A preliminary study on the teratogenesis of dexamethasone and the preventive effect of vitamin B₁₂ on murine embryonic palatal shelf fusion in vitro

A preliminary study on the teratogenesis of dexamethasone and the preventive effect of vitamin B 12 on murine embryonic palatal shelf fusion in vitro 1 2 first-author 1 2 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:shibingcn@sina.comtianmeng_scu@yahoo.com.cn †E-mail:shibingcn@sina.comtianmeng_scu@yahoo.com.cn shibingcn@sina.com 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:shibingcn@sina.comtianmeng_scu@yahoo.com.cn †E-mail:shibingcn@sina.comtianmeng_scu@yahoo.com.cn shibingcn@sina.com 1 1 State Key Laboratory of Oral Disease, West China College of Stomatology, Sichuan University, Chengdu 610041, China State Key Laboratory of Oral Disease, West China College of Stomatology, Sichuan University, Chengdu 610041, China Department of Cleft Lip and Palate Surgery, West China Stomatological Hospital, Sichuan University, Chengdu 610041, China Department of Cleft Lip and Palate Surgery, West China Stomatological Hospital, Sichuan University, Chengdu 610041, China Excessive dexamethasone (Dex) administrated into pregnant mice during critical periods of palatal development can produce a high incidence of cleft palate. Its mechanisms remain unknown. Vitamin B 12 has been shown to antagonize the teratogenic effects of Dex, which, however, remains controversial. In this study, we investigated the effects of Dex and vitamin B 12 on murine embryonic palatal shelf fusion using organ culture of murine embryonic shelves. The explanted palatal shelves on embryonic day 14 (E14) were cultured for 24, 48, 72 or 96 h in different concentrations of Dex and/or vitamin B 12 . The palatal shelves were examined histologically for the morphological alterations on the medial edge epithelium (MEE) and fusion rates among different groups. It was found that the palatal shelves were not fused at 72 h or less of culture in Dex group, while they were completely fused in the control and vitamin B 12 -treated groups at 72 and 96 h, respectively. The MEE still existed and proliferated. In Dex+vitamin B 12 group the palatal shelves were fused at each time point in a similar rate to controls. These results may suggest that Dex causes teratogenesis of murine embryonic palatal shelves and vitamin B 12 prevents the teratogenic effect of Dex on palatogenesis on murine embryos in vitro. Dexamethasone Vitamin B 12 Organ culture Cleft palate Medial edge epithelial cells INTRODUCTION 1 Cleft lip and palate is one of the most frequent congenital malformations in humans. Its etiology is complex and multifactorial. Both genetic and environmental factors are involved and regulated spatially and temporally by complicated molecular mechanisms (Gritli-Linde, 2007 2007 ; Rice, 2005 2005 ). The formation of the mammalian secondary palate requires several critical steps, including growth, elevation, contact, medial edge epithelium (MEE) disappearance, and, finally, bilateral palatal shelves fusion. Interruption of any of these steps may cause cleft palate. B-vitamins are a group of water-soluble vitamins, including B 6 , B 12 , folic acid, etc. Deficiency of vitamins is associated with many illness and birth defects (Botto et al., 2004 2004 ; Herrmann et al., 2007 2007 ; Krapels et al., 2004 2004 ). Epidemiological studies of cleft lip and palate showed that a pregnant female taking certain doses of vitamin B 12 may decrease the incidence of cleft and palate in her offspring (van Rooij et al., 2003 2003 ). However, others also reported that children with congenital malformations did not show any serum vitamin deficiencies compared with a normal population (Stoll et al., 1999 1999 ). It was reported that palatal development in vitro was similar to in vivo (Koch and Smiley, 1981 1981 ; Shimizu