Food and Chemical Toxicology 56 (2013) 67–74

Contents lists available at SciVerse ScienceDi rect

Food and Chemi cal Toxic ology

journal homepage: www.elsevier .com/locate / foodchemtox

Dexamethasone alters epithelium proliferation and survival and suppresses Wnt/b-catenin signaling in developing cleft palate

Xiao Hu a, Jian Hua Gao a,⇑, Yun Jun Liao a, Shi Jie Tang b, Feng Lu a,⇑a Department of Plastic and Cosmetic Surgery, Nan Fang Hospital, Guangzhou 510515, People’s Republic of China b Department of Plastic and Burn Surgery, Second Affiliated Hospital, Shantou University Medical College, Shantou 515041, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Received 25 November 2012 Accepted 4 February 2013 Available online 13 February 2013

Keywords:Cell apoptosis Cell proliferation Cleft palate DexamethasoneWnt/b-catenin signaling

0278-6915/$ - see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.fct.2013.02.003

Abbreviations: Dex, dexamethasone; E, embryonimedial edge epithelium; MES, midline epithelial seam⇑ Corresponding authors. Tel.: +86 20 61641866; fa

E-mail addresses: gaopsnf@yahoo.com.cn (J.H. Gao(F. Lu).

Dexamethasone (Dex) contributes to a cleft palate, but the cellular and molecular mechanisms responsi- ble for the deleterious effect on the developing palate are unclear. Wnt signaling is a causal mechanism of Dex-induced osteoporosis, so this study was conducted to determine whether Dex-induce d cleft palate may result from altered Wnt signaling. Administration of Dex to mice completely inhibited canonical Wnt/b-catenin signaling and altered cell proliferation and apoptosis of the craniofacial epithelium in developing embryos. Thus, downregulated Wnt/ b-catenin signaling was associated with Dex-induced cleft palate. Moreover, altered cell fate by Dex responsible for small palates, delaying shelf elevation and unfused palates was a crucial mechanism in cleft palate. Our findings help in elucidating the mech- anisms of Dex-induced cleft palate.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction subsequent ly degenerates to allow mesenchymal continuity across

Cleft palate, the congenit al craniofaci al birth defects in humans, arises from genetic or environmental perturbations in the multi- step process of palate developmen t. The frequent occurrence and significant healthcare burden imposed by cleft palate highlight the need to dissect the etiology and molecular pathogenes is of this condition. Because mice and humans share similar craniofacial morphologic features during developmen t (Tamarin and Boyde, 1977; Gritli-Linde, 2008 ), the mouse is a suitable model to study these processes .

The first sign of development of the murine secondary palate occurs on embryonic day 11.5 (E11.5) with the outgrowth from the maxillary processes of paired palatal shelves, consisting of acentral core of neural-crest-cel l-derived mesenchym e surrounded by an epithelium compose d of a basal layer of cuboidal ectodermal cells and a surface layer of flattened periderm cells that initially grows vertically down the sides of the developing tongue (E12.5and E13.5). During E14.5, the palatal shelves are rapidly elevated to a horizontal position above the dorsum of the tongue. The med- ial edge epithelium (MEE) of the apposed shelves contact and ad- here to form a midline epithelial seam (MES), which

ll rights reserved.

c day; KM, Kun Ming; MEE, .

x: +86 20 87743734. ), doctorlufeng@hotmail.com

the palate (E15.5). At E17.5, the palatal fusion is complete d. Glucocorticoi ds are immunosupp ressive drugs widely used to

treat acute and chronic autoimm une diseases and malignancies de- spite their known non-specificity and multiple side effects (Green-stein et al., 2002; Rhen and Cidlowski, 2005 ). Glucocorticoi ds at physiolog ical levels are necessary for craniofaci al developmen t in early embryos; however, high-dose exogenous glucocorti coids are harmful to fetuses (Fraser et al., 1954; Pinsky and Digeorge, 1965; Abbott, 1995 ). In 1954, Fraser and colleagues successfully in- duced cleft palate in 100% of A/J mice exposed to cortisone and found that exposure delayed shelf elevation, producing smaller palatal shelves that could not contact and fuse (Fraser et al., 1954). Pinsky and Digeorge used hydrocortis one, prednisolone and dexamet hasone (Dex) to produce cleft palate in A/J mice and concluded that Dex was 300 times more teratogenic than hydro- cortisone in the cleft-palate production (Pinsky and Digeorge, 1965). Early work showed that expression of epiderma l growth factor and Tgf- b (transforming growth factor b) in the epithelium of palatal shelves was associated with Dex-induced cleft palate (Abbott, 1995 ). Dex suppressed Wnt (wingless) protein signaling and induced cell apoptosis , thus resulting in osteopor osis, and Dex-indu ced osteocyte apoptosis could be rescued by Wnt signal- ing (Hamidouche et al., 2008; Hayashi et al., 2009; Kitase et al., 2010; Almeida et al., 2011 ). However, the detailed mechanism of Dex-indu ced cleft palate has not been fully elucidated.

