp and Root Canal during Postnatal Tooth Development

Abstract

The dentin matrix protein-1 (DMP-1) gene is identified in odontoblasts during both embryonic and postnatal development. In vitro study suggests that this noncollagen acidic phosphoprotein plays a role in mineralization. However, deletion of the Dmp-1 gene has little effect on tooth development during embryogenesis. To address the role of DMP-1 in tooth during postnatal development, we analyzed changes of dentinogenesis in Dmp-1 null mice from 3 days after birth to 1 year. Here we show that Dmp-1 null mice postnatally develop a profound tooth phenotype characterized by a partial failure of maturation of predentin into dentin, enlarged pulp chambers, increased width of predentin zone with reduced dentin wall, and hypomineralization. The tooth phenotype of these mice is strikingly similar to that in dentin sialophosphoprotein (Dspp) null mice and shares some features of the human disease dentinogenesis imperfecta III. We have also demonstrated that DSPP levels are reduced in Dmp-1 null mice, suggesting that DSPP is probably regulated by DMP-1 during dentinogenesis. Finally, we show the absence or delayed development of the third molar in Dmp-1 null mice, which is probably secondary to defects in Dmp-1 null bone. Taken together, these studies suggest that DMP-1 is essential for later dentinogenesis during postnatal development. The dentin matrix protein-1 (DMP-1) gene is identified in odontoblasts during both embryonic and postnatal development. In vitro study suggests that this noncollagen acidic phosphoprotein plays a role in mineralization. However, deletion of the Dmp-1 gene has little effect on tooth development during embryogenesis. To address the role of DMP-1 in tooth during postnatal development, we analyzed changes of dentinogenesis in Dmp-1 null mice from 3 days after birth to 1 year. Here we show that Dmp-1 null mice postnatally develop a profound tooth phenotype characterized by a partial failure of maturation of predentin into dentin, enlarged pulp chambers, increased width of predentin zone with reduced dentin wall, and hypomineralization. The tooth phenotype of these mice is strikingly similar to that in dentin sialophosphoprotein (Dspp) null mice and shares some features of the human disease dentinogenesis imperfecta III. We have also demonstrated that DSPP levels are reduced in Dmp-1 null mice, suggesting that DSPP is probably regulated by DMP-1 during dentinogenesis. Finally, we show the absence or delayed development of the third molar in Dmp-1 null mice, which is probably secondary to defects in Dmp-1 null bone. Taken together, these studies suggest that DMP-1 is essential for later dentinogenesis during postnatal development. Dentin is a mineralized tissue that closely resembles bone in composition and mechanism of formation. The mechanisms for mineralization are largely unclear, although two hypotheses are proposed to explain initiating mineralization: matrix vesicles in mantle dentin and collagen-phosphophoryn complexes in circumpulpal dentin (1Garant P.R. Dickson A. Oral Cells and Tissues. Quintessence Publishing, Chicago, IL2003: 25-52Google Scholar). The in vitro studies also suggest that phosphorylated extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; DMP, dentin matrix protein; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; RT, reverse transcriptase; micro-CT, micro-computed tomography. 