et al., 2001 2001 ). In this study we have investigated the teratogenic effect of dexamethasone (Dex) on the palate and employed vitamin B 12 to antagonize the teratogenic effects of Dex on palate fusion in mouse embryos in vitro, using the organ culture method (Hassell, 1975 1975 ). MATERIALS AND METHODS 1 Animals 2 Adult C57BL/6J mice, weighting 15~20 g, one male and two females per cage, were mated overnight in a temperature-controlled (22 °C) SPF (specified-pathogens free) room, water and food ad libitum. Vaginal plugs appeared in the following morning, which was designated as the embryonic day 0 (E0). Dissection and organ culture 2 On E14, mouse embryos were quickly immersed in GMF-PBS (Ca 2+ and Mg 2+ free-phosphate buffed solution, pH 7.2) and flushed lightly several times. Then the palatal shelves were removed using microsurgical shears and forceps under a dissecting microscope. Isolated palatal shelves were cultured according to the previous methods (Hassell, 1975 1975 ). Briefly, the palatal shelves were placed in pairs on 0.4 μm porosity millipore filters (Millipore Corporation, USA), nasal epithelium down, media edges in contact, on 35 mm-tissue culture dishes (FALCON, France). The culture medium was composed of DMEM/F12 (Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham’s) (Hyclone Corporation, USA), 10% fetal bovine serum (FBS, Hyclone Corporation), and 100 U/ml Penicillin-Streptomycin, with or without 20 ng/ml Dex (Sigma, USA) and/or 10 ng/ml vitamin B 12 (Sigma, USA). Transplants were divided into the control, the Dex-treated, the vitamin B 12 -treated, and the Dex+vitamin B 12 -treated groups, with 8 samples in each group. Samples were cultured for 24, 48, 72 or 96 h at 37 °C in an incubator with 5% CO 2 (Leica, Germany). All the medium and treatments were replaced every 24 h. Histology 2 At the end of incubation, transplants were immediately immersed in 4% neutral paraformaldehyde and embedded in paraffin. Then, 5 μm-thick serial sections were cut and stained with eosin and haematoxylin. These sections were then photographed using a Leica photographic system (Germany). Scanning electron microscopy (SEM) preparation 2 Transplants were immersed in 2.5% glutaraldehyde solution for 2 h at room temperature, and then followed were ethanol gradient dehydration, isoamylacetate replacement, critical point drying, and gold plating. At last, the samples were scanned to observe surface changes of the palatal shelves. Statistical analysis 2 Statistical analysis was performed with the chi-square test and variance analysis (SPSS 13.0). Different groups in the same designated time and in the same group across different incubation times were compared. Values of P < 0.05 were considered significant. RESULTS 1 The numbers of palatal shelf fusion in various experimental groups are presented in Table 1 1 . The palatal shelves were not fused at the 72 h or less of culture in Dex group, compared with controls ( P < 0.0025), indicating the teratogenesis of Dex. In Dex+vitamin B 12 group the palatal shelves were fused at each time point in a similar rate to the control and vitamin B 12 groups, significantly different from the Dex group ( P < 0.05). These results indicate the antagonistic effect of vitamin B 12 against Dex. 2 Group 4 Number of palatal shelf fusion 24 h 48 h 72 h 96 h Control 3 7 8 8 Dex 0 1 2 2 Vitamin B 12 4 6 6 8 Dex+vitamin B 12 5 6 7 7 Light microscope 2 Transplants were classified in three groups according to Vargas ( 1967 1967 ) method: no fusion, partial fusion, and complete fusion. Complete fusion or mesenchymal coalescence means no MEE is observed, and the epithelial laminae have been broken down and resorbed. All of the palates in the control group completely fused after 48 h (Fig. 1b 1b ), and no MEE was observed. All of the palates in the vitamin B 12 -treated group