Journal of Histochemistry& Cytochemistry
http://jhc.sagepub.com/

Planar Cell Polarity Protein Localization in the Secretory Ameloblasts of rat Incisors
Sumio Nishikawa and Tadafumi Kawamoto
J Histochem Cytochem published online 29 February 2012
DOI: 10.1369/0022155412438887
The online version of this article can be found at:
http://jhc.sagepub.com/content/early/2012/02/29/0022155412438887.citation

Published by:
http://www.sagepublications.com

On behalf of:

Official Journal of The Histochemical Society

Additional services and information for Journal of Histochemistry & Cytochemistry can be found at:
Email Alerts: http://jhc.sagepub.com/cgi/alerts
Subscriptions: http://jhc.sagepub.com/subscriptions
Reprints: http://www.sagepub.com/journalsReprints.nav
Permissions: http://www.sagepub.com/journalsPermissions.nav>> Accepted Manuscript - Feb 29, 2012
What is This?

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Planar Cell Polarity Protein Localization in the Secretory Ameloblasts of Rat
Incisors
Sumio Nishikawa and Tadafumi Kawamoto
Department of Biology (SN), Radioisotope Research Institute (TK), Tsurumi
University School of Dental Medicine, Yokohama, Japan10.1369/0022155412438887

Address for correspondence:
Sumio Nishikawa
Department of Biology, Tsurumi University School of Dental Medicine
2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
phone: +81-45-580-8466
FAX: +81-45-573-9599
e-mail: nishikawa-s@tsurumi-u.ac.jp
1

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Abstract
The localization of the planar cell polarity proteins Vangl2, frizzled-3, Vangl1 andCelsr1 in the rat incisors was examined using immunocytochemistry. The results
showed that Vangl2 was localized at two regions of the Tomes’ processes of inner
enamel-secretory ameloblasts in rat incisors: a proximal and a distal region. In
contrast, frizzled-3 was localized at adherens junctions of the proximal and distal
areas of inner enamel- and outer enamel-secretory ameloblasts, where N-cadherin
and β-catenin were localized. frizzled-3 was also localized in differentiating inner
enamel epithelial cells. Vangl1 was localized sparsely in differentiating
preameloblasts and extensively at the cell boundary of stratum intermedium.
Celsr1 was not localized in ameloblasts but localized in odontoblasts extensively.
These results suggest the involvement of planar cell polarity proteins inodontogenesis.

Key words: ameloblasts, Vangl2, frizzled-3, Vangl1, Celsr1, planar cell polarity,
immunofluorescence, enamel, rat incisor

2

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Introduction
Epithelial cells are known to be polarized along the apical-basal axis. In
addition to this apical-basal polarity, most epithelial cells have another polarity
which is referred to as planar cell polarity (PCP). PCP genes and their expression
have been extensively studied using Drosophila wings and eyes (Klein and Mlodzik,
2005; Wu and Mlodzik, 2009). Drosophila wing cells are polarized along the
distal-proximal axis and Drosophila eye cells are polarized along the dorsal-ventral
axis. PCP proteins are located in the noncanonical Wnt signaling pathway (vanAmerogen and Berns, 2006; Klein and Mlodzik, 2005). The core PCP genes are;frizzled, disheveled, Van Gogh (Vang) (also known as strabismus), prickle, diego andflamingo. Disheveled is also shown to have a role in canonical Wnt/β-catenin
pathway. These core PCP genes are present in all organisms from Drosophila tomammals, and are therefore evolutionarily conserved (Fanto and McNeill, 2004;
Klein and Mlodzik, 2005; van Amerogen and Berns, 2006; Lawrence et al., 2007;
Seifert and Mlodzik, 2007; Wang and Nathans, 2007; Vlader et al., 2009; Wu andMlodzik, 2009; McNeill, 2010; Skoglund and Keller, 2010; Wallingford, 2010;
Goodrich and Strutt, 2011; Wallingford and Mitchell, 2011; Wansleeben and
Meijlink, 2011). The presence of Vangl2 and frizzled-3, which are vertebrate genes
3

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

of Vang and frizzled, has been previously detected in dental tissue by using in situ
hybridization and microarray gene expression analysis (Tissir and Goffinet, 2006).
However, incisor which has been long studied because of examination of wholeamelogenesis in a tooth, has not been examined. And no information of protein
distribution of PCP in dental tissues is available.
PCP proteins are also involved in convergent extension of spinal cord epithelia
and notochordal and somatic tissues in Xenopus and zebrafish (Torban et al., 2004;
Keller, 2002; Keller et al., 2000; Wallingford et al., 2002; Seifert and Mlodzik, 2007).
In these notochord or somite elongation processes, cells with lamellipodia at their
medial or lateral end, where the actin cytoskeleton is abundant, are intercalated bypulling each other medially or laterally.
An enamel rod is a basic unit of enamel structure.

