Med Mol Morphol (2010) 43:102–106
DOI 10.1007/s00795-009-0482-7© The Japanese Society for Clinical Molecular Morphology 2010

ORIGINAL PAPER
Masaki Hata · Tadafumi Kawamoto · Mariko Kawai
Toshio Yamamoto

Differential expression patterns of the tight junction-associated proteins
occludin and claudins in secretory and mature ameloblasts in mouse incisor

Received: August 10, 2009 / Accepted: October 28, 2009

Abstract Tight junctions (TJs) function primarily as abarrier against paracellular transport between epithelial
cells and are composed mainly of occludin (OLD) and claudins (CLDs). The CLD family consists of 24 members thatshow tissue- or cell-speciﬁc expression. Ameloblasts, which
originate from the oral epithelium, form enamel, and enamel
proteins and minerals are transported across the ameloblastic layer during amelogenesis. We immunohistochemicallyexamined the distribution patterns of TJs in ameloblasts byobserving the expression patterns of OLD and CLDs
(CLD-1 to CLD-10). Secretory ameloblasts contained OLD
and CLD-1, -8, and -9 at the distal end of the cell. In mature
ameloblasts, OLD and CLD-1, -6, -7, -8, -9, and -10 were
present mainly at both the distal and proximal ends of thecell, regardless of whether the ameloblasts were rufﬂeended or smooth-ended. Mature ameloblasts in which only
the proximal ends were stained for OLD and CLDs werealso found. These results indicate that the expression patterns of CLDs and the distribution patterns of TJs change
drastically between the secretory and mature ameloblaststages, suggesting that these patterns reﬂect the different
functions of these cells, speciﬁcally in the transport of proteins and ions for enamel formation.
Key words Tight junction · Occludin · Claudin ·Ameloblast · Tooth

M. Hata1 · M. Kawai · T. Yamamoto (*)
Department of Oral Morphology, Okayama University Graduate
School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1
Shikata-cho, Okayama 700-8525, Japan
Tel. +81-86-235-6630; Fax +81-86-235-6634
e-mail: ytoshio@md.okayama-u.ac.jp
T. Kawamoto
Radioisotope Research Institute, Tsurumi University School of
Dental Medicine, Yokohama, Japan
1

Present address: Department of Pathology, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo, JapanA part of this study was reported at the 47th Annual Meeting of theJapanese Association for Oral Biology, held at Sendai, Japan, in 2005

Introduction
Ameloblasts, which are differentiated from the inner enamel
epithelium of the enamel organ, form tooth enamel and line
the enamel surface as a simple columnar epithelium, throughamelogenesis. Amelogenesis is divided into two distinct
stages, the secretory stage and the maturation stage, andameloblasts undergo morphological and functional changes
during the different stages of amelogenesis. At the secretory
stage, ameloblasts, called secretory ameloblasts, characteristically exhibit Tomes’ processes and well-developed cellorganellae, such as rough endoplasmic reticulum and Golgi
apparatus. This cell type primarily functions to secrete
abundant enamel proteins and participates in the partial
calciﬁcation of the secreted enamel matrix. After the organic
matrix formation is completed, ameloblasts drastically modulate their morphological characteristics to become maturation ameloblasts: the cell shortens, the cell organellae suchas rough endoplasmic reticulum and Golgi apparatus
decrease, and the Tomes’ process disappears. At this stage,
the organic matrix that was secreted at the secretory stage
is degraded and resorbed, and calcium (Ca) inﬂux acrossthe ameloblastic layer into the enamel matrix increases.
These qualitative changes in the enamel matrix are achieved
by both rufﬂe-ended ameloblasts (R-As) and smooth-ended
ameloblasts (S-As) at the maturation stage. These two typesof mature ameloblasts periodically undergo dramatic modulations of their morphological characteristics, switching
between the R-A and S-A phenotypes. The sequence of
amelogenesis is described in several reviews.1–3
It is well accepted that the transportation of materials
across the epithelial cell layer is carried out by paracellular
and/or transcellular pathways. The tight junction (TJ) is anintercellular junction that acts as a seal between epithelial
cells to prevent the diffusion of materials via the paracellular pathway. The molecular architecture of the TJ has
recently been clariﬁed: it is composed mainly of the integral
transmembrane proteins occludin (OLD) and claudin
family members (CLDs). OLD was the ﬁrst identiﬁed component of TJs4; the CLDs were found later as TJ-associated

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5,6

adhesion molecules. The CLDs comprise a gene familywith 24 members and are expressed in tissue-speciﬁc distribution patterns, where they presumably play speciﬁc rolesin the paracellular transport between epithelial cells in eachorgan or tissue.7 Freeze-fracture studies reveal TJs between
ameloblasts,8–13 and proteins, minerals, and water are transported across the ameloblastic layer in amelogenesis; thus,
it has been suggested that the TJs between ameloblasts playa role for the transportation of solutes and Ca.1–3 However,the distribution patterns of the TJs and the molecular architecture of the junction at each ameloblast stage have notbeen fully clariﬁed. In the present study, we used the immunohistochemical analysis of ameloblasts from mouse incisors to identify the distribution pattern of TJs and theirmolecular architecture at each ameloblast stage, focusing on
the contributions of the claudin family.