Mutation s in the Wnt3 and Wnt9b genes are associated with cleft lip and palate in both humans and mice, so Wnt signaling plays acritical role in craniofacial development (Juriloff et al., 2001, 2006;

Table 1Incidence of cleft palate with different concentrations of dexamethasone in mice.

Group Dose (mg/kg/day)

Period of administration

No. of pregnant females

Viable embryos

Cleft palate (%)

Control – E8.5–13.5 10 105 01 0.8 a E10.5–12.5 11 100 2 (2)2 2.5 b E10.5–13.5 9 84 35 (41.7)3 4b E8.5–13.5 5 43 27 (62.8)4 8b E8.5–13.5 14 116 95 (81.9)

E, embryonic day. a Once a day. b Twice a day.

68 X. Hu et al. / Food and Chemical Toxicology 56 (2013) 67–74

Niemann et al., 2004; Carroll et al., 2005 ). The Wnt family of signal- ing molecules comprises 19 members in humans and is highly con- served in humans and mice (reviewed by Mikels and Nusse, 2006 ).During early craniofacial developmen t in the mouse embryo, mem- bers of the Wnt family are expresse d at tooth initiation sites of the oral epithelium , in the mesenchy me of the elevating palatal shelves, and at sites of future bone formatio n of the maxilla and mandible. Wnt ligands activate the Wnt/ b-catenin or canonical pathway and b-catenin-indepen dent pathway or non-canonical pathway. In the absence of a Wnt ligand, cytoplasmic b-catenin is associate d with adenomatou s polyposis coli and axin, phosphorylated by Gsk-3 b(glycogen synthase kinase 3b) and casein kinase I and polyubiquiti- nated by the b-transducin repeat-co ntaining protein complex, tar- geting it for proteosoma l degradation . Under these condition s, Lef/ Tcf (lymphoid enhancer factor/T-cell factor) transcription factors in the nucleus associate with transcriptional co-repressors, such as Groucho, to repress transcription of Wnt target genes. In contrast, in the presence of a Wnt ligand, the enhanced phosphoryla tion of Gsk-3b inhibits its activity, thus leading to inhibited phosphoryla -tion and degradation of b-catenin, which allows it to accumulate in the cytoplasm and translocate into the nucleus. Nuclear b-catenininteracts with the Lef/Tcf family transcriptio n factors and several other transcriptional co-activators to initiate transcriptio n of target genes such as c-Jun. When Wnts such as Wnt1, Wnt3a, and Wnt8 bind to cell-surfa ce co-receptors, cytoplasmic b-catenin is stabilized and enters the nucleus. Some Wnts, such as Wnt5a and Wnt11, acti- vate a b-catenin-indepen dent pathway, as initially observed during embryogenes is (reviewed by Veeman et al., 2003 ). Wnt3A, Wnt5A and Wnt11 were significantly associated with non-syndromic cleft lip with or without cleft palate (Chiquet et al., 2008 ). The Wnt mol- ecules act through specific receptors to activate intracellular signals that dictate various cell actions, including proliferation , migration, growth, differentiation , and apoptosis.

The developing organism is a master at using simple paradigms to generate complexity. This is typified by the use, at least during early organogenes is, of the same molecular pathways to drive the developmen t of organs and tissues as diverse as the brain, limbs, palate, bone, teeth, and skin. We hypothesize d that Dex-in- duced cleft palate is associated with Wnt signaling by altering the palatal cell fate in the crucial time of craniofacial developmen t. We analyzed the morphologic features in palatogenesis and the pro- tein expression of Wnt signaling molecule s with Dex exposure and investigated cell proliferation and survival from E13.5 to E15.5.

2. Materials and methods

2.1. Animals

All animal experiments were approved by the Nan Fang Hospital Institutional Animal Care and Use Committee. Females of Kun Ming (KM) mice were crossed with fertile males overnight. The morning when a vaginal plug was observed was defined as E0.5. Pregnant females at E8.5 were randomly divided into 2 groups for treatment: a total daily dose of 8 mg/kg Dex (two doses of 4 mg/kg at approxi- mately 12 h intervals; MP Biomedicals, OH, USA) or normal sodium chloride subcu- taneously, twice a day, from E8.5 to E13.5. Embryos were harvested during palatogenesis (E13.5–E15.5, E17.5).