The course along which anenamel rod runs varies between different regions of enamel and also between
different animal teeth, including human teeth (Kawai,1955; Boyde, 1971; Osborn,
1968a,b). As expected, the movement of secretory ameloblasts during enamel
secretion dictates enamel rod arrangement and orientation (Nishikawa, 1992,
Hanaizumi et al., 2010). Actin and actin-related cytoskeletal components such as
myosin and tropomyosin are abundant at ameloblast junctions (Nishikawa and4

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Kitamura, 1985a; 1986; Nishikawa et al., 1988; Nishikawa, 1992). The localizationof adherens junction proteins such as N-cadherin (A-CAM), E-cadherin, β-catenin,
and p120-catenin at these ameloblast junctions was previously examined(Nishikawa et al., 1990; Obara et al., 1998; Sorkin et al., 2000; Bartlett et al., 2010).
However, directional movement of ameloblasts may require ameloblast planar cell
polarization as well as a distinct localization of actin-based filament bundles. In fact,polarized distribution of actin filament bundles has been reported (Nishikawa andKitamura, 1985b; 1986; Nishikawa et al., 1988). Since the downstream signaling
pathway of planar cell polarity involves actin cytoskeleton organization, it wastherefore of interest to examine the localization of PCP proteins in ameloblasts todetermine if they play a role in odontogenesis.

5

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Materials and Methods
Seventeen male Jcl Wistar rats (4-5 weeks old, 73-145 g, obtained from Clea
Japan, Tokyo, Japan) were used for the present study. Institutional guidelines foranimal care were followed for all experimental procedures. The animals weresacrificed by decapitation under deep anesthesia. The maxillae and mandibles weredissected and were then fixed with 4% paraformaldehyde at 4 °C overnight.
Subsequently, the maxillae and mandibles were demineralized for 3 weeks at 4 °C
with 5% EDTA, pH 7.2 (adjusted with concentrated NaOH solution). After washingwith PBS and infusing with a 25% sucrose solution in PBS, the tissues were rapidly
frozen and cryosections were made using a cryotome (HM505E, Microm, Walldorf,
Germany).
Cryosections were labeled with rabbit polyclonal antibodies against a synthetic
peptide corresponding to the N-terminal extracellular domain of human frizzled-3(FZD3, LS-A4434, MBL International, Woburn, MA, USA) and goat polyclonal
antibodies against a synthetic peptide corresponding to a region within the
N-terminal domain of Vangl2 (N-13)(sc-46561, Santa Cruz, Santa Cruz, CA, USA).
Cryosections were incubated with anti-frizzled-3 Ab diluted 1:10 with 1% bovine
serum albumin in PBS (BSA-PBS) at RT for 30 min, followed by labeling with Alexa
6

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

488-conjugated donkey anti-rabbit IgG or Alexa 555-conjugated donkey anti-rabbit
IgG (Invitrogen, Carmarillo, CA, USA) diluted 1:100 at RT for 30 min.

Sections as

negative control for stainings were incubated with normal rabbit immunoglobulin(Dako, Glostrup, Denmark) instead of the primary antibody, using an equivalent
dilution, and were processed in the same way as described above. Some cryosections
were labeled with anti-Vangl2 Ab at a concentration of 20 µg/ml diluted with 1%
BSA-PBS, at 4 °C overnight, followed by labeling with FITC-conjugated anti-goat
IgG (Santa Cruz) diluted 1:20 with 1% BSA-PBS at RT for 30 min. For staining
control on sections, the anti-Vangl2 antibody solution was first incubated withexcess blocking peptide (sc-46561P, Santa Cruz) for 2 hr, according to the
manufacturer’s instructions. Sections as negative control for stainings were thenincubated with the resulting antibody-absorbed solution and processed in the same
way as described above. Some other sections were labeled with anti-Vangl2 antibody,followed by ImmunoCruz Staining System (goat LSAB, Santa Cruz, sc-2053) anddeveloped by diaminobenzidine (DAB) as chromogen.
Other cryosections were labeled with antibodies against adherens junction
proteins: rabbit monoclonal anti-N-cadherin antibody (C-terminus clone EPR1792Y,
Millipore, Temecula, CA, USA) and rabbit polyclonal anti-β-catenin antibody
7

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

(Millipore). Sections were labeled with anti-N-cadherin and anti-β-catenin Abs,
diluted 1:50 or 1:100 with 1% BSA-PBS, at 4 °C for 30 min followed by labeling with
Alexa 488-conjugated anti-rabbit IgG (Invitrogen).
For F-actin detection, sections were labeled with rhodamine-phalloidin(Invitrogen), diluted 1:50, at RT for 30 min. Cell nuclei were labeled by incubation ofsections with 1 µg/ml Hoechst 33342 (Invitrogen) at RT for 30 min.
Some of the animals (nine day-old Jcl Wistar rats, Japan Clea, Tokyo, Japan)
were sacrificed under deep anesthesia and rapidly frozen and freeze-dried sections
were obtained without fixation and demineralization according to the method of
Kawamoto (Kawamoto, 2003). Freeze-dried sections (5-µm thick) of mandibles
including incisors were labeled with anti-frizzled-3 antibodies followed by Alexa
488-conjugated anti-rabbit IgG in the manner described above. Freeze-dried
sections were also labeled with anti-Vangl1 (G-17, sc-46557, Santa Cruz) or
anti-Celsr1 (M-125, sc-99198, Santa Cruz) diluted 1:20, followed by labeling with
FITC-conjugated anti-goat IgG (Santa Cruz) or Alexa 488-conjugated anti-rabbit
IgG (Invitrogen), respectively. Some sections were also labeled with Alexa Fluor
647 phalloidin (Invitrogen) diluted 1:50 for F-actin detection. Sections as negative
control for stainings were incubated with 1% BSA-PBS alone for anti-Vangl1
8Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