1The abbreviations used are: ECM, extracellular matrix; DMP, dentin matrix protein; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; RT, reverse transcriptase; micro-CT, micro-computed tomography. proteins localized within collagen gap zones can bind calcium and phosphate ions in an appropriate conformation to nucleate the formation of apatite crystals (2Hunter G.K. Hauschka P.V. Poole A.R. Rosenberg L.C. Goldberg H.A. Biochem. J. 1996; 317: 59-64Google Scholar, 3Glimcher M.J. Anat. Rec. 1989; 224: 139-153Google Scholar). One of the noncollagenous proteins that appears to play an important role in dentin ECM formation and mineralization is dentin matrix protein-1 (DMP-1). DMP-1, an acidic phosphorylated extracellular matrix protein (4George A. Sabsay B. Simonian P.A. Veis A. J. Biol. Chem. 1993; 268: 12624-12630Google Scholar), is expressed in odontoblasts that secrete matrix proteins to form dentin. Using protein chemistry approaches, some progress has been made in identifying the normally processed forms of DMP-1 in mineralized tissues (5Qin C. Brunn J.C. Cook R.G. Orkiszewski R.S. Malone J.P. Veis A. Butler W.T. J. Biol. Chem. 2003; 278: 34700-34708Google Scholar). Although full-length DMP-1 has been cloned and sequenced, the corresponding intact protein has not been isolated from mineralized tissues. However, two proteolytic fragments, a 37-kDa N-terminal fragment and a 57-kDa C-terminal fragment, have been isolated from bone and dentin extracts (5Qin C. Brunn J.C. Cook R.G. Orkiszewski R.S. Malone J.P. Veis A. Butler W.T. J. Biol. Chem. 2003; 278: 34700-34708Google Scholar). Recent studies suggest that DMP-1 can be cleaved by bone morphogenetic protein-1/tolloid-like proteinases (6Steiglitz B.M. Ayala M. Narayanan K. George A. Greenspan D.S. J. Biol. Chem. 2004; 279: 980-986Google Scholar). In vitro studies suggest that overexpression of Dmp-1 induces differentiation of mesenchymal cells to odontoblast-like cells and enhances mineralization (7Narayanan K. Srinivas R. Ramachandran A. Hao J. Quinn B. George A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4516-4521Google Scholar) and that DMP-1 can bind to Ca2+ and initiate mineral deposition in vitro (8He G. Dahl T. Veis A. George A. Nat. Mater. 2003; 2: 552-558Google Scholar). However, effects of recombinant DMP-1 on in vitro mineralization are controversial and depend on the phosphorylation status of DMP-1. 2Tartaix, P. H., Doulaverakis, M., George, A., Fisher, L. W., Butler, W. T., Qin, C., Salih, E., Tan, M., Fujimoto, Y., Spevak, L., and Boskey, A. L. (February 9, 2004) J. Biol. Chem. 10.1074/jbc.M314114200. 2Tartaix, P. H., Doulaverakis, M., George, A., Fisher, L. W., Butler, W. T., Qin, C., Salih, E., Tan, M., Fujimoto, Y., Spevak, L., and Boskey, A. L. (February 9, 2004) J. Biol. Chem. 10.1074/jbc.M314114200. In addition, ectopic application of the recombinant DMP-1 showed no apparent effect on mineralization (9Hassan A.H. Evans C.A. Zaki A.M. George A. Connect. Tissue Res. 2003; 44: 30-41Google Scholar). To determine the in vivo role of DMP-1 in dentinogenesis and mineralization, we have cloned and sequenced the Dmp-1 gene (GenBank™ accession number AJ242625) and have generated Dmp-1 null mice, in which exon 6 of Dmp-1 was replaced by a lacZ reporter gene and a neo-cassette (10Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Google Scholar). The expression of Dmp-1, reflected by X-gal staining, is not only identified in odontoblasts but also in pulpal cells, precursor cells of odontoblasts during embryonic development. However, there is no apparent early tooth phenotype observed in the Dmp-1 null embryos and newborns, suggesting that DMP-1 may be redundant or nonessential for early dentinogenesis and mineralization (10Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Google Scholar). The purpose of this study was to determine whether DMP-1 is required for dentinogenesis and mineralization during postnatal development by characterization of the Dmp-1 lacZ knock-in mice from day 3 to 1 year old. Here we show that Dmp-1 null mice postnatally develop a profound tooth phenotype characterized by enlarged pulp chambers, increased width of predentin zone with reduced dentin thickness, and hypomineralization. The tooth phenotype of these mice is strikingly similar to dentin sialophosphoprotein (Dspp) null mice (11Sreenath T. Thyagarajan T. Hall B. Longenecker G. D'Souza R. Hong S. Wright J.T. MacDougall M. Sauk J. Kulkarni A.B. J. Biol. Chem. 2003; 278: 24874-24880Google Scholar) and shares some features of the human