also completely fused at 96 h. There was no significant difference between control and vitamin B 12 -treated groups ( P > 0.5) after 48, 72 and 96 h, which suggests that vitamin B 12 had no effect on normal palatal fusion (Figs. 1e and 1f 1e and 1f ). In the Dex-treated group at 24 h no fusion was observed (Fig. 1c 1c ) and the MEE existed; at 48 h only 1 out of 8 (Fig. 1d 1d ) and at 72 h 2 out of 8 fused (Fig. 1g 1g ), and MEE did not disappear but thickened. In Dex+vitamin B 12 group at 24 (Fig. 1g 1g ), 48 (Fig. 1h 1h ), 72 and 96 h, 5, 6, 7 and 7 out of 8 samples fused, respectively, indicating the antagonism of vitamin B 12 against Dex. Black arrows show the MEE; Scale bars=50 µm Scanning electron microscopy 2 Figs. 2a and 2b 2a and 2b showed situation of the palatal shelves in the control group, and incubated after 24 h, bilateral palatal shelves have achieved completely fusion. The spheroidal structures in MES (medial epithelial seam) seemed likely deciduous perithelial cells from MEE and migrated to surface. The observations of palatal shelves of Dex-treated group showed in Figs. 2c and 2d 2c and 2d . Opposing palatal shelves seemed fused incompletely and retained seam between bilateral palates. The spheroidal structures in MES and palatal surface were less than the control group, and the spheroidal structures trapped, which suggested perithelial cells that underwent slough and migration were less than the control group. The perithelial cells did not slough and migrate, which resulted in basal cell of MEE and could not contact and induced cleft palate. Some fibroblast-like cells were observed on the surface of palatal shelves after vitamin B 12 treatment (Figs. 2e and 2f 2e and 2f ), but fusion of shelves was normal (Fig. 2g 2g ). Possibly, function of vitamin B 12 involved in cell morphous was needed further study. In vitamin B 12 +Dex-treated group, surface morphous of palatal shelves was similar to that of the control group, and bilateral shelves fused but retained some feature like vitamin B 12 -treated group (Figs. 2h, 2i 2h, 2i and 2j 2j ). (a), (b) Scale bars=100 µm; (c), (d), (e), (i), (j) Scale bars=50 µm; (g), (h) Scale bars=30 µm; (f) Scale bars=5 µm DISCUSSION 1 Bilateral palatal fusion includes two phases: the first phase in which palates move from a vertical position to a horizontal position, and the second phase in which the two palates contact and fuse to form the secondary palate. The potential of the palatal shelves to fuse in vitro depends on the age of embryo, which varies among species. Pourtois ( 1966 1966 ) found that in the rat the potential to fuse was acquired 24 h before the actual time of the secondary palatal fusion, whereas Vargas ( 1967 1967 ) found that in the mouse it was at least 40 h before the actual time of fusion in vivo. Teratogens that disturb any of these developmental events may induce a failure of the palates to fuse. Studies both in vivo and in vitro demonstrated that environmental toxicant 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), a cleft palate teratogen, not only disrupts the differentiation and proliferation of the bilateral MEE in mice, but also has effects on the expression of some growth factors (Abbott and Birnbaum, 1990a 1990a ; 1990b 1990b ; Abbott et al., 1989a 1989a ; 1992 1992 ; 2003 2003 ; 2005b 2005b ; Bock and Köhle, 2006 2006 ; Hassoun and Dencker, 1982 1982 ; Miettinen et al., 2004 2004 ; Ryan et al., 1989 1989 ). Other teratogens also showed effects on the fusion of the palatal shelves, such as retinoic acid (RA), which induces cleft palate when administered in excess to the maternal mouse, depending on the stage of embryonic development exposed (Abbott et al., 1989b 1989b ; Newall and Edwards, 1981 1981 ). Both in vivo and in vitro studies showed that RA changed the regulation of cell proliferation, which was