antibody and normal rabbit IgG in 1% BSA-PBS for anti-Celsr1 antibody, resulting
in no specific labelings in the dental tissues of incisors.
Fluorescent images were acquired using an Olympus AX80 fluorescencemicroscope equipped with a CCD camera (Quantix KAF1401E, Photometrics,
Tucson, AR, USA) and using MetaMorph software (Universal imaging, Downington,
PA, USA).

9

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012Results
Adherens junction protein localization in ameloblasts.
Since downstream effector of noncanonical pathway of Wnt signaling is actin
cytoskeleton, actin-associated adherens junction proteins were examined. Adherens
junctions of secretory ameloblasts were localized by staining for the junction
markers β-catenin and N-cadherin.

An N-cadherin signal was detected at theproximal and distal end of inner enamel-secretory and outer enamel-secretory
ameloblasts (Fig. 1A-C). Inner enamel-secretory and outer enamel-secretory
ameloblasts were classified by the shape of distal junction of each cell andarrangement of Tomes’ processes of cells: the former had rectangular junction andherring bone pattern of Tomes’ processes, the latter had flattened hexagonal
junction and uniformly running pattern of Tomes’ processes (Nishikawa et al., 1988).
Based on this labeling pattern, the labeled structures were considered to beadherens junctions that are located at proximal and distal junctional complexes(Nishikawa et al., 1990; Bartlet et al., 2010). Another adherens junction protein,β-catenin, was localized at the proximal and distal junctions of inner
enamel-secretory, outer enamel-secretory and early maturation ameloblasts (Figs.
1D-G and 2B,C).
10

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Localization of frizzled-3, Vangl2 and Vangl1 in incisor enamel organ.
The localization of two PCP proteins, Vangl2 and frizzled-3, in rat incisor
sections was then analyzed by immunofluorescence (Figs. 2-4).

The anti-frizzled-3

Ab labeled proximal and distal junctional complexes in inner enamel-secretory
ameloblasts (Figs. 2H, I, 3A, C and 4), whereas the anti-Vangl2 Ab labeled only
secretory ameloblasts. The localization of frizzled-3 in freeze-dried specimens thatwere not fixed or demineralized was then analyzed, and frizzled-3 localization at
different stages of ameloblast differentiation and enamel secretion was determined.
An anti-frizzled-3 Ab signal was localized at the proximal junctional area ofdifferentiating inner enamel epithelial cells (Fig. 4A,B). At a later stage ofdifferentiation it became localized at the distal junctional area in addition to theproximal junctional area (Fig. 4C). In secretory ameloblasts, the anti-frizzled-3 Ab
clearly labeled both the proximal and distal junctional areas (Fig. 4D-F), whereas it
did not label ameloblasts at the transition zone or at maturation (Fig. 4G,H).
Sections as negative control for stainings showed no specific labeling (Fig. 4I). The
anti-Vangl2 antibodies strongly labeled Tomes’ processes of inner enamel-secretory
ameloblasts, displaying a linear staining pattern, but only weakly labeled outer
enamel-secretory ameloblasts (Figs. 2A, D, G and 3B, D, F, G).
11

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Double labeling of ameloblasts with anti-Vangl2 and anti-β-catenin Abs
indicated that these proteins were localized in different regions of ameloblasts.
When incisors were longitudinally sectioned, the anti-Vangl2 Ab signal was
localized

6-8

µm

below

the

anti-β-catenin-positive

signal.

This

anti-β-catenin-positive signal was detected as a distal dotted line, and indicatedadherens junctions at the distal end of the cell body (Fig. 2B, C). The anti-Vangl2
reactive signal appeared as a horizontally extended fluorescent line (Fig. 2A, C).
Vertical fluorescent lines extended downwards from the horizontal linear
fluorescence, towards the dentino-enamel junction, and reached up to 15 µm inheight (Fig. 2A, C). Longitudinal sections were also double labeled with anti-Vangl2
and rhodamine-phalloidin (Rh-Ph), which stains F-actin at the adherens junction
and at the Tomes’ processes of the ameloblasts. This staining indicated a horizontal
fluorescent anti-vangl2 signal at the proximal region of Tomes’ processes that was
similar in appearance to the staining at the distal region. This anti-Vangl2 signal
was located 6 µm below the Rh-Ph-labeled distal junctional area, which appeared asa dotted line (Fig. 2D-F). Furthermore, vertical fluorescent lines were again
detected with the anti-Vangl2 Ab. These lines were inclined in the apical direction of
the incisor, were always attached to Rh-Ph-positive Tomes’ processes (Fig. 2E, F).
12