2.2. Scanning electron microscopy

Trimmed fetuses at E17.5 were fixed with 2.5% glutaraldehyde at 4 �C for 12 h. After dehydration with a graded acetone series, specimens were dried with liquid CO2 and coated with a thin layer of gold before being viewed under an S-3000N scanning electron microscope.

2.3. Histochemical staining

Embryonic heads were fixed in 4% paraformaldehyde and dehydrated through an ethanol series and embedded in paraffin for sectioning by routine procedures. For general morphology, deparaffinized sections (5 lm) were stained with hema- toxylin and eosin by standard procedures.

2.4. Western blot analysis

The palatal shelves and tongue extracts of controls and treatment with Dex through E13.5–E15.5 were examined by standard protocols with antibodies for Wnt3a, Wnt5a, and Wnt11 (all R&D Systems, MN, USA); Gsk-3 b, b-catenin, and Lef-1 (all Cell Signaling, MA, USA); c-Jun and phospho-c-Jun (pS63; both Epitomics, Hangzhou, China); and a-Tubulin (Santa Cruz Biotechnology, CA, USA).

2.5. BrdU analysis and TUNEL

Pregnant females during E13.5–E15.5 were injected with 1 ml BrdU (5-bromo-20-deoxyuridine; Invitrogen, CA, USA) per 100 g body weight intraperitoneally. After 2 h, embryos were removed, trimmed and fixed with 4% paraformaldehyde. Anti- body detection of BrdU involved standard immunostaining protocols that included an antigen retrieval step of 30 min in 2 N HCl at 37 �C. Staining involved an Alexa Fluor� 555 conjugate antibody recognizing BrdU. The proportion of BrdU-positive cells to total cells in the palatal shelf mesenchyme and epithelium was counted from 5 randomly selected sections per sample. Five palate samples of 3–5 dams were evaluated from each experimental group.

Apoptotic cells were assayed by TUNEL (terminal deoxynucleotidyl transferase- mediated dUTP-biotin nick end labeling) with the FragEL™ DNA Fragmentation Detection Kit, Fluorescein (Calbiochem, Darmstadt, Germany).

2.6. Statistical analysis

Data are expressed as mean ± SD and were analyzed by Student’s t test. Results were considered significant at p < 0.05. Each experiment was at least performed in triplicate.

3. Results

3.1. Embryos with Dex treatment exhibited cleft palate

We tested various doses and periods of administration of Dex for the optimal concentratio n and time. Unexpected ly, low dose and short period Dex administration did not produce a high inci- dence of cleft palate, with no linear correlation by concentr ation or time (Table 1). Therefore, we considered that Dex would not in- duce 100% cleft palate and used 8 mg/kg/da y, twice a day, from E8.5 to E13.5; 82% of embryos showed cleft palate. No control em- bryos showed cleft palate.

With Dex treatment, at E17.5, the gap between the unfused pal- atal shelves was wider in the anterior and posterior regions than in the middle region, and the rugae were more apparent than with control treatment (Fig. 1). The opposing palatal shelves of Dex- treated embryos were elevated but could not fuse (Fig. 1B), which is consisten t with morphology staining.

To gain greater insight into the pathogenesis of Dex-indu ced cleft palate, we examined the anterior and posterior craniofacial morphologi c features from E13.5 to E15.5 and E17.5. At E13.5, the anterior palatal shelves of Dex-treated embryos were similar to those of littermate controls (Fig. 2A and B). However , the open space in the oral-nasal cavity of posterior regions was wider for controls than Dex-treated embryos (Fig. 2C and D) at all times. Therefore, we considered that it was not an artifact resulting from the paraffin-embedding process (Greene and Kochhar, 1973 ). E14.5

Fig. 1. Scanning electron microscopy of mouse embryonic day 17.5 (E17.5) palate after dexamethasone (Dex) treatment. Control palate (A) and Dex-treated palate (B). Arrows point to rugae. Con, control; ps, palatal shelf; sp, secondary palate. Bars, 1 mm.

X. Hu et al. / Food and Chemical Toxicology 56 (2013) 67–74 69

embryos showed various shelf morphologic features, from bilater- ally elevated or even contacted shelves of controls (Fig. 2E and G)to vertically oriented shelves of Dex-treated embryos (Fig. 2F and H). As compared with the open space in the oral-nasal cavity of the posterior region of Dex-treated embryos at E13.5, that at E14.5 was relatively wider, to prepare enough space for possible elevation (Fig. 2D and H). By E15.5, Dex-treated shelves were ele- vated but were not contacted and fused (Fig. 2J and L), whereas control shelves contacted, adhered and began to fuse (Fig. 2I and

Fig. 2. Palate development in Dex-treated embryos. E13.5 (A–D), E14.5 (E–H), E15.5 (I–L)F, H, J, L, N and P). Histological frontal sections in anterior and posterior (at the level of ththe open space of the oral-nasal cavity. Con, control; f, floor of mouth; ns, nasal septum

K). As compare d with the rapidly developing other craniofacial tis- sue, shelves of Dex-treated embryos were relatively clumsy and displayed a large gap that eventually manifest ed as the cleft palate phenotyp e at E17.5 (Fig. 2N and P), whereas controls developed acomplete secondary palate (Fig. 2M and O).