dentin disease dentinogenesis imperfecta III. In addition, we demonstrate that DSPP is reduced in Dmp-1 null mice, suggesting that DSPP could be directly or indirectly controlled by DMP-1 during dentinogenesis. Generation of Dmp-1 Null Mice—Mice deficient in Dmp-1 were generated by gene targeting in embryonic stem cells as described previously (10Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Google Scholar). To obtain Dmp-1-deficient teeth, heterozygous Dmp-1 null mice were interbred to generate homozygotes in the C57BL/6 background (>95%) or CD-1 outbred background in the expected Mendelian ratio. Males and females were both fertile. All experiments were performed using a protocol approved by the Institutional Animal Care and Use Committees of University of Missouri-Kansas City and NIEHS, National Institutes of Health, laboratory animal facilities. Tail PCR Genotyping—Genotyping of Dmp-1 null mice was determined by PCR analysis of genomic DNA extracted from tail with primers p01 (5′-CTTGACTTCAGGCAAATAGTGACC-3′) and p02 (5′-GCGGAATTCGATAGCTTGGCTG-3′) to detect the targeted allele (280 bp) and primers p01 (5′-CTTGACTTCAGGCAAATAGTGACC-3′) and 5′-CTGTTCCTCACTCTCACTGTGC-3′ to detect the wild-type allele (410 bp). β-Galactosidase (lacZ) Expression Assay—β-Galactosidase staining was assessed from 5-week-old heterozygous jaw using the method described previously (12Zhang J. Tan X. Contag C.H. Lu Y. Guo D. Harris S.E. Feng J.Q. Biochem. Biophys. Res. Commun. 2002; 293: 1412-1419Google Scholar). Briefly, mandibles were fixed by immersion in ice-cold 4% paraformaldehyde for 30 min, and then washed three times with phosphate-buffered saline for 5 min each. The specimens were then stained overnight in freshly prepared X-gal solution (1 mg/ml) at 32 °C, followed by refixation, decalcification, paraffin-embedding, sectioning, and counterstaining. In Situ Hybridization—The digoxigenin-labeled Dmp-1 cRNA probe from exon 6 was prepared by using an RNA Labeling Kit (Roche Applied Science). Preparation of a 0.6-kb mouse Dmp-1 RNA probe and in situ hybridization on paraffin sections were carried out essentially as described previously (13Feng J.Q. Zhang J. Dallas S.L. Lu Y. Chen S. Tan X. Owen M. Harris S.E. MacDougall M. J. Bone Miner. Res. 2002; 17: 1822-1831Google Scholar). Digoxigenin-labeled Dmp-1 probe was detected in an enzyme-linked immunoassay with a specific anti-digoxigenin-AP antibody conjugate and the color substrates nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate according to the manufacturer's instructions (14Thompson D.L. Sabbagh Y. Tenenhouse H.S. Roche P.C. Drezner M.K. Salisbury J.L. Grande J.P. Poeschla E.M. Kumar R. J. Bone Miner. Res. 2002; 17: 311-320Google Scholar). Tooth Isolation and High Resolution Radiography—For better imaging of the teeth on radiographs, fresh mandibles dissected from wild-type and Dmp-1 null mice were incubated in lysis buffer for on the of the teeth were the mandibles were washed in phosphate-buffered and were extracted using a and isolated were then on a with a of from wild-type and Dmp-1 null mice were with a also to as were with using a at and from and Dmp-1 null mice were dissected and fixed in paraformaldehyde and in buffer solution at for and then to buffer in of the were a and on with the with and with Preparation of from and Dmp-1 null mice were dissected and fixed in 4% paraformaldehyde at followed by in solution 3 and then in and at of DSPP and J.T. Scholar) were by from National Institutes of was performed on paraffin and the sections were in to and with 1 for 30 min at were then with at for The DSPP or antibody was at a of or in phosphate-buffered and the sections were incubated overnight at sections were with antibody at a of and then incubated at for The sections were washed and incubated with the at for The was used to were with and on were by the antibody with RNA and from wild-type and Dmp-1 null mice were the RNA was extracted by using and 30 of were performed using according to the manufacturer's The primers used in the study are the Dmp-1 and reverse and reverse and and reverse Expression of Dmp-1 in during the for studies of DMP-1 in the expression of Dmp-1 in embryonic teeth were in situ a Dmp-1 was observed in the at In addition, a of Dmp-1 was in pulpal precursor cells of as as a Dmp-1 expression in the we whether Dmp-1 is expressed postnatally in teeth using the Dmp-1 lacZ knock-in mice a lacZ neo-cassette was used to exon 6 of the Dmp-1 gene (10Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Google Scholar). The of this is that lacZ expression Dmp-1 this reporter gene is the of the Dmp-1 (10Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Google Scholar). The mice two of this be null for the Dmp-1 which was used for studies of tooth phenotype in mice that the Dmp-1 gene The molar of 5-week-old heterozygous mouse was for Dmp-1 Dmp-1 lacZ is detected in both and Dmp-1 Null an in Dentin and we showed that Dmp-1 null embryos or and could not be from by and (10Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Google Scholar). To determine the role of DMP-1 in postnatal tooth development, we the mineralization and of the Dmp-1 null teeth from birth to 1 year of To changes analysis and were were observed wild-type and heterozygous mice, suggesting that of a of the Dmp-1 gene or of a DMP-1 protein has no effect on tooth development. However, Dmp-1 null and were by and staining in Dmp-1 null teeth are The of predentin dentin dentin mineralized dentin and of mineralized by were reduced in Dmp-1 null In the mineral in bone appears increased in the Dmp-1 null with and Dentin in Dmp-1 Null during address the in vivo of DMP-1 in mineralization of the from the wild-type and Dmp-1 null mice were from the for from 3 to in there is no apparent in tooth and the wild-type and the Dmp-1 null mice, suggesting that DMP-1 has no effect on tooth formation. The in mineralization was by a in of the of tooth 1 of the Dmp-1 null mice showed an in both the pulp and the similar was also in Dmp-1 null at the of and and by the we analyzed changes in mineralization and dentin using and that of mineralized tissues. dentin is characterized by the of dentin by a of dentin and of and In little mineral was in the Dmp-1 null dentin and suggesting that DMP-1 is required for mineralization during postnatal dentinogenesis. In addition, the dentin is with an increased The of the is replaced by a and dentin in Dmp-1 null mice is not that the matrix is the or the of a in mineralization during dentinogenesis. of into Dentin in Dmp-1 Null the ECM is predentin mineral is later and to dentin, a mineralized To better increased pulp and reduced dentin in Dmp-1 null mice we a of analysis in both molar and at sections from and show that the predentin zone is the dentin zone is in the Dmp-1 null mice with the may in the increased of the pulp and the as as the reduced of the dentin observed by In addition, we have also the in Dmp-1 null mice, which is with that in the mice that a of Dmp-1 is expressed by that The of a in in Dmp-1 Null to DMP-1, there is important noncollagenous protein in dentin, DSPP M. D. X. J. Feng J. J. Biol. Chem. Scholar, J.Q. X. J. D. T. Kulkarni A.B. D'Souza C.A. MacDougall M. J. Biol. Chem. Scholar). DMP-1 and DSPP are of the of phosphorylated B. Biochem. Biophys. Res. Commun. 2001; Scholar, B. J. Biol. Chem. Scholar). a similar genomic and are in the on the are and both to be processed into N-terminal and C-terminal (5Qin C. Brunn J.C. Cook R.G. Orkiszewski R.S. Malone J.P. Veis A. Butler W.T. J. Biol. Chem. 2003; 278: 34700-34708Google Scholar, B.M. Ayala M. Narayanan K. George A. Greenspan D.S. J. Biol. Chem. 2004; 279: 980-986Google Scholar). null mice a strikingly similar phenotype in teeth as observed in Dmp-1 null reduced of dentin, increased of enlarged pulp and hypomineralization. we whether there was a DMP-1 and DSPP during postnatal dentinogenesis. we DSPP protein in Dmp-1 null from 1 to in with a antibody