Materials and methods
C57BL6/J mice at 5–6 weeks of age were used as experimental animals. A 0.04% 3,3-bis[N,N-di(carboxymethyl)aminomethyl] ﬂuorescein (calcein) solution was administered to some mice via the left ventricle 10 min before
death, to distinguish the R-As and S-As, as reported elsewhere.14,15 The animal experiment in this study was approvedby the Animal Research Control Committee of Okayama
University.
Immunohistochemistry
Freshly frozen undecalciﬁed sections of mandibular incisors
were used for the immunohistochemical detection of OLD
and CLDs. The sections were prepared according to themethod of Kawamoto.16 Brieﬂy, mice were decapitated after
being anesthetized, and the heads were immediately frozen
in liquid nitrogen-cooled isopentane and freeze-embedded
in 5% carboxymethyl cellulose. Undecalciﬁed fresh-frozen
sections of entire incisors of the mandible approximately
5 μm thick were cut with a cryostat (Leica CM3050S;
Nussloch, Germany) at a temperature range of −23° to−25°C and mounted on adhesive ﬁlm on glass slides. The
sections were kept at −80°C until use. Before immunohistochemical reactions, the sections were ﬁxed in cooled 95%
ethyl alcohol at −20°C for 30 min and immersed in 100%
acetone for 1 min at room temperature. The sections werethen rinsed in phosphate-buffered saline (PBS) containing
1% bovine serum albumin (BSA) several times, and incubated with antibodies to OLD or the CLDs for 4 h at 4°C.
The sections were rinsed brieﬂy with PBS containing 1%
BSA and then incubated with Cy3-conjugated secondary
antibodies (diluted to 1:250 with PBS containing 1% BSA)
for 1 h at room temperature. These antibodies were subjected to centrifugation at 15 000 rpm before use to reduce
nonspeciﬁc staining. The incubation was terminated bywashing the sections several times with PBS. In some sections, the nuclei of ameloblasts were counterstained with
Hoechst (bis-benzamide trihydrochloride). The slides werethen mounted with 95% glycerin containing 0.1% pphenylenediamine. The sections were observed with a ﬂuorescence microscope, and phase-contrast images were also
obtained (Leica, New York).
Antibodies for occludin and claudins
Primary antibodies for detecting OLD and CLDs were an
antimouse OLD monoclonal antibody and antimouse polyclonal antibodies for CLD-1, -2, -3, -4, -5, -6, -7, -8, -9, and-10, respectively. The anti-CLD-1, -3, and -5 polyclonal antibodies were purchased (Zymed Laboratories, San Francisco, CA, USA). The others were kindly provided by thelate Prof. Shoichiro Tsukita (Kyoto University Graduate
School of Medicine, Kyoto, Japan).

Results
In the present study, three types of ameloblasts wereobserved: secretory ameloblasts, mature R-As and mature
S-As. The observed regions of each incisor are illustrated inFig. 1.
In the secretory ameloblasts, OLD staining was observedat the proximal end of the cells, as were CLD-1, -8, and -9(Figs. 2, 3). In contrast, CLD-2, -3, -4, -5, -6, -7, and -10, were
not detected in these cells. OLD and CLD-1, -6, -7, -8, -9,
and -10 were detected, however, in maturation-stage cells;i.e., CLD-6, -7, and -10 were newly expressed by the cells at
this stage. OLD and these CLDs also showed interesting
localization patterns in the mature ameloblasts. OLD ﬂuorescence was mainly localized to both the proximal anddistal ends of the cells, forming a railroad-like pattern in thealigned cells (Fig. 4). The CLDs showed the same distribution pattern as OLD. For example, as shown in Fig. 5, CLD-1
was detected at both ends of the mature ameloblastic layer,
and this pattern was not associated speciﬁcally with the R-A
or S-A region, as judged by calcein ﬂuorescence. In addition
to this distribution pattern, some groups of mature ameloblasts showed labeling only at their proximal ends (Fig. 6).
In these cells, CLD-6, -7, -8, -9, and -10 showed the same

Fig. 1. Diagram of the regions observed for the three types of ameloblasts: secretory ameloblasts, rufﬂe-ended ameloblasts (R-As), andsmooth-ended ameloblasts (S-As)

