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Human Molecular Genetics Advance Access originally published online on January 13, 2005
Human Molecular Genetics 2005 14(5):575-583; doi:10.1093/hmg/ddi054
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Human Molecular Genetics, Vol. 14, No. 5 © Oxford University Press 2005; all rights reserved

Enamelin (Enam) is essential for amelogenesis: ENU-induced mouse mutants as models for different clinical subtypes of human amelogenesis imperfecta (AI)

Hiroshi Masuya1, Kunihiko Shimizu2, Hideki Sezutsu3,4, Yoshiyuki Sakuraba3, Junko Nagano1, Aya Shimizu1, Naomi Fujimoto3, Akiko Kawai1, Ikuo Miura1, Hideki Kaneda1, Kimio Kobayashi1, Junko Ishijima1, Takahide Maeda2, Yoichi Gondo3, Tetsuo Noda1, Shigeharu Wakana1 and Toshihiko Shiroishi1,*

1Mouse Functional Genomics Research Group, RIKEN GSC, 3-1-1 Kouyadai, Tsukuba, Ibaraki 305-0074, Japan, 2Department of Pediatric Dentistry, Nihon University School of Dentistry at Matsudo, Japan, 3Population and Quantitative Genomics Team, RIKEN GSC, Japan and 4Insect Gene Engineering Laboratory, Insect Biotechnology and Sericology Department, Institute of Insect and Animal Sciences, National Institute of Agrobiological Sciences, Japan

* To whom correspondence should be addressed. Tel: +81 298369017; Fax: +81 298363616; Email: tshirois{at}lab.nig.ac.jp

Received November 11, 2004; Revised December 22, 2004; Accepted January 4, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Amelogenesis imperfecta (AI) is a group of commonly inherited defects of dental enamel formation, which exhibits marked genetic and clinical heterogeneity. The genetic basis of this heterogeneity is still poorly understood. Enamelin, the affected gene product in one form of AI (AIH2), is an extracellular matrix protein that is one of the components of enamel. We isolated three ENU-induced dominant mouse mutations, M100395, M100514 and M100521, which caused AI-like phenotypes in the incisors and molars of the affected individuals. Linkage analyses mapped each of the three mutations to a region of chromosome 5 that contained the genes encoding enamelin (Enam) and ameloblastin (Ambn). Sequence analysis revealed that each mutation was a single-base substitution in Enam. M100395 (EnamRgsc395) and M100514 (EnamRgsc514) were putative missense mutations that caused S to I and E to G substitutions at positions 55 and 57 of the translated protein, respectively. EnamRgsc395 and EnamRgsc514 heterozygotes showed severe breakage of the enamel surface, a phenotype that resembled local hypoplastic AI. The M100521 mutation (EnamRgsc521) was a T to A substitution at the splicing donor site in intron 4. This mutation resulted in a frameshift that gave rise to a premature stop codon. The transcript of the EnamRgsc521 mutant allele was degraded, indicating that EnamRgsc521 is a loss-of-function mutation. EnamRgsc521 heterozygotes showed a hypomaturation-type AI phenotype in the incisors, possibly due to haploinsufficiency of Enam. EnamRgsc521 homozygotes showed complete loss of enamel on the incisors and the molars. Thus, we report here that the Enam gene is essential for amelogenesis, and that mice with different point mutations at Enam may provide good animal models to study the different clinical subtypes of AI.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Amelogenesis imperfecta (AI) is a group of congenital disorders, which primarily affects the dental enamel, a highly mineralized tissue mainly (~95%) composed of hydroxyapatite crystals (1Go). In addition to hydroxyapatite crystals, enamel contains a small amount of protein. Amelogenin, ameloblastin and enamelin are structural proteins secreted from ameloblasts, epithelium-derived cells that produce enamel matrix. AI has clinically diverse forms with varying degrees of abnormality in the amount, structure, and composition of the enamel. On the basis of the clinical appearance, cases of AI are roughly classified into hypoplastic, hypomaturation and hypocalcified (hypomineralized) types (2Go,3Go). Hypoplastic-type AI is characterized by a reduced thickness of the enamel. The appearance of the enamel surface allows further classification of hypoplastic AI into pitted, local, smooth and rough subtypes. Hypomaturation-type AI is characterized by opaque white enamel with a ground-glass appearance. Although the thickness of the enamel is not affected, it is slightly softer than normal often leading to abrasions on the incisal and occlusal surfaces. In hypocalcified-type AI, the enamel initially develops with a normal thickness, but the enamel matrix is poorly calcified.