Consisten t with previous reports (reviewed by Bush and Jiang, 2012), elevation of both anterior and posterior palatal shelves of- ten occurred asynchro nously in Dex-treated embryos at E14.5, with one palatal shelf elevating before the other (Fig. 3A and B).

and E17.5 (M–P) of control (A, C, E, G, I, K, M and O) and Dex-treated embryos (B, D, e first lower molar tooth) regions of developing palate. Bidirectional arrows point to ; ps, palatal shelf; t, tongue. Bars, (A–L) 200 lm and (M–P) 250 lm.

Fig. 3. One palatal shelf keeping vertical outgrowth at all times in Dex-treated embryos. One palatal shelf was elevated, and the other was not in E14.5 (A and B),E15.5 (C and D) and E17.5 embryos (E and F) of anterior (A, C and E) and posterior (B, D and F) portions. ps, palatal shelf; t, tongue. Bars, (A–D) 250 lm and (E and F)1000 lm.

70 X. Hu et al. / Food and Chemical Toxicology 56 (2013) 67–74

Because palatal shelf elevation was too rapid to be easily observed ,we discovered this phenomeno n only with Dex treatment, and Dex significantly delayed one palatal shelf to elevate or even stopped it (Fig. 3C–F).

3.2. Wnt/ b-catenin signaling was inhibited by Dex from E13.5 to E15.5

Wnt signaling proteins were detected by immunoblo tting in developing maxilla tissue of Dex-treated embryos and controls during E13.5–E15.5 (Fig. 4). Surprisingly , Gsk-3 b expressionshowed a downward shift with Dex as compared with controls, which indicated the sharply reduced level of phospho -Gsk-3 band the increased activity of Gsk-3 b. Consistent with altered Gsk- 3b expression, the downstream molecules of Wnt/ b-catenin signal- ing, including b-catenin, Lef-1, and c-Jun and phospho-c-Jun , were completely downregulated by Dex treatment, whereas the expres- sion of Wnt3a, Wnt5a and Wnt11 was not markedly different from that of controls (Fig. 4).

3.3. Epithelium proliferat ion and survival in Dex-induced cleft palate

Because Wnt/ b-catenin signaling with Dex treatment was downregulated in developing craniofacial tissues and this pathway has a crucial role in cell fate such as cell prolifera tion, different ia- tion, adhesion and survival, we examined cell turnover by BrdU incorporation (Fig. 5, red fluorescence) and TUNEL staining (Figs. 6

and 7, green fluorescence). Consisten t with previous investigatio ns, we chose 3 stages to analyze: E13.5–E15.5 with anterior and pos- terior regions. At E13.5, the MEE proliferation was lower in Dex- treated palatal shelves in both anterior (right palatal shelves, p = 0.001) and posterior regions than in controls (Fig. 5A–H; col- umn 1 of S–V), but at E14.5, MEE proliferation was greater in Dex-treated anterior (right palatal shelves, p = 0.04) and posterior (right palatal shelves, p = 0.029 and left palatal shelves, p = 0.032)regions than in controls (Fig. 5I–N; column 2 of S–V). By E15.5, the proliferation of MEE was still increased in both anterior (rightpalatal shelves, p = 0.015) and posterior (right palatal shelves, p = 0.041) regions as compare d with controls (Fig. 5O–R; column 3 of S–V). During E13.5–E15.5, the cell prolifera tion of palatal mes- enchyme and oral-nasal side epithelium of the anterior and poster- ior regions did not differ between Dex and control treatment (Fig. 5). Intriguingly at E14.5, the mesenchym e proliferation of the Dex-treated anterior palatal shelf was near the oral-nasal walls as compared with controls (Fig. 5I–K, arrows). We found no differ- ences in proliferation of epithelium and mesenchym e of the tongue in both Dex-treated embryos and controls during E13.5–E15.5 (data not shown).

