ORIGINAL PAPER

The expression of TGF-b3 for epithelial-mesenchymetransdifferentiated MEE in palatogenesis

Akira Nakajima • Eiji Tanaka • Yoshihiro Ito •

Masao Maeno • Koichi Iwata • Noriyoshi Shimizu •

Charles F. Shuler

Received: 27 July 2010 / Accepted: 7 September 2010 / Published online: 22 October 2010

� Springer Science+Business Media B.V. 2010

Abstract The fate of the palatal medial edge epithelial

(MEE) cells undergoes programming cell death, migration,

and epithelial-mesenchymal transdifferentiation (EMT)

coincident with the process of palatal fusion and disap-

pearance of MEE. Mesenchymal cells in the palate have

both cranial neural crest (CNC) and non-CNC origins. The

objectives of this study were to identify the populations of

palatal mesenchymal cells using b-galactosidase (b-gal)

and DiI cell lineage markers, and to determine whether

MEE-derived cells continued to express transforming

growth factor-b3 (TGF-b3) and transforming growth fac-

tor-b type III receptor (TbR-III), which were specific for

MEE. A model has been developed using Wnt1 tissue

specific expression of Cre-recombinase to activate b-gal

solely in the CNC. The expressions of TGF-b3 and TbR-III

in MEE were temporally correlated with critical events in

palatogenesis. Three cell populations could be distin-

guished in the palatal mesenchymal CNC-derived, non-

CNC derived and MEE-derived. After fusion, b-gal (-)

and DiI (?) mesenchymal cells continued to express TGF-

b3, however TbR-III was expressed only in the epithelial

MEE, as well as keratin expression. In addition, we per-

formed laser capture microdissection to identify mRNA

expression of isolated DiI (?) MEE cells. Both epithelial

and transdifferentiated MEE have expressed TGF-b3,

however, TbR-III was only expressed in epithelium.

Extracellular matrix, especially MMP13 has been expres-

sed coincident with fused stage which can be strongly

associated with TGF-b3. These results demonstrate that

combining a heritable marker and a cell lineage dye can

distinguish different populations of mesenchymal cells in

the developing palate. Furthermore, TGF-b3 and MMP13

could be strongly associated with EMT in palatogenesis.

Keywords Palatal fusion � Medial edge epithelial �Epithelial-mesenchyme transdifferentiation � Cranial

neural crest cell � Transforming growth factor � Transgenic

Cre-recombinase mouse � Laser capture microdissection

Introduction

The medial edge epithelium (MEE) has an important role

in the fusion of the secondary palate. A multi-layer epi-

thelial seam is formed by the adhesion of the two MEE

populations covering the tips of the palatal shelves at E14

in the mouse (Ferguson 1988). Thereafter the MEE seam

thins to a single cell layer at E14.5, while at the same time

MEE cells accumulate at the oral and nasal aspects to

form epithelial triangle (Carette and Ferguson 1992).

A. Nakajima (&) � N. Shimizu

Department of Orthodontics, Nihon University School

of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku,

Tokyo 1018314, Japan

e-mail: nakajima-a@dent.nihon-u.ac.jp

A. Nakajima � M. Maeno � K. Iwata � N. Shimizu

Dental Research Center, Nihon University School of Dentistry,

1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 1018310, Japan

E. Tanaka

Department of Orthodontics and Dentofacial Orthopedics,

The University of Tokushima Graduate School of Oral Sciences,

3-18-15 Kuramoto-cho, Tokushima 7708504, Japan

Y. Ito

The Brodie Laboratory for Craniofacial Genetics,

School of Dentistry, University of Illinois at Chicago,

801 South Paulina Street, MC 841, Chicago, IL 60612, USA

C. F. Shuler

School of Dentistry, University of British Colombia,

2199 Westbrook Mall, Vancouver, BC V6T 1Z3, Canada

123

J Mol Hist (2010) 41:343–355

DOI 10.1007/s10735-010-9296-0

The epithelial seam eventually disappears so that only

mesenchymal cells are observed in the midline of the

palate, with epithelial remnants absent by E15 (Ferguson

1988).

The fate of the MEE has been attributed to three

different mechanisms, which are migration, epithelial-

mesenchymal transdifferentiation (EMT) (Carette and

Ferguson 1992; Fitchett and Hay 1989; Shuler et al. 1991,

1992; Griffith and Hay 1992; Kang and Svoboda 2005;

Ahmed and Nawshad 2007) and programming cell death

(PCD) (Mori et al. 1994; Martinez-Alvarez et al. 2000;

Cuervo et al. 2002; Vaziri Sani et al. 2005; Xu et al. 2006).

Additionally, some MEE retain their epithelial phenotype

and migrate to merge with the nasal and oral surface epi-

thelia (Carette and Ferguson 1992; Martinez-Alvarez et al.

