Environmental Factors (Dexamethasone, Retinoic Acid,2,3,7,8-tetrachlorodibenzo-p-dioxin) In The Etiology Of Cleft Palate

BACKGROUNDCleft palate, the congenital craniofacial birth defects in humans, arises from genetic or environmental perturbations in the multi-step process of palate development, however the detailed mechanism of cleft palate has not been fully elucidated. The frequent occurrence and significant healthcare burden imposed by cleft palate highlight the need to dissect the etiology and molecular pathogenesis of this condition. Because mice and humans share similar craniofacial morphologic features during development, the mouse is a suitable model to study these processes.Glucocorticoids are immunosuppressive drugs, and at physiological levels they are necessary for craniofacial development in early embryos; however, high-dose exogenous glucocorticoids are harmful to fetuses. In1954, Fraser and colleagues successfully induced cleft palate in100%of A/J mice exposed to cortisone and found that exposure delayed shelf elevation, producing smaller palatal shelves that could not contact and fuse. Pinsky and Digeorge used hydrocortisone, prednisolone and dexamethasone (Dex) to produce cleft palate in A/J mice and concluded that Dex was300times more teratogenic than hydrocortisone in the cleft-palate production. Therefore, Dex is a suitable environmental factor for producing cleft palate. Dex suppressed Wnt protein signaling and induced cell apoptosis, thus resulting in osteoporosis, and Dex-induced osteocyte apoptosis could be rescued by Wnt signaling.Retinoic acid (RA), a hormone-like signal derived from vitamin A, plays an essential role during embryonic development by regulating morphogenesis, cell proliferation and differentiation, and extracellular matrix production. Excess exogenous RA can adversely affect craniofacial development in rodents and humans. Cleft palate is one of the major malformations induced by RA in mouse fetuses. RA is highly effective in inducing cleft palate in mice and, and strain-specific sensitivity is not an issue, so RA is an ideal inductive agent to investigate the pathogenesis of cleft palate. Notably in recent years, RA was linked to Wnt signaling in organ development and disease therapy.Abnormal cleft palate may result from fetal exposure to toxic factors or environmental contaminants such as dioxins. Exposure to2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) led to high incidences of cleft palate and hydronephrosis in sensitive strains of mice but had no other teratogenic effects. In recent years, some studies reported that TCDD was associated with Wnt signaling. However, the detailed mechanism of TCDD-induced cleft palate has not been fully elucidated.Although not yet implicated by genome-wide association studies, variants within WNT genes were found associated with cleft lip and palate, and mutations in WNT3 underlie autosomal-recessive tetra-amelia with cleft lip and palate. Mutations in the Wnt3and Wnt9b genes are associated with cleft lip and palate in both humans and mice, so Wnt signaling plays a critical role in craniofacial development. These findings have led to further analyses of genes in the WNT signaling pathway as candidates for normal development of the lip and palate. During early craniofacial development in the mouse embryo, members of the Wnt family are expressed at lip and palate. Wnt ligands activate at least3known pathways:the Wnt/β-catenin or canonical pathway, the non-canonical Wnt/Ca2+pathway involving protein kinase A, and the planar cell polarity pathway. Wnt3A, Wnt5A and Wntll were significantly associated with non-syndromic cleft lip with or without cleft palate. The Wnt molecules act through specific receptors to activate intracellular signals that dictate a variety of cell actions, including proliferation, migration, growth, differentiation, and apoptosis.OBJECTIVESWe hypothesized that Dex, RA and TCDD-induced cleft palate was associated with Wnt signaling by altering palatal cell fate in the crucial time of craniofacial development (E13.5to E15.5). The findings may help in elucidating the mechanisms of environmental factors-induced cleft palate.MATERIALS AND METHODS(一) AnimalsFemales of Kun Ming mice were crossed with fertile males overnight (3females:1male, from Center for