often associated with the altered expression of epidermal growth factor receptor (EGFR) (Abbott and Birnbaum, 1990c 1990c ; Abbott et al., 2005a 2005a ) and transforming growth factor beta (TGF-β) (Degitz et al., 1998 1998 ; Nugent et al., 1998 1998 ). Dex, one of the glucocorticoids (GC) that are well-known teratogens, is a fat-soluble hormone that penetrates the cell membrane and binds to GC receptor (GR) in the cytoplasm, inhibiting palatal mesenchymal proliferation, and resulting in smaller palates that fail to contact and fuse (Greene and Kochhar, 1975 1975 ; Hackney, 1980 1980 ; Pratt et al., 1984 1984 ; Shah et al., 1989 1989 ). Sensitivity to Dex varies by species and stages of pregnancy, and the species differences in susceptibility are related to H-2 histocompatility (Kusanagi, 1984 1984 ; Montenegro and Palomino, 1989 1989 ). A population-based case-control study of cleft lip and palate showed that maternal corticosteroid use during pregnancy is associated with moderately increased risk of delivering an infant with an orofacial cleft (Carmichael et al., 2007 2007 ). Various in vitro methods have been employed in the investigation on the effects of Dex on the palate, including mesenchymal culture, or palatal epithelial and mesenchymal co-incubation, and organ culture. The methods vary in their ability to stimulate palate development and fusion, and the organ culture has significant advantages in this regard (Chou et al., 2004 2004 ). In the current study, we observed that Dex appeared to stimulate the thickening of the MEE. When exposed to 20 ng/ml Dex for 24 h, the palatal shelves from E14 pregnant mice retained the MEE, and even after 96 h the MEE still existed. SEM images show that the spheroidal structures did not disappear and seemed trapped in the medial edge seam. Compared with the control group, few filopodia were observed. Vitamin B 12 is one of the most important coenzymes and participates in many biochemical reactions in vivo (Selhub, 2002 2002 ). Population-based epidemiological investigations showed that pregnant women taking certain doses of vitamin B 12 could reduce the risk of non-syndromic craniofacial clefts (van Rooij et al., 2003 2003 ; Vujkovic et al., 2007 2007 ). It was demonstrated that the vitamin B 12 concentration of 185 pmol/L or less and the pyridoxal-5′-phosphate (PLP) concentration of 44 nmol/L or less in mothers increased the risk of having a child with cleft lip with or without cleft palate (van Rooij et al., 2003 2003 ). Animal experiments showed that when vitamin B 12 level decreased, the level of amniotic homocysteine, a risk factor for the malformation of the palate, increased (Weingärtner et al., 2007 2007 ). Experiments on mice from E10~E13 demonstrated that vitamin B 12 could antagonize the Dex-induced cleft palate (Natsume et al., 1986 1986 ). However, the main mechanism of how vitamin B 12 antagonized the Dex-induced craniofacial clefts is still unknown. In the current studies, in the vitamin B 12 -treated group 10 ng/ml vitamin B 12 was added in the medium, and the palate fuse rate and SEM images were similar to the control, suggesting that vitamin B 12 had no effect on palatal MEE. With exposure to 20 ng/ml Dex+10 ng/ml vitamin B 12 , at 48 h incubation, the fuse rate significantly increased to near the one in controls and SEM images became similar to the control as well. These results demonstrate that vitamin B 12 could antagonize the Dex-induced failure of the palatal fusion in mouse embryos. CONCLUSION 1 In summary, our results may suggest that Dex causes teratogenesis of murine embryonic palatal shelves and vitamin B 12 prevents the teratogenic effect of Dex on palatogenesis on murine embryos in vitro. We, therefore, propose that a proper concentration of vitamin B 12 may effectively antagonize the effects of Dex that leads to MEE thickening, which is possibly a consequence of excessive MEE proliferation or reduced cell death. Investigations into the molecular mechanisms of how Dex interacted with vitamin B 12 were not examined and further studies are needed to clarify the preventive effects of vitamin B 12 on cleft lip and palate. References BD Abbott , LS Birnbaum . TCDD-induced altered expression of growth factors may have a role in producing cleft palate and enhancing the incidence of clefts after coadministration of retinoic acid and TCDD . Toxicol Appl Pharmacol , 1990 . 