Enamel Defects and Ameloblast-specific Expression in Enam Knock-out/lacZ Knock-in Mice

Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam+/- mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam-/- mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam-/- mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization. Enamelin is critical for proper dental enamel formation, and defects in the human enamelin gene cause autosomal dominant amelogenesis imperfecta. We used gene targeting to generate a knock-in mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene replacing the Enam translation initiation site and gene sequences through exon 7. Correct targeting of the transgene was confirmed by Southern blotting and PCR analyses. No enamelin protein could be detected by Western blotting in the Enam-null mice. Histochemical 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining demonstrated ameloblast-specific expression of enamelin. The enamel of the Enam+/- mice was nearly normal in the maxillary incisors, but the mandibular incisors were discolored and tended to wear rapidly where they contacted the maxillary incisors. The Enam-/- mice showed no true enamel. Radiography, microcomputed tomography, and light and scanning electron microscopy were used to document changes in the enamel of Enam-/- mice but did not discern any perturbations of bone, dentin, or any other tissue besides the enamel layer. Although a thick layer of enamel proteins covered normal-appearing dentin of unerupted teeth, von Kossa staining revealed almost a complete absence of mineral formation in this protein layer. However, a thin, highly irregular, mineralized crust covered the dentin on erupted teeth, apparently arising from the formation and fusion of small mineralization foci (calcospherites) in the deeper part of the accumulated enamel protein layer. These results demonstrate ameloblast-specific expression of enamelin and reveal that enamelin is essential for proper enamel matrix organization and mineralization. Enamel crystals have a unique shape and organization. They are many times longer than they are wide and are organized into rods (prisms), each comprising about 10,000 individual crystallites (1Daculsi G. Kerebel B. J. Ultrastruct. Res. 1978; 65: 163-172Crossref PubMed Scopus (133) Google Scholar, 2Nanci, A. (ed) (2003) in Ten Cate's Oral Histology Development, Structure, and Function, 6th Ed., pp. 218-224, Mosby, St. Louis, MOGoogle Scholar). Each rod is the product of a single ameloblast in which crystals are formed under the control of specialized enamel proteins secreted by ameloblasts (3Fincham A.G. Moradian-Oldak J. Simmer J.P. J. Struct. Biol. 1999; 126: 270-299Crossref PubMed Scopus (496) Google Scholar). These proteins assemble immediately subjacent to the secretory pole of the ameloblast cell membrane such that mineralization occurs immediately at this cell-matrix interface (4Ronnholm E. J. Ultrastruct. Res. 1962; 6: 249-303Crossref PubMed Scopus (137) Google Scholar). Because enamel crystals grow longer at this mineralization front, the ameloblasts are displaced away from the growing tooth as the enamel layer as a whole thickens. Once the enamel crystals are fully elongated and the enamel layer has reached its final thickness, the secretion of enamel proteins is terminated or greatly reduced, accumulated extracellular proteins are degraded and reabsorbed, and the crystals grow in width and thickness until adjacent crystals come into contact. Thus, the secretion of enamel proteins is associated with the early part of crystal formation, when the crystals are growing primarily in length and the growing crystal tips are in close proximity to the secretory surface of the ameloblast. There are three major secretory stage enamel proteins: amelogenin (5Snead M.L. Zeichner-David M. Chandra T. Robson K.J. Woo S.L. Slavkin H.C. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 7254-7258Crossref PubMed Scopus (93) Google Scholar, 6Snead M.L. Lau E.C. Zeichner-David M. Fincham A.G. Woo S.L. Slavkin H.C. Biochem. Biophys. Res. Commun. 1985; 129: 812-818Crossref PubMed Scopus (177) Google Scholar), ameloblastin (7Krebsbach P.H. Lee S.K. Matsuki Y. Kozak C.A. Yamada K. Yamada Y. J. Biol. Chem. 1996; 271: 4431-4435Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), and enamelin (8Hu C.-C. Fukae M. Uchida T. Qian Q. Zhang C.H. Ryu T. Y. M. Simmer J.P. J. Res. PubMed Scopus Google Scholar, Qian Q. Zhang C.H. Simmer J.P. J. Res. PubMed Scopus Google Scholar). to the secretory gene K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), and are critical for proper dental enamel is the secreted enamel protein and is from the or the and the E.C. Slavkin H.C. M.L. PubMed Scopus Google Scholar, E.C. P.H. K. J. Google Scholar). cause amelogenesis in that the enamel layer of the J. A. PubMed Scopus Google Scholar, T. Simmer J.P. PubMed Scopus Google Scholar). mice amelogenesis B. G. E. T. T. S. G. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Although amelogenin expression has detected in many the Y. J. Sci. PubMed Scopus Google Scholar, A. A. Y. B. Y. E. E. B. S. J.P. J. Sci. PubMed Scopus Google Scholar), no defects in have in with amelogenesis imperfecta. No of amelogenesis by human ameloblastin have but mice enamel S. T. B. T. G. P.H. A. Yamada Y. J. Biol. PubMed Scopus Google Scholar). enamelin gene cause autosomal dominant amelogenesis K. PubMed Scopus Google Scholar, B. K. Simmer J.P. J. Res. PubMed Scopus Google Scholar, Y. Oral Biol. PubMed Scopus Google Scholar). a that a single allele a of amelogenesis defects in the enamel layer Ryu S. G. E. J. PubMed Scopus Google Scholar, E. G. Ryu J. Res. PubMed Scopus Google Scholar). in the mouse enamelin gene have with the and Enam have the site in exon and K. Y. J. A. A. K. J. T. Y. T. S. T. PubMed Scopus Google Scholar, M. U. M. J. Res. PubMed Scopus Google Scholar). The enamel a and enamel surface in mice and enamel in the null to the three secretory are secreted into the enamel matrix used matrix microcomputed scanning electron and Simmer J.P. J. H.C. E.C. 1996; PubMed Scopus Google Scholar, J.P. Fukae M. T. Y. Uchida T. J. H.C. M. J. Res. PubMed Scopus Google Scholar). is secreted in the early stage with and is in the stage Zhang S. Simmer J.P. J. Oral Sci. PubMed Scopus Google Scholar, J. J. and J. C.-C. in of the on and pp. Scholar). in the cause autosomal amelogenesis in Ryu S. J. PubMed Scopus Google Scholar, Simmer J.P. J. PubMed Scopus Google Scholar, Ryu M. E. J. Res. PubMed Scopus Google Scholar). mice have a enamel that is than normal and to the surface J. Y. Simmer J.P. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, E. Lee J. Res. PubMed Scopus Google Scholar). expression to be to Ryu J. Simmer J.P. H.C. Res. Scopus Google Scholar), is in many but is in and in K. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar, Zhang Y. Ryu Simmer J.P. PubMed Scopus Google Scholar, S. M. and E. Chem. Scholar). is that and are for proper dental enamel formation, but they are not in and for about of of amelogenesis Simmer J.P. J. Sci. PubMed Scopus Google Scholar). the of amelogenesis of the proteins in the enamel layer of have and is that not of the major extracellular have J. and J. (2003) in of the on and pp. Scholar). The secretion of with the enamel proteins that enamelin and ameloblastin be from These enamel proteins have and other M. T. Uchida T. M. Res. 1996; PubMed Scopus Google Scholar, C.-C. Fukae M. Uchida T. Qian Q. Zhang C.H. Ryu T. Y. M. Simmer J.P. J. Res. PubMed Scopus Google Scholar), and to generate with the have to in any T. Y. P.H. Simmer J.P. J. Res. PubMed Scopus Google Scholar). is to the secretory stage extracellular matrix and the formation of enamel crystals in into the dental enamel formation and to the normal and of Enam have used gene targeting to normal Enam replacing the Enam with that of a reporter in mice. We have demonstrated that Enam is and by ameloblasts and that Enam-null mice not enamel of a complete to at the in the of a mineralization at the secretory surface of the ameloblast. were and by the on and of at the of of the targeting with Western a a lacZ reporter with a mouse of a a into the unique site in the of the The was by that could be by The Enam was by PCR as and into the site of The Enam in and in was into the The final targeting was by and by with and into by of in the that was for by Southern were and into The were to and the were for by Southern three The was with and and the and the knock-in were The was with and and the and the knock-in were was with and and to for targeting the for the was of the were with mice to the and mice were that The or absence of a or was by PCR or and or were by PCR of by for Enam and was a control a Enam at demonstrated the of at Enam lacZ at demonstrated the of at knock-in The mice were for which a mice were on for and in a and three mouse enamelin were from for of and were from mice for each of the three and a of was at the of the of on the were from mice to of in and for staining M. A. Biol. 1999; PubMed Scopus Google Scholar, Y. Y. J. A. PubMed Google Scholar, Y. Zhang Y. 129: PubMed Google Scholar). The were in for to to the of the mice and of the tissue with three times for and in for to to the of the mice PubMed Scopus Google Scholar). The were three times in and in were at thickness, for in three times in and at for in staining and Biochem. PubMed Scopus Google Scholar). were and in for with and under a or a light were a and and of mouse were and the were and on in a The incisors and were tissue under a and the and enamel were and in on and for each enamelin were and with of and at and the were by and with The were a and and of protein was in of Each was by on three was with the were to and with proteins for Western analyses. membrane was amelogenin J.P. Lau E.C. T. M. Zeichner-David M. M.L. Slavkin H.C. Fincham A.G. PubMed Scopus