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Double labeling of ameloblasts with anti-Vangl2 and anti-frizzled-3 antibodies
confirmed that these antibodies labeled different parts of the ameloblasts; theanti-Vangl2 Ab labeled Tomes’ processes as vertical and horizontal fluorescent lines,
and the anti-frizzled-3 Ab labeled the distal junctional complexes as dotted lines(Fig. 2G-I). Tangential sections of the incisor were also examined by double labelingwith anti-frizzled-3 and anti-Vangl2 Abs. These sections provided profiles oftransverse

sectionsofameloblasts,

whichallowedanalysis

of

inner

enamel-secretory ameloblasts. The antibody signals were clearly detected at
different locations in these sections: the anti-frizzled-3 Ab signal was localized at
the distal junctional complexes in the form of rectangular outlines and theanti-Vangl2 Ab signal was localized at Tomes’ processes (Fig. 3A-C). In regions of
the section in which enamel secretion was being initiated, an anti-Vangl2 antibody
signal that had the appearance of a brown deposit was localized at the periphery of
newly formed Tomes’ processes (Fig. 3D). Staining control on sections that wereincubated with antibody that had been pre-absorbed with excess antigen showed no
specific reactivity (Fig. 3E). Double labeling with anti-Vangl2 and Rh-Ph showed
that, in the middle region of the area of inner enamel secretion, anti-vangl2 was
localized both around the middle part, and at the end of the distal part of Tomes’
13

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

processes that were labeled with Rh-Ph (Fig. 3F). On the other hand, little
anti-Vangl2 signal could be detected at Tomes’ processes in the outer
enamel-secretory ameloblasts (Fig. 3G). As another Vang gene of vertebrates,

Vangl1 is known (Wu and Mlodzik, 2008). Anti-Vangl1 localization was, therefore,
examined to know whether or not Vangl1 and Vangl2 play a redundant role inodontogenesis, using the freeze-dried specimens which were not fixed ordemineralized. Anti Vangl1 Ab was localized in the stratum intermedium fromdifferentiating enamel epithelia to papillary layer of enamel maturation zone in theincisors (Fig. 5A-D). In close examination, boundary of stratum intermedium cells
was labeled as bright fluorescent dots (Fig5B, D). Inner enamel epithelial cells inthe differentiating zone were also labeled as sparsely fluorescent dots (Fig. 5A, B).
Immunofluorescent localization of Celsr1 in dental tissues of the incisors.
As Celsr1 and Vangl2 are known to be necessary for orienting hair follicles andto be interdependent for their polarized hair follicle distribution (Devenport andFuchs, 2008), Celsr1 localization was examined using the freeze-dried, unfixed andnon-demineralized specimens. Anti-Celsr1-derived fluorescence was brightly
localized in the cell bodies of odontoblasts but not in the ameloblasts (Fig. 5E-H).

14

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Discussion
The presence of Vangl2 and frizzled-3 in dental tissue has been previously
detected by using in situ hybridization and microarray gene expression analysis.
Tissir and Goffinet (2006) reported strong expression of frizzled-3 and Vangl2 byusing oligonucleotide probes in an in situ hybridization study of mouse molar tooth
germ. Pemberton et al. (2007) reported that the developing mouse molar tooth
showed increased expression of Vangl2 along with increased frizzled-6 expression,compared with control tissues. Thus, the mRNA expression of PCP genes including

Vangl2, frizzled-3 and frizzled-6 in tooth germ has been shown. In the present study,
the localization of Vangl2 and frizzled-3 protein was examined in rat incisor
ameloblasts by immunohistochemistry. The results showed that an anti-Vangl2 Ab
signal was localized at Tomes’ processes of secretory ameloblasts but not inpresecretory or maturation ameloblasts. In contrast, an anti-frizzled-3 Ab signal
was localized at both presecretory and secretory ameloblasts, but not at maturation
ameloblasts.

The localization of the anti-frizzled-3 Ab signal at presecretory andsecretory ameloblasts was similar to the localization previously reported for F-actin(Nishikawa et al., 1986). F-actin first appears at the proximal junctional area ofdifferentiating ameloblasts and later appears at the distal junctional area ofdifferentiating ameloblasts (Nishikawa and Kitamura, 1986). In secretory

15

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

ameloblasts, F-actin is continuously localized both at the proximal and distal
junctional areas (Nishikawa and Kitamura, 1986). The anti-frizzled-3 Ab showed asimilar localization pattern in the present study. Furthermore, F-actin filament
bundles are components of adherens junctions. N-cadherin and β-catenin, which are
adherens junction proteins, have been previously shown to be localized inameloblasts (Nishikawa et al., 1990; Bartlet et al., 2010). In the present study,frizzled-3 is localized both at proximal and distal junctions, which are stained
positive by antibodies of adherens junction proteins, anti-N-cadherin andanti-β-catenin.
Vangl2 and actin filament bundles are abundant in Tomes’ processes of secretory
ameloblasts but the frizzled-3 protein has not been localized in these processes. In
the present study, Vangl2 was localized in two different regions of Tomes’ processes:
a proximal area and a distal area. Both of these regions appear to be associated withthe secretory surface of the membrane of Tomes’ processes, although ultrastructural
localization of Vangl2 is necessary to clarify the stained structure. Thus, planarcell polarity (PCP) proteins appear to be involved in enamel rod formation. Vangland frizzled-type proteins are known to antagonize each other and to function at
different cell membrane areas of the cell (Seifert and Mlodzik, 2007). PCP proteins16