AI also shows genetic variation because families can exhibit autosomal dominant, autosomal recessive or X-linked inheritance patterns. Genetic analyses of affected human families have revealed the causative genes for AI. Recessive and dominant X-linked forms of hypoplastic and hypomaturation AI, designated as AIH1, have been linked to mutations affecting amelogenin, the most abundant protein component of enamel (4Go–17Go). Depending on the type and location of the mutation in the amelogenin gene (AMELX, mapped to chromosome Xp22.3), AIH1 can cause a diverse set of phenotypes (ranging from smooth hypoplastic to hypomineralized/hypomaturation) (15Go,16Go).

Another dominant form of AI, AHI2, is caused by mutations in the enamelin gene (ENAM) mapped to chromosome 4q13.2 (18Go–22Go). Enamelin is the largest and least abundant of the three principal enamel matrix proteins, representing between 1 and 5% of the total matrix protein (23Go). Proteolytic processing gives rise to multiple enamelin cleavage products, which accumulate in different parts of the enamel matrix (23Go–26Go). Recently, it was reported that a novel mutation in ENAM is responsible for a recessive form of AI (ARAI) in humans (22Go). As in the case for AHI1, diverse phenotypes (ranging from generalized hypoplastic to pitted hypoplastic) might be attributable to different mutations of ENAM. This may be more difficult to prove, however, as there are fewer mutant ENAM alleles reported, when compared with AMELX.

Mouse mutants have been used as animal models for human AI, as forward and reverse genetic approaches allow studies of gene functions. Mice with targeted knockouts of amelogenin (Amelx) and enamelysin (Mmp20), a gene that codes for a protease that is active during the secretory stage of enamel development, have been generated. Amelogenin-null mice revealed that amelogenin is not essential for the initiation of mineral crystal formation, but is required for organizing crystal patterns and regulating the enamel thickness (27Go). Enamelysin-deficient mice cannot process amelogenin properly, resulting in serious enamel defects (28Go).

In this study, we report three new mutations of the mouse enamelin gene (Enam), which were obtained from a large-scale phenotype-driven screen of mice mutagenized with a chemical mutagen, N-ethyl-N-nitrosourea (ENU). Analyses of these three mutants provided useful clues in understanding the function of enamelin in enamel formation and the phenotypic variation observed in human AI.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Three dominant mutant alleles of Enam were obtained following ENU mutagenesis
During a large-scale screen of ENU-mutagenized mice, we screened 10 236 G1 (founder mice generated from crosses of ENU-mutagenized males and wild-type females) animals for dominant abnormal phenotypes of the incisor surface. Identified phenodeviants (animals that exhibited abnormal phenotypes) were subsequently backcrossed to DBA/2J mice for inheritance testing. The inheritance tests confirmed that six independent phenotypes were clearly transmitted to the next generation. Heterozygous carriers of each of the six mutations, M100268, M100395, M100448, M100514, M100521 and M100888, exhibited abnormalities of their incisor surfaces. While the incisors of wild-type mice were smooth with a yellowish tint, the incisor surfaces of M100395 and M100514 heterozygotes appeared rough and rugged (Fig. 1A, B and C). M100521 mutants were detected by the ground-glass-like rough surface of their abnormally white incisors. These mice also had abnormal macroscopic abrasions at the sites of incisor occlusion (Fig. 1D). All three mutations exhibited dominant inheritance patterns and no other abnormalities were observed in mutant mice. Information about the other three mutations that produced AI-like phenotypes, M100268, M100448 and M100888, is available on our website (http://www.gsc.riken.go.jp/Mouse/).



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  		Figure 1. ENU-induced dominant mutants that exhibited AI-like phenotypes. (A) Wild-type mice showed smooth and yellowish surfaces of incisors in macroscopic observations. M100395 (B) and M100514 (C) heterozygotes showed markedly rough surfaces of the incisors. The anterior side of the incisors, where enamel was located, was affected. A M100521 heterozygote (D) exhibited a whitish ground-glass-like surface in the anterior portion of the incisor. Highly abnormal wear was observed at the occlusions.