2000; Vaziri Sani et al. 2005). Using a fluorescent dye,

DiI (l,l0-dioctadecyl-3,3,3,30-tetramethylindo-carbocyanine

perchlorate), as a cell lineage tracer, Shuler et al. (1991,

1992) confirmed the ultra-structural findings of EMT and

showed EMT does take place during seam disintegration.

Recent study established palatal chimeric culture system

using Rosa26 transgenic and C57BL/6 mice, in which a

Rosa26-originated ‘blue’ palatal shelf was paired with a

C57BL/6-derived ‘white’ palatal shelf (Jin and Ding 2006).

Using this organ culture system, the results could be

observed the migration of MEE cells to the nasal side, but

not to the oral side. They also observed an anteroposterior

migration of MEE cells, which may play an important role

in posterior palate fusion. b-Galactosidase (b-gal) staining

provided extensive signals in the palatal mesenchymal

region during and after palate fusion, demonstrating the

occurrence of an EMT mechanism during palate fusion (Jin

and Ding 2006).

These EMT have been considered to be strongly asso-

ciated with transforming growth factor-b3 (TGF-b3) and

their receptors in palatogenesis (Kaartinen et al. 1997; Cui

and Shuler 2000; Cui et al. 2005; Blavier et al. 2001; Ito

et al. 2003; Nakajima et al. 2007; Ahmed et al. 2007). The

gene expressions of TGF-b3 in the MEE are temporally

correlated with critical events in palatogenesis and the

expression of TGF-b during palatal fusion is coincident

with tracking transdifferentiated MEE (Cui et al. 2005).

Thus, most of the fusing MEE have been shown to be

linked to the expression of TGF-b3 (Fitzpatrick et al. 1990;

Pelton et al. 1990; Proetzel et al. 1995; Kaartinen et al.

1995, 1997; Taya et al. 1999; Cui and Shuler 2000; Cui

et al. 2005; Blavier et al. 2001; Ito et al. 2003; Dudas et al.

2004; Pungchanchikul et al. 2005; Nakajima et al. 2007;

Ahmed et al. 2007). In addition, TGF-b3 null mutants are

characterized by clefting of the secondary palate (Proetzel

et al. 1995; Kaartinen et al. 1995).

We previously showed the distribution of TGF-b3 and

TGF-b type III receptor (TbR-III) expressions in the MEE

cells (Cui and Shuler 2000; Nakajima et al. 2007). A key

observation is the presence of TGF-b3 (?) mesenchymal

cells in the region of the midline epithelial seam (Cui and

Shuler 2000). The persistence of expressions of TGF-b3

and linked extracellular matrix (ECM) with TGF-b3 in the

MEE-derived mesenchymal cells is unknown due to the

inability to definitively identify this population of cells in

the fused palate. The mechanism of EMT has been linked

to the remodeling of ECM through the function of MMPs

and TIMPs (Morris-Wiman et al. 2000; Blavier et al. 2001;

Brown et al. 2002). TGF-b belongs to a family of growth

factors that have a broad range of regulatory activities,

including control of cell proliferation, regulation of ECM

deposition, MMPs and TIMPs, cell migration, differentia-

tion, and EMT (Miettinen et al. 1994; Morris-Wiman et al.

2000; Blavier et al. 2001; Brown et al. 2002; Kang and

Svoboda 2002, 2005; Nawshad and Hay 2003; Nawshad

et al. 2004, 2007).

We used the two-component Wnt1-Cre/R26R model

(Jiang et al. 2000; Chai et al. 2000) to mark the cranial

neural crest (CNC) cell derived mesenchymal cells in the

secondary palate. This model has permitted the identifica-

tion of non-CNC-derived palatal mesenchyme cells. In

addition, we identified DiI (?) cells heritable marker

(Shuler et al. 1991, 1992) and Immuno-reaction gene maker

using Wnt1-Cre/R26R model. Mesenchymal cells derived

from MEE will not express b-gal and thus this marker could

potentially aid in the identification of MEE-derived cells

following palatal fusion. Therefore, the purposes of this

study were to identify the populations of palatal mesen-

chymal cells using b-gal and DiI cell lineage markers, and

to determine whether MEE-derived cells continued to

express TGF-b3 which is specific for the MEE. In addition,

we investigated the expressions of such ECM in the isolated

transdifferentiated MEE cells as MMP-2, 13 and TIMP-2, 4,

which were associated with TGF-b expression and EMT.

Materials and methods

Animal and tissue preparation of Wnt1-Cre/R26R

double transgenic mice

Timed pregnant Wnt1-Cre/R26R mice (Jiang et al. 2000;

Chai et al. 2000) were used for histological analysis in vivo

(E13, E14.5 and E15), and for palatal shelf organ culture in

vitro (E13). The Wnt1-Cre transgene conditional reporter

allele has been described previously (Danielian et al.

