tretinoin and Cleft-Palate

Palatogenesis involves various developmental events such as growth, elevation, elongation and fusion of opposing palatal shelves. Extrinsic factors such as mouth opening and subsequent tongue withdrawal are also needed for the horizontal elevation of palate shelves. Failure of any of these steps can lead to cleft palate, one of the most common birth defects in humans. It has been shown that retinoic acid (RA) plays important roles during palate development, but excess RA causes cleft palate in fetuses of both rodents and humans. Thus, the coordinated regulation of retinoid metabolism is essential for normal palatogenesis. The endogenous RA level is determined by the balance of RA-synthesizing (retinaldehyde dehydrogenases: RALDHs) and RA-degrading enzymes (CYP26s). Cyp26b1 is a key player in normal palatogenesis. In this review, we discuss recent progress in the study of the pathogenesis of RA-induced cleft palate, with special reference to the regulation of endogenous RA levels by RA-degrading enzymes.

Topics: Animals; Cleft Palate; Cytochrome P-450 Enzyme System; Embryo, Mammalian; Embryonic Development; Humans; Organogenesis; Palate; Retinal Dehydrogenase; Retinoic Acid 4-Hydroxylase; Rodentia; Signal Transduction; Tongue; Tretinoin

Nutritional factors such as vitamin intake contribute to the etiology of cleft palate. Vitamin A is a regulator of embryonic development. Excess vitamin A can cause congenital malformations such as spina bifida and cleft palate. Therefore, preventive nutritional strategies are required. This review identifies putative biological mechanisms underlying the association between maternal vitamin A intake and cleft palate. Excessive vitamin A may disturb all three stages of palatogenesis: 1) during shelf outgrowth, it may decrease cell proliferation and thus prevent tissue development; 2) it may prevent shelf elevation by affecting extracellular matrix composition and hydration; and 3) during shelf fusion, it may affect epithelial differentiation and apoptosis, which precludes the formation of a continuous palate. In general, high doses of vitamin A affect palatogenesis through interference with cell proliferation and growth factors such as transforming growth factor β and platelet-derived growth factor. The effects of lower doses of vitamin A need to be investigated in greater depth in order to improve public health recommendations.

Topics: Animals; Cleft Palate; Embryonic Development; Female; Humans; Male; Maternal Nutritional Physiological Phenomena; Nutrition Policy; Palate; Pregnancy; Teratogens; Tretinoin; Vitamin A

Technical advances are radically altering our concepts of normal prenatal craniofacial development. These include concepts of germ layer formation, the establishment of the initial head plan in the neural plate, and the manner in which head segmentation is controlled by regulatory (homeobox) gene activity in neuromeres and their derived neural crest cells. There is also a much better appreciation of ways in which new cell associations are established. For example, the associations are achieved by neural crest cells primarily through cell migration and subsequent cell interactions that regulate induction, growth, programmed cell death, etc. These interactions are mediated primarily by two groups of regulatory molecules: "growth factors" (e.g., FGF and TGFalpha) and the so-called steroid/thyroid/retinoic acid superfamily. Considerable advances have been made with respect to our understanding of mechanisms involved in primary and secondary palate formation, such as growth, morphogenetic movements, and the fusion/merging phenomenon. Much progress has been made on the mechanisms involved in the final differentiation of skeletal tissues. Molecular genetics and animal models for human malformations are providing many insights into abnormal development. A mouse model for the fetal alcohol syndrome(FAS), a mild form of holoprosencephaly, demonstrates a mid-line anterior neural plate deficiency which leads to olfactory placodes being positioned too close to the mid-line, and other secondary changes. Work on animal models for the retinoic acid syndrome (RAS) shows that there is major involvement of neural crest cells. There is also major crest cell involvement in similar syndromes, apparently including hemifacial microsomia. Later administration of retinoic acid prematurely and excessively kills ganglionic placodal cells and leads to a malformation complex virtually identical to the Treacher Collins syndrome. Most clefts of the lip and/or palate appear to have a multifactorial etiology. Genetic variations in TGF alpha s, RAR alpha s, NADH dehydrogenase, an enzyme involved in oxidative metabolism, and cytochrome P-450, a detoxifying enzyme, have been implicated as contributing genetic factors. Cigarette smoking, with the attendant hypoxia, is a probable contributing environmental factor. It seems likely that few clefts involve single major genes. In most cases, the pathogenesis appears to involve inadequate contact and/or fusion of the facial prominences or palatal s

