ARTICLE |
Correspondence to: Hiroaki Nakamura, First Dept. of Oral Anatomy, Niigata U. School of Dentistry, Gakkocho-dori-2, Niigata 951, Japan.
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Summary |
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We studied the immunohistochemical localization of CD44 and the ezrin-radixin-moesin (ERM) family of actin binding proteins in mouse enamel organ, using confocal laser scanning microscopy and transmission electron microscopy to clarify their role in cytoskeletal organization. At the differentiation stage of ameloblasts, immunoreactivity to CD44 was detected on the plasma membrane of the inner enamel epithelium, the stellate reticulum, the stratum intermedium, and the external enamel epithelium. In accordance with the differentiation of preameloblasts into secretory ameloblasts, immunoreactivity increased in the stratum intermedium cells. At the maturation stage, intense immunoreactivity was observed on the papillary layer cells. For the ERM family, the stratum intermedium and the papillary layer cells were stained with anti-ezrin and -radixin monoclonal antibodies but not with the anti-moesin antibody. Electron microscopic observations revealed that CD44, ezrin, and radixin were localized in the region at which preameloblasts came into contact with the stratum intermedium at the differentiation stage. At the secretory and maturation stages, they were concentrated in the microvilli of the stratum intermedium and the papillary layer cells. These findings suggest that the CD44-ezrin-radixin-actin filament system is involved in cell-cell interaction between preameloblasts and the stratum intermedium, and in the cytoskeletal organization of the cells in the stratum intermedium and the papillary layer. (J Histochem Cytochem 45:1481-1492, 1997)
Key Words: CD44, ERM family, immunohistochemistry, enamel, confocal laser scanning microscopy, transmission electron microscopy
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Introduction |
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Enamel formation and maturation are closely related to the differentiation and morphological changes of ameloblasts, the stratum intermedium, the stellate reticulum, the outer enamel epithelium, and the papillary layer. Epithelial-mesenchymal interaction, reciprocal tissue interaction, also has a regulatory effect on the morphogenesis and the differentiation of the enamel organ (
We previously reported that the plasma membranes of stratum intermedium cells and papillary layer cells contain carbohydrates (
In this study we investigated the localization of CD44 and the ERM family in the mouse incisor enamel organ to clarify their relationships, using monoclonal antibodies directed against mouse CD44, ezrin, radixin, and moesin. (
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Materials and Methods |
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Animals and Tissue Preparation
Ten 4-week-old male ddY mice, weighing approximately 17 g each, were immunohistochemically investigated. The mice were anesthetized with sodium pentobarbital and perfused through the left ventricle, first with Ringer's solution. Mice used for CD44 detection were perfused with 4% paraformaldehyde, 0.1% glutaraldehyde, and 0.1% tannic acid in 0.05 M phosphate buffer (pH 7.3) for 10 min. For detection of the ERM family, mice were perfused with 4% paraformaldehyde in 0.05 M phosphate buffer. Their mandibles were dissected, immersed in the same fixative for 4 hr at 4C, and decalcified in 4.13% EDTA (pH 7.3) for 5 days at 4C. Incisor enamel organs were used in the following ways.
Immunohistochemical Studies
Cryosections were preincubated in PBS containing 1% BSA for 15 min at room temperature (RT) and then incubated with fluorescein isothiocyanate (FITC)-conjugated rat monoclonal antibody to mouse CD44 (Pharmingen; San Diego, CA) diluted 1:100 for 12 hr at 4C. For the ERM family, sections were incubated with rat monoclonal antibodies against mouse ezrin, radixin, and moesin, (kindly provided by Dr. Tsukita, Kyoto University). After being washed with PBS, they were incubated with FITC-conjugated goat anti-rat IgG (Kirkegaard & Perry Laboratories; Gaithersburg, MD) diluted 1~40 for 1 hr, rinsed with PBS, and examined by CLSM (Olympus, LSM-GB 200; Tokyo, Japan).
Immunocytochemical Studies
Microslicer sections or cryosections were preincubated in PBS containing 1% BSA for 6 hr at 4C and incubated with biotin-conjugated rat anti-mouse CD44 monoclonal antibody (Pharmingen) diluted 1~100 for 24-48 hr at 4C, followed by horseradish peroxidase (HRP)-conjugated streptavidin (Nichirei; Tokyo, Japan) or 10-nm gold-conjugated streptavidin for 24 hr at 4C and washed with PBS. For the ERM family, sections were incubated with rat monoclonal antibodies against mouse ezrin, radixin, and moesin for 24 hr at 4C, followed by HRP-conjugated goat anti-rat immunoglobulins (Biosource International; Camarillo, CA). As for the observation through the HRP method, specimens were first immersed in a DAB solution (0.05% diaminobenzidine in 0.05 M Tris-HCl buffer, pH 7.6), and then in a DAB-H2O2 solution for 10 min at RT. After postfixation with 1% OsO4 in 0.1 M phosphate buffer (pH 7.4) for 1 hr at 4C, the tissue slices were dehydrated in graded acetone and then embedded in Poly/Bed 812 (Polyscience; Warrington, PA). Ultrathin sections were stained with lead citrate and observed under an H-7100 TEM (Hitachi; Tokyo, Japan) at an accelerating voltage of 75 kV.
Controls
Control sections were not incubated with primary antibody, nor were they incubated with normal rat serum.