1998). Cross breeding Wnt1-Cre?/- mice (Danielian et al.

1998) with R26R?/- mice (Soriano 1999) generated the

Wnt1-Cre/R26R double transgenic mice with b-gal label-

ing of the CNC and all derivatives. The genotype of the

mice was determined by PCR. Genomic DNA was isolated

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from fetuses, newborns and adult. The 50 and 30 primers

were used for detecting genotype of Wnt1-Cre and R26R

transgenic animals as previously reported (Danielian et al.

1998; Soriano 1999).

Histological analysis for b-gal labeling

Tissue was dissected in Hanks solution (Gibco), and fixed

by immersion in 0.2% glutaraldehyde solution for 30 min

at room temperature. Fixed samples were washed three

times (0.005% Nonidet P-40 and 0.01% sodium deoxy-

cholate in PBS). Embryos were stained with b-gal staining

solution consisting of 5 mM potassium ferricyanide, 2 mM

MgCl, and 0.4% X-gal (Sigma) in PBS overnight at room

temperature in the dark, rinsed twice in PBS, and post-fixed

in 3.7% formaldehyde. These embryos were sectioned to

observe b-gal expression at the cellular level. Frozen sec-

tions were cut at 10 lm thickness, and counterstained with

Nuclear Fast Red for in vivo samples.

Immunohistochemistry

Immunostaining was conducted by the following standard

procedures (Nakajima et al. 2007). Briefly, serial sections

(10 lm) of cultured palates were cut and mounted onto the

same slide to ensure exposure to the same antibody con-

centration. Double labeling (b-gal labeling and Immuno-

staining) in vivo and triple labeling (b-gal labeling, DiI

labeling and Immunostaining) methods in vitro for FITC

immunohistochemistry were completed on the palatal

tissues isolated at defined stages of palatal fusion. The

sections were placed in peroxidase blocking solution (50%

methanol, 50% H2O2), placed in citrate buffer (2% citric

acid solution, 8% sodium citrate solution; pH 6.0) for 30 s,

and then washed with PBS 3 times. Sections for in vivo

double staining were incubated with anti-TGF-b3 (Santa

Cruz) and anti-TbR-III (Santa Cruz), and in vitro triple

labeling sections were incubated with above antibodies and

anti-Keratin (Sigma). FITC-conjugated secondary antibody

was used to localize the primary antibody. FITC fluores-

cence was identified with a Zeiss fluorescent microscope at

an excitation wavelength of 490 nm and an emission

wavelength of 520 nm.

DiI labeling and organ culture in vitro

In order to identify the fate of the MEE during fusion and

compare these findings with the in vivo results, we used in

vitro palatal organ culture. The epithelium of the palatal

shelves was labeled with DiI (Molecular Probes) for cell

lineage analysis. The mandibles were removed and the

maxillary region of the heads at E13 was submerged in

0.025% DiI (in normal saline with 1% ethanol) for 30 min

at room temperature. The tissues were rinsed with culture

medium to remove unincorporated DiI and the palatal

shelves dissected. The palatal shelves were placed in

pairs with their medial edges in contact at the air-medium

interface in Grobstein organ culture dishes with BGjb

medium (Gibco) at 37�C in a 5% CO2-air atmosphere. The

organ cultures could be maintained for more than 72 h,

however the events critical to palatal fusion used as end

points in this study occurred within the first 72 h of culture.

The explanted palatal shelves were examined by stan-

dard histological techniques in order to determine the time

required for in vitro recapitulation of the in vivo palatal

fusion events. In vitro-maintained palatal shelves required

72 h to complete fusion; therefore sampling times were

identified in the first 72 h of organ culture, which provided

palatal shelf tissue representative of critical stages in the

process of palatal fusion (Nakajima et al. 2007). The stages

of palatal fusion selected were adhesion of the medial

edges, reduction of the MEE to a single layer of cells which

occurred by 24 h in vitro, discontinuity and breakdown of

the MEE into clumps of cells which occurred from 48 to

72 h in vitro, and mesenchymal continuity which achieved

a complete at 72 h in vitro (Nakajima et al. 2007).

Quantitative evaluation of TGF-b3, TbR-III

and extracellular matrix expressions in DiI (?) MEE

cells

To examine TGF-b3, TbR-III and ECM gene expressions

in the MEE during palatogenesis requires obtaining pure

epithelial and mesenchymal cell populations. We isolated

DiI (?) MEE cells using laser capture microdissection

(LCM) (Arcturus, Mountain View, CA) (Fig. 6A a–d). The

section (10 lm thick) was coated with plastic foil. After

LCM, the cells were placed in the collecting tube con-

taining RLT buffer (Qiagen, CA) with 10% b-metocapta-

nol and homogenized. RNeasy mini kit (Qiagen, CA) was

used for total RNA extraction and purification. Extracted

RNA was purified and quantified by spectrophotometry.