 
We mapped the mutant loci by using the backcross progeny to carry out linkage analysis. All three of the mutations described above were mapped to the same region of chromosome 5 (Fig. 2). Haplotype analysis allowed us to map M100395 to the interval, between the genetic markers D5Mit112 and D5Mit361 on chromosome 5 (Fig. 2A and D). Subsequent genotyping of the backcross progeny revealed that M100395 and M100514 were fully penetrant while the penetrance of M100521 was close to 95% (22/23).



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  		Figure 2. Genetic mapping of three dominant mutations that cause AI-like phenotypes. The upper panels show haplotype analysis of the backcross progeny of the G1 mice carrying M100395 (A), M100514 (B) and M100521 (C). Genetic markers are indicated on the left of each panel (rs3023044, rs3090699, rs3090397, rs3023051 and rs3023057 are cluster IDs from dbSNP (http://www.ncbi.nlm.nih.gov/SNP/)). The boxes represent the genotype of the progeny. Open boxes represent the DBA/2J allele, and black box corresponds to the C576BL/6J allele. Numbers of recombinants are shown at the bottom of each panel. Lower panels are genetic maps constructed using the progeny of G1 mice carrying M100395 (D), M100514 (E) and M100521 (F). Because the penetrance of the M100521 phenotype was not complete, linkage analysis was performed using only progeny that express abnormalities of the incisors (C and F). All mutants were mapped closely to D5Mit112 and D5Mit361 on mouse chromosome 5.

 
A search of the public genome database (http://www.ensembl.org/) revealed that two genes encoding enamel matrix proteins, ameloblastin (Ambn) and Enam, were located in the interval between D5Mit112 and D5Mit361. To test whether the three mutant phenotypes were caused by genetic alterations in these two genes, we used genomic DNA from each of the mutant lines to sequence the coding regions and splice junction sites of Ambn and Enam. To exclude the possibility that base changes were solely preexisting polymorphisms, we compared the sequences from the mutant mice with those from wild-type (DBA/2JxC57BL/6J)-F1 mice. A single-base substitution, G to T, at position 6489 of Enam (AF303737, NCBI) was identified in the M100395 mutant allele. The base change caused an amino acid substitution, S to I, at position 55 of the translated protein. A single-base substitution, A to G, at position 6495 was identified in M100514, which led to an amino acid substitution, E to G, at position 57. Both of these substitutions were found in exon 5 of Enam, and the amino acid changes were located in the N-terminal portion of the enamelin protein. A single-base substitution, T to G, at position 6341 of Enam was identified in M100521. This base substitution disrupted a GT splicing donor signal (summarized in Fig. 3), which resulted in a splicing error and a frameshift that introduced a premature stop codon. We designated the three new Enam mutant alleles, M100395, M100514 and M100521, as EnamRgsc395, EnamRgsc514 and EnamRgsc521, respectively (MGI accession nos are MGI: 3055582, MGI: 3055586 and MGI: 3055587, respectively). We found no other alterations in the Enam and Ambn coding regions of mutant mice.



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  		Figure 3. Sequence analysis of three Enam mutations. (A) Exon–intron organization of the mouse Enam gene. All the mutations were found in the region between the exon 4/intron 4 boundary and exon 5. (B) Direct sequence analyses of M100395 (EnamRgsc395), M100514 (EnamRgsc514) and M100521 (EnamRgsc521). Upper panels are comparisons of sequence chromatograms from wild-type mice and mice heterozygous for each of the three Enam mutations. The point mutations are indicated by the arrowheads on the chromatograms. Confirmed sequence is shown below each chromatogram. The lower panel shows genomic sequence of a wild-type mouse at the boundaries of Enam exon 4/intron 4 and intron 4/exon 5. Numbers above the sequence indicate the positions in the retrieved genomic sequence AF303737 (NCBI). Mutation sites in the upper chromatograms are indicated in red and with arrows. The sequences in the chromatograms are underlined in the genome sequence in the lower panel. M100395 and M100514 heterozygotes have nucleotide substitutions, G to T at position 6489 and A to G at position 6495, respectively. The M100521 heterozygote has a substitution of T to A at the splicing donor signal. Consensus sequences of the splicing acceptor and donor sites of intron 4 are depicted by green squares. Exon–intron structure is shown under the genomic sequence. (C) Translated amino acid sequence. Upper panel is a comparison of amino acid sequences of the human and mouse enamelin proteins. Amino acids identical in the two species are indicated by dots in the human sequence. A vertical broken line indicates the boundary of exons 4 and 5. The substituted amino acids caused by M100395 (EnamRgsc395) and M100514 (EnamRgsc514) are indicated in red. The lower panel shows the amino acid sequence of the three mutants. M100395 and M100514 have single amino acid substitutions. M100521 (EnamRgsc521) affects splicing out of intron 4 and results in a frameshift in exon 5, giving rise to a stop codon (asterisk).