At E13.5, clustered TUNEL-pos itive cells were detected in the oral-side epithelium of the anterior and posterior regions of the palatal shelf in both Dex-treated embryos and controls (Fig. 6A–H, white boxes). However at E14.5, apoptotic cells were still de- tected in the oral-side epithelium of unelevated anterior palatal shelves of Dex-treated embryos but not elevated anterior palatal shelves of controls (Fig. 6I–L, arrows). Posterior palatal shelves of Dex-treated samples and controls did not differ in TUNEL staining (Fig. 6M–O). By E15.5, the anterior and posterior palatal shelves of both Dex-treated embryos and controls were elevated to a hori- zontal position above the dorsum of tongue. The remnants of MES in controls showed TUNEL-posi tive staining to degenerate MEE for continuity mesenchyme to form the secondary palate, but Dex-treated embryos showed no apoptotic cells in MEE or MES (Fig. 6P–S, asterisks). Interestin gly, Dex treatment produced no TUNEL-positiv e cells in the epithelium of the floor of mouth of the anterior (Fig. 7B, white boxes) and posterior regions (Fig. 7D, white skew boxes) as compare d with controls (Fig. 7Aand C). However , at E14.5, extensive apoptotic cells were detected in this position of Dex-treated embryos (data not shown). Notably, the mesenchym e of the palatal shelf during E13.5–E15.5 showed no TUNEL-pos itive cells, but the bend region between the cranial base and the palatal shelf showed clusters of apoptotic cells in both Dex-treated samples and controls at E14.5 (Fig. 7E and F, white boxes).

4. Discussion

4.1. Morphog enesis of Dex-induced cleft palate

KM mice showed 82% incidence of cleft palate with 8 mg/kg/da yDex twice a day from E8.5 to E13.5. We observed no other external anomalie s and complications in craniofacial tissues with Dex treat- ment, so the cleft palate did not develop from other craniofacial malformati ons. We found a dose-related incidence of cleft palate with Dex treatment (Table 1). Differences in strains affect the rate of Dex-indu ced cleft palate in mice (reviewed by Abbott, 2010 ), so our use of KM mice may be the primary reason why Dex could not induce a 100% incidence of cleft palate. The middle palatal shelves approach ed first, and the anterior and posterior palatal shelves showed a relatively larger gap (Fig. 1B). Intriguingly, rugae were more apparent in Dex-treated than control embryos (Fig. 1).

Shelf development is achieved by distinct mechanis ms in differ- ent regions along the anteroposter ior axis (Hilliard et al., 2005 ),

Fig. 4. Dex inhibited Wnt/ b-catenin signaling in developing palate. Western analysis of protein levels in mouse maxilla tissue after Dex treatment at E13.5–E15.5. Wnt signaling protein levels are relative to that of a-Tubulin. Samples were run on the same gel and were contiguous. Con, control.

Fig. 5. Cell proliferation in palatal shelves with Dex treatment. Immunostaining for incorporated BrdU (red fluorescence) in developing palate from the anterior (E13.5, A and C; E14.5, I and E15.5, O) and posterior (E13.5, E and G; E14.5, L and E15.5, Q) portions of controls and anterior (E13.5, B and D; E14.5, J and K; E15.5, P) and posterior (E13.5, Fand H; E14.5, M and N; E15.5, R) regions of Dex-treated embryos. White lines demarcate the palatal region for counting BrdU-labeled cells in epithelium and mesenchyme. DAPI stained cell nuclei (blue fluorescence). Red asterisk indicates significantly reduced medial edge epithelium (MEE). Green asterisks represent statistically increased MEE. M, mesenchyme; mee, medial edge epithelium; nc, nasal cavity; ns, nasal septum; nse, nasal side epithelium; ose, oral side epithelium; ps, palate shelf; t, tongue. Bars, 50 lm. Proportion of BrdU-positive cells in the medial edge (S–V) of fixed area in the palatal shelves of controls and Dex-treated embryos at E13.5 to E15.5. Data are mean ± SD. �p < 0.05. Con, control. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

X. Hu et al. / Food and Chemical Toxicology 56 (2013) 67–74 71

and left and right shelf development is controlle d by different mechanism s, such as unilateral cleft lip or palate, accordin g to clin- ical observations. Therefore, we analyzed morphologi c changes in

anterior and posterior regions with Dex and control treatment. Dex delayed the palatal shelf elevation for approximat ely 24 h, from completely elevated palatal shelves for controls (Fig. 2E and

