SEARCH

SEARCH BY CITATION

Keywords:

  • amelotin;
  • ameloblast;
  • enamel;
  • immunohistochemistry;
  • RT-PCR

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Amelotin is expressed and secreted by ameloblasts in tooth development, but amelotin distribution during enamel development is not clear. In this report, we first investigated amelotin expression in developing teeth by immunohistochemistry. Amelotin was detected in the enamel matrix at the secretion and maturation stages of enamel development. Amelotin was also observed at Tomes' processes on the apical ends of secretory ameloblasts. We then compared amelotin gene expression with those of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT-PCR. The expression of amelotin was detected as early as in postnatal day 0 mandibles and amelotin was coexpressed with amelogenin, ameloblastin, and enamelin during tooth development. These data strongly suggest that amelotin is an enamel matrix protein expressed at the secretion and maturation stages of enamel development. Anat Rec, 2010. © 2009 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Tooth enamel, the hardest calcified tissue of the body, is composed of highly organized hydroxyapatite crystals contributing more than 90% to the total weight of mature enamel. The formation of apatite crystals is a complex biomineralization process, in which enamel matrix proteins are involved (Zeichner-David et al., 1988). Enamel biomineralization is characterized by enamel matrix protein secretion and enamel maturation. During the secretary stage of enamel development, ameloblasts secrete matrix proteins, which induce crystal nucleation and provide an adequate microenvironment for the formation of large apatite crystals. The full thickness of the enamel is laid down mainly at this stage. The maturation stage of enamel development is characterized by gradually losing the secreted enamel matrix proteins, which are replaced by apatite crystals.

Ameloblasts, which form tooth enamel, synthesize and secrete two major classes of structural proteins, amelogenin, and nonamelogenin (Paine et al., 2001). Amelogenin is a major component of the developing enamel matrix. Amelogenin studies have shown that the self-assembly of amelogenin monomers into “nanospheres” is an important process for apatite crystal growth (Moradian-Oldak and Goldberg et al., 2005). In amelogenin-null mice, the enamel is defective, disorganized, and hypoplastic (Gibson et al., 2001), suggesting that amelogenin may play a critical role in enamel biomineralization. There are two nonamelogenin matrix proteins in the enamel, enamelin, and ameloblastin, which are tooth-specific and expressed by ameloblasts. Enamelin is processed into several smaller functional products localized to the layers of mature enamel (Uchida et al., 1991). Mutations in enamelin create variability in tooth enamel thickness and are responsible for heritable enamel development disorders (Pavlic et al., 2007). Ameloblastin is also a part of the organic enamel matrix. It is expressed at high levels by ameloblasts at the secreting stage of enamel development. In ameloblastin-null mice, the enamel develops severe enamel hypoplasia and displays the detachment of ameloblasts from the enamel matrix (Fukumoto et al., 2004).

Recently, a new ameloblast-specific gene, amelotin, is discovered (Iwasaki et al., 2005). Studies on rat and mouse tissues have revealed that amelotin is highly expressed by mature ameloblasts and is present in the basal lamina between the ameloblasts and the enamel matrix, and the junctional epithelial cells (Moffatt et al., 2006). The amelotin gene is located on the chromosomal locus 4q in human close to ameloblastin and enamelin on the same chromosome. Previous studies suggest that the chromosomal locus 4q13-21 in human is strongly associated with various forms of amelogenesis imperfecta (Forsman et al., 1994; Karrman et al., 1996). Investigating the spatiotemporal expression of amelotin would likely give insights into the biological functions of amelotin in enamel development. In the present report, we first investigated amelotin expression in developing teeth by immunohistochemistry and then compared the expression of amelotin with the expression of amelogenin, enamelin, and ameloblastin in the mandibles of postnatal mice by RT-PCR.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Kunming strain mice were used for all experiments. All protocols involving mice were reviewed and approved by the ethical committee of the Institute of Zoology, Chinese Academy of Sciences. Mandibles were obtained from mice at different postnatal developmental stages.