Total RNA samples of epithelial MEE and mesenchymal

MEE cells from different stages of palatogenesis were

prepared and reversely transcribed into cDNA, and real-

time RT–PCR was performed as described by Scanlan et al.

(2002). Aliquots containing equal amounts of mRNA were

subjected to RT–PCR. First-strand cDNA synthesis was

carried out using 1 lg of DNase-treated total RNA in 20 ll

of a solution containing first-strand buffer, 50 ng random

primers, 10 mM dNTP mixture, 1 mM DTT, and 0.5 U

reverse transcriptase at 42�C for 60 min. The cDNA mix-

tures were diluted five fold in sterile distilled water, and

2 ll aliquots were subjected to real-time RT–PCR using

J Mol Hist (2010) 41:343–355 345

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SYBR Green I dye. The real-time quantitative PCR was

performed in 25 ll of a solution containing 19 real-time

PCR buffer, 1.5 mM dNTP mixture, 19 SYBR Green I,

15 mM MgCl2, 0.25 U ExTaq polymerase real-time RT–

PCR version (TaKaRa, Tokyo Japan), and 20 mM specific

primers. The primer pairs were described in Table 1.

The primers were designed using Primer3 software

(Whitehead Institute for Biomedical Research, Cambridge,

Table 1 Real-time RT–PCR primer pairs and product size

Primer Forward Reverse Product size (bp)

TGF-P3 50-ACA ACA CCT GAA CCC AGAG-30 50-ACT GCA GTG AGC AAG CTGA-30 103

TGFp-RIII 50-GAG GAT CCT GAG GTG GTC AA-30 50-GGC TCT CTG TGG TCT GGA AG-30 105

MMP2 50-GCC GCC TTT AAC TGG AGC AA-30 50-TCC CAG GCA TCT GCG ATG AG-30 98

MMP13 50-GTC TTC CCC GTG TCC AAA AGA-30 50-TGA CCT GGG ATT TCC AAA AGA-30 105

TIMP2 50-GCA TCA CCC AGA AGA AGA GC-30 50-GTC CAT CCA GAG GCA CTC AT-30 106

TIMP4 50-TCC TGC AAG TCC CCT GAT AC-30 50-AAC CTG GAG GGA AAA TGC TT-30 102

GAPDH 50-CAA TGA CCC CTT CAT TGA CC-30 50-GAC AAG CTT CCC GTT CTC AG-30 106

Fig. 1 The distribution of CNC-derived and non-CNC-derived cells

in palate mesenchyme at different anterior-posterior positions as

detected by b-galatosidase (b-gal). At E13 CNC-derived cells were

detected as b-gal (?) at anterior (a), middle (b) and posterior

(c) positions of the palatal shelves. Non-CNC-derived mesenchymal

cells were b-gal (-) and stained pink (a–c single arrows). The

epithelial cells were b-gal (-). At E14.5 palatal shelves began to fuse

in the anterior region (d), middle region (e), and posterior region of

palate (f) leaving a midline epithelial seam (double arrows). The

transdifferentiated MEE were b-gal (-) and could not be distin-

guished from the non-CNC mesenchymal cells (single arrows). At

E15, the palatal shelves were fused in all three regions (g–i) and

transdifferentiated MEE could not be definitely distinguished from

other b-gal (-) cells. Bars a (100 lm); d and g (50 lm)

346 J Mol Hist (2010) 41:343–355

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MA). PCR was carried out in a thermal cycler (Smart

Cycler, Cepheid, Sunnyvale, CA), and the data were ana-

lyzed using Smart Cycler software (ver. 1.2d). The PCR

conditions were 95�C for 3 s and 60�C for 20 s for 35

cycles, and measurements were taken at the end of the

annealing step at 60�C in each cycle. All real-time

Fig. 2 The distribution of CNC-derived cells and TGF-b3 and TbR-

III expressions during palatal fusion in vivo. At E13 CNC-derived

cells were detected as b-gal (?) in the palatal mesenchyme (a, c), and

TGF-b3 detected in the epithelium (b), however, TbR-III expression

was low level at E13 (d). TGF-b3 expression is only detected in

epithelial cells at this stage. At E14.5 palatal shelves began to fuse (e–

h). The arrows indicated b-gal (-)/TGF-b3 (?) palatal mesenchyme

(f) consistent with transdifferentiated MEE and TbR-III was only

localized in the MEE midline seam at E14.5 (h). The rectangularframe placed on panel f showed a superimposed image of both b-gal

and FITC immunohistochemistry, and the arrowheads indicated b-gal

(-)/TGF-b3 (?) mesenchyme cells. At E15, the palatal shelves were

fused in all three regions (i–l) and transdifferentiated MEE could be

observed as b-gal (-)/TGF-b3 (?) cells (i, h). The rectangular frameplaced on panel j showed a superimposed image of b-gal staining

and FITC immunohistochemistry. The arrowheads also indicated the

b-gal (-)/TGF-b3 (?) mesenchyme cells around the midline seam of

secondary palate. After fusion, TbR-III expression is decreased at E15

(h). The single arrow indicated b-gal (-)/TGF-b3 (-) non-CNC-

derived mesenchymal cells. Bars a and c (100 lm); e, g, i and

k (50 lm)