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Fig. 4. Occludin detection in mature ameloblasts. A Phase-contrast
photograph showing the occludin-positive area. Nuclear staining for
ameloblasts by the ﬂuorescent dye Hoechst (arrow) is overlaid. B
Occludin ﬂuorescence appeared as two parallel lines in mature ameloblasts. C Phase-contrast image and occludin ﬂuorescence image are
overlaid. Occludin was detected at both the distal and proximal endsof the cells. E, enamel; D, dentin. Bar 100 μm

Fig. 2. Phase-contrast image and claudin-1 localization of secretory
ameloblasts in the same section. A Secretory ameloblasts are identiﬁedby their tall and columnar cell proﬁles (brackets). Asterisk indicates theenamel of the secretory stage. B Claudin-1 was detected at the distal
end of each secretory ameloblast, and the positive ﬂuorescence ran asa single line along the enamel (arrow). The ﬂuorescence of the enamel
matrix was from the nonspeciﬁc adsorption of antibodies. C Merged
image composed of phase-contrast and ﬂuorescence images. D, dentin.
Bars 100 μm

Fig. 5. Claudin-1 detection in mature ameloblasts. Calcein was administered before death to distinguish the S-A region from the R-A region.
A Phase-contrast image of an observed area. B Same area showing the
S-A region labeled with green ﬂuorescence (arrows) and the R-A area
as negative for ﬂuorescence (arrowheads). C Orange ﬂuorescence indicates claudin-1 localized to both the distal and proximal ends of thecells. D Overlaid image composed of B and C. The localization patternof claudin-1 showed no difference between S-As and R-As. E, enamel.
Bar 50 μm
Fig. 3. Claudin-8 and -9 in secretory ameloblasts. Both Claudin-8 (A)and claudin-9 (B) were detected at the distal end of secretory ameloblasts (arrows). Bars 20 μm

distribution patterns as OLD and CLD-1 (Fig. 7). As asummary, the localization of positively detected CLDs in
ameloblasts is shown in Table 1. CLD-2, -3, -4, and -5 were
not detected in mature ameloblasts in the present study.

Discussion
Previous immunohistochemical studies have shown thatameloblasts begin to express OLD at the distal cell membrane as their differentiation progresses,17 and that OLD
and CLD-1 are detected at the proximal end of ameloblasts

at the late stage of differentiation.18 In addition, a recent insitu hybridization study of claudin mRNAs in murine
embryonic tooth germ cells showed that CLD-1, -3, -4, -6,-7, and -10 have restricted expression patterns in the tooth
epithelium at the bud stage that correlate with the patternsof epithelial cell differentiation; only CLD-2 mRNA expression is observed in preameloblasts at the early bell stage.19
These reports mainly examined the expression patterns of
OLD and CLDs in differentiating ameloblasts. In thepresent study, OLD and CLDs (CLD-1 to -10) were immunohistochemically examined in functional ameloblasts, i.e.,secretory ameloblasts and mature ameloblasts in mouseincisors during amelogenesis. In the secretory ameloblasts,
OLD and CLD-1, -8, and -9 were localized to the distal endof the cells. In mature ameloblasts, CLD-6, -7, and 10 wereexpressed in addition to OLD and the CLDs observed at

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Table 1. Distribution patterns of claudins in ameloblast stages
Ameloblast stages

Localization

Claudin 1

Claudin 6

Claudin 7

Claudin 8

Claudin 9

Claudin 10

Secretory ameloblasts

Proximal end
Distal end

−
+

−
−

−
−

−
+

−
+

−
−

Mature R-Asa

Proximal end
Distal end

+
+

+
+

+
+

+
+

+
+

+
+

Mature S-Asa

Proximal end
Distal end

+
+

+
+

+
+

+
+

+
+

+
+

R-As, rufﬂe-ended ameloblasts; S-As, smooth-ended ameloblasts
Signals of immunohistochemistry: −, not detected; +, detectedaMature ameloblasts in which only proximal ends were positive for occludin and claudins were also found in a few cases

Fig. 6. Claudin-1 localization in mature ameloblasts. A Mature ameloblasts showing claudin-1 at only the proximal end were sometimes
observed (arrows). B Phase-contrast image of A. E, enamel; brackets,
mature ameloblasts. Bar 50 μm