Immunolocalization of Amelotin in Mouse Mandibles

The mandibles at various developmental stages from Kunming mice were dissected and fixed with 4% paraformaldehyde in PBS for 20 hr at 4°C. Mandibles were decalcified at 4°C in a 10% (w/v) Na2-EDTA solution (pH7.0) for 10 days, and paraffin samples were prepared as described (Gao et al., 2005). The sections were pretreated with 10 mM sodium citrate (pH 6.0) for 40 min at 80°C, and then treated with 3% hydrogen peroxide for 10 min. The sections were incubated in blocking solution (1% BSA, 10% normal goat serum) for 1 hr. The dilution of antiamelotin antibody (Ct antibody, which recognizes the C-terminal residues 187–200, ATHTTEGTTIDPPN of amelotin, Proteintech Group, Chicago) used for immunohistochemistry was 1:50. Antibody binding was visualized using the Vectastain ABC Elite Kit and peroxidase substrate kit (Vector Laboratories, Burlingame). For the control section, the primary antibody was replaced with normal rabbit IgG. Sections were counterstained with hematoxylin and visualized under a light microscope (Olympus, Japan).

RNA Isolation and RT–PCR Analyses

Total RNA from the mandibles of postnatal day 0, 1, 3, 7, 12, and 19 mice was isolated using Trizol Reagent (Invitrogen) according to the manufacturer's specifications. Total RNA was reverse-transcribed with super-script II reverse transcriptase and oligo(dT) (Fermentas MBI). PCR was performed by MyCycler Thermal Cycler (BIO-RAD) using mouse specific primers of amelogenin, ameloblastin, enamelin, amelotin, and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase). The sequences for the primers used in this study are shown in Table 1. Parallel reactions with GAPDH-specific primers were performed to monitor the integrity and uniformity of the RNA. PCR products were subjected to 1% agarose gels and stained with ethidium bromide.

Table 1. Primer sequences and conditions used in this study for RT–PCR analyses
GeneUpper primersLower primersProduct size (bp)
  1. The PCR parameters were: 94°C for 4 min, 30 cycles of 94°C for 30 see 55–58°C for 30–90 sec, 72°C for 1 min, and 72°C for 10 min.

Amelotin5′-CCGAGTAAAGTGGAGAAG-3′5′-AGGTCTGTCTGAAGTGGC-3′969
Enamelin5′-CTATGGGAGGCAAGACGA-3′5′-AGCCACCAATACAGCAAGA-3′878
Ameloblastin5′-GAACTGTTTTGATTGGCATC-3′5′-AAGAAGGGACCTACACTATTT-3′1431
Amelogenin5′-GCTTCAGACAGAAACTCACT-3′5′-GAACACTACTACATGCCATTAT-3′759
GAPDH5′-TGCCCAGAACATCATCCCT-3′5′-TGAAGTCGCAGGAGACAACC-3′254

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

Immunolocalization of Amelotin in Mouse Incisor

To investigate amelotin distribution in teeth, we first assessed the sequential expression of amelotin in the developing incisors by immunohistochemistry. As shown in Fig. 1A, amelotin labeling was detected in the enamel organ of incisors in postnatal day 3 mice. Under the high magnification, a thick layer of dentin was observed, but no obvious staining was detected in ameloblast layer or on the dentin surface (Fig. 1B). When the enamel layer was apparent, a faint immunostaining was detected on the surface of the dentin layer (red arrow). At the secretion stage of enamel development, ameloblasts were characterized by Tomes' processes projecting into the developing enamel matrix. The entire enamel matrix, including the Tomes' process layer, was intensely immunostained (Fig. 1C). At the early maturation stage of enamel development (Fig. 1D), the inner layer of enamel matrix was mostly degraded. Tomes' processes of ameloblasts continuously secreted the enamel matrix, which was obviously immnostained.

thumbnail image

Figure 1. Immunohistochemical detection of amelotin protein in mouse mandibular incisors. Amelotin was localized to the enamel organ of the lower incisor (A). Pictures (B, C, D) are magnified regions boxed in (A) (from left to right, respectively). In Fig. 1B, no specific immunostaining is visible in the ameloblast layer and on the dentin surface (arrowhead). Gradually, a faint staining was detected on the surface of the dentin layer (arrow). At the secretion stage, the enamel matrix and the apical ends of ameloblasts were intensely immunostained (Fig. 1C). With the maturation of the enamel (Fig. 1D), the inner part of the enamel matrix was degraded (asterisk indicates the degraded enamel space), and specific staining was detected in the outer layer of the enamel matrix. eo, enamel organ; am, ameloblast; de, dentin; emp, enamel matrix proteins; T, Tomes' processes. Original magnifications: A, ×100; B, C, and D, ×400.

