Cloning Human Enamelin cDNA, Chromosomal Localization, and Analysis of Expression during Tooth Development

Hu, C.-C.; Hart, Thomas C.; DuPont, Barbara R.; Chen, J.J.; Sun, Xiaoling; Qin, Qian; Zhang, C.H.; Jiang, Heping; Mattern, Vicki; Wright, J. Timothy; Simmer, James P.

doi:10.1177/00220345000790040501

Cited by 71 publications

(64 citation statements)

References 29 publications

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“…1D). These results correlate well with previous studies of enamelin expression by in situ hybridization (9,56). The only difference is that maturation stage ameloblasts are positive for X-gal staining but negative for in situ hybridization.…”

Section: Generation Of the Enam-nullsupporting

confidence: 91%

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

Smith

et al. 2008

Journal of Biological Chemistry

151

193

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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-␤-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.

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Section: Generation Of the Enam-nullsupporting

confidence: 91%

“…There are three major secretory stage enamel proteins: amelogenin (5,6), ameloblastin (7), and enamelin (8,9). All belong to the secretory calcium-binding phosphoprotein (SCPP) gene family (10), and all are critical for proper dental enamel formation.…”

Section: Correct Targeting Of the Transgene Was Confirmed By Southementioning

confidence: 99%

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

Smith

et al. 2008

Journal of Biological Chemistry

151

193

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“…The translation initiation site has not been determined experimentally for ENAM, but we found an authentic SP sequence of 16 aa consisting of a canonical tripartite structure: short, positively charged n-region, a central hydrophobic h-region, and a more polar c-region with a cleavage site (26,27). Mouse Enam has an additional 5Ј-untranslated exon, which was detected within the human first intron but has not been identified in the transcripts (28).…”

Section: Resultsmentioning

confidence: 70%

Mineralized tissue and vertebrate evolution: The secretory calcium-binding phosphoprotein gene cluster

Kawasaki

Weiss

2003

Proc. Natl. Acad. Sci. U.S.A.

284

249

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Gene duplication creates evolutionary novelties by using older tools in new ways. We have identified evidence that the genes for enamel matrix proteins (EMPs), milk caseins, and salivary proteins comprise a family descended from a common ancestor by tandem gene duplication. These genes remain linked, except for one EMP gene, amelogenin. These genes show common structural features and are expressed in ontogenetically similar tissues. Many of these genes encode secretory Ca-binding phosphoproteins, which regulate the Ca-phosphate concentration of the extracellular environment. By exploiting this fundamental property, these genes have subsequently diversified to serve specialized adaptive functions. Casein makes milk supersaturated with Ca-phosphate, which was critical to the successive mammalian divergence. The innovation of enamel led to mineralized feeding apparatus, which enabled active predation of early vertebrates. The EMP genes comprise a subfamily not identified previously. A set of genes for dentine and bone extracellular matrix proteins constitutes an additional cluster distal to the EMP gene cluster, with similar structural features to EMP genes. The duplication and diversification of the primordial genes for enamel͞dentine͞bone extracellular matrix may have been important in core vertebrate feeding adaptations, the mineralized skeleton, the evolution of saliva, and, eventually, lactation. The order of duplication events may help delineate early events in mineralized skeletal formation, which is a major characteristic of vertebrates.

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“…The major secretory stage enamel proteins are amelogenin (21,22), ameloblastin (23)(24)(25), and enamelin (26,27). These proteins function specifically during enamel formation, and the disease phenotypes exhibited by mice lacking these genes are confined to the developing teeth and include enamel agenesis (28 -30).…”

mentioning

confidence: 99%

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

Simmer

Lertlam

et al. 2009

Journal of Biological Chemistry

134

184

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Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) staining demonstrated expression of ␤-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentinoenamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.Dental enamel is composed of highly ordered, very long crystals of calcium hydroxyapatite (Ca 10 (PO 4 ) 6 (OH) 2 ). Mature enamel crystallites are about 70 nm wide and 30 nm thick, but are of unmeasurable length (1), probably extending all the way from the dentin layer to the surface of the tooth (2). Enamel crystallites are organized into bundles called rods, with about 10,000 parallel crystals in a rod (3). Each enamel rod is the product of a single ameloblast, the cell type that forms a continuous sheet over the developing enamel and orchestrates its formation. Dental enamel of erupted teeth is ϳ95% mineral (by weight) (4), with most of the non-mineral component being water. Protein comprises Ͻ1% of its weight. Forming enamel, however, is over 30% protein (5). Much of the protein is reabsorbed by ameloblasts and degraded in lysosomes (6, 7), but extracellular proteases also play a role in matrix protein removal (8 -10).Dental enamel formation is divided into secretory, transition, and maturation stages (11,12). During the secretory stage, enamel crystals grow primarily in length. As the crystals extend, the enamel layer expands. Enamel crystallites lengthen along a mineralization front at the secretory surface of the ameloblast cell membrane. There, mineral deposits rapidly on the crystallite tips, and very slowly on their sides...

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Cloning Human Enamelin cDNA, Chromosomal Localization, and Analysis of Expression during Tooth Development

Cited by 71 publications

References 29 publications

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

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

Mineralized tissue and vertebrate evolution: The secretory calcium-binding phosphoprotein gene cluster

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

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