106 ( 3 ): 418 - 432 . 2260090 . TCDD-induced altered expression of growth factors may have a role in producing cleft palate and enhancing the incidence of clefts after coadministration of retinoic acid and TCDD BD Abbott , LS Birnbaum . Rat embryonic palatal shelves respond to TCDD in organ culture . Toxicol Appl Pharmacol , 1990 . 103 ( 3 ): 441 - 451 . 2339417 DOI: 10.1016/0041-008X(90)90317-N http://doi.org/10.1016/0041-008X(90)90317-N . Rat embryonic palatal shelves respond to TCDD in organ culture BD Abbott , LS Birnbaum . Retinoic acid-induced alterations in the expression of growth factors in embryonic mouse palatal shelves . Teratology , 1990 . 42 ( 6 ): 597 - 610 . 2087681 DOI: 10.1002/tera.1420420604 http://doi.org/10.1002/tera.1420420604 . Retinoic acid-induced alterations in the expression of growth factors in embryonic mouse palatal shelves BD Abbott , JJ Diliberto , LS Birnbaum . 2,3,7,8-tetrachlorodibenzo- p -dioxin alters embryonic palatal medial epithelial cell differentiation in vitro . Toxicol Appl Pharmacol , 1989 . 100 ( 1 ): 119 - 131 . 2763295 DOI: 10.1016/0041-008X(89)90096-3 http://doi.org/10.1016/0041-008X(89)90096-3 . 2,3,7,8-tetrachlorodibenzo-p-dioxin alters embryonic palatal medial epithelial cell differentiation in vitro BD Abbott , MW Harris , LS Birnbaum . Etiology of retinoic acid-induced cleft palate varies with the embryonic stage . Teratology , 1989 . 40 ( 6 ): 533 - 553 . 2623642 DOI: 10.1002/tera.1420400602 http://doi.org/10.1002/tera.1420400602 . Etiology of retinoic acid-induced cleft palate varies with the embryonic stage BD Abbott , JJ Diliberto , LS Birnbaum . Mechanisms of TCDD-induction of cleft palate: insights from in vivo and in vitro approaches . Chemosphere , 1992 . 25 ( 1-2 ): 75 - 78 . DOI: 10.1016/0045-6535(92)90483-8 http://doi.org/10.1016/0045-6535(92)90483-8 . Mechanisms of TCDD-induction of cleft palate: insights from in vivo and in vitro approaches BD Abbott , AR Buckalew , MJ DeVito , RP David , L Bryant , JE Schmid . EGF and TGF-α expression influence the developmental toxicity of TCDD: dose response and AhR phenotype in EGF, TGF-α, and EGF+TGF-α knockout mice . Toxicol Sci , 2003 . 71 ( 1 ): 84 - 95 . 12520078 DOI: 10.1093/toxsci/71.1.84 http://doi.org/10.1093/toxsci/71.1.84 . EGF and TGF-α expression influence the developmental toxicity of TCDD: dose response and AhR phenotype in EGF, TGF-α, and EGF+TGF-α knockout mice BD Abbott , DS Best , MG Narotsky . Teratogenic effects of retinoic acid are modulated in mice lacking expression of epidermal growth factor and transforming growth factor-alpha . Birth Defects Research Part A Clinical and Molecular Teratology , 2005 . 73 ( 4 ): 204 - 217 . DOI: 10.1002/bdra.20117 http://doi.org/10.1002/bdra.20117 . Teratogenic effects of retinoic acid are modulated in mice lacking expression of epidermal growth factor and transforming growth factor-alpha BD Abbott , AR Buckalew , KE Leffler . Effects of epidermal growth factor (EGF), transforming growth factor (TGF), and 2,3,7,8-tetrachlorodibenzo- p -dioxin on fusion of embryonic palates in serum-free organ culture using wild-type, EGF knockout, and TGF-α knockout mouse strains . Birth Defects Research Part A Clinical and Molecular Teratology , 2005 . 