Laboratory Animal Sciences of Southern Medical University). The morning when a vaginal plug was observed was defined as E0.5.Pregnant females at E8.5were randomly divided into2groups for treatment:8 mg/kg Dex (MP Biomedicals, OH, USA) or10ml/kg normal sodium subcutaneously, twice a day, from E8.5to E13.5.Pregnant females at E12were randomly divided into2groups for treatment:RA (Sigma, MO, USA) dissolved in sesame oil by gavage at70mg/kg, or10ml/kg sesame oil only as a control.Pregnant females at E12were randomly divided into2groups for treatment: TCDD (Cerilliant, MA, USA) dissolved in sesame oil by gavage at64μg/kg and10ml/kg sesame oil only.Embryos were harvested during palatogenesis (E13.5to E15.5, E17.5).(二) Scanning electron microscopyTrimmed fetuses at E17.5(Dex and the controls, RA and the controls, TCDD and the controls) were fixed with2.5%glutaraldehyde at4℃for12hr. After dehydration with a graded acetone series, specimens were dried with liquid CO2and coated with a thin layer of gold before being viewed under an S-3000N scanning electron microscope.(三) Histochemical stainingEmbryonic heads (Dex and the controls, RA and the controls, TCDD and the controls) were fixed in4%paraformaldehyde and dehydrated through an ethanol series and embedded in paraffin for sectioning by routine procedures. For general morphology, deparaffinized sections (5μm) were stained with hematoxylin and eosin by standard procedures.(四) Western blot analysisThe maxilla tissue extracts (Dex and the controls, RA and the controls, TCDD and the controls) though E13.5to E15.5were examined by standard protocols with antibodies for Wnt3a, Wnt5a, and Wntll (all R&D Systems, MN, USA); Gsk-3β, P-catenin, and Lef-1(all Cell Signaling, MA, USA); c-Jun and phospho-c-Jun (pS63; both Epitomics, Hangzhou, China); and α-Tubulin (Santa Cruz Biotechnology, CA, USA).(五) BrdU analysisPregnant females during E13.5to E15.5were injected with1ml BrdU (5-bromo-2’-deoxyuridine; Invitrogen, CA, USA) per100g body weight intraperitoneally. After2hr, embryos (Dex and the controls, RA and the controls, TCDD and the controls) were removed, trimmed and fixed with4%paraformaldehyde. Antibody detection of BrdU involved standard immunostaining protocols that included an antigen retrieval step of30min in2N Hcl at37℃. Staining involved an Alexa Fluor(?)555conjugate antibody recognizing BrdU.(六) TUNELEmbryos (Dex and the controls, RA and the controls, TCDD and the controls) were removed during E13.5to E15.5, trimmed and fixed with4%paraformaldehyde. Apoptotic cells were assayed by TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling) with the FragELTM DNA Fragmentation Detection Kit, Fluorescein (Calbiochem, Darmstadt, Germany).(七) Statistical analysisData were analyzed by independent sample t test. Data analysis involved use of Windows SPSS v13.0(SPSS, Chicago, IL, USA). Results were considered significant at p<0.05. Each experiment was at least performed in triplicate.RESULTS(一) Dex-induced cleft palateA Embryos with Dex treatment exhibited cleft palateWe used8mg/kg, twice a day, from E8.5to E13.5;82%of embryos showed cleft palate. No control embryos showed cleft palate. With Dex treatment, at E17.5, the apposing palatal shelves of Dex-treated embryos were elevated but could not fuse. At E13.5, the anterior palatal shelves of Dex-treated embryos were similar to those of littermate controls. E14.5embryos showed vertically oriented shelves of Dex-treated embryos. By E15.5, Dex-treated shelves were elevated or even contacted and adhered but were unfused, whereas control shelves contacted, adhered and began to fuse. Shelves of Dex-treated embryos were relatively clumsy and displayed a large gap that eventually manifested as the cleft palate phenotype at E17.5, whereas controls developed a complete secondary palate.B Wnt/β-catenin signaling was inhibited by Dex from E13.5to E15.5Wnt signaling proteins were detected by immunoblotting in developing maxilla tissue of Dex-treated embryos and controls during E13.5to E15.5(Fig.4). Surprisingly, Gsk-3β expression showed a downward shift with Dex. Consistent with altered Gsk-3β expression, the downstream molecules of Wnt/β-catenin signaling, including P-catenin, Lef-1, and c-Jun and phospho-c-Jun, were completely downregulated by Dex treatment.C Epithelium proliferation and