Topics: Animals; Cleft Lip; Cleft Palate; Congenital Abnormalities; Embryonic and Fetal Development; Female; Gene Expression Regulation, Developmental; Genes, Homeobox; Germ Layers; Growth Substances; Head; Holoprosencephaly; Humans; Neural Crest; Pregnancy; Prenatal Exposure Delayed Effects; Skull; Steroids; Tretinoin

Craniofacial dysmorphologies are common, ranging from simple facial disfigurement to complex malformations involving the whole head. With the advent of gene mapping and cloning techniques, the genetic element of both simple and complex human craniofacial dysmorphologies can be investigated. For many of the dysmorphic syndromes, it is possible to find families that display a particular phenotype in either an autosomal dominant, recessive, or X-linked manner. This article focuses on a subgroup of craniofacial dysmorphologies, covering these three main inheritance patterns, that are being studied using molecular biology techniques: DiGeorge syndrome, Treacher Collins syndrome, Greig cephalopolysyndactyly syndrome, acrocallosal syndrome, amelogenesis imperfecta, and X-linked cleft palate with ankyloglossia. Once the mutated or deleted gene or genes for each syndrome have been cloned, patterns of normal and abnormal craniofacial development should be elucidated. This should enhance both diagnosis and treatment of these common and disfiguring disorders.

Topics: Animals; Chromosome Aberrations; Chromosome Disorders; Cleft Palate; Cloning, Molecular; DiGeorge Syndrome; Facial Bones; Fetal Alcohol Spectrum Disorders; Forecasting; Genetic Linkage; Head; Humans; Mandibulofacial Dysostosis; Mice; Skull; Syndrome; Tongue; Tretinoin

Cleft lip and/or palate (CLP) is a common craniofacial birth defect caused by genetic as well as environmental factors. The phenotypic spectrum of CLP also includes submucous clefts with a defect in the palatal bone. To elucidate the contribution of vitamin A, we evaluated the effects of the vitamin A metabolite all-trans retinoic acid (ATRA) on the osteogenic differentiation and mineralization of mouse embryonic palatal mesenchymal cells (MEPM).. MEPM cells were isolated from the prefusion palates of E13 mouse embryos from three different litters.. MEPM cells were cultured with and without 0.5 μM ATRA in osteogenic medium. Differentiation was analysed by the expression of osteogenic marker genes and alkaline phosphatase (ALP) activity after 1, 2, and 7 days. The expression of Wnt marker genes was also analysed. Mineralization was assessed by alizarin red staining after 7, 14, 21, and 28 days.. The bone marker genes Sp7, Runx2, Alpl, and Col1a1 were inhibited 10% ± 2%, 59% ± 7%, 79% ± 12% and 57% ± 20% (P < .05) at day 7. ALP activity was inhibited at days 1 and 7 by 35 ± 0% (P < .05) and 23 ± 6% (P < .001). ATRA also inhibited mineralization at 3 and 4 weeks. Finally, expression of the universal Wnt marker gene Axin2 was strongly reduced, by 31 ± 18% (P < .001), at day 7.. Our data indicate that ATRA (vitamin A) inhibits bone formation by reducing Wnt signalling. This might contribute to the molecular aetiology of submucous clefting.

Topics: Animals; Cell Differentiation; Cells, Cultured; Cleft Lip; Cleft Palate; Mice; Osteogenesis; Tretinoin; Vitamin A; Wnt Proteins

Cleft palate is a common maxillofacial congenital malformation, and its mechanism still has not been fully illustrated. Recently, lipid metabolic defects have been observed in cleft palate. Patatin-like phospholipase domain-containing 2 (Pnpla2) is an important lipolytic gene. However, its effect on the formation of cleft palate remains unknown. In this research, we explored the expression of Pnpla2 in the palatal shelves of control mice. We also studied mice with cleft palates induced by retinoic acid and its effect on the embryonic palatal mesenchyme (EPM) cells phenotype. We found that Pnpla2 was expressed in the palatal shelves of both the cleft palate and control mice. Pnpla2 expression was lower in cleft palate mice than in the control mice. Experiments with EPM cells showed that knockdown of Pnpla2 inhibited cell proliferation and migration. In conclusion, Pnpla2 is linked to palatal development. We have indicated that low expression of Pnpla2 affects palatogenesis by inhibiting the proliferation and migration of EPM cells.