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RT–PCR reactions were performed in triplicate, and the

levels of mRNA expression were calculated and normal-

ized to the level of GAPDH mRNA at each time point. The

results from multiple groups were compared with ANOVA

and Tukey’s-HSD multiple comparison tests. The accept-

able level of significance was set at P* \ 0.05. Data were

analyzed with the SPSS software.

Results

Palatal development and distribution of MEE

and CNC cells in vivo

The Wnt1-Cre/R26R double transgenic samples were not

distinguishable developmentally from the non-transgenic

control mice at similar gestational ages with respect to the

process of palatal fusion. The palatal shelves were vertical

at E13 (Fig. 1a–c). CNC mesenchyme was b-gal (?) while

epithelial cells and non-CNC mesenchyme cells were b-gal

(-). These of non-CNC-derived mesenchymal cells were

present in the palate from anterior to posterior regions with

no apparent changes in frequency or distribution in any

region at E13 (Fig. 1a–c).

At E14.5, the palatal shelves were adherent and in some

areas the MEE was only a single cell layer with some

discontinuities (Fig. 1d–f). A key observation was the

presence of b-gal (-) mesenchymal cells in the region of

the midline epithelial seam, that could include the MEE-

derived mesenchymal cells. However, since E13 was prior

to the transformation of the MEE, these b-gal (-) cells

represent a population of mesenchymal cells that were not

CNC-derived and would be indistinguishable from the b-

gal (-) transdifferentiated MEE that are present after

E14.5.

By E15 palatal fusion was complete in the posterior

region, and epithelial cells were not present in the midline

region (Fig. 1g–i). The b-gal (-) cells in the midline

position could be derived from either the transdifferenti-

ated MEE or the non-CNC mesenchyme observed at E13.

Distribution of CNC-derived mesenchyme with TGF-

b3 and TbR-III expression during palatal fusion in vivo

The CNC mesenchyme was b-gal (?) while epithelial cells

were TGF-b3 (?)/b-gal (-), while the non-CNC mesen-

chyme cells b-gal (-)/TGF-b3 (-) at E13 (Fig. 2a, b). A

key observation was the presence of b-gal (-) mesenchy-

mal cells in the region of the midline seam prior to palatal

fusion (Fig. 2a, b). These non-CNC-derived mesenchymal

cells were present in the palate with no apparent changes in

frequency or distribution at E13, even though TbR-III

expression was weak in the epithelial cells at same stage

(Fig. 2c, d).

At E14.5, b-gal (-) MEE epithelial cells strongly

expressed TGF-b3 (Fig. 2e, f) and TbR-III (Fig. 2g, h).

Figure 2f shows clearly superimpose of b-gal and TGF-b3

expression. The arrowheads locating around the midline

epithelial seam, indicate b-gal (-)/TGF-b3 (?) mesen-

chyme.

By E15 palatal fusion was complete in the posterior

region, and epithelial cells were not present in the midline

region. Figure 2j shows the merged b-gal staining and

FITC of TGF-b3 expressions image, and the arrowheads

indicate the b-gal (-) and TGF-b3 (?) mesenchyme cells,

whose population might be the transdifferentiated MEE.

The b-gal (-) mesenchyme cells in the midline position

could be derived from two different cells possibility, which

were either the TGF-b3 (?)/TbR-III (-) MEE cells or the

TGF-b3 (-)/TbR-III (-) non-CNC mesenchyme cells

observed at E13 (Fig. 2i–l).

Comparison of TGF-b3 and TbR-III in the mesenchy-

mal cells following palatal fusion leads to option that these

cells were either the b-gal (-)/TGF-b3 (?) that were

originally MEE or b-gal (-) mesenchyme after E14.5

(Fig. 2e, f). TbR-III expression was present only in epi-

thelial MEE cells (Fig. 2g, h). Prior to palatal fusion the b-

gal (-) cells in the mesenchyme were neither TGF-b3 (?)

nor TbR-III (?), and additionally, TbR-III expression was

weaker than TGF-b3 in the MEE (Fig. 2b, d). The evalu-

ation process could not exclude the possibility that the b-

gal (-)/TGF-b3 (?)/TbR-III (-) mesenchymal cells were

not originally the MEE but rather represented new

expression of TGF-b3 in non-CNC-derived palatal mes-

enchymal cells (Fig. 2e–h). Determination of the original

origin of the TGF-b3 (?) mesenchymal cells required the

use of additional cell lineage markers by which whether

tracking MEE was epithelial-mesenchymal transdifferen-

tiation can be confirmed (Fig. 2i, j).