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

have been shown to play roles in tissue formation such as in alignment of mouse
inner ear hair cells and Drosophila eyes (Seifert and Mlodzik, 2007). All PCP
proteins examined in this study were localized in the incisors. As Wnt5a is known to
be a representative of noncanonical Wnt pathway which is upstream signaling
pathway of PCP proteins (Moon et al., 1993; Kilian et al., 2003) and Wnt5a-deficient
mice exhibit small tooth germ (Lin et al., 2011), it is likely that PCP proteins are
deeply related to tooth formation.
Mouse body hairs incline in a head-to-tail direction. The underlying molecularbasis of this inclination was shown to depend on PCP proteins such as Vangl2 andthe atypical cadherin Celsr1 (Devenport and Fuchs, 2008). Another member offrizzled, frizzled-6 is localized in a pattern indistinguishable from those of Vangl2
and Celsr1 in the hair follicles (Devenport and Fuchs, 2008). In incisors of rats,
anti-Celsr1 was localized in different tooth-forming cells, odontoblasts. Thus, it ispossible that some family proteins are associated with enamel and dentin formation
to remain to be clarified. To ask the possibility of another Vang protein in theameloblast junctions, Vangl1 was examined. Anti Vangl1 Ab was localized in thestratum intermedium. It suggests that Vangl1 may function in the formation andmaintenance of cell-to-cell junction in stratum intermedium. In amelogenesis
17

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

anti-Vangl1 Ab labeled differentiating inner enamel epithelial cells as sparse brightdots, but rarely labeled in secretory ameloblasts. These distribution patterns aredifferent from that of the frizzled-3 in this study. Thus, interaction of Vangl1 andfrizzled-3 proteins is unlikely in ameloblasts. It would be interesting to knowodontogenesis of knockout mice of PCP genes. Although many kind of knockout mice
have been shown (van Amerongen and Berns, 2006), looptail and crash mutant micefor Vangl2 and Celsr1, respectively, are not viable after birth (Devenport and Fuchs,
2008). Knockout mice of frizzled-3 gene are also not viable after birth (vanAmerongen and Berns, 2006). Experiments of odontogenesis using these mutant
mice, thus, may be limited.
In Drosophila wing cells, small vesicles containing frizzled are transported toadherens junctions at the cell boundary via microtubules that run in a horizontal
direction (Shimada et al., 2006), suggesting that frizzled proteins are transported by
microtubules that are oriented horizontally to the cell membrane. In secretory
ameloblasts, most microtubules run along the long axis of the cell, that is theapical-basal axis, but some microtubules do run horizontally, i.e., along theorthogonal plane of the apical-basal axis at the level of the distal junctional complex(Nishikawa and Kitamura, 1985a). It is therefore possible that material important
18

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

for junctional membranes, including frizzled-3, may be transported by microtubules
in secretory ameloblasts. Since frizzled is a Wnt receptor, and a noncanonicaldownstream signaling pathway of frizzled is actin cytoskeleton reorganization (vanAmerongen and Berns, 2006; Klein and Mlodzik, 2005), the presence of frizzled inameloblast junctional complexes may induce the formation of, and maintain, actin
cytoskeleton structure via the regulation of a small G protein such as RhoA. The
small G proteins RhoA and Rac, as well as the Rho kinase Rock, are known
downstream components of frizzled receptor signaling (van Amerongen and Berns,
2006; Klein and Mlodzik, 2005). Inhibition of Rho kinase was reported to mimic
PCP-linked defects (Yates et al., 2010). Furthermore, Rho GTPases such as RhoA
and Rac, as well as their regulator molecules, have been shown to function in
amelogenesis (Li et al., 2005; Hatakeyama et al., 2009; Biz et al., 2010; Ohtsu et al.,2010; Li et al., 2011).
Although the characteristic localization of frizzled-3 and Vangl2 in ameloblasts
strongly suggests the involvement of PCP proteins in tooth enamel formation,
further studies are obviously needed, to confirm their involvement and tounderstand their mechanism of action.

19

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

References
Bartlett JD, Dobeck JM, Tye CE, Perez-Moreno M, Stokes N, Reynolds AB, Fuchs E,
Skobe Z. 2010. Targeted p120-catenin ablation disrupts dental enamel development.
PLoS ONE 5(9): e12703. doi:10.1371/journal.pone.0012703.

Biz MT, Marques MR, Crema VA, Moriscot AS, dos Santos MF. 2010. GTPases RhoA
and Rac1 are important for amelogenin and DSPP expression during differentiationof ameloblasts and odontoblasts. Cell Tissue Res 340:459-470.

Boyde A. 1971. Comparative histology of mammalian teeth. In: Dental morphology
and evolution, AA Dahlberg, ed. University of Chicago Press. Chicago. p.81-94.

Devenport D, Fuchs E. 2008. Planar polarization in embryonic epidermis
orchestrates global asymmetric morphogenesis of hair follicles. Nat Cell Biol.
11:1257-1268.

Fanto M, McNeill H. 2004. Planar polarity from flies to vertebrates. J Cell Sci
117:527-533.
20

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Goodrich LV, Strutt D. 2011. Principles of planar polarity in animal development.
Development 138:1877-1892.

Hanaizumi Y, Yokota R, Domon T, Wakita M, Kozawa Y. 2010. The initial process ofenamel prism arrangement and its relation to the Hunter-Schreger bands in dog
teeth. Arch Histol Cytol 73:23-36.

Hatakeyama J, Fukumoto S, Nakamura T, Haruyama N, Suzuki S, Hatakeyama Y,
Shum L, Gibson CW, Yamada Y, Kulkarni AB. 2009. Synergistic roles of amelogenin
and ameloblastin. J Dent Res 88:318-322.

Kawai N. 1955. Comparative anatomy of the bands of Schreger. Okajimas Folia
Anat Jpn. 27:115-131.

Kawamoto T. 2003 Use of a new adhesive film for the preparation of multi-purpose
fresh-frozen sections from hard tissues, whole-animals, insects and plants. ArchHistol Cytol. 66:123-143
21

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Keller R. 2002. Shaping the vertebrate body plan by polarized embryonic cell
movements. Science. 298:1950-1954.

Keller R, Davidson L, Edlund A, Elul T, Ezin M, Shook D, Skoglund P. 2000.
Mechanisms of convergence and extension by cell intercalation. Phil Trans R Soc
Lond B. 355:897-922.

Kilian B, Mansukoski H, Barbosa FC, Ulrich F, Tada M, Heisenberg CP. 2003. The
role of Ppt/Wnt5a in regulating cell shape and movement during zebrafish
gastrulation. Mech Dev 120:467-476.

Klein TJ, Mlodzik M. 2005. Planar Cell polarization: an emerging model points in
the right direction. Annu Rev Cell Dev Biol. 21:155-176.

Lawrence PA, Struhl G, Casal J. 2007. Planar cell polarity: one or two pathways?
Nat Rev Genet 8:555-563.

22

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Li Y, Decker S, Yuan Z, DenBesten PK, Aragon MA, Jordan-Sciutto K, Abrams WR,
Huh J, McDonald C, Chen E, MacDougall M, Gibson CW. 2005. Effects of sodium
fluoride on the actin cytoskeleton of murine ameloblasts. Archs Oral Biol
50:681-688.

Li Y, Pugach MK, Kuehl MA, Peng L, Bouchard J, Hwang SY, Gibson CW. 2011.
Dental enamel structure is altered by expression of dominant negative RhoA in
ameloblasts. Cells Tissues Organs 194:2-4

Lin M, Li L, Liu C, Liu H, He F, Yan F, Zhang Y, Chen Y. 2011. Wnt5a regulates
growth, patterning, and odontoblast differentiation of developing mouse tooth. Dev
Dyn 240:432-440.

McNeill H. 2010. Planar cell polarity: keeping hairs straight is not so simple. Cold
Spring Harb Perspect Biol 2:a003376.

Moon RT, Campbell RM, Christian JL, McGrew LL, Shih J, Fraser S. 1993.

Xwnt-5a: a maternal Wnt that affects morphogenetic movements after
23

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

overexpression in embryos of Xenopus laevis. Development 119:97-111.

Nishikawa S. 1992. Correlation of the arrangement pattern of enamel rods andsecretory ameloblasts in pig and monkey teeth: a possible role of the terminal webs
in ameloblast movement during secretion. Anat Rec 232:466-478.

Nishikawa S, Kitamura H. 1985a. Three-dimensional network of microtubules insecretory ameloblasts of rat incisors. Archs Oral Biol. 30:1-11.

Nishikawa S, Kitamura H. 1985b. Actin filaments in the terminal webs of secretory
ameloblasts of rats. Archs Oral Biol. 30:13-21.

Nishikawa S, Kitamura H. 1986. Localization of actin during differentiation of theameloblast, its related epithelial cells and odontoblasts in the rat incisor using
NBD-phallacidin. Differentiation 30:237-243

Nishikawa S, Fujiwara K, Kitamura H. 1988. Formation of the tooth enamel rod
pattern and the cytoskeletal organization in secretory ameloblasts of the rat incisor.
24

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Eur J Cell Biol 47:222-232.

Nishikawa S, Tsukita S, Tsukita S, Sasa S. 1990. Localization of adherens junction
proteins along the possible sliding interface between secretory ameloblasts of therat incisor. Cell Struct Funct. 15:245-249.

Obara N, Suzuki Y, Nagai Y, Takeda M. 1998. Expression of E- and P-cadherin
during tooth morphogenesis and cytodifferentiation of ameloblasts. Anat Embryol.
197:469-475.

Osborn JW. 1968a. Evaluation of previous assessments of prism directions inhuman enamel. J Dent Res. 47:217-222.

Osborn JW. 1968b. Directions and interrelationships of enamel prisms from thesides of human teeth. J Dent Res 47:223-232.