Google the other membrane was the enamelin and with the were with to was detected by and enamelin was detected by of mandibular incisors from each of the three at were Oral Biol. PubMed Scopus Google Scholar, J. Res. PubMed Scopus Google Scholar). at the at for for the of was immediately the for dental with a was used to a in in the mandibular and the enamel and dentin of the was by a and The mice were and the were and at The incisors were on a and the the on the and the on the was with a three times The the in the and the on the was by the of that the were the of was of and Enam+/- mice were from and a at from three mice from each of the three were and under was on at the of the from three of each The was at and at were a by a to the a of scanning was for each which of were on surface to a of the to of the and of the were a of of at microscopy of at the of the unerupted was on and mice at of were with in and in were with a on to Kossa staining for mineral was by to the and to light for were for tissue and cell were a on of at from mice for each of the three were with a at the of the and with were with a to and with a scanning electron in the electron at of of and at mandibular from and knock-in mice were in in and in a at and were and and with and a of Enamel at and mice at of were and for tissue and were of and in in The were for at The and enamel the incisors were and the enamel were with The enamel layer on each maxillary and mandibular was with a into a of from the the Each was in a small and at The were to and on a to the of the enamel Each was to a and and at for The were and each enamel was to the of the and any mineral in each enamel H.C. J. Res. PubMed Scopus Google Scholar). of this the protein of a was as the the final The mineral by in each was by the from were from a of maxillary and mandibular incisors or incisors and in of three of enamel early and nearly of for that normal of were by Enamelin and in the of enamelin on crystal crystals were in a G. Biochem. J. PubMed Scopus Google in the of or enamelin. were from the and to with a and a highly J. Res. PubMed Scopus Google Scholar). of the used gene targeting to generate a mouse carrying a null allele of enamelin (Enam) that has a lacZ reporter gene into the of the Enam the targeting in Enam are to as Enam-/- or as to the of Enam expression or the of expression the of the enamelin The lacZ which was to a mouse and into the Enam exon replacing the Enam translation initiation of the Enam through was that the lacZ enamelin the part of Enam through The sequences for the were with and by mice with mice. Correct targeting and were confirmed by Southern and for Enam and and the lacZ reporter replacing the Enam translation initiation site and with the translation initiation site and Enam expression in expression in its Because the is in the as expression a reporter for Enam of the Enam with the as a reporter for enamelin The is from of its and its is a that is at be detected J. 1999; PubMed Scopus Google Scholar). is a that is by to The of the on a be by the length of the The for the of expression a to the times are to demonstrate the absence of reporter in from the of with the of the the and into immediately adjacent We for the expression of from the enamelin in knock-in mice by at staining of mouse the in and No was in any other tissue in the or the of the not the the in to ameloblasts and were in showed no These results with of enamelin expression by in Qian Q. Zhang C.H. Simmer J.P. J. Res. PubMed Scopus Google Scholar, Zhang Simmer J.P. J. Oral Sci. PubMed Scopus Google Scholar). The is that stage ameloblasts are for staining but for in is the of the protein than Enam to in the generate expression of the protein has Enam expression was detected by and by no other cell the highly The close the lacZ staining in the mice and normal ameloblast-specific expression enamelin as by in Zhang Simmer J.P. J. Oral Sci. PubMed Scopus Google that the knock-in was Western the that Enam was by with the targeting is by the results of Western analyses. Enamel proteins were from incisors and and of proteins from and Enam-/- mice were by and Western Enamelin protein was detected in from and Enam+/- mice but not from the enamelin null mice when the proteins from the Enam-/- were in times the that could be detected in Enam+/- no enamelin protein could be detected and of the mouse at changes in tooth shape and the and Enam mice in the Enam-/- mice enamel and the Enam+/- mice defects that are The mandibular incisors of the Enam+/- mice are in the erupted and the maxillary incisors are from normal to The mandibular enamel the part of the is apparently rapidly a wear on the Enamel is on the of the Enam+/- mice The of wear is on the Enam-/- which are and at the The maxillary incisors of Enam+/- mice normal to have in the enamel with and the wear on the tips of were not or as as on the maxillary incisors of The mandibular incisors of Enam+/- in with of and and the of enamel the of the incisors. wear on were with enamel and dentin the in to the on erupted of the in tips that a shape than the shape of incisors. of enamel were on the maxillary and the mandibular incisors of Enam-/- mice. These a and showed dentin the and of the The erupted of the incisors were with a layer of that and in The of the were with a layer of and showed of were in the and of showed the mandibular with and in to the of the and There to be tissue in the under the There was for tissue in to the of the incisors. was in the enamel the but was in the the the enamel were than was and from the and of were in the stage at the and the There was a small on the incisors where the and Enam+/- and Enam-/- mice could not be from in of of of and were on and the and mice did not any in or when with There was no mice of the three in a of the of and to be in critical mineralization and a in a The that the targeting Enam in lacZ was by Southern PCR and the dental in the Enam+/- and Enam-/- mice. The results that was no in and the the expression of Enam by the that the targeting a of the dental incisors as they and the for the enamel layer to J. C.A. Res. PubMed Scopus Google Scholar). from with the tooth its of S. M. Res. PubMed Scopus Google Scholar). Although enamelin is not to a in tooth from wear or a of to in changes in the of mandibular were in mice the enamel and adjacent and the the a of was not to the enamel in this of the of the enamel layer in the Enam a was through the and and the were on to in the and enamelin mice were The for mandibular incisors of mice was The for the Enam-null mice was The for the mice was three were than for mouse mandibular incisors Oral Biol. PubMed Scopus Google Scholar), of the of and The mandibular for the Enam+/- mice to in the the maxillary and mandibular incisors are maxillary incisors of Enam+/- mice normal The incisors of Enam+/- in a of and with enamel and dentin at the the mandibular incisors of the Enam+/- mice maxillary incisors the wear of the mandibular incisors and this for The in the and Enam-/- mice are not to cause in the of mineralization of the mice under demonstrated that the of the mouse mandibular dental at from the to the Enam+/- to the Enam-/- mice with the in tooth mineralization in the The mineralized of the Enam-/- mice were and than in the that the Enam-null mice enamel and at the of the mandibular and in the the and that the mineralized enamel layer in the Enam-/- mice is to be detected or enamel is in the and Enam+/- mice. of mandibular through the and of by and that could be used to the mineralized tissue and tooth of the Enam mice. The revealed a of the mineralized tissue a of mineralized in the and from Enam-/- mice to the and Enam+/- no were in and The and a or of dental enamel in the Enam-/- mice. No in or tooth could be the and Enam+/- mice. mandibular of the mice were von Kossa staining and which mineralized to but not or was that the secretory stage enamel layer of the Enam-/- mice was von for small that in the No von Kossa staining was at the mineralization front, which is the secretory of the membrane of the ameloblasts the surface of the enamel The secretory stage enamel of the and Enam+/- mice the enamel of the mice did not as as that of the mice. that enamel proteins in the enamel but the of enamel of the mineral dentin in the Enam-/- mice is true which by the of crystal 10,000 each at the mineralization that grow in width and thickness to the enamel (1Daculsi G. Kerebel B. J. Ultrastruct. Res. 1978; 65: 163-172Crossref PubMed Scopus (133) Google Scholar). electron of mouse and incisors revealed early enamel layer with the of and enamel S. J. 1996; Google Scholar). the accumulated enamel matrix of the Enam-null mice showed of enamel rods that the and enamel of at showed enamel in the stage of enamel rods were in the and Enam+/- mice and but were from the Enam-null mice. the Enam-/- mice the accumulated enamel proteins were and a layer of mineral the The surface of the was and to or The in Enam+/- mice but that the enamel on than and were of and of the enamel surface that were the of the and of the and of of enamel of length were from the to the of mouse incisors. Each was and its Each was which the and the of the The protein is the the The mineral the The were by major and for the and Enam-/- mice The and mineral of enamel on maxillary incisors from the and Enam+/- mice were the mandibular incisors of Enam+/- mice and mineral that were almost of for enamel at in mice and The mineral to protein in enamel on mandibular incisors of the Enam+/- mice was normal the secretory normal early when enamel crystals growing in and normal at a when the enamel in mice was almost to with a were for the mineral by was to enamel from the erupted of teeth, of normal with enamel on mandibular incisors of mice not We were to of the length of the maxillary and mandibular incisors in Enam-null mice. The maxillary and mandibular incisors showed for and mineral the of the incisors and about of the for mice. The mineral to protein were of normal and the mineral by at stage of was the nearly enamel in mice was mineral by The mineral to protein and mineral by for the Enam-null mice were almost to for normal not Enamelin and of the enamelin protein is degraded and from the enamel with the of a product that to about of enamel protein T. T. E.C. Fukae M. M. PubMed Scopus Google Scholar). crystals were in in the of enamelin. showed that crystal length was in the absence of enamelin and with protein to at crystal did not with enamelin and at and enamelin the length of crystals in not the crystal that enamelin is the protein the enamel matrix of The proteins and are in and are to and Because of its and from the the is the used for enamel proteins Y. P.H. Simmer J.P. PubMed Scopus Google Scholar). enamelin is secreted as a that is rapidly by in the extracellular Y. Oral Biol. PubMed Scopus Google Scholar). The secreted protein is from its to generate and but not and are the enamel surface M. T. Uchida T. M. Res. 1996; PubMed Scopus Google Scholar, T. Y. Fukae M. Y. K. M. Simmer J.P. Uchida T. Res. PubMed Scopus Google Scholar). and of into ameloblasts the of the matrix that mineralization. The of enamelin is that for the enamelin at the mineralization where they are and no is deeper in the enamel (8Hu C.-C. Fukae M. Uchida T. Qian Q. Zhang C.H. Ryu T. Y. M. Simmer J.P. J. Res. PubMed Scopus Google Scholar). to other of the enamelin protein the thickness of enamel T. T. E.C. Fukae M. M. PubMed Scopus Google Scholar, T. T. Fukae M. M. PubMed Scopus (93) Google Scholar, T. T. Fukae M. M. Yamada M. K. S. PubMed Scopus Google Scholar). The of the enamelin protein the at the mineralization has that enamelin is critical for enamel crystal the of a thick layer of enamel proteins in the extracellular secretory ameloblasts in the Enam-/- no mineralization occurs at the mineralization that enamelin is a critical of the mineralization that or the of enamel crystals J.P. Fincham A.G. Oral Biol. 6: PubMed Scopus Google Scholar). the enamel crystals that at the are to at the mineralization (4Ronnholm E. J. Ultrastruct. Res. 1962; 6: 249-303Crossref PubMed Scopus (137) Google Scholar, A. (ed) in Ten Cate's Oral Histology Development, Structure, and Function, Ed., pp. Mosby, St. Louis, MOGoogle and the of enamel crystal formation to be in the Enam-/- be that the initiation and of enamel crystallites occurs by the or a of the and mineralization of of the of a that is in The small of that dentin in the Enam-/- mice to be than true is of not and is the product of ameloblasts the mineral to protein and mineral by are to for We that is no true enamel the dentin in the Enam-/- mice. The mineral that by a than for true enamel. to the enamel protein layer as that and a crust the dentin that These the of the mineralization at the secretory surface of the ameloblast membrane as to the formation of true enamel. The shape of the enamel crystals and organization into rods is at the mineralization and is the enamelin as the enamel layer and in of the enamelin have for enamel crystals and in the the enamel with the of ameloblastin T. Y. Fukae M. Y. K. M. Simmer J.P. Uchida T. Res. PubMed Scopus Google Scholar). The enamelin product is the enamelin that the crystallites enamel the thickness of secretory stage enamel T. T. E.C. Fukae M. M. PubMed Scopus Google Scholar, T. T. Fukae M. M. PubMed Scopus (93) Google Scholar). and three Y. PubMed Scopus Google Scholar, Y. T. Fukae M. M. Res. PubMed Scopus Google Scholar). The enamelin not in with amelogenin and J. in and and pp. but for to Y. T. S. Simmer J.P. Fukae M. Oral Biol. PubMed Scopus Google Scholar). part of enamelin to its to by Y. Fukae M. T. Simmer J.P. J. Oral Sci. PubMed Scopus Google Scholar), which is the in secretory stage enamel Fincham A.G. Zhang Qian Q. Simmer J.P. J. Res. 1999; PubMed Scopus Google Scholar). the enamelin the length but not the of crystals in These results that the enamelin a in enamel crystallites in The reporter in of enamelin the highly of Enam enamelin is by any cell other than be at to secretory highly of expression with which is by ameloblasts but be detected in many other to that enamelin expression in other be and amelogenin expression be mouse was that a Enam of the translation initiation site expression of the mouse enamelin gene at than normal a Enam was by in the and not by The and for that the gene expression of enamelin J. J. and J. C.-C. J. in Scholar). have and Enam knock-in The results that enamelin is by ameloblasts and that enamelin expression is for proper of the mineralization associated with the ameloblast secretory enamelin is no true enamel. Enamelin defects are in as for enamelin is to the enamel formation tooth We of of and and of for and was by at and of the knock-in and of the mouse were by with

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