Fig. 3 Localization of b-galactosidase, DiI and TGF-b3 expression

during palatal fusion. The distribution of these three markers occurs at

four time points in vitro 24 h (a–d) and 72 h (e–k). At E13 ? 24 h,

the midline MEE cells detect as b-gal (-) (a), DiI (?) (b), and TGF-

b3 (?) (c). Superimposition of DiI and TGF-b3 are shown to identify

the cells at E13 ? 24 h (d). At E13 ? 72 h organ culture, both

palatal shelves are completely fused and most of mesenchyme cells

are observed b-gal (?) cells (e). However, some mesenchyme cells

are shown b-gal (-) and DiI (?) (f, h), and b-gal (-) and TGF-b3

(?) (g, i) in the midline seam at E13 ? 72 h. The merge of DiI and

TGF-b3 expression is shown in j, and yellow cell population is both

expressions. The arrows indicated b-gal (-)/DiI (?)/TGF-b3 (?)

cells that represent transdifferentiated MEE (h–j). Bars a and

e (50 lm)

c

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Fate of MEE based on two sets of markers and gene

expressions in vitro

We also examined the both CNC and DiI heritable marking

and then analyzed these tissues with immunohistochemis-

try to characterize the MEE related gene expression of

TGF-b3 and TbR-III in vitro palatal organ culture. We

observed E13 palatal shelf fusion at 24 and 72 h of organ

culture. DiI labeled only the epithelium and the midline

seam after 24 h of culture (Fig. 3b). The mesenchymal

cells were either b-gal (?) or b-gal (-) with a distribution

similar to that observed in vivo (Fig. 3a). All the epithelial

cells, including those in the midline seam, were b-gal (-)

(Fig. 3a). The b-gal (-)/DiI (?) MEE were positive for

TGF-b3, TbR-III and Keratin at this point and neither of

these two molecules were detected in the mesenchyme

(Fig. 3a–d, 4a–d, 5a–d).

The palatal shelves were completely fused following

72 h of organ culture and the cells with an epithelial

morphology could not be identified in the midline, as

mentioned above. DiI (?) MEE cells were b-gal (-) and

present in the midline region of the palatal mesenchyme

(Figs. 3, 4, 5). These non-CNC derived mesenchymal cells

represent MEE with continued TGF-b3 expression (Fig. 3).

However, TbR-III and Keratin was not expressed in the

mesenchyme (Figs. 4, 5g). TbR-III characterized the MEE

that still had an epithelial phenotype and were associated

with a basement membrane. Only b-gal (-)/DiI (?) mes-

enchymal cells had TGF-b3 expression. The use of the

heritable b-gal marker, the DiI cell lineage marker, and

TGF-b3 allowed the mesenchymal cells and the MEE to be

characterized at different stages of palatal fusion and

mesenchymal MEE (Fig. 3).

TGF-b3 and TbR-III expressions in isolated DiI (?)

MEE cells during palatal fusion

In order to characterize the pattern of TGF-b3 and TbR-III

expressions during palatal fusion in the Dil (?) MEE cells,

we examined DiI (?) MEE cells in palatal organ culture

isolated by LCM from 24 to 72 h. The effectiveness of

LCM to recover the DiI labeled MEE was confirmed by

comparing the images before LCM (Fig. 6A-a at

E14 ? 24 h and Fig. 6A-c at E14 ? 72 h) with the images

after LCM (Fig. 6A-b at E14 ? 24 h and Fig. 6A-d at

E14 ? 72 h). Furthermore, we examined TGF-b3 and

TbR-III mRNA expressions of Dil (?) MEE cells using

real-time RT–PCR.

TbR-III expression showed a significantly higher level

at E14 ? 24 h compared to that at E13, and it decreased

thereafter (Fig. 6B). Furthermore, at E14 ?72 h, TbR-III

showed a marked drop in expression level, which was

significantly lower than that at E13. TGF-b3 expression

also showed a peak level at E14 ? 24 h, which expression

was significantly higher than that at E13. The post-fused

DiI (?)/TGF-b3 (?) mesenchymal cells were consistent

with MEE undergoing EMT (Fig. 6B).

The expression level of MMP4 was low but almost

constant during palatal fusion, and there was no significant

difference compared to that at E13. Meanwhile, MMP13

expression of DiI (?) cells revealed a significantly higher

level at E13 ? 24 h than at E13, and drastically decreased

at E13 ? 72 h (Fig. 6B). Moreover, MMP13 mRNA was

very intensely and precisely expressed at the site of contact

between the palatal shelves. The expression of TIMP2

during the formation of the secondary palate revealed a

slight increase with time. TIMP2 mRNA was diffusely

expressed in the MEE cells, while no specific signal was

detected with a probe for TIMP4 (Fig. 6B).