 
The transcript of the EnamRgsc521 (M100521) allele
To examine whether the mutation in EnamRgsc521 (M100521) caused splicing defects, we analyzed the transcript by RT–PCR. Total RNA was prepared from the lower jaw of adult homozygous wild-type and EnamRgsc521 mutant mice as well as from an EnamRgsc521 heterozygote. Using a primer set that flanked exon 5 in the transcript, we investigated whether a splicing defect occurred in EnamRgsc521 transcripts (Fig. 4A). We were able to amplify a longer 256 bp cDNA fragment that contained intron 4 from EnamRgsc521 heterozygous and homozygous mice (Fig. 4B). Sequencing this fragment confirmed the presence of the intron 4 sequence (Fig. 4C) and an in-frame nonsense codon in exon 5 (Fig. 3C). All these results indicated that EnamRgsc521 was a splicing error mutation. In the RT–PCR analysis, we seemed to amplify far less of the 256 bp product than of the 150 bp wild-type fragment. We thought that this might be due to activation of nonsense mRNA decay. To test this possibility, we attempted to measure the amount of transcript derived from the mutated allele by means of quantitative real-time PCR. When specific PCR primer sets were used to amplify regions of exons 2, 3 and 4, the heterozygote showed a reduced level (71.3% of the wild-type level) of the transcript, while the homozygous mutant showed a much lower level (1.9%) of transcript (Fig. 4C).



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  		Figure 4. RT–PCR analysis and mRNA quantification of the EnamRgsc521 allele. (A) Positions of primers used in fragment analysis by RT–PCR (solid arrows) and quantification of the Enam mRNA by real-time PCR (open arrows) are indicated on the diagram of the partial transcript derived from the wild-type allele and EnamRgs521. Exons are indicated by open boxes and numbers. (B) RT–PCR of the Enam transcripts isolated from wild-type mice and EnamRgsc521 heterozygotes and homozygotes. A PCR product of 150 bp was amplified from the wild-type mouse. In EnamRgsc521 heterozygotes and homozygotes, a 256 bp product was amplified using the same primer pair. No fragments were amplified from the genomic DNA template. (C) Sequence chromatograms of the RT–PCR product amplified from a wild-type mouse and an EnamRgsc521 homozygote. Exons are indicated under the confirmed sequences. In EnamRgsc521 homozygote, the sequence of intron 4 immediately follows that of exon 4. The EnamRgsc521 mutation is indicated by the arrowhead. (D) Comparison of the amount of Enam transcript in wild-type mice, EnamRgsc521 heterozygotes and EnamRgsc521 homozygotes (two individual animals were subjected to experiments in triplicate). The values obtained were normalized to internal levels of ß-actin. Each column represents the average of three experiments with a SE error bar. Homozygotes showed significantly lower levels of mRNA than wild-type mice and heterozygotes.

 
Characterization of the phenotypes of Enam mutants
For detailed characterization of the phenotypes of adult Enam mutants, we observed the enamel surfaces of the molars and incisors of EnamRgsc395 (M100395), EnamRgsc514 (M100514) and EnamRgsc521 heterozygotes using scanning electron microscopy (SEM) (Fig. 5). In addition, to examine dental structure, X-ray micro-computerized tomography (CT) analyses of the lower jaws were also performed on all three heterozygous mutants as well as EnamRgsc521 homozygotes (Fig. 6). Two mutants carrying amino acid substitutions in exon 5, EnamRgsc395 and EnamRgsc514 heterozygotes, showed a similar hypoplastic phenotype (Fig. 5). The entire enamel surface of the molars exhibited a rough appearance with microscopic cracks and the delamination. A cross-section by X-ray micro-CT showed enamel of normal thickness and an abnormal gap between the enamel and the dentin core (Fig. 6). These defects were more prominent in the incisors, and the enamel surfaces were affected more severely. The X-ray micro-CT analyses showed local hollowing of the enamel in the incisors before eruption. These abnormalities were observed macroscopically in the initial observation of the incisors after eruption (Fig. 1). On the other hand, the molars and incisors of EnamRgsc521 heterozygotes had a slightly rougher surface than those of the wild-type mouse. The enamel had a normal thickness, but, as was the case with EnamRgsc395 and EnamRgsc521 heterozygotes, there were gaps between the enamel and the dentin core. In macroscopic observation of the incisor occlusion surface, EnamRgsc521 heterozygotes showed abnormal wear in the region of occlusion (Fig. 1). X-ray fluoroscopy revealed that the angle of wear at the occlusion of the upper and lower incisors was obviously different from that of the wild-type mouse (Fig. 7). These results indicated that the enamel of the incisors of the EnamRgsc521 heterozygote was softer than that of the wild-type mouse. The X-ray micro-CT analysis of EnamRgsc521 homozygotes revealed a complete loss of enamel of the molars and incisors, which resulted in a decrease in the size of the tooth (Fig. 6). The absence of enamel-like structures was observed in all sectional images of the lower jaws by X-ray micro-CT analyses (data not shown).



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  		Figure 5. SEM analysis of the enamel surface of molars and incisors of the three mutants. Molar surfaces at lower (A, E, I and M) and higher (B, F, J and N), and incisor surfaces at lower (C, G, K and O) and higher (D, H, L and P) magnification are shown with the scale bar below each panel. A wild-type mouse (A–D), EnamRgsc395 heterozygote (E–F), EnamRgsc514 heterozygote (I–J) and EnamRgsc521 heterozygote (M–P) are shown. EnamRgsc395 and EnamRgsc514 heterozygotes resembled the hypoplastic phenotype in their molars (F and J) and incisors (H and L), with cracks and peelings. The EnamRgsc521 heterozygote exhibited a slightly rougher surface than the wild-type mouse in the molars (M) and incisors (O). Small pits in the enamel surface were observed at higher magnifications (N and P).

 


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  		Figure 6. Transverse sections of lower jaws at the level of the first molar (A–E) and the root of the incisor (F–J) of an adult mouse by X-ray CT analysis. A wild-type mouse (A and F) shows well-developed enamel (En) that is tightly connected to the dentin core (De). EnamRgsc395 (B and G) and EnamRgsc514 (C and H) heterozygotes revealed normal thickness of the enamel and an abnormal gap between the enamel and the dentin core (arrow heads) in the molars. In the pre-eruption incisors, the enamel was locally deficient (arrows). An EnamRgsc521 heterozygote (D and I) showed normal thickness of the enamel in the molars and incisors. An abnormal gap was also observed between the enamel and dentin. An EnamRgsc521 homozygote exhibited complete loss of enamel in the molars and the pre-eruption incisors (E and J).

 


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  		Figure 7. Abnormal wear of the upper and the lower incisors in the EnamRgsc521 heterozygote, which was observed by X-ray fluoroscopy. The wild-type mouse (A) shows a sharp edge of the enamel in the lower incisors and depressed wear of the upper incisors in the occlusion (arrows). The EnamRgsc521 heterozygote (B) shows an obtuse edge of the lower incisors and no obvious dent in the occlusion of the upper incisors (arrows).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
ENU mutagenesis caused various mutations in the Enam gene
ENU is a powerful chemical mutagen that randomly induces point mutations in genomic DNA at a high frequency (29Go–32Go). Phenotype-based screening allows comprehensive collection of mutations in genes involved in specific biological pathways. Because human genetic diseases are often caused by point mutations rather than gross alterations of the genome, ENU mutagenesis is suitable for production of mutants that can serve as models for human disease. In addition, large-scale ENU mutagenesis allows the production of multiple mutant alleles of a given gene, thereby enabling the study of multiple functional domains of the gene product. This is essential if we are to understand the heterogeneity and complexity of the phenotypes of many human diseases.

In this study, we screened >10 000 G1 animals for dominant mutations that affect the dental surface of the incisors. While rodent molars show a structure similar to that of human teeth, rodent incisors show a more primitive structure than that of human teeth. It was, however, recently reported that knockout mutants of enamel matrix proteins such as amelogenin exhibited a prominent incisor phenotype similar to human AI (28Go,29Go,33Go), suggesting that observation of the mouse incisor surface is suitable for screening for animal models of human AI. Indeed, in our screening we obtained three alleles of Enam, which had different mutation types. In this study, the mutation rate at the Enam locus was calculated to be 0.29x10–3. This rate was somewhat lower than values estimated for other loci (1.37x10–3 at myo5a, 0.73x10–3 at tyrp2 and 1.17x10–3 at Gck) (34Go and unpublished data) in our study, but seems to be comparable with the values previously reported (31Go,32Go). In addition to the three Enam mutants, we also obtained other mutants that exhibited an abnormal dental surface, and were not mapped to chromosome 5. More specific screens focusing on amelogenesis may lead to the discovery of more genes involved in enamel formation.

Multiple mouse mutant alleles revealed that enamelin is essential for amelogenesis
Enamel matrix proteins are considered to control multiple steps (nucleation, growth and organization) in the crystallization of hydroxyapatite during enamel formation. These proteins and their cleavage products are generally localized into different compartments of developing enamel (22Go). Enamelin protein is cleaved into 186, 155, 142, 89, 32, 25 and 34 kDa subproducts. These cleavage products are localized in different regions of developing enamel (23Go,24Go,26Go,33Go,35Go,36Go). The distribution patterns may reflect the mineralization process of developing enamel. In this study, we showed that one of the ENU-induced Enam mutations, EnamRgsc521, is a one base substitution at the consensus splicing donor sequence in intron 4. This mutation affects the splicing out of intron 4, which leads to a frameshift after the 41st amino acid, and results in truncation of the peptide at the 86th amino acid by the appearance of a nonsense codon (Fig. 3). The induced nonsense codon causes the loss of 97% of the primary structure of the enamelin protein. All known enamelin cleavage products should be deleted as a result of the EnamRgsc521 allele. Furthermore, we demonstrated that amount of transcripts from the EnamRgsc521 allele was reduced to <2% of the transcripts derived from the wild-type allele (Fig. 5C). The EnamRgsc521 transcript is most likely degenerated by activation of nonsense-mediated mRNA decay. Thus, these results strongly suggest that EnamRgsc521 is a loss-of-function mutation, which leads to almost complete loss of intact enamelin protein and its cleavage products. Surprisingly, homozygotes of EnamRgsc521 showed complete loss of enamel even in the incisors before eruption as well as in the molars. Taking all of these results into account, enamelin has been shown to be a key molecule for enamel formation, and is required for the initiation of mineral crystal formation in the early stages of tooth development. In contrast, knockout mouse of another enamel matrix protein, amelogenin, had enamel with a reduced thickness, suggesting that amelogenin is not required for the initiation of mineral crystal formation, but is essential for the growth of enamel crystals (27Go). Thus, enamelin and amelogenin are functionally segregated in the processes of mineralization in enamel development.

EnamRgsc521 heterozygotes showed a hypomaturation-type AI-like phenotype with apparently soft enamel on the incisors. Because EnamRgsc521 appeared to be a loss-of-function allele, this abnormality should result from haploinsufficiency of enamelin. This result indicates that enamelin also functions in assuring the hardness of the enamel on mouse incisors. Normal mineralization of the enamel may require at least enamelin protein and/or its cleavage products. In SEM analysis, the EnamRgsc521 heterozygote showed a slightly rougher enamel surface with small pits in the molars as well as the incisors, suggesting that haploinsufficiency of enamelin affects amelogenesis in mouse molars. More detailed analyses of the dental structure of the EnamRgsc521 heterozygote may disclose the function of enamelin in the later stage of molar development, which possibly resembles that of humans.

The two mutations, EnamRgsc395 and EnamRgsc514, had amino acid substitutions at positions 55 and 57, respectively. Mice heterozygous for these two alleles showed a similar local hypoplastic AI-like phenotype (scraping and local loss of enamel), which differs from that of EnamRgsc521 heterozygotes. The amino acid substitutions in the mutants affected the 186, 155, 142 and 89 kDa cleavage subproducts. The abnormal cleaved products of enamelin or combinations of them must be responsible for these phenotypes. Further analyses are needed to elucidate the molecular mechanism underlying these abnormalities. EnamRgsc395, EnamRgsc514 and EnamRgsc521 heterozygotes commonly exhibited abnormal gaps between the enamel and the dentin core. In the case of EnamRgsc521 heterozygotes, poor supply of enamelin and/or its cleavage products might cause the abnormality. Enamelysin is known to be one of the proteolytic enzymes that cleaves amelogenin and enamelin during the secretory stage (37Go,38Go). Although separation of the enamel and dentin is not obvious in the amelogenin-deficient mouse, it is induced in the enamelysin-deficient knockout mouse (27Go). Considering the results of the present study, it is possible that enamel–dentin separation in the enamelysin knockout mouse was due to a deficiency in cleavage activity of the enamelin protein.

Enam mutants serve as mouse models for different clinical subtypes of human AI
Human AMELX mutations lead to some variations in the clinical phenotypes of AIH1 (15Go,16Go). A loss-of-function mutation of mouse Amelx generated by gene targeting showed a phenotype similar to a subtype of AIH1. Likewise, mouse ENU-induced Enam alleles showed phenotypes reminiscent of some subtypes of AIH2. EnamRgsc395 and EnamRgsc514 heterozygotes exhibited a phenotype similar to local hypoplastic AI, which was shown to be caused by an introduced stop codon at amino acid position 53 of human enamelin (23Go). EnamRgsc521 homozygotes have a generalized hypoplastic AI-like phenotype observed in patients homozygous for a mutation that causes a frameshift at amino acid position 422 (22Go). EnamRgsc521 heterozygotes had some characteristics of hypomaturation-type AI, e.g. a ground-glass appearance and soft enamel with normal thickness. Phenotypes caused by human ENAM mutations and AMELX mutations are different, with some overlaps. This is consistent with the concept that enamelin functions mainly in the early stage of enamel development and may be involved in the initiation of crystallization of hydroxyapatite. Enamelin and amelogenin may be involved in subsequent steps that control enamel hardness and growth. Phenotypic overlap resulting from ENAM and AMEL mutations suggests cooperative function or interaction of enamelin and amelogenin. It was reported that amelogenin and the 32 kDa cleavage product of enamelin cooperate in the induction of apatite crystal formation in vitro (42Go).

We report here that multiple mutations of the Enam gene, which were generated by ENU mutagenesis, provide significant insight into the function of enamelin. Collection of more mutant alleles of Enam and other enamel matrix genes and characterization of their phenotypes should serve as useful models to study the molecular mechanism of amelogenesis and to understand the heterogeneity and complexity of clinical subtypes of human AI.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
ENU mutagenesis, phenotype screening, inheritance testing and gene mapping
Large-scale mouse ENU mutagenesis was conducted using a protocol modified from previous reports (29Go,30Go). The detailed protocol for ENU mutagenesis is available at http://www.gsc.riken.go.jp/Mouse/ and in a previous report (34Go). C57BL/6J and DBA/2J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). C57BL/6J males administered ENU (total dosage of 150–250 mg/kg) were crossed to DBA/2J females. The resultant F1 hybrids (referred to as G1 animals in this report) were subjected to screening for various dominant phenotypes at 8 weeks of age (34Go,40Go).

Examination of oral cavities was performed as a subtest of the Modified-SHIRPA protocol, a comprehensive package of screens for morphological and behavioral phenotypes (40Go,41Go). A complete list of the subtests is available on our website (http://www.gsc.riken.go.jp/Mouse/). To examine the morphology of the G1 incisors, X-ray imaging of adult mice was performed using a micro-focus X-ray system (DFX- 100K; Pony Industry Co., Ltd). This system can take multiple radiographs of mice from different angles at different zoom values.