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Results |
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Immunohistochemical Localization of CD44 in Enamel Organ
At the ameloblast differentiation stage, the enamel organ consisted of four types of cells: pre-ameloblasts, stratum intermedium, stellate reticulum, and outer enamel epithelium. Pre-ameloblasts were column-shaped and faced the odontoblasts. Stratum intermedium cells, roughly polyhedral in shape, were located at the proximal end of the pre-ameloblasts and formed two or three lines. Immunoreactivity to CD44 was detected on the plasma membrane of all cells in the enamel organ (Figure 1a). Preameloblasts and the stratum intermedium showed stronger reactivity than other cells. From the late differentiation stage to the early secretory stage, immunoreactivity on the stratum intermedium gradually increased. At the secretory stage, the staining was most pronounced on the stratum intermedium in the enamel organ (Figure 1b). At the maturation stage of ameloblasts, the stratum intermedium, stellate reticulum, and outer enamel epithelium turned into irregularly shaped polygonal cells, called papillary layer cells. These three types of cells were no longer discernible as separate layers. Papillary layer cells exhibited intense immunoreactivity (Figure 1c and Figure 1d). Minimal immunoreactivity was detected on the lateral plasma membrane of ruffle-ended ameloblasts (RA) and smooth-ended ameloblasts (SA). The ruffled border of RA and the distal plasma membrane of SA did not appear to show any immunoreactivity (Figure 1c and Figure 1d).
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Immunohistochemical Localization of the ERM Family in Enamel Organ
Immunoreactive patterns of ezrin and radixin in the stratum intermedium and the papillary layer were similar to each other (Figure 2 and Figure 3). At the differentiation stage, immunoreactivity to ezrin and radixin was restricted to the region of contact between pre-ameloblasts and the stratum intermedium (Figure 2a and Figure 3a). The intensity was gradually increased on two or three layers of the stratum intermedium cells. The stratum intermedium cells at the secretory stage and the papillary layer at the maturation stage were strongly immunolabeled by anti-ezrin and anti-radixin antibodies (Figure 2b-d and Figure 3b-d). The lateral plasma membrane of SA and RA also showed weak reactivity to radixin and moesin. The ruffled border of RA also showed immunoreactivity to the ERM family. No apparent reactivity to moesin was seen on the cells of the stratum intermedium and the papillary layer, although blood capillaries showed immunoreactivity to moesin (Figure 4). Odontoblasts showed immunoreactivity to radixin and moesin (Figure 3a and Figure 4a).
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Immunocytochemical Localization of CD44 and the ERM Family
At the differentiation stage, immunoreactivity to CD44 was observed on the plasma membranes of ameloblasts and stratum intermedium. Reactivity was also detected in the region at which the stratum intermedium cells and pre-ameloblasts came into contact with each other (Figure 5a). Electron microscopy showed that ezrin and radixin were localized in the cytoplasmic processes of pre-ameloblasts where they came into contact with the stratum intermedium (Figure 5b). At the secretory and the maturation stage, the plasma membranes of the stratum intermedium and the papillary layer cells were intensely immunolabeled by anti-CD44 monoclonal antibody (Figure 6a and Figure 6b). In addition, gold particles indicating localization of CD44 were mainly detected on the extracellular side of the plasma membrane of the microvilli, containing well-developed actin filaments (Figure 7a). Immunoreactivity to ezrin and radixin was seen in the cytoplasm of the microvilli (Figure 7b).
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Control
No specific immunoreactivity was observed in control sections incubated without any primary antibody or with normal rat serum.
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Discussion |
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We used mouse incisor enamel organ in this study because monospecific monoclonal antibodies to the mouse ERM family now available. We confirmed that CD44 was also localized in epithelial cells of the mouse incisor enamel organ, as previously reported in rats (
The stratum intermedium and the papillary layer have been believed to be involved in the transport of mineral and organic materials because of their characteristics, such as alkaline phosphatase (
Inner and outer enamel epithelia form a distinct multiple layer at the secretory stage, consisting of secretory ameloblasts, the stratum intermedium, the stellate reticulum, and the outer enamel epithelium. At the maturation stage, the latter three layers become the relatively homogeneous papillary layer, with an extensive network of microvilli. The ameloblast layer also shows morphological alterations, such as and ruffle- and smooth-ended ameloblasts. All of these events necessitate extensive rearrangements of the cell membrane and cytoskeleton. Recently, the cytoplasmic domain of CD44 was believed to bind to actin filaments via either the ERM family (Tsukita et al. 1994) or ankyrin-like molecules (
Adhesion and cytoskeletal changes among secretory ameloblasts are believed to be related to their sliding and the production of sinuous enamel prisms (
In conclusion, the CD44-ezrin-radixin-actin filament system is involved in cell-cell interaction between pre-ameloblasts and the stratum intermedium and in the cytoskeletal organization in the cells of the stratum intermedium and the papillary layer.
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Acknowledgments |
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Supported in part by a research grant from the Ministry of Education, Science, Sports and Culture of Japan.
We would like to thank all the members of our laboratory (1st Department of Oral Anatomy, Niigata University School of Dentistry) for assistance, especially S. Ejiri for helpful discussions and Ms S. Maekawa for expert secretarial assistance. We are also grateful to Dr Sh. Tsukita (Department of Cell Biology, Faculty of Medicine, Kyoto University) for his generous gift of ant-mouse ERM monoclonal antibodies.
Received for publication October 31, 1996; accepted May 27, 1997.
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