Download figure to PowerPoint

Immunolocalization of Amelotin in Developing Mouse Molars

Immunostaining presented in Fig. 2 illustrates the pattern and localization of amelotin in the first lower molars of various developmental stages. In the first molar of postnatal day 1 mouse mandible, predentin was synthesized by odontoblasts as arrow indicated, but no enamel matrix layer was observed, and no specific staining was detected by immunohistochemistry (Fig. 2A). In the first molar of postnatal day 3 mandibles, the enamel matrix appeared on the dentin surface and the inner part of enamel matrix on the cusp tips started to be degraded (Fig. 2B). Amelotin protein was strongly expressed in the entire enamel layer. In postnatal day 7 mandibles (Fig. 2C), from tips to bases on the cusps in the first molar, the inner part of the enamel matrix was degraded and the full thick layer of enamel protein was only observed in the cervical loops of the first molar (black arrow). Amelotin was detected in the outer layer of the enamel matrix. In postnatal day 12 mandibles (Fig. 2D), most of the enamel matrix was degraded and the enamel matrix, which was intensely immunostained by amelotin antibody, was only observed around the cervical loops in the first molar. On the control section, no specific staining was detected (Fig. 2E,F).

thumbnail image

Figure 2. Immunohistochemical detection of amelotin in developing mandibular molars. In the first molar of a postnatal day 1 mandible, a thin layer of predentin was formed (arrow), but amelotin was not detected (Fig. 2A). In the first molar of a Day 3 mandible (Fig. 2B), amelotin was exclusively expressed in the enamel matrix. The arrow indicates the dentin layer; the arrowhead indicates the degraded enamel matrix. In the first molar of a Day 7 mandible (Fig. 2C), the red arrow indicates the space formed by the degraded enamel matrix and the black arrow indicates enamel matrix proteins. In the first molar of a Day 12 mandible (Fig. 2D), the enamel matrix was still visible at the cervical loop and immunostained strongly. The control sections were from a Day 3 mandible (Fig. 2E) and a Day 7 mandible (Fig. 2F). eo, enamel organ; dp, dental papilla; de, dentin; emp, enamel matrix proteins; sm, second molar. Original magnification: A, E, and F ×100; B, C, D, ×40.

Download figure to PowerPoint

We also examined amelotin expression under high magnification in cervical loops of the first molars at various developmental stages (Fig. 3). In the first molar of Day 3 mandible where no obvious Tomes' process structure was observed at the apical end of ameloblasts (Fig. 3A, arrowhead), no specific immunostaining was detected in the thin enamel layer on the dentin surface (black arrow). With the appearance of Tomes' processes, a high level of immunoreactivity in the ameloblast layer and the enamel matrix was observed (Fig. 3A, red arrow). In the first molar of Day 7 mandibles, the inner dental epithelium cells in cervical loops differentiated to ameloblasts, which started to secrete the enamel matrix, but no specific staining was observed in the enamel matrix layer (Fig. 3B, black arrow), and only a weak staining was observed in the ameloblast layer (Fig. 3B, red arrow). An intense staining was observed in the secreted enamel matrix surrounding Tomes' processes. In the first molar of postnatal day 12 mandible, ameloblasts in the cervical loop were shortened and Tomes' processes disappeared (Fig. 3C). The existing enamel matrix was specifically stained, and a faint staining was detected on the apical ends of ameloblasts (red arrow). In the first molar of Day 19 mandible, the ameloblasts at the cervical loop were replaced by junctional epithelium (Fig. 3D). Amelotin staining was only observed in the tissues at the cervical loop (red arrow).

thumbnail image

Figure 3. Immunohistochemical detection of amelotin protein at the cervical loops of molars. In (A), the arrowhead indicates the differentiating ameloblasts; the black arrow indicates the enamel layer; the red arrow indicates the secreting ameloblasts layer. In (B), the black arrow indicates the enamel layer; the red arrow indicates secreting ameloblasts. In (C), the red arrow indicates the apical ends of ameloblasts. In (D), the red arrow indicates the tissues at the cervical loop and the asterisk indicates the enamel space after demineralization. am, ameloblasts; de, dentin; od, odontoblast; emp, enamel matrix proteins; T, Tomes' processes. Original magnifications: ×400.