Otsu K, Kishigami R, Fujiwara N, Ishizeki K, Harada H. 2010. Functional role of
Rho-kinase in ameloblast differentiation. J Cell Physiol 226:2527-2534.
25

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Pemberton TJ, Li F-Y, Oka S, Mendoza-Fandino GA, Hsu Y-H, Bringas, Jr P, Chai Y,
Snead ML, Mehrian-Shai R, Patell PI. 2007. Identification of novel genes expressed
during mouse tooth development by microarray gene expression analysis. Dev Dyn
236:2245-2257.

Seifert JRK, Mlodzik M. 2007. Frizzled/PCP signaling: a conserved mechanism
regulating cell polarity and directed motility. Nat Rev Genet. 8:126-138.

Shimada Y, Yonemura S, Ohkura H, Strutt D, Uemura T. 2006. Polarized transportof Frizzled along the planar microtubule arrays in Drosophila wing epithelium. Dev
Cell. 10:209-222.

Skoglund P, Keller R. 2010. Integration of planar cell polarity and ECM signaling inelongation of the vertebrate body plan. Curr Opin Cell Biol 22:589-596.

Sorkin BC, Wang MY, Dobeck JM, Albergo KL, Skobe Z. 2000. The cadherin-catenincomplex is expressed alternately with the adenomatous polyposis coli protein
26

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

during rat incisor amelogenesis. J Histochem Cytochem. 48:397-406.

Tissir F, Goffine AM. 2006. Expression of planar cell polarity genes during
development of the mouse CNS. Eur J Neurosci. 25:597-607.

Torban E, Kor C, Gros P. 2004. Van Gogh-like2 (Strabismus) and its role in planarcell polarity and convergent extension in vertebrates. Trends Genet. 20:570-577.

van Amerongen R, Berns A. 2006. Knockout mouse models to study Wnt signaltransduction. Trends Genet. 22:678-689.

Vladar EK, Antic D, Axelrod JD. 2009. Planar cell polarity signaling: the developingcell’s compass. Cold Spring Harb Perspect Biol 1:a002964.

Wallingford JB. 2010. Planar cell polarity signaling, cilia and polarized ciliary
beating. Curr Opin Cell Biol 22:597-604.

Wallingford JB, Mitchell B. 2011. Strange as it may seem: the many links between
27

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Wnt signaling, planar cell polarity, and cilia. Genes Dev 25:201-213.

Wallingford JB, Fraser SE, Harland RM. 2002. Convergent extension: the molecular
control of polarized cell movement during embryonic development. Dev Cell.
2:695-706.

Wang Y, Nathans J. 2007. Tissue/planar cell polarity in vertebrates: new insights
and new questions. Development 134:647-658

Wansleeben C, Meijlink F. 2011. The planar cell polarity pathway in vertebrate
development. Dev Dyn 240:616-626.

Wu J, Mlodzik M. 2009. A quest for the mechanism regulating global planar cell
polarity of tissues. Trends Cell Biol.19:295-305.

Wu J, Klein TJ, Mlodzik M. 2004. Subcellular localization of frizzled receptors,
mediated by their cytoplasmic tails, regulates signaling pathway specificity PLos
Biol. 2:E158.
28

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Yates LL, Schnatwinkel C, Murdoch JN, Bogani D, Formstone CJ, Townsend S,
Greenfield A, Niswander LA, Dean CH. 2010. The PCP genes Celsr1 and Vangl2 arerequired for normal lung branching morphogenesis. Hum Mol Genet. 19:2251-2267.

29

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Fig. 1. Immunofluorescent localization of anti-N-cadherin and anti-β-catenin
antibody signals in the ameloblasts of rat mandibular incisors. Sections of rat
mandibular incisors were immunohistochemically stained with anti-N-cadherin (A,B) or anti-β-catenin (D, E, F) antibodies. Sections as negative control for
anti-N-cadherin (C) and anti-β-catenin (G) are also shown. Inner enamel-secretory(A, C, D, G), outer enamel secretory- (E), outer enamel-secretory to transitional (B)and transitional (F) ameloblasts are shown. An anti-N-cadherin antibody signal was
detected at the proximal and distal junctions of inner enamel- and outer
enamel-secretory ameloblasts (A, B). The Sections as negative control shows nospecific labeling (C). An anti-β-catenin antibody signal was also detected at theproximalanddistaljunctional

complexes

in

inner

enamel-

and

outer

enamel-secretory ameloblasts as well as at the distal cell end of transitional
30

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

amelobasts (D, E, G). No specific reactivity was observed in the section as negative
control (G). Bars=50 µm.

31

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Fig. 2. Triple labeling of inner enamel-secretory ameloblasts with anti-Vangl2 andHoechst 33342 (H33342), and either anti-β-catenin, rhodamine-phalloidin or
anti-frizzled-3. Sections of rat incisors were stained with anti-Vangl2 Ab (A, C-G,I) (green), anti-β-catenin (B, C) (red), rhodamine-phalloidin (Rh-Ph) (E, F) (red), or
anti-frizzled-3 (H, I) (red), and nuclei were stained with Hoechst 33342 (H33342)(A-E,G-I) (blue). A merged image of (A) and (B) is shown in (C). An anti-Vangl2
Ab-labeled horizontal fluorescent line (arrows in A, D, F, G) was localized at the32

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

proximal part of Tomes’ processes. This region was located 5-10 µm below the distal
junctional complexes that were labeled with anti-β-catenin (B, C) or F-actin (E, F).
Anti-frizzled-3

antibodies

labeleddistaljunctional

complexes

(H,

I).