Fig. 6. Cell survival in palatal shelves with Dex treatment. TUNEL staining (green fluorescence) at E13.5 for controls (A, C, E and G) and anterior (B and D) and posterior (F and H) regions of Dex-treated palatal shelves. White boxes indicate the cluster of TUNEL-labeled cells in oral-side epithelium (A–H). At E14.5, TUNEL-positive cells in the oral-side epithelium of anterior portions of unelevated palatal shelves with Dex treatment (J and L, arrows) and controls (I and K, arrows). At E14.5, posterior regions of controls (M)and Dex-treated samples (N and O). At E15.5, midline epithelial seam of anterior (P, white asterisks) and posterior (R, white asterisks) adhered and fused palate of controls and anterior (Q, white asterisks) and posterior (S, white asterisk) regions of MEE of Dex-treated unapproaching palatal shelves. DAPI stained cell nuclei (blue fluorescence).Con, control; ps, palatal shelf; t, tongue. Bars, 50 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 7. Significantly reduced TUNEL-positive cells in the floor of mouth of Dex- treated embryos. At E13.5, TUNEL staining of apoptotic cells (green fluorescence) in the epithelium of the floor of mouth of anterior (A, white box) and posterior (C,white skew box) regions of developing control embryos and same position of Dex- treated embryos (B, white box and D, white skew box). At E14.5, TUNEL-positive cells in the bend between the cranial base and the palate shelf of both controls (E,white box) and Dex-treated embryos (F, white box). DAPI stained cell nuclei (bluefluorescence). Con, control; f, the floor of mouth; nc, nasal cavity; ps, palatal shelf; t,tongue. Bars, 50 lm. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

72 X. Hu et al. / Food and Chemical Toxicology 56 (2013) 67–74

G) to completely unelevated palate shelves with Dex (Fig. 2F and H) at E14.5 and almost all palatal shelves of Dex-treated embryos complete ly elevated to a horizontal position above the dorsum of tongue at E15.5 (Fig. 2J and L). Interestingl y, the open space of the oral-nasal cavity was larger with control than Dex treatment at E13.5 (Fig. 2C and D, bidirectional arrows). Moreover, in Dex-treated embryos, the open space of the oral-nasal cavity was larger at E14.5 than E13.5 (Fig. 2H and D, bidirectional arrows),so the tongue descended to prepare enough space for palatal shelf elevation. However at E15.5, Dex-treated palatal shelves could not contact and adhere, and failed to fuse (Fig. 2J and L). At E17.5, Dex-treated embryos showed small palatal shelves with a gap be- tween them, which could not form a barrier for independen tbreathing and feeding (Fig. 2N and P). We did not find a difference between anterior and posterior palatal shelves with Dex exposure.

Interestin gly, the left and right palatal shelves seemed to differ in elevation (Fig. 3; column 2 of Fig. S1I–L). Dex exposure perma- nently interrupted one palatal shelf in elevation (Fig. 3). One pala- tal shelf may elevate before the other and is aggravated by Dex exposure. Collectively, pregnant females exposed to Dex showed delayed elevation of palatal shelves and interrupted descent of the tongue to impede the rotation of the shelves; however, these Dex effects were not vital to palate formation. The significant event was failure of fusion after Dex exposure.

4.2. Cellular and molecula r mechanisms of Dex-induced cleft palate

Because Dex induced cleft palate through molecular pathways to control cell fate, we wondered whether Wnt signaling was asso- ciated with Dex-induced cleft palate. We examined the Wnt signal- ing molecule s Wnt3a, Wnt5a, Wnt11, Gsk-3 b, b-catenin, Lef-1, c- Jun and phospho-c-Jun . Dex completely inhibited canonica l Wnt/ b-catenin signaling pathway (Fig. 4). Thus, the Wnt/ b-catenin

Fig. 8. Possible molecular cross-talk after Dex exposure in developing mouse palate. Scheme depicts the interaction between canonical Wnt/ b-catenin signaling and possible downstream signaling to control cell fate in craniofacial tissues during E13.5–E15.5. Arrows represent inductive relationships. Arrows following molecules or cell fate indicate up- or downregulation and numbers of arrows represent the degree of regulation. Dash lines arrows represents possible downstream molecule. Broken lines represent cell membrane and solid lines indicate nuclear membrane.

X. Hu et al. / Food and Chemical Toxicology 56 (2013) 67–74 73

signaling pathway may be the main molecular target with Dex treatment of embryos and the fate of the craniofaci al epithelium and mesenchyme mainly regulated by Wnt/ b-catenin signaling.