Download figure to PowerPoint

Analyses of Gene Expression Patterns by RT-PCR

To further explore the expression profile of amelotin during amelogenesis, we compared amelotin gene expression with other enamel-specific genes by RT-PCR analyses. As shown in Fig. 4, all genes examined in mandibles showed a similar expression pattern: a very low level of signals in postnatal day 0 mandibles and a high level of signals in postnatal day 3 mandibles. With the development of mandibles, gene expression levels, especially for amelogenin, decreased obviously.

thumbnail image

Figure 4. RT-PCR analyses of ameloblast-specific genes in mouse mandibles. Total RNA was extracted from postnatal day 0, 1, 3, 7, 12, and 19 mouse mandibles and used as templates to detect amelotin, enamelin, ameloblastin, amelogenin, and GAPDH in two-step RT-PCR reactions. The PCR products were resolved on 1% agarose gels and stained with ethidium bromide. The primers for amelotin produce a band at 969 bp, enamelin primers produce a band at 878 bp, ameloblastin primers produce a band at 1431 bp, and amelogenin primers produce a band at 759 bp. GAPDH primers, yielding a 254 bp product, were used in parallel reactions to assess the integrity and uniformity of the RNAs. RT-PCR, Reverse transcription polymerase chain reaction; D, Day; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED

The formation of enamel, a calcium phosphate substance on the tooth surface, involves the secretion of enamel matrix proteins (amelogenin, ameloblastin, and enamelin) and the formation of an enamel matrix microenvironment that initiates nucleation and mediates growth of apatite crystals (Paine et al., 2000; Ravindranath et al., 2004; Margolis et al., 2006; Fan et al., 2007). In this report, we presented new evidence for the localization of amelotin. In the early developmental stage of incisors (Fig. 1B) and molars (Fig. 2A), the enamel matrix formation was not obvious and no specific amelotin staining was detected in the enamel organ, which suggests that the function of amelotin in tooth development is not linked to the differentiation of ameloblasts. We showed that at the secretion stage of enamel development, amelotin expression in the enamel layer was intense with the appearance of Tomes' processes on the apical ends of ameloblasts. On the basis of these results, we concluded that amelotin is an enamel matrix protein synthesized by ameloblasts. With the development of the enamel, the enamel matrix secreted at the early stage of enamel development was gradually degraded, no amelotin was detected in the enamel space, but amelotin was still detected in the outer part of the enamel matrix and in the layer of Tomes' process. All these studies suggested that amelotin, like other structural enamel proteins such as amelogenin, ameloblastin, and enamelin (Hu et al., 1997; Lee et al., 2003), is secreted to the enamel matrix layer from the secretion stage to the early maturation stage of enamel development. These results contrast with a previous report showing that amelotin is not one of enamel matrix proteins and exclusively locates to the basal lamina between ameloblasts and the enamel matrix layer (Moffatt et al., 2006). A possible explanation for this discrepancy is the different methodology of sample processing for immunohistochemistry. We found that without antigen retrieval treatment in sodium citrate solution (pH 6.0), no obvious immunostaining can be detected in the enamel matrix layer (data not shown). One study has been shown that amelotin is expressed with other enamel matrix proteins in developing teeth (Trueb et al, 2007). In newborn mice (postnatal day 0), ameloblasts in incisors are differentiated and began to synthesize and secrete enamel matrix. Our RT-PCR study showed that amelotin expression was detected in the mandibles as early as postnatal day 0, increased significantly in postnatal 3 mandibles, and exhibited a similar expression pattern to amelogenin, ameloblastin, and enamelin in tooth development, which presented strong evidence that amelotin starts to secret at an early stage of enamel development. Our studies on various stages of mouse low molars also presented evidence that amelotin was not expressed in ameloblasts at the differentiating stage and the post-maturation stage, but it was transiently expressed during enamel development.

As a gene that encodes a newly identified enamel matrix protein, amelotin is located close to ameloblastin and enamelin on the chromosome locus in human and mouse. On the long arm of human chromosome 4 and mouse chromosome 5, amelotin, ameloblastin, and enamelin are also located close to the SIBLING (small integrin-binding ligand N-linked glycoproteins) gene family, which is involved in the biomineralization of dentin and bone (Fisher et al., 2003; Qin et al., 2004; Huang et al., 2008). Our studies have presented the first evidence for the expression of amelotin in the enamel matrix layer, which strongly suggests that amelotin may be involved in the biomineralization of the enamel. In tooth development, the expressions of amelotin, ameloblastin, enamelin, and amelogenin were exclusively defined to dental tissues. The coexpression of amelotin, ameloblastin, enamelin, and amelogenin in tooth development suggests that the enamel matrix proteins may promote the process of enamel biomineralization cooperatively. Further studies on amelotin in vivo and in vitro would advance our knowledge of amelotin protein in enamel development.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. LITERATURE CITED