Topics: Animals; Cell Proliferation; Cleft Palate; Mice; Palate; Tretinoin

While exposure to high levels of all-trans retinoic acid (atRA) during pregnancy is known to suppress murine embryonic palate mesenchymal (MEPM) cells proliferation and to result in cleft palate (CP) development, the underlying mechanisms are poorly understood. Accordingly, this study was designed with the goal of clarifying the etiological basis for atRA-induced CP. A murine model of CP was established via the oral administration of atRA to pregnant mice on gestational day (GD) 10.5, after which transcriptomic and metabolomic analyses were performed with the goal of clarifying the critical genes and metabolites associated with CP development through an integrated multi-omics approach. MEPM cells proliferation was altered by atRA exposure as expected, contributing to CP incidence. In total, 110 genes were differentially expressed in the atRA treatment groups, suggesting that atRA may influence key biological processes including stimulus, adhesion, and signaling-related activities. In addition, 133 differentially abundant metabolites were identified including molecules associated with ABC transporters, protein digestion and absorption, mTOR signaling pathway, and the TCA cycle, suggesting a link between these mechanisms and CP. Overall, combined analyses of these transcriptomic and metabolomic results suggested that the MAPK, calcium, PI3K-Akt, Wnt, and mTOR signaling pathways are particularly important pathways enriched in the palatal cleft under conditions of atRA exposure. Together, these integrated transcriptomic and metabolomic approaches provided new evidence with respect to the mechanisms underlying altered MEPM cells proliferation and signal transduction associated with atRA-induced CP, revealing a possible link between oxidative stress and these pathological changes.

Topics: Animals; Cell Proliferation; Cleft Palate; Female; Mice; Mice, Inbred C57BL; Phosphatidylinositol 3-Kinases; Pregnancy; TOR Serine-Threonine Kinases; Transcriptome; Tretinoin

Topics: Animals; Cleft Palate; Mice; Osteogenesis; Tretinoin; Wnt Signaling Pathway

All-trans retinoic acid (atRA) is a teratogen that can induce cleft palate formation. During palatal development, murine embryonic palate mesenchymal (MEPM) cell proliferation is required for the appropriate development of the palatal frame, with Meg3 serving as a key regulator of the proliferative activity of these cells and the associated epithelial-mesenchymal transition process. DNA methylation and signaling via the TGFβ/Smad pathway are key in regulating embryonic development. Here, the impact of atRA on MEPM cell proliferation and associations between Tgfβ2 promoter methylation, Meg3, and signaling via the Smad pathway were explored using C57BL/6 N mice treated with atRA (100 mg/kg) to induce fetal cleft palate formation. Immunohistochemistry and BrdU assays were used to detect MEPM proliferation and DNA methylation assays were performed to detect Tgfβ2 promoter expression. These analyses revealed that atRA suppressed MEPM cell proliferation, promoted the upregulation of Meg3, and reduced the levels of Smad2 and Tgfβ2 expression phosphorylation, whereas Tgfβ2 promoter methylation was unaffected. RNA immunoprecipitation experiments indicated that the TgfβI receptor is directly targeted by Meg3, suggesting that the ability of atRA to induce cleft palate may be mediated through the Tgfβ/Smad signaling pathway.

Topics: Animals; Cell Proliferation; Cleft Palate; DNA Methylation; Female; Mice; Mice, Inbred C57BL; Palate; Pregnancy; Signal Transduction; Transforming Growth Factor beta; Tretinoin

Embryonic craniofacial development depends on the coordinated outgrowth and fusion of multiple facial primordia, which are populated with cranial neural crest cells and covered by the facial ectoderm. Any disturbance in these developmental events, their progenitor tissues, or signaling pathways can result in craniofacial deformities such as orofacial clefts, which are among the most common birth defects in humans. In the present study, we show that

Topics: Animals; Cleft Lip; Cleft Palate; Craniofacial Abnormalities; Embryonic Development; Hedgehog Proteins; Mice; Neural Crest; Tretinoin

The etiology of cleft lip with or without cleft palate (CL/P), a common congenital birth defect, is complex, with genetic and epigenetic, as well as environmental, contributing factors. Recent studies suggest that fetal development is affected by maternal conditions through microRNAs (miRNAs), a group of short noncoding RNAs. Here, we show that miR-129-5p and miR-340-5p suppress cell proliferation in both primary mouse embryonic palatal mesenchymal cells and O9-1 cells, a neural crest cell line, through the regulation of Sox5 and Trp53 by miR-129-5p, and the regulation of Chd7, Fign and Tgfbr1 by miR-340-5p. Notably, miR-340-5p, but not miR-129-5p, was upregulated following all-trans retinoic acid (atRA; tretinoin) administration, and a miR-340-5p inhibitor rescued the cleft palate (CP) phenotype in 47% of atRA-induced CP mice. We have previously reported that a miR-124-3p inhibitor can also partially rescue the CP phenotype in atRA-induced CP mouse model. In this study, we found that a cocktail of miR-124-3p and miR-340-5p inhibitors rescued atRA-induced CP with almost complete penetrance. Taken together, our results suggest that normalization of pathological miRNA expression can be a preventive intervention for CP.