survival in Dex-induced cleft palateAt E13.5, the medial edge epithelium proliferation was lower in Dex-treated palatal shelves in both anterior and posterior regions than in controls, but at E14.5, medial edge epithelium proliferation was greater in Dex-treated anterior and posterior regions than in controls. By E15.5, the proliferation of medial edge epithelium was still increased in both anterior and posterior regions as compared with controls.At E13.5, clustered TUNEL-positive 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. However at E14.5, apoptotic cells were still detected in the oral-side epithelium of unelevated anterior palatal shelves of Dex-treated embryos but not elevated anterior palatal shelves of controls. By E15.5, the remnants of midline epithelial seam in controls showed TUNEL-positive staining to degenerate medial edge epithelium for continuity mesenchyme to form the secondary palate, but Dex-treated embryos showed no apoptotic cells in medial edge epithelium or MES. Dex treatment produced no TUNEL-positive cells in the epithelium of the floor of mouth of the anterior and posterior regions as compared with controls. However, at E14.5, extensive apoptotic cells were detected in this position of Dex-treated embryos. 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.(二) RA-induced cleft palateA Embryos exposed to RA showed100%incidence of cleft palateWe chose70mg/kg RA at E12. The100%of fetuses showed cleft palate with litter death and few other malformations. RA-treated E17.5embryos showed a wider, complete secondary cleft palate than did controls because of unelevated palate, which was manifested by our morphology results of RA-treated embryos. Residues of rugae remained and were more apparent in RA-treated than control embryos.At E13.5, RA-exposed embryos showed no grooves between the palatal shelf and the body of maxilla in the anterior palatal shelves, but grooves were observed in controls. All E14.5RA-exposed embryos showed vertically oriented anterior and posterior shelves. Notably, the grooves between the palatal shelf and the body of maxilla were not observed in the anterior palatal shelves in RA-treated embryos as compared with unelevated controls at E14.5. As compared with the open space of the oral-nasal cavity in the posterior portions between control E13.5and E14.5embryos and between RA-exposed E14.5and control E14.5embryos, the space for E14.5controls was larger than others. All control shelves had completely contacted and adhered by E15.5, whereas the anterior and posterior palatal shelves of RA-treated embryos were still unelevated. This condition was observed at E17.5; as compared with the complete secondary palate formation for controls, a large gap eventually manifested as a wider cleft phenotype with RA treatment.B Wnt/β-catenin signaling was inhibited by RA in E13.5and E14.5Wnt signaling pathway proteins were detected in developing maxilla tissue of RA-treated embryos and controls from E13.5to E15.5. In E13.5and E14.5, Gsk-3β expression showed a downward shift in RA-treated embryos as compared with controls. Consistent with altered Gsk-3β level, the downstream molecules of Wnt signaling, included β-catenin, Lef-1, and c-Jun and phospho-c-Jun, were completely downregulated in E13.5and E14.5embryos. However, the protein level of Gsk-3β did not differ from that of controls at E15.5. Consistent with this alteration, the downstream molecules of Wnt signaling, β-catenin and Lef-1, were slightly upregulated with RA at E15.5, with no difference in protein level of c-Jun and phospho-c-Jun between RA samples and controls.C Anterior palatal mesenchyme proliferation was significantly increased in RA-treated embryosDuring E13.5to E15.5, BrdU-positive cells in the mesenchyme of the right and left anterior palatal shelves were higher for RA-treated embryos than controls.D Apoptosis of epithelium and mesenchyme was altered by RA at E13.5and E14.5At E13.5, clustered TUNEL-positive cells were detected in the oral-side epithelium of anterior regions of palatal shelves in controls but not in RA-treated embryos. As well, the amount of TUNEL-positive cells in the mesenchyme of the bend between the palatal shelf and the cranial base was greater for RA-treated embryos than controls; and the amount of TUNEL-labeled cells in the epithelium of the floor of mouth was lower for RA-treated embryos than controls at E13.5. A substantial amount of TUNEL-positive cells was detected in the anterior mesenchyme of the primordial frenulum and posterior mesenchyme of the primordial genioglossus muscle with RA than control treatment. At E14.5, apoptotic cells were evident in the oral-side epithelium of the anterior portions of palatal shelves for controls but not RA-treated embryos. We detected apoptotic cells in the epithelium of the floor of mouth for controls but not RA-treated embryos. However, we detected clustered TUNEL-labeled cells in the mesenchyme of the primordial frenulum of RA-treated embryos but not controls. At E15.5, TUNEL-positive cells were detected in the anterior medial edge epithelium of palatal shelves with RA treatment and control treatment.(三) TCDD-induced cleft palateA Embryos with TCDD treatment exhibited cleft palateWe used64μg/kg TCDD at E12, with89%of embryos exhibiting cleft palate. We observed no cleft palates in controls. The apposing palatal shelves with TCDD had elevated but could not fuse.At E13.5, the anterior and posterior palatal shelves of TCDD-treated embryos were similar to those of controls. All E14.5TCDD-treated embryos showed vertically oriented shelves. At E15.5, all control shelves were elevated and contacted, to adhere and fuse; however, with TCDD treatment, only some of the shelves were elevated and the others were still vertically oriented. As compared with the open space of the oral-nasal cavity in the posterior portions between the E13.5and E14.5controls and between E14.5embryos exposed to TCDD and E14.5controls, that of the E14.5controls was larger than others. As compared with the open space between TCDD-exposed E13.5and E14.5embryos, E14.5embryos were larger and that of E15.5TCDD-treated embryos was larger than that of E14.5embryos. By E17.5, almost all TCDD-treated shelves were elevated and contained a large gap that eventually manifested as the cleft palate, whereas controls developed a complete secondary palate.B Wnt5a and Lef-lwere downregulated by TCDD from E13.5to E15.5Wnt signaling pathway proteins were detected by immunoblotting in developing mouse palatal shelves and tongue extracts of TCDD-treated embryos and controls from E13.5to E15.5. From E13.5to E15.5, the protein level of Gsk-3β, β-catenin and c-Jun did not differ between controls and TCDD-treated embryos; however, the expression of the transcript factor Lef-1was downregulated with TCDD treatment as compared with controls. The protein level of phospho-c-Jun downstream of Lef-1was upregulated by TCDD treatment as compared with controls at E14.5and E15.5. The expression of Wnt3a and Wntll, Wnt ligands, was not markedly different from that of controls, whereas Wnt5a level was downregulated by TCDD treatment from E13.5to E15.5C Cell proliferation and survival in TCDD-treated embryosCell proliferation and apoptosis in the epithelium and mesenchyme of the anterior and posterior portions of palatal shelves, the floor of mouth and tongue did not differ between TCDD-treated embryos and controls from E13.5toE15.5.CONCLUSIONSThe adhesion and fusion of palatal shelves in mammals are essential mechanisms during the development of the secondary palate; failure of these processes leads to cleft palate. Our findings suggest that the mechanisms of the proliferation and apoptosis of medial edge epithelium can be interrupted by Dex exposure during palate development, which results in failure to fuse. Downregulation of canonical Wnt/p-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.Administration of RA at E12of mouse embryos impeded the elevation of palatal shelves, thus leading to cleft palate. Our study has identified a previously unknown molecular mechanism that the downregulation of canonical Wnt/β-catenin signaling is associated with RA-induced cleft palate, and the altered cell fate by signaling molecules directly shaped the morphologic features of RA-induced cleft palate.Our results show that the non-canonical Wnt/Ca2+signaling pathway is inhibited by TCDD exposure in the developing palate, and delayed elevation of palatal shelves mainly contributed to the high incidence of cleft palate.These findings are helpful for highlighting palatogenesis and the teratogenic action of Dex, RA and TCDD.