73 ( 6 ): 447 - 454 . DOI: 10.1002/bdra.20133 http://doi.org/10.1002/bdra.20133 . Effects of epidermal growth factor (EGF), transforming growth factor (TGF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fusion of embryonic palates in serum-free organ culture using wild-type, EGF knockout, and TGF-α knockout mouse strains KW Bock , C Köhle . Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions . Biochem Pharmacol , 2006 . 72 ( 4 ): 393 - 404 . 16545780 DOI: 10.1016/j.bcp.2006.01.017 http://doi.org/10.1016/j.bcp.2006.01.017 . Ah receptor: dioxin-mediated toxic responses as hints to deregulated physiologic functions LD Botto , SJ Richard , D Erickson . Vitamin supplements and the risk for congenital anomalies other than neural tube defects . Am J Med Genet Part C: Semin Med Genet , 2004 . 125C ( 1 ): 12 - 21 . DOI: 10.1002/ajmg.c.30004 http://doi.org/10.1002/ajmg.c.30004 . Vitamin supplements and the risk for congenital anomalies other than neural tube defects SL Carmichael , GM Shaw , C Ma , MM Werler , SA Rasmussen , EJ Lammer . Maternal corticosteroid use and orofacial clefts . Am J Obstet Gynecol , 2007 . 197 ( 6 ): 585.e1 - 585.e7 . DOI: 10.1016/j.ajog.2007.05.046 http://doi.org/10.1016/j.ajog.2007.05.046 . Maternal corticosteroid use and orofacial clefts MJ Chou , T Kosazuma , T Takigawa , S Yamada , S Takahara , K Shiota . Palatal shelf movement during palatogenesis: a fate map of the fetal mouse palate cultured in vitro . Anat Embryol , 2004 . 208 ( 1 ): 19 - 25 . 14986130 DOI: 10.1007/s00429-004-0379-0 http://doi.org/10.1007/s00429-004-0379-0 . Palatal shelf movement during palatogenesis: a fate map of the fetal mouse palate cultured in vitro SJ Degitz , D Morris , L George , B Foley , M Francis . Role of TGF-β in RA-induced cleft palate in CD-1 mice . Teratology , 1998 . 58 ( 5 ): 197 - 204 . 9839358 DOI: 10.1002/(SICI)1096-9926(199811)58:5<197::AID-TERA6>3.0.CO;2-8 http://doi.org/10.1002/(SICI)1096-9926(199811)58:5<197::AID-TERA6>3.0.CO;2-8 . Role of TGF-β in RA-induced cleft palate in CD-1 mice RM Greene , DM Kochhar . Some aspects of corticosteroid-induced cleft palate: a review . Teratology , 1975 . 11 ( 1 ): 47 - 55 . 166456 DOI: 10.1002/tera.1420110106 http://doi.org/10.1002/tera.1420110106 . Some aspects of corticosteroid-induced cleft palate: a review A Gritli-Linde . Molecular control of secondary palate development . Dev Biol , 2007 . 301 ( 2 ): 309 - 326 . 16942766 DOI: 10.1016/j.ydbio.2006.07.042 http://doi.org/10.1016/j.ydbio.2006.07.042 . Molecular control of secondary palate development JF Hackney . A glucocorticoid receptor in fetal mouse: its relationship to cleft palate formation . Teratology , 1980 . 21 ( 1 ): 39 - 51 . 7385055 DOI: 10.1002/tera.1420210106 http://doi.org/10.1002/tera.1420210106 . A glucocorticoid receptor in fetal mouse: its relationship to cleft palate formation JR Hassell . The development of rat palatal shelves in vitro: An ultrastructural analysis of the inhibition of epithelial cell death and palate fusion by the epidermal growth factor . Dev Biol , 1975 . 45 ( 1 ): 90 - 102 . 1235782 DOI: 10.1016/0012-1606(75)90244-4 http://doi.org/10.1016/0012-1606(75)90244-4 . The development of rat palatal shelves in vitro: An ultrastructural analysis of the inhibition of epithelial cell death and palate fusion by the epidermal growth factor EM Hassoun , L Dencker . TCDD embryo toxicity in the mouse may be enhanced by β-naphthoflavone, another ligand of the Ah-receptor . Toxicol Lett , 1982 . 12 ( 2-3 ): 191 - 198 . 6287677 DOI: 10.1016/0378-4274(82)90185-0 http://doi.org/10.1016/0378-4274(82)90185-0 . TCDD embryo toxicity in the mouse may be enhanced by β-naphthoflavone, another ligand of the Ah-receptor M Herrmann , J Schmidt , N Umanskaya , G Colaianni , F Al Marrawi , T Widmann , A Zallone , B Wildemann , W Herrmann . Stimulation of osteoclast activity by low B-vitamin concentrations . Bone , 2007 . 