Discussion

The fate of the MEE following palatal fusion remains an

intriguing area. In the many other instances of (1) MEE

retaining their epithelial phenotype and migrated to merge

with the nasal and oral surface epithelia (Carette and

Ferguson 1992; Martinez-Alvarez et al. 2000; Vaziri Sani

et al. 2005; Jin and Ding 2006), (2) PCD (Mori et al. 1994;

Martinez-Alvarez et al. 2000; Cuervo et al. 2002; Vaziri

Sani et al. 2005; Xu et al. 2006) and (3) EMT (including

transformation) (Carette and Ferguson 1992; Fitchett and

Hay 1989; Shuler et al. 1991, 1992; Griffith and Hay 1992;

Kang and Svoboda 2005; Jin and Ding 2006), the new

population of mesenchymal cells is required to complete an

activity important for some developmental events; however

it has yet to be determined whether a specific role exists for

the transdifferentiated MEE (Takahara et al. 2004; Takig-

awa and Shiota 2004; Nawshad 2008). To identify the fate

of remaining MEE, we previously detected both prolifer-

ating cells and apoptotic cells in palatal mesenchyme and

epithelial including MEE cells (Nakajima et al. 2007).

In the present study, we proved an analysis using mul-

tiple strategies to define the mesenchymal cell populations

after palatal fusion. In addition, we could isolate and

analyze labeled DiI cells by using LCM and real time

RT–PCR to provide additional data about the specific

Fig. 4 Localization of b-galactosidase, DiI and TbR-III expression

during palatal fusion. The distribution of b-gal and DiI makers and

TbR-III expression occurs at E13 ? 24 h (a–d) and 72 h (e–j). b-gal

(?) cells are blue (a, e), DiI (?) cells fluoresce red (b, f) and TbR-III

(?) fluoresce green (c, g). Merged b-gal and DiI at E13 ? 72 h was

shown in h, and merged b-gal and TbR-III was shown in i. Super-

imposition of DiI with TbR-III (g, j) is shown to identify of remaining

midline seam MEE that cells population is shown fluoresce red. The

arrows indicated DiI (?)/TbR-III (-) cells that remaining palatal that

cells population is shown fluoresce red. Bars a and e (50 lm)

c

350 J Mol Hist (2010) 41:343–355

123

transdifferentiated MEE cells. The Wnt1-Cre/R26R trans-

genic labeling system has been shown to be a very effective

means of labeling the CNC cells and those cells and tissues

derived from the Neural Crest (Jiang et al. 2000; Chai et al.

2000). This system enable to distinguish two different

mesenchymal cell populations, CNC-derived b-gal (?) and

J Mol Hist (2010) 41:343–355 351

123

Fig. 5 Localization of b-galactosidase, DiI and Keratin expression

during palatal fusion. The distribution of two markers and Keratin

expression occurs at 24 h (a–d) and 72 h (e–j). b-gal (?) cells are

blue (a, e), DiI (?) cells fluoresce red (b, f) and Keratin (?)

fluoresce green (c, g). Superimposition of DiI with Keratin (d, j) is

shown to distinguish between the epithelial and mesenchyme cells

in the midline seam. Panel h showed superimposed b-gal and

DiI staining, and panel i a merged image of b-gal and FITC

immunohistochemistry. The arrows indicated b-gal (-)/DiI (?)/

Keratin (?) cells that represent MEE that retain an epithelial

phenotype (h–j). Bars a and e (50 lm)

352 J Mol Hist (2010) 41:343–355

123

non-CNC-derived b-gal (-), although the non-CNC mes-

enchyme has multiple origins. This observation required

the development of a triple labeling strategy in vivo using

both cell lineage markers and proteins specific for the MEE

during palatal fusion. The present result has shown that

three populations of palatal mesenchymal cells can be

Fig. 6 TGF-b3, TbR-III and ECM mRNA quantified in DiI positive

cells isolation by laser capture microdissection. Before (A a and

A c) and after (A b and A d) laser capture microdissection (LCM) at

E13 ? 24 h (A a and A b) and 72 h (A c and A d) organ culture in

vitro. Arrowhead indicates DiI (?) MEE before capture (A a and

A c) and after capture (A b and A d). TGF-b3 and TbR-III mRNA are

expressed at 24, 48 and 72 h after placing the palatal tissues in organ

culture (b). TGF-b3 expression is detected continuously during

palatal fusion, and there are significant differences comparing with

E13 expression. TbR-III with DiI was peaked at E13 ? 24 h and

there after the expression is decrease by E13 ? 72 h. Extracellular

matrix of MMP13 and TIMP2 expressions are detected during palatal

fusion. MMP13 is indicated at fusing stage and TIMP2 mRNA

expression is continuously expressing in DiI(?) palatal MEE and

transdifferentiated MEE. Bars A a and A c (80 lm). * P \ 0.05%

(comparing with E13 expression)

J Mol Hist (2010) 41:343–355 353

123

identified following palatal fusion using the three different

types of markers.

TGF-b3 has a critical role in palatogenesis (Fitzpatrick

et al. 1990; Pelton et al. 1990). The timing of TGF-b3

expression is temporally correlated with the critical events

surrounding palatal shelf adhesion. In the null mutant mice

the palatal shelves fail to adhere properly, the basement

membrane is not degraded and the MEE does not trans-

differentiated (Kaartinen et al. 1995, 1997). The concept of

EMT during palatogenesis has been used extensively as a

basis to assign biological roles to a number of factors,

including TGF-b3 (Kaartinen et al. 1997; Nawshad and

Hay 2003; Nawshad et al. 2004, 2007; Nawshad 2008;

Nogai et al. 2008), Snail (Martınez-Alvarez et al. 2004),

Lef1 and/or Smads (Nawshad et al. 2004, 2007; Dudas

et al. 2004). We could identify b-gal (-) mesenchyme

cells observed in the palatal mesenchyme as DiI (?)

mesenchyme around midline seam after palatal fusion. In

addition, TGF-b3 was expressed DiI (?) epithelial/mes-

enchymal cells at fusing stage and after fusion. Therefore

our result was almost consistent with previous studies that

some MEE can include EMT which strongly associates

with TGF-b3 expressions during palatal development

(Shuler et al. 1991, 1992; Kaartinen et al. 1997; Cui and

Shuler 2000; Nawshad et al. 2007; Gordon et al. 2008;

Nogai et al. 2008).

TGF-b3 belongs to a family of growth factors that have

a broad range of regulatory activities, including control of

cell proliferation, regulation of extracellular matrix (ECM)

deposition such as MMPs and TIMPs that are associated

with cell migration, differentiation, and EMT (Griffith and

Hay 1992; Blavier et al. 2001; Nawshad et al. 2004, 2007;

Kang and Svoboda 2005). In these cases the TGF-b3

expression has either a paracrine or autocrine signaling

mechanism closely linked to the cell/tissue physiologic

event, thus the finding that TGF-b3 remains to express in

the transdifferentiated MEE is of interest. MMPs, espe-

cially MMP13, were known to be strongly expressed in

MEE cells during palatal fusion (Blavier et al. 2001). The

ability to identify and recover MEE cells from the mes-

enchyme following phenotypic transformation will provide

the opportunity to more closely evaluate the different

mechanistic events regulated by ECM associating TGF-b3

at different stages of palatogenesis.

The present study have provided the possibility of the

occurring EMT in the MEE cells using Cre/lox system, but

it is neither against to PCD nor migration (Vaziri Sani et al.

2005; Xu et al. 2006). However, our results could support

the recent study in which EMT palatal mesenchyme was

established in the chimeric culture system using Rosa26

transgenic and C57BL/6 mice (Jin and Ding 2006) and

using cell line (Nawshad et al. 2007; Gordon et al. 2008;

Nogai et al. 2008). The MEE undergoing EMT represents a

small but distinct subpopulation of palatal mesenchymal

cells. The contribution to subsequent developmental events

or involvement in pathologic processes is unknown due to

the lack of suitable methods to identify these cells. Using a

heritable marker for CNC cells (b-gal), a cell lineage

marker for MEE (DiI) and molecules specific to the MEE

during palatal fusion (TGF-b3, TbR-III and Keratin), we

have been able to characterize three distinct populations of

palatal mesenchymal cells. These findings will permit

future studies structured to specifically isolate the cells,

examine signaling processes and identify MEE-specific

markers and gene expressions. The present results will

enable a precise technique for the characterization of MEE-

derived mesenchymal cells to determine their post-palatal

fusion developmental fate.

Acknowledgments The author thanks our colleagues at the Center

for Craniofacial Molecular Biology University of Southern California

and at Nihon University School Dentistry for their continuous strong

support. Also we thank the Jackson Laboratory this works was pro-

vided Wnt1-Cre mice and R26R-Cre mice and their licenses from the

Jackson laboratory resources. This work was supported by NIDCR

grants PO1DE-12941 and RO1DE-12711, and MEXT Grant for

multi-disciplinary research projects, MEXT Grant-in-Aid for Scien-

tific Research (C) 16592055 and 20592415, A Grant from the Min-

istry of Education, Culture, Sports, Science and Technology to

promote multi-disciplinary research projects, Nihon University

Research Individual Grant for 2005 and 2008, and Grant from Dental

Research Center, Nihon University.

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