Anti-Vangl2-labeled vertical fluorescent lines were also seen below the horizontal
fluorescent line in Tomes’ processes (A, C-G, I). Double-labeling of Tomes’ processes(TP in F) with anti-Vangl2 Ab and rhodamine phalloidin showed that these vertical
fluorescent lines were localized at the lateral surface of Tomes’ process on the apicalside of the incisor (F). The arrows indicate the incisal direction. AM, ameloblast; EN,
enamel; DTW, distal terminal web; TP, Tomes’ process. (C, D, I) Bars=50 µm. (F)Bar=10 µm.

33

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Fig. 3. Labeling of Vangl2, frizzed-3 and actin in tangential sections of ameloblasts.
Tangential sections of rat incisors at the level of Tomes’ processes and the distal
terminal web at the zone of inner enamel secretion (A-F) and of outer enamel
secretion (G) are shown. A-C: Double labeling with anti-frizzled-3 and anti-Vangl2.
The anti-frizzled-3 Ab labeled distal junctional complexes (DTW) but not Tomes’
34

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

processes (A, C), whereas the anti-Vangl2 Ab labeled proximal and distal parts ofTomes’ processes (TPP and TPD, respectively) but not distal junctional complexes (B,C).

D, E: Anti-Vangl2 localization in early enamel formation visualized byimmunoperoxidase followed by DAB staining, showed brown positive reactivity
around the newly formed Tomes’ processes (D). The staining control on section
which was incubated with anti-Vangl2 antibodies preabsorbed with excess antigen,showed no specific reactivity (E). DT. dentin; AM, ameloblast.

F, G: Double

labeling with the anti-Vangl2 antibody and with rhodamine phalloidin for F-actin
detection. Whole Tomes’ processes were labeled with rhodamine-phalloidin (red).
Distal Parts of Tomes’ processes were shown by small arrows (F). In the area of
inner enamel secretion (F), the anti-Vangl2 Ab (green) labels the periphery of theproximal part of Tomes’ processes (F, asterisk) and also labels one side of the distal
part of Tomes’ processes (F, arrowheads), whereas in the area of outer enamel
secretion (G), only long slender fluorescent Tomes’ processes (red) and weak anti
Vangl2 Ab fluorescence (green) were observed. Large double pointed arrows (F, G)
show the long axis of the incisor. (C) Bar= 50 µm. (D, E) Bars= 20 µm. (F, G) Bars=
10 µm.

35

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Fig.4Frizzled-3 labeling of freeze-dried sections of rat incisors without fixationand demineralization. Anti-frizzled-3 labeling (A-H) of inner enamel epithelia andameloblasts

atthe following stages

are shown:proliferation (A),earlydifferentiation (B), late differentiation (C), early inner enamel secretion (D), middle
inner enamel secretion (E), outer enamel secretion (F), transition (G), and earlymaturation (H). A section for negative control is also shown (I). Initially, theproximal part of the inner enamel epithelia was anti frizzled-3-Ab-positive (A, B).
The distal part then became positive in late differentiation (C). Both proximal anddistal junctional complexes in the region of inner and outer enamel secretion werepositive (D-F), but fluorescence gradually diminished at the transition zone (G), and36

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

disappeared in the zone of maturation (H). The section for negative control showedno specific labeling (I). Odontoblasts showed no labeling with the anti-frizzled-3 Ab(C, D). The arrow in A shows incisal direction. IEE, inner enamel epithelial cell; AM,ameloblast; EN, enamel; OD, odontoblast. Bars=50 µm.

37Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012

Fig.5 Vangl1 and Celsr1 labeling of freeze-dried sections of rat incisors without
fixation and demineralization. Double labeling of differentiating inner epithelia (IE)
and provisional stratum intermedium (PSI) (A, B) and inner enamel secretory
ameloblasts (AM) and stratum intermedium (SI) (C, D) with anti-Vangl1 (A-D,
green) and Alexa 647 phalloidin (B, D, red). Double labeling of inner enamel
secretory ameloblasts (AM) and odontoblasts (OD) with anti-Celsr1 (E, F, green)and Alexa 647 phalloidin (F, red). Section as negative control for staining was
incubated with normal rabbit IgG followed with Alexa 488-conjugated anti-rabbit
IgG (G, H, green) and Alexa 647 phalloidin (H, red). Anti-Vangl1 labels
differentiating preameloblasts and provisional stratum intermedium as brightfluorescent dots (A, B), and stratum intermedium but not secretory ameloblasts (C,38

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012D). Anti-Celsr1 labels odontoblasts extensively but does not label ameloblasts (E, F),
whereas the section as negative control for staining incubated with normal rabbit
antibody shows no specific labeling in odontoblasts or ameloblasts (G, H). Bars=50µm.

39

Downloaded from jhc.sagepub.com at TSURUMI UNIV LIB on March 22, 2012