Topics: Animals; Cell Proliferation; Cleft Lip; Cleft Palate; Mice; MicroRNAs; Tretinoin

Cleft palate (CP) is one of the most common congenital birth defects in the craniofacial region, retinoic acid (RA) gavage is the most common method for inducing cleft palate model. Although several mechanisms have been proposed to illuminate RA-induced cleft palate during embryonic development, these findings are far from enough. Many efforts remain to be devoted to studying the etiology and pathogenesis of cleft palate. Recent research is gradually shifting the focus to the effect of retinoic acid on gut microbiota. However, few reports focus on the relationship between the occurrence of CP in embryos and gut microbiota.. In our research, we used RA to induce cleft palate model for E10.5 the feces of 5 RA-treated pregnant mice and 5 control pregnant mice were respectively metagenomics analysis.. Compared with the control group, Lactobacillus in the gut microbiome the RA group was significantly increased. GO, KEGG and CAZy analysis of differentially unigenes demonstrated the most abundant metabolic pathway in different groups, lipopolysaccharide biosynthesis, and histidine metabolism.. Our findings indicated that changes in the maternal gut microbiome palatal development, which might be related to changes in Lactobacillus and These results provide a new direction in the pathogenesis of CP induced by RA.

Topics: Animals; Cleft Palate; Female; Gastrointestinal Microbiome; Mice; Microbiota; Pregnancy; Tretinoin

Abnormally high concentrations of all-trans retinoic acid (atRA) induce cleft palate, which is accompanied by abnormal migration and proliferation of mouse embryonic palatal mesenchyme (MEPM) cells. Hormone-sensitive lipase (HSL) is involved in many embryonic development processes. The current study was designed to elucidate the mechanism of HSL in cleft palate induced by atRA. To establish a cleft palate model in Kunming mice, pregnant mice were administered atRA (70 mg/kg) by gavage at embryonic Day 10.5 (E10.5). Embryonic palates were obtained through the dissection of pregnant mice at E15.5. Hematoxylin and eosin (H&E) staining was used to evaluate growth changes in the palatal shelves. The levels of HSL in MEPM cells were detected by immunohistochemistry, quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting. RNAi was applied to construct vectors expressing HSL small interference RNAs (siRNAs). The vectors were transfected into MEPM cells. Cell proliferation and migration were evaluated by the cell counting kit-8 (CCK-8) assay and wound healing assay, respectively. The palatal shelves in the atRA group had separated at E15.5 without fusing. In MEPM cells, the expression of HSL was reversed after atRA treatment, which caused cleft palate

Topics: Animals; Cleft Palate; Female; Mice; Palate; Pregnancy; RNA, Small Interfering; Sterol Esterase; Tretinoin

Palatal mesenchymal cell proliferation is essential to the process of palatogenesis, and the proliferation of mouse embryonic palate mesenchymal (MEPM) cells is impacted by both all-trans retinoic acid (atRA) and the TGF-β/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been shown to activate TGF-β/Smad signaling and to thereby regulate cell proliferation, differentiation, and related processes. Herein, we found that atRA treatment (100 mg/kg) promoted Meg3 upregulation in MEPM cells, and that such upregulation was linked to the suppression of MEPM cell proliferation in the context of secondary palate fusion on gestational day (GD) 13 and 14. Moreover, the demethylation of specific CpG sites within the lncRNA Meg3 promoter was detected in atRA-treated MEPM cells, likely explaining the observed upregulation of this lncRNA. Smad signaling was also suppressed by atRA treatment in these cells, and RNA immunoprecipitation analyses revealed that Smad2 can directly interact with Meg3 in MEPM cells following atRA treatment. Therefore, we propose a model wherein Meg3 is involved in the suppression of MEPM cell proliferation, functioning at least in part via interacting with the Smad2 protein and thereby suppressing Smad signaling in the context of atRA-induced cleft palate.

Topics: Animals; Cleft Palate; Female; Gene Expression Regulation, Developmental; Keratolytic Agents; Mice; Palate; Pregnancy; RNA, Long Noncoding; Smad Proteins; Tretinoin

Mesenchymal cell proliferation is critical for the growth of the palate shelf. All-trans retinoic acid (atRA), as well as pathways associated with TGF-β/Smad signaling, play crucial roles in the proliferation of mouse embryonic palate mesenchymal (MEPM) cells. We have found that MEPM-cell proliