(Received for publication, September 18, 1995; and in revised form, November 6, 1995)
From the
We report the full-length sequencing, cell type-specific expression, and immunolocalization of a novel gene expressed in rat incisors, which we have designated ameloblastin. Northern blot analysis of RNA from multiple rat and mouse tissues demonstrated high levels of expression of two distinct transcripts of approximately 2.0 and 1.6 kilobase pairs that were expressed only in teeth. In situ hybridization using a digoxigenin-labeled RNA probe showed that the tissue distribution of ameloblastin was limited to the ameloblast in rat incisors. Immunohistochemical staining of rat incisors using a polyclonal antibody raised against a fusion protein revealed a unique localization pattern. Ameloblastin was found to be expressed during the differentiation of inner enamel epithelium into ameloblasts, with intense localization in the Tomes' processes of secretory ameloblasts. In contrast to amelogenin, only modest amounts of ameloblastin were detected in enamel matrix. The ameloblastin gene encodes an open reading frame of 422 amino acids corresponding to a putative protein of 45 kDa. The predicted protein is acidic (pI = 5.54) and the most abundant amino acids are Pro (15.2%), Gly (9.9%), and Leu (9.9%). We have also mapped the ameloblastin gene, Ambn, to a locus on mouse chromosome 5 near other genes associated with mineralized tissues. Thus, ameloblastin represents a unique ameloblast-specific gene product that may be important in enamel matrix formation and mineralization.
The mammalian tooth is a specialized structure that develops through a series of reciprocal epithelial-mesenchymal interaction culminating in the formation of three different mineralized tissues: enamel, dentin, and cementum(1, 2, 3) . The tooth has provided a valuable model system for understanding specific gene regulation, morphogenesis, and mineralization. Enamel is an oral epithelial-derived mineralized tissue, while dentin and cementum are formed by cranial neural crest-derived ectomesenchyme(4, 5, 6) . This cranial neural crest-derived ectomesenchyme is derived from the neuroectoderm of rhombomere 2 of the vertebrate hindbrain(7, 8) . As in bone, the premineralized matrix of dentin and cementum contains a collagen-rich matrix that is maintained throughout the mature tissue. However, enamel differs from these other mineralized tissues in two important ways: 1) the epithelial-derived enamel matrix does not contain type I collagen; and 2) the organic matrix of enamel is essentially lost during tissue maturation, resulting in a tissue that is more than 98% mineral in the form of hydroxyapatite(9, 10) .
Amelogenesis, the formation of tooth enamel, is divided into discrete developmental stages(11) . Following a presecretory stage in which the inner enamel epithelium receives developmental cues from the dental papillae, the inner enamel epithelium differentiates into secretory ameloblasts. The enamel matrix, secreted by the ameloblast, is composed of two classes of proteins termed the amelogenins and the enamelins (12) . The amelogenins are hydrophobic proteins rich in proline, histidine, and glutamic acid and comprise about 90% of the enamel matrix. Diversity of the amelogenins found in enamel matrix is due to alternative mRNA splicing(13) . The remaining 10% of enamel matrix is made up of acidic enamelins (14) and other non-amelogenin proteins such as amelogeninase(15, 16) . Once the full thickness of enamel matrix is formed, significant morphological and functional changes occur within the ameloblast as it passes through the transition, maturation, and protective stages of development. Thus, the developing tooth is a unique mineralizing organ and provides an interesting developmental system.
Although a few tooth-specific genes have been identified, the precise mechanisms of tooth morphogenesis, mineralization and the pathophysiology of inherited enamel and dentin defects are not well defined. We have therefore initiated a genome project with an ultimate goal of identifying novel genes involved in tooth formation which may help explain the mechanisms of odontogenesis(17) . We chose to use the continuously erupting rat incisor as a model system, because the complete developmental sequence of odontogenesis can be analyzed in a single tooth. As an initial approach, we constructed a unidirectional cDNA library from the non-calcified portion of incisors of 3-4-week-old rats, sequenced clones, and classified sequences based on their homology to known genes(17) . One clone termed Y224 was the most abundant novel gene expressed and was partially sequenced(17) . Here, we report the full-length sequencing, cell-specific expression, and protein localization pattern of a newly described ameloblast-specific gene which we have designated ameloblastin.
We previously constructed a unidirectional cDNA library from the non-calcified tissues of rat incisors and partially sequenced the 5` portion of the coding strands of about 400 randomly selected clones (17) . Sequence homology searches through GenBank(TM) data base and PIR protein data base revealed that the majority of the clones (about 56%) represented previously unidentified genes. To identify novel genes involved in tooth development we have first focused on the most frequently expressed genes from this previously unidentified cDNA population.
Figure 1:
Tissue-specific expression of
ameloblastin mRNA. RNA extracted from rat (A) or mouse (B) tissues was subjected to Northern blot analysis. A
10-µg sample of total RNA was separated on a 1%
agarose-formaldehyde gel and transferred to a nylon membrane by
capillary action. Filters were hybridized with a
[P]dCTP-labeled cDNA (Y224). The lower
panel shows ethidium bromide staining of 18 and 28 S ribosomal
RNAs.
Figure 2:
Ameloblastin expression in rat incisors
( 400, no counterstain). A, in situ hybridization using
the Y224 sense RNA probe (negative control). B, in situ hybridization using the Y224 antisense probe. Ameloblastin is
localized to the cytoplasm of secretory ameloblasts (Am). C, immunohistochemical staining of ameloblastin. Ameloblastin
is localized to the distal cytoplasm and Tomes' processes of
secretory ameloblasts (closed arrow). Extracellular staining
is evident at the dentino-enamel junction (open arrow). E represents enamel matrix.
Figure 3: Nucleotide sequence and deduced amino acid sequence of rat ameloblastin. The 1929-base pair nucleotide sequence of the full-length cDNA is shown, including untranslated 5` and 3` sequences. The numbers on the left refer to nucleotide positions. The numbers on the right refer to amino acid positions. Potential polyadenylation signals are boxed. The presumptive signal sequence is underlined and the closed arrow represents the predicted signal sequence cleavage site(25) .
Figure 4: Map location of the ameloblastin gene on mouse chromosome 5. To the right of the map are recombination fractions for adjacent loci: the first fraction represents data from the M. m. musculus crosses, and the second fraction is from the M. spretus crosses. In parentheses are recombinational distances and standard errors calculated according to Green(35) , Map locations of the human homologs are indicated to the left.
As a result of our initial strategy designed to identify new genes involved in odontogenesis, we obtained partial DNA sequence on over 400 cDNA clones from a rat incisor library(17) . One clone that appeared multiple times during random sequencing, Y224, was further characterized and named ameloblastin. We have completed the sequencing of the cDNA for ameloblastin, characterized its expression in the continuously erupting rat incisor, and mapped its locus within the mouse genome. Four other clones that share partial sequence with ameloblastin were identified and are currently being investigated as potential alternatively spliced transcripts.
The cDNA for ameloblastin encodes an open reading frame of 422 amino acids that corresponds to a predicted 45-kDa protein with no significant homology to any reported gene except Y224. The predicted protein is rich in proline and glycine, but it does not display a primary structure consistent with reported collagens. Thus, ameloblastin is a novel tooth-specific gene. Although ameloblastin does not share sequence homology with other reported genes, it does contain potential phosphorylation sites, including a casein kinase II phosphorylation site that is shared by proteins involved in mineralization such as in osteopontin(27) , bone sialoprotein(28) , bone acidic glycoprotein-75(29) , and dentin phosphoprotein(30) , as well as a tyrosine kinase site and three protein kinase C sites.
Northern blot analysis of multiple rat and mouse tissues showed that ameloblastin expression was restricted to the developing tooth. Two transcripts were observed at 2.0 and 1.6 kb. It is not yet determined if the two transcripts result from alternative mRNA splicing, the use of multiple transcription start sites, or the use of two different polyadenylation sites.
The immunohistochemical analysis of ameloblastin was consistent with the results obtained by in situ hybridization. The finding that ameloblastin became condensed in the Tomes' processes of secretory ameloblasts and in the distal cytoplasm of mature ameloblasts may imply that the production of ameloblastin is closely related to the process of secretion. Diffuse staining was seen in the superficial enamel layer near the mature ameloblasts, a region that is mainly composed of homogenous enamel matrix. Irregular positive immunolocalization of ameloblastin was also observed in the dentino-enamel junction in a pattern similar to that reported for tuftelin(14) . The persistent expression of ameloblastin in the cytoplasm of mature ameloblasts and the dispersed positive reaction of ameloblastin in the superficial enamel layer near post-secretory ameloblasts support the notion that ameloblastin could play a role in enamel mineralization. However, the precise function of ameloblastin during amelogenesis remains to be determined.
Previous studies have demonstrated that the region of the Ambn locus on mouse chromosome 5, and the corresponding region on human chromosome 4 contain a number of genes important in mineralization, including osteopontin, bone sialoprotein, and bone morphogenetic protein 3(31) . Ambn is not, however, clustered with these more distally located genes. Recent studies, however, place the human disorder dentinogenesis imperfecta type II in close linkage with human osteopontin(32, 33) . Since both osteopontin and bone sialoprotein have been excluded as candidates for this disorder(23, 33) , we are pursuing the possibility that a defect in Ambn might be responsible for this phenotype as well as an autosomal dominant form of amelogenesis imperfecta, which has been linked to human chromosome 4q(34) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U35097[GenBank].