To study the role of Wnt signaling in cell fate during the devel- opment of the palate, we investiga ted cell proliferation and apop- tosis of craniofacial tissue from E13.5 to E15.5 with Dex exposure. Cell proliferation appeared in all craniofacial tissue, including the epithelium and mesenchym e, and was increased in the early em- bryo developmen t but gradually reduced from E13.5 to E15.5. However, cell apoptosis appeared only in a specific location and time and was required for craniofacial developmen t. Thus, cell pro- liferation and apoptosis of the epithelium and mesenchym e have adistinct effect on the developing craniofacial tissue. Collectively, the proliferation and apoptosis of the epithelium coordinated the fusion and the movement direction of organs (more proliferation for lack of fusion and to change direction, and vice versa), whereas the prolifera tion and apoptosis of the mesenchym e influenced the size and movement capability of organs (more prolifera tion with large size and strong movement capability, and vice versa). At E13.5, the reduced epithelium apoptosis in the floor of Dex-treated embryo mouths mainly contributed to the failure of the tongue to descend for impeding palate elevation (Fig. 7A–D). At E14.5, Dex- treated palatal shelves were elevated because of the normal cell apoptosis mechanism in the oral-side epithelium (Fig. 6J and L)and in the epithelium of the floor of mouth (data not shown). With Dex exposure, the reduced MEE proliferation at E13.5, increased proliferation at E14.5 and E15.5, and reduced MEE apoptosis deter- mined that palatal shelves were not capable of fusing (Figs. 5A–Rand 6P–S).

Epithelial proliferation and apoptosis are regulated by signaling molecules induced by Dex exposure in developing palates resulting in cleft palate. Several studies support a critical role for Tgf- b3 in normal palate fusion involving the MEE (Fitzpatrick et al., 1990; Pelton et al., 1990 ). Tgf- b3 may induce MEE apoptosis (Martinez-Alvarez et al., 2000 ). Interestingl y, Wnt expression often coincides with the expression of molecules of the hedgeho g and Tgf- b fami-lies (Paiva et al., 2010 ). Wnt/ b-catenin signaling plays an essential role in the epithelial component during palate fusion by controlling the expression of Tgf- b3 in the MEE (He et al., 2011 ). Thus, Tgf- b3may be a downstream molecule regulated by Wnt/ b-catenin sig- naling in MEE of E14.5 and E15.5 embryos. Tgf- b3 is downregu- lated by reduced expression of Wnt/ b-catenin signaling with exposure to Dex at E14.5 and E15.5, thus increasing MEE prolifer- ation and reducing apoptosis (Fig. 8).

In summary, the adhesion and fusion of palatal shelves in mam- mals are essential mechanis ms during the developmen t of the sec- ondary palate; failure of these processes leads to cleft palate. Our findings suggest that the mechanisms of the proliferation and apoptosis of MEE can be interrupted by Dex exposure during palate developmen t, which results in failure to fuse. Downregulati on of canonica l Wnt/ b-catenin signaling is involved in the Dex-induced cleft palate, and the altered cell fate by signaling molecules directly shaped the morphologic features of the Dex-induced cleft palate.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowled gements

We thank Dr. Zhenguo Chen and Professors Shenqiu Luo, Xiaoc- hun Bai, Hong Shen for thoughtful discussion and advice. This work was supported by National Natural Science Foundation of China (81071589 and 81171834) and by Natural Science Foundation of Guangdo ng Province, China (S2011010003058).

Appendi x A. Supplementar y material

Supplement ary data associate d with this article can be found, in the online version, at http://dx.doi.o rg/10.1016/ j.fct.2013.02.00 3.

References

Abbott, B.D., 1995. Review of the interaction between TCDD and glucocorticoids in embryonic palate. Toxicology 105, 365–373.

Abbott, B.D., 2010. The etiology of cleft palate: a 50-year search for mechanistic and molecular understanding. Birth Defects Res. B Dev. Reprod. Toxicol. 89, 266–274.

Almeida, M., Han, L., Ambrogini, E., Weinstein, R.S., Manolagas, S.C., 2011. Glucocorticoids and tumor necrosis factor a increase oxidative stress and suppress Wnt protein signaling in osteoblasts. J. Biol. Chem. 286, 44326–44335.

Bush, J.O., Jiang, R., 2012. Palatogenesis: morphogenetic and molecular mechanisms of secondary palate development. Development 139, 231–243.

Carroll, T.J., Park, J.S., Hayashi, S., Majumdar, A., McMahon, A.P., 2005. Wnt9b plays acentral role in the regulation of mesenchymal to epithelial transitions underlying organogenesis of the mammalian urogenital system. Dev. Cell 9, 283–292.

Chiquet, B.T., Blanton, S.H., Burt, A., Ma, D., Stal, S., Mulliken, J.B., Hecht, J.T., 2008. Variation in WNT genes is associated with non-syndromic cleft lip with or without cleft palate. Hum. Mol. Genet. 17, 2212–2218.

Fitzpatrick, D.R., Denhez, F., Kondaiah, P., Akhurst, R.J., 1990. Differential expression of TGF beta isoforms in murine palatogenesis. Development 109, 585–595.

74 X. Hu et al. / Food and Chemical Toxicology 56 (2013) 67–74

Fraser, F.C., Kalter, H., Walker, B.E., Fainstat, T.D., 1954. The experimental production of cleft palate with cortisone and other hormones. J. Cell. Physiol. Suppl. 43, 237–259.

Greene, R.M., Kochhar, D.M., 1973. Palatal closure in the mouse as demonstrated in frozen sections. Am. J. Anat. 137, 477–482.

Greenstein, S., Ghias, K., Krett, N.L., Rosen, S.T., 2002. Mechanisms of glucocorticoid- mediated apoptosis in hematological malignancies. Clin. Cancer Res. 8, 1681–1694.

Gritli-Linde, A., 2008. The etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models. Curr. Top. Dev. Biol. 84, 37–138.

Hamidouche, Z., Hay, E., Vaudin, P., Charbord, P., Schule, R., Marie, P.J., Fromigue, O., 2008. FHL2 mediates dexamethasone-induced mesenchymal cell differentiation into osteoblasts by activating Wnt/ b-catenin signaling-dependent Runx2 expression. FASEB J. 22, 3813–3822.

Hayashi, K., Yamaguchi, T., Yano, S., Kanazawa, I., Yamauchi, M., Yamamoto, M., Sugimoto, T., 2009. BMP/Wnt antagonists are upregulated by dexamethasone in osteoblasts and reversed by alendronate and PTH: potential therapeutic targets for glucocorticoid-induced osteoporosis. Biochem. Biophys. Res. Commun. 379, 261–266.

He, F., Xiong, W., Wang, Y., Li, L., Liu, C., Yamagami, T., Taketo, M.M., Zhou, C., Chen, Y., 2011. Epithelial Wnt/ b-catenin signaling regulates palatal shelf fusion through regulation of Tgf b3 expression. Dev. Biol. 350, 511–519.

Hilliard, S.A., Yu, L., Gu, S., Zhang, Z., Chen, Y.P., 2005. Regional regulation of palatal growth and patterning along the anterior-posterior axis in mice. J. Anat. 207, 655–667.

Juriloff, D.M., Harris, M.J., Brown, C.J., 2001. Unravelling the complex genetics of cleft lip in the mouse model. Mamm. Genome 12, 426–435.

Juriloff, D.M., Harris, M.J., McMahon, A.P., Carroll, T.J., Lidral, A.C., 2006. Wnt9b is the mutated gene involved in multifactorial nonsyndromic cleft lip with or without

cleft palate in A/WySn mice, as confirmed by a genetic complementation test. Birth Defects Res. A Clin. Mol. Teratol. 76, 574–579.

Kitase, Y., Barragan, L., Qing, H., Kondoh, S., Jiang, J.X., Johnson, M.L., Bonewald, L.F., 2010. Mechanical induction of PGE 2 in osteocytes blocks glucocorticoid- induced apoptosis through both the b-catenin and PKA pathways. J. Bone Miner. Res. 25, 2657–2668.

Martinez-Alvarez, C., Tudela, C., Perez-Miguelsanz, J., O’Kane, S., Puerta, J., Ferguson, M.W., 2000. Medial edge epithelial cell fate during palatal fusion. Dev. Biol. 220, 343–357.

Mikels, A.J., Nusse, R., 2006. Wnts as ligands: processing, secretion and reception. Oncogene 25, 7461–7468.

Niemann, S., Zhao, C., Pascu, F., Stahl, U., Aulepp, U., Niswander, L., Weber, J.L., Muller, U., 2004. Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family. Am. J. Hum. Genet. 74, 558–563.

Paiva, K.B., Silva-Valenzuela, M.G., Massironi, S.M., Ko, G.M., Siqueira, F.M., Nunes, F.D., 2010. Differential Shh, Bmp and Wnt gene expressions during craniofacial development in mice. Acta Histochem. 112, 508–517.

Pelton, R.W., Dickinson, M.E., Moses, H.L., Hogan, B.L., 1990. In situ hybridization analysis of TGF beta 3 RNA expression during mouse development: comparative studies with TGF beta 1 and beta 2. Development 110, 609–620.

Pinsky, L., Digeorge, A.M., 1965. Cleft palate in the mouse: a teratogenic index of gluocorticoid potency. Science 147, 402–403.

Rhen, T., Cidlowski, J.A., 2005. Antiinflammatory action of glucocorticoids-new mechanisms for old drugs. New Engl. J. Med. 353, 1711–1723.

Tamarin, A., Boyde, A., 1977. Facial and visceral arch development in the mouse embryo: a study by scanning electron microscopy. J. Anat. 124, 563–580.

Veeman, M.T., Axelrod, J.D., Moon, R.T., 2003. A second canon. Functions and mechanisms of beta-catenin-independent Wnt signaling. Dev. Cell 5, 367–377.