Fig. 7. Claudin-6, -8, -9, and -10 were detected at both the distal andproximal ends of mature ameloblasts. A Claudin-6 localization.
B Claudin-8 localization. The photograph is overlaid on the phasecontrast image. C Claudin-9 localization. Among the positive cells, agroup showing positive reactivity at only the proximal end is indicated(arrow). D Claudin-10 localization. Bars 100 μm

the secretory stage, and OLD and these CLDs were localized to both the distal and proximal ends of mature ameloblasts, although in some areas only the distal end of the cells
was positively stained. Thus, this report is the ﬁrst to identify
the expressions of OLD and CLD-1, -8, and -9 in secretory

ameloblasts and those of OLD and CLD-1, -8, -9 plus -6, -7,
and -10 in mature ameloblasts. With regard to OLD and
CLDs expression in functional incisors, a recent immunohistochemical study reported that secretory ameloblasts
were positive for CLD-4 but negative for OLD and CLD-1,
whereas in mature ameloblasts OLD, CLD-1, and CLD-4
were positively detected using rat incisors.20 In addition, theother CLDs examined in the study such as CLD-2, -3, -5, -6,-7, -8, -12, and -15 were negative in ameloblasts. The CLD-1
expression in mature ameloblasts is as in a previous report,20
i.e., CLD-1 was positively stained in mature ameloblasts.
However, the cause of the differences is still uncertain,
although it may be pointed out that the animals used andthe methods for specimen preparation are different in eachstudy.
It is tempting to speculate that the expression of CLD-6,-7, and -10 in maturation ameloblasts reﬂects a functional
difference between secretory ameloblasts and mature ameloblasts, which would suggest that the functions of the TJs
shift in association with ameloblast differentiation. However,the functional signiﬁcance of the newly expressed CLDs
(CLD-6, -7, and -10) in maturation ameloblasts is not known.
We point out only that mature ameloblasts primarily function in the absorption of degraded enamel proteins, dehydration, and the further mineral deposition of enamel.1–3
Further examination will be necessary, for example, using
CLD knockout mice, to determine the relationship between
amelogenesis and the functions of each CLD. In addition,
the expression of the other CLDs, CLD-11 to -24, in ameloblasts still remains to be studied.7
Mature ameloblasts consist of two cell types, R-A and
S-A. The former mainly functions in the transport of Ca intoenamel and the latter in the absorption of enamel matrix.1–3
In the present study, we examined whether there were differences between R-As and S-As with regard to the expression patterns of CLDs. We used a proven method,administering calcein ﬂuorescence, to identify regions containing the R-A and S-A ameloblasts in frozen sections.14,15
Interestingly, the expression patterns of the CLDs examined showed no difference between the R-A and S-A cell
types. Therefore, it appears that mature ameloblasts do not
express different sets of CLDs (or at least of CLD-1 to -10),
regardless of their functional state. It may be necessary that
the TJs be maintained while the R-A and S-A cyclicallychange their functions and morphological characteristics,
i.e., from R-A to S-A and from S-A to R-A.

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Freeze-fracture studies have shown TJs to be located at
the distal end of secretory ameloblasts and mature R-As
but at the proximal end of mature S-As.8–13 In the present
study, TJs were localized to the distal end of secretory ameloblasts, in agreement with previous reports.8–13 However, inmature ameloblasts, TJs were mostly localized to both the
distal and proximal ends of the cells, regardless of whether
they were R-As or S-As, although some mature ameloblasts
showed TJs only at the proximal end of the cells. Regarding
the localization of OLD and CLDs in secretory and mature
ameloblasts, a previous immunohistochemical study showed
the bipolar localization of OLD, CLD-1, and CLD-4 andstated that CLD-1 was detected at both ends of all ameloblasts except for the distal ends of S-As, and further that
CLD-4 and OLD were detected at both ends of mature
ameloblasts except for the distal ends of S-As.20 In thepresent study it was shown that OLD and CLD-6, -7, -8, -9,
and -10 also localized to both the distal and proximal ends
of mature ameloblasts, in addition to previously reported
OLD and CLDs localization.20 Generally, TJs localize to theapical side of cells in epithelia, and no report is available
that describes a bipolar localization of TJs, i.e., on both theapical and basal sides, of cells other than ameloblasts. Thus,
the bipolar localization of TJs may be unique to ameloblasts. The reason for the discrepancy in the localization of
TJs in mature ameloblasts between the present immunohistochemical study and freeze-fracture studies,8–13 and/or aprevious immunohistochemical study,20 is unclear. However,to explain the complicated ion transport that is observed
physiologically during amelogenesis, it is hypothesized thatTJs modulate their localizations, i.e., at both the proximaland distal ends or solely at the proximal end.3 Our immunohistochemical study may support this hypothesis.3 Further
examination is necessary to elucidate the functions of the
CLDs and the distribution patterns of the TJs during
amelogenesis.
Acknowledgments The authors thank Prof. Mikio Furuse, Division of
Cellular and Molecular Medicine, Kobe University Graduate Schoolof Medicine, for advice on occludin and claudin immunohistochemistry.
This study was supported in part by a Grant-Aid for Scientiﬁc Research(C) from the Ministry of Education, Science, and Culture of Japan toT. Yamamoto (No. 17591909).

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