Phenodeviants that showed anomalies in their incisors were crossed to the wild-type DBA/2J mice to test for phenotype transmission and for genetic mapping of the affected genes. Thirty-nine, 40 and 42 G2 progeny were produced from the M100395, M100514 and M100521 founder G1 animals, respectively. Genomic DNA was prepared from the tail tips of the G2 progeny using the NA-2000 automatic nucleic acid isolation system (KURABO Industries, Ltd., Japan). We used the db-SNP website (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=snp) to detect single nucleotide polymorphisms (SNPs) and microsatellite markers on the MGI website (http://www.informatics.jax.org/) to detect simple sequence length polymorphisms. Polymorphic loci were examined using the TaqMan MGB assay (Applied Biosystems, USA) and ABI 7700 and ABI 7900 sequence detection systems. Information about the mouse mutants described in this report can be found on our website (http://www.gsc.riken.jp/Mouse/). The mutant lines are cryopreserved at the RIKEN BioResource Center (BRC) (Tsukuba, Japan), and are available through BRC under a Material Transfer Agreement.

Sequence analysis and genotyping
To search for mutations in Ambn and Enam, we sequenced coding regions and splice sites of these two genes from each of the mutant lines. We designed primer pairs using the genomic sequence available for Ambn (NW_000231) and Enam (AF303737) in the NCBI database. Primer sequences are listed in the Supplementary Material.

Electron-microscopic analysis and X-ray micro-CT analysis
For the electron-microscopic (SEM) analysis, the heads were soaked in 2% KOH at 43°C for 48 h, soft tissue was removed and the maxilla and mandible were washed with water. The first molars and the incisors were extracted from the left mandible. The teeth were carefully cleaned by sonication. After drying overnight the teeth were sputter-coated with 20 nm gold–palladium and observed with a Hitachi S-2150 scanning electron microscope operated at 20 kV.

X-ray micro-CT was performed as follows. The right side of the mandible that was not dried was examined using a TOSCANER 31300 µhd (Toshiba IT and Control Systems, Japan). Parameters selected for this study were a source energy of 74 kVp and 119 mA to obtain the best contrast between bone and enamel tissue. For each sample, 100 micro-tomographic slices with a thickness of 0.04 mm were acquired, covering the body of the mandible and the first molar (the width between micro-tomographic slices was 0.05 mm).

Analyses of the enamelin gene transcript
RT–PCR analyses were performed to examine mRNA transcribed from the EnamRgsc521 mutant allele. Total RNA was extracted from the lower jaw of adult mice (about 8 weeks of age) by the following procedure. We eviscerated the lower jaw and removed the distal part of incisors. The samples were immediately placed in a microtube and soaked in liquid nitrogen. The cold samples were crushed quickly using a multibead shocker (Yasui Kikai Co., Ltd.). Total RNA was isolated using TRIZOL reagent (Gibco GRL). RT–PCR was performed using the total RNA and the mRNA Selective PCR Kit Ver. 1.1 (TaKaRa) that prevents amplification of genomic DNA (43Go,44Go). For the PCRs, primers specific for Enam transcripts were 5'-GAT GAG TCT CCT TGT TTT CC-3', which bound in exon 4 and 5'-ATC ATT GGT GGG GCA TTC AT-3', which was specific for the sequence at the boundary of exons 6 and 7.

Real-time PCR analysis
To quantitate the levels of Enam transcripts in EnamRgsc521 homozygotes and heterozygotes, real-time PCR analyses were performed using the Quantitect SYBR Green RT–PCR kit (Qiagen) and the ABI PRISM 7700 Sequence Detection System (ABI). The method was essentially the same as described elsewhere with inclusion of primers specific for mouse ß-actin transcript as the endogenous control for each sample (34Go). Separate calibration (standard) curves for ß-actin and Enam were constructed using serial dilutions of mRNA from wild-type animals. For the amplification of 193 bp fragment we used the following primers: 5'-AAG TGG CAT TGG CTC TCA TC-3' and 5'-CAG ACC CAG GAA AAC AAG GA-3'.


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Ms. Lisa Shoji, Akiko Shinogi and Harumi Ayuzawa for supporting gene mapping and the technical staff of the Mouse Functional Genomics Research Group of RIKEN GSC for animal caretaking and phenotype screening. We thank Dr. Maki Inoue for technical advice for the real-time PCR experiment.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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