the mouse In the wild-type a DSPP is detected in both the dentin and the a of DSPP is observed in Dmp-1 null molar we whether this was by a of DSPP expression or an in of DSPP in mice the method at 30 we the in Dmp-1 null teeth with that in the in are reduced in Dmp-1 null teeth, suggesting a of is by which showed a of in Dmp-1 null with the We that DSPP can be regulated directly or indirectly by Dmp-1 and that some of dentinogenesis defects in Dmp-1 null mice are probably by a of DSPP with the Dentin in Dmp-1 Null of the is by of the and of mineral crystals in vitro J. Dent. Res. Scholar). null mice and bone T. P. Longenecker G. S. J. A. A.M. B. K. P. C. Kulkarni A.B. Nat. Scholar), ectopic is observed in and null mice L. D. K. A. T. J. 2002; Scholar). To address whether there is a in expression in Dmp-1 null mice, a antibody to mouse was used for of the from and mice of in the predentin of Dmp-1 null mice was observed in the which has been in null mice (11Sreenath T. Thyagarajan T. Hall B. Longenecker G. D'Souza R. Hong S. Wright J.T. MacDougall M. Sauk J. Kulkarni A.B. J. Biol. Chem. 2003; 278: 24874-24880Google Scholar). However, there was no in not suggesting that there is no in in Dmp-1 null The in Dmp-1 Null null phenotype appears postnatally and formation of the third molar is during the after We a effect of the absence of Dmp-1 on development of the third molar from to 1 year in in the third molar is or delayed in development in Dmp-1 null on analysis Dmp-1 null mice with of Dmp-1 null mice this In the defects described a The mechanism is and is odontoblasts secrete extracellular a precursor of dentin mineralized predentin the mineralization and the the predentin is to the mineralized tissue apatite crystals are within and collagen (1Garant P.R. Dickson A. Oral Cells and Tissues. Quintessence Publishing, Chicago, IL2003: 25-52Google Scholar, W.T. H. J. Biol. Scholar, A. Oral and Scholar). mechanisms that the and of apatite formation. In the of formation of the precursor be the as that of mineralization. of these to as of the predentin and in the dentin as observed in some in some human dentin Oral Scholar). Although the mechanisms for this are largely noncollagenous proteins in dentin have been proposed to be for this during dentinogenesis W.T. H. J. Biol. Scholar). In this we in vivo to DMP-1 in of dentinogenesis with a Dmp-1 lacZ knock-in null animal as described Dmp-1 is expressed in dentin after birth and and Dmp-1 null defects with and of dentin at day 3 after birth enlarged pulp chambers, and in the dentin the Dmp-1 null mouse although are only to 1 year 3 and suggesting that a of a in later of dentinogenesis. a in maturation of predentin to dentin, as reflected by an predentin zone and reduced of dentin, is in both and in the the dentin is and with little mineral in suggesting that DMP-1 is required for both the and in dentin. All of the phenotype is suggesting that of the can for the role of DMP-1 in later of dentinogenesis and mineralization. there is no apparent phenotype observed in Dmp-1 null embryos or newborns, although Dmp-1 is in the during this (10Feng J.Q. Huang H. Lu Y. Ye L. Xie Y. Tsutsui T.W. Kunieda T. Castranio T. Scott G. Bonewald L.B. Mishina Y. J. Dent. Res. 2003; 82: 776-780Google Scholar). is of that mineralization in dentin, DMP-1 plays a after of mineralization in dentin and after birth may explain for defects observed in later of dentinogenesis. To determine and are in dentinogenesis could be in the Dmp-1 null mice, we have and matrix proteins proteins and 1 bone and by both and this there is little in Dmp-1 null mice not for a in DSPP and the of in dentin matrix The of these is described as DMP-1, is a of the and expressed in odontoblasts during dentinogenesis A. G. J. T. Kulkarni A.B. MacDougall M. J. Bone Miner. Res. Scholar). are on the and similar and genomic DNA features J.Q. X. J. D. T. Kulkarni A.B. D'Souza C.A. MacDougall M. J. Biol. Chem. Scholar, B. Biochem. Biophys. Res. Commun. 2001; Scholar, B. J. Biol. Chem. Scholar). In vitro studies have that dentin a cleaved of is in mineralization in vitro M. S. Sabsay B. Veis A. Bone Miner. Scholar, A. A. Tissue 1989; 44: Scholar). in the human DSPP gene are with the disease dentinogenesis imperfecta X. J. C. S. C. P. G. B. Y. Nat. 2001; Scholar, S. C. X. W. Y. L. G. M. J. Y. L. B. Huang W. J. Chen G. X. Nat. 2001; Scholar). Finally, both Dmp-1 null mice and null mice (11Sreenath T. Thyagarajan T. Hall B. Longenecker G. D'Souza R. Hong S. Wright J.T. MacDougall M. Sauk J. Kulkarni A.B. J. Biol. Chem. 2003; 278: 24874-24880Google Scholar) a similar in later of suggesting a these two Here we show that DSPP is reduced in Dmp-1 null mice at both the and protein levels is in with an in vitro study by Narayanan (7Narayanan K. Srinivas R. Ramachandran A. Hao J. Quinn B. George A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4516-4521Google Scholar), in which overexpression of Dmp-1 also DMP-1 in the K. Ramachandran A. Hao J. G. M. George A. J. Biol. Chem. 2003; 278: Scholar). Taken together, DSPP is probably of the targeted by DMP-1, and changes in DSPP may in some of the defects observed in Dmp-1 null mice for of However, null mice not show tooth phenotype of (11Sreenath T. Thyagarajan T. Hall B. Longenecker G. D'Souza R. Hong S. Wright J.T. MacDougall M. Sauk J. Kulkarni A.B. J. Biol. Chem. 2003; 278: 24874-24880Google Scholar). null mice a of pulp a not in Dmp-1 null suggests that the absence of DMP-1 or changes of to of DMP-1 may play a role in the development of defects in Dmp-1 null dentinogenesis. in the in Dmp-1 null predentin matrix are probably of levels in mineralized tissues is a of the and of mineral crystals in vitro J. Dent. Res. Scholar). of to and bone T. P. Longenecker G. S. J. A. A.M. B. K. P. C. Kulkarni A.B. Nat. Scholar) or to ectopic with L. D. K. A. T. J. 2002; Scholar). In null mice, both by and protein by levels were increased (11Sreenath T. Thyagarajan T. Hall B. Longenecker G. D'Souza R. Hong S. Wright J.T. MacDougall M. Sauk J. Kulkarni A.B. J. Biol. Chem. 2003; 278: 24874-24880Google Scholar). (11Sreenath T. Thyagarajan T. Hall B. Longenecker G. D'Souza R. Hong S. Wright J.T. MacDougall M. Sauk J. Kulkarni A.B. J. Biol. Chem. 2003; 278: 24874-24880Google Scholar) demonstrated the increased and of by in null In there is no in the of not the of protein in Dmp-1 null predentin suggests that the of DMP-1 is required for of in predentin matrix directly or In addition, there is no in or protein levels of in Dmp-1 null mice not that DMP-1 and DSPP may have in dentinogenesis. One of the in this study is that the third molar is or in some of Dmp-1 null mice Tooth is a and has been in some to in or H. Nat. 1996; Scholar, P. M. D'Souza Nat. Scholar, M.J. M. Nat. Scholar, Guo A. L. Evans B. T. D. D'Souza J. Dent. Res. 2002; Scholar, A.R. J. Dent. Res. 2003; 82: Scholar). of or to tooth in mice R. Nat. Scholar, H. A. K. R. Scholar), suggesting that these early are required for tooth formation and In Dmp-1 is in the a and deletion of Dmp-1 has no effect on tooth and 3 and tooth only for of Dmp-1 null mice in which bone defects suggesting that this is probably secondary to that of the bone In by Dmp-1 null mice, studies demonstrated that DMP-1 is required for mineralization and maturation of predentin into dentin during later of dentinogenesis. The Dmp-1 tooth phenotype is and in both background and outbred CD-1 background not that tooth defects observed in Dmp-1 null mice are of In this we have also a effect of the neo-cassette on the Dmp-1 null tooth the tooth phenotype is in Dmp-1 null mice after of the neo-cassette by with mice not The defects could not be by a calcium not suggesting that the effect of DMP-1 on mineralization is mechanism studies show that DSPP can be directly or indirectly controlled by DMP-1. However, of DSPP can only explain in the defects in Dmp-1 null We for mice and Kunieda and W. Tsutsui for of animal We and for and on the We also for and