41 ( 4 ): 584 - 591 . 17681876 DOI: 10.1016/j.bone.2007.06.005 http://doi.org/10.1016/j.bone.2007.06.005 . Stimulation of osteoclast activity by low B-vitamin concentrations WE Koch , GR Smiley . In-vivo and in-vitro studies of the development of the avian secondary palate . Arch Oral Biol , 1981 . 26 ( 3 ): 181 - 187 . 6947725 DOI: 10.1016/0003-9969(81)90128-X http://doi.org/10.1016/0003-9969(81)90128-X . In-vivo and in-vitro studies of the development of the avian secondary palate IPC Krapels , IALM van Rooij , MC Ocké , BAGL van Cleef , AM Kuijpers-Jagtman , RPM Steegers-Theunissen . Maternal dietary B vitamin intake, other than folate, and the association with orofacial cleft in the offspring . Eur J Nutr , 2004 . 43 ( 1 ): 7 - 14 . 14991264 DOI: 10.1007/s00394-004-0433-y http://doi.org/10.1007/s00394-004-0433-y . Maternal dietary B vitamin intake, other than folate, and the association with orofacial cleft in the offspring T Kusanagi . Sensitive stages and dose-response analyses of palatal slit and cleft palate in C57BL/6 mice treated with a glucocorticoid . Teratology , 1984 . 29 ( 2 ): 281 - 286 . 6740512 DOI: 10.1002/tera.1420290214 http://doi.org/10.1002/tera.1420290214 . Sensitive stages and dose-response analyses of palatal slit and cleft palate in C57BL/6 mice treated with a glucocorticoid HM Miettinen , H Huuskonen , AM Partanen , P Miettinen , JT Tuomisto , R Pohjanvirta , J Tuomisto . Effects of epidermal growth factor receptor deficiency and 2,3,7,8-tetrachlorodibenzo- p -dioxin on fetal development in mice . Toxicol Lett , 2004 . 150 ( 3 ): 285 - 291 . 15110080 DOI: 10.1016/j.toxlet.2004.02.009 http://doi.org/10.1016/j.toxlet.2004.02.009 . Effects of epidermal growth factor receptor deficiency and 2,3,7,8-tetrachlorodibenzo-p-dioxin on fetal development in mice MA Montenegro , H Palomino . Inhibition of palatal fusion in vitro by indomethacin in two strains of mice with different H-2 backgrounds . Arch Oral Biol , 1989 . 34 ( 12 ): 949 - 955 . 2610630 DOI: 10.1016/0003-9969(89)90051-4 http://doi.org/10.1016/0003-9969(89)90051-4 . Inhibition of palatal fusion in vitro by indomethacin in two strains of mice with different H-2 backgrounds N Natsume , T Narukawa , T Kawai . Teratogenesis of dexamethasone and preventive effect of vitamin B 12 . Int J Oral Maxillofac Surg , 1986 . 15 ( 6 ): 752 - 755 . 3100678 . Teratogenesis of dexamethasone and preventive effect of vitamin B12 DR Newall , JRG Edwards . The effect of vitamin A on fusion of mouse palates. I. Retinyl palmitate and retinoic acid in vivo . Teratology , 1981 . 23 ( 1 ): 115 - 124 . 7245083 DOI: 10.1002/tera.1420230114 http://doi.org/10.1002/tera.1420230114 . The effect of vitamin A on fusion of mouse palates. I. Retinyl palmitate and retinoic acid in vivo P Nugent , L Ma , RM Greene . Differential expression and biological activity of retinoic acid-induced TGFβ isoforms in embryonic palate mesenchymal cells . J Cell Physiol , 1998 . 177 ( 1 ): 36 - 46 . 9731743 DOI: 10.1002/(SICI)1097-4652(199810)177:1<36::AID-JCP4>3.0.CO;2-F http://doi.org/10.1002/(SICI)1097-4652(199810)177:1<36::AID-JCP4>3.0.CO;2-F . Differential expression and biological activity of retinoic acid-induced TGFβ isoforms in embryonic palate mesenchymal cells M Pourtois . Onset of the acquired potentiality for fusion in the palatal shelves of rats . J Embryol Exp Morphol , 1966 . 16 ( 1 ): 171 - 182 . 5969708 . Onset of the acquired potentiality for fusion in the palatal shelves of rats RM Pratt , EL Perry , LM Chapman , EH Goulding . Glucocorticoid teratogenesis in mouse whole embryo culture . Teratology , 1984 . 30 ( 1 ): 71 - 81 . 6484854 DOI: 10.1002/tera.1420300110 http://doi.org/10.1002/tera.1420300110 . Glucocorticoid teratogenesis in mouse whole embryo culture DP Rice . Craniofacial anomalies: from development to molecular pathogenesis . Curr Mol Med , 2005 . 5 ( 7 ): 699 - 722 . 16305494 DOI: 10.2174/156652405774641043 http://doi.org/10.2174/156652405774641043 . Craniofacial anomalies: from development to molecular pathogenesis RP Ryan , GI Sunahara , GW Lucier , LS Birnbaum , KG Nelson . Decreased ligand binding to the hepatic glucocorticoid and epidermal growth factor receptors after 2,3,4,7,8-pentachlorodibenzofuran and 1,2,3,4,7,8-hexachlorodibenzofuran treatment of pregnant mice . Toxicol Appl Pharmacol , 1989 . 98 ( 3 ): 454 - 464 . 2718174 DOI: 10.1016/0041-008X(89)90174-9 http://doi.org/10.1016/0041-008X(89)90174-9 . Decreased ligand binding to the hepatic glucocorticoid and epidermal growth factor receptors after 2,3,4,7,8-pentachlorodibenzofuran and 1,2,3,4,7,8-hexachlorodibenzofuran treatment of pregnant mice J Selhub . Folate, vitamin B 12 and vitamin B 6 and one carbon metabolism . J Nutr Health Aging , 2002 . 6 ( 1 ): 39 - 42 . 11813080 . Folate, vitamin B12 and vitamin B6 and one carbon metabolism RM Shah , YP Chen , DN Burdett . Growth of the secondary palate in the hamster following hydrocortisone treatment: shelf area, cell number, and DNA synthesis . Teratology , 1989 . 40 ( 2 ): 173 - 180 . 2772852 DOI: 10.1002/tera.1420400211 http://doi.org/10.1002/tera.1420400211 . Growth of the secondary palate in the hamster following hydrocortisone treatment: shelf area, cell number, and DNA synthesis N Shimizu , H Aoyama , N Hatakenaka , M Kaneda , S Teramoto . An in vitro screening system for characterizing the cleft palate-inducing potential of chemicals and underlying mechanisms . Reprod Toxicol , 2001 . 15 ( 6 ): 665 - 672 . 11738519 DOI: 10.1016/S0890-6238(01)00175-7 http://doi.org/10.1016/S0890-6238(01)00175-7 . An in vitro screening system for characterizing the cleft palate-inducing potential of chemicals and underlying mechanisms C Stoll , B Dott , Y Alembik , C Koehl . Maternal trace elements, vitamin B 12 , vitamin A, folic acid, and fetal malformations . Reprod Toxicol , 1999 . 13 ( 1 ): 53 - 57 . 10080300 DOI: 10.1016/S0890-6238(98)00058-6 http://doi.org/10.1016/S0890-6238(98)00058-6 . Maternal trace elements, vitamin B12, vitamin A, folic acid, and fetal malformations IA van Rooij , DW Swinkels , HJ Blom , HM Merkus , RP Steegers-Theunissen . Vitamin and homocysteine status of mothers and infants and the risk of nonsyndromic orofacial clefts . Am J Obstet Gynecol , 2003 . 189 ( 4 ): 1155 - 1160 . 14586370 DOI: 10.1067/S0002-9378(03)00592-1 http://doi.org/10.1067/S0002-9378(03)00592-1 . Vitamin and homocysteine status of mothers and infants and the risk of nonsyndromic orofacial clefts VI Vargas . Palatal fusion in vitro in the mouse . Arch Oral Biol , 1967 . 12 ( 11 ): 1283 - 1288 . 5234234 DOI: 10.1016/0003-9969(67)90130-6 http://doi.org/10.1016/0003-9969(67)90130-6 . Palatal fusion in vitro in the mouse M Vujkovic , MC Ocke , PJ van der Spek , N Yazdanpanah , EA Steegers , RP Steegers-Theunissen . Maternal western dietary patterns and the risk of developing a cleft lip with or without a cleft palate . Obstet Gynecol , 2007 . 110 ( 2 Pt 1 ): 378 - 384 . 17666614 . Maternal western dietary patterns and the risk of developing a cleft lip with or without a cleft palate J Weingärtner , S Maile , P Proff , C Reicheneder , V Bienengräber , J Fanghänel , T Gedrange . Secondary palatal closure in rats in association with relative maternofetal levels of folic acid, vitamin B 12 , and homocysteine . Ann Anat , 2007 . 189 ( 3 ): 229 - 233 . 17534029 . Secondary palatal closure in rats in association with relative maternofetal levels of folic acid, vitamin B12, and homocysteine 10.1631/jzus.B0710625 Project (No. 30530730) supported by the National Natural Science Foundation of China 2007-12-24 2008-02-14 2022-01-05 Copyright © 2008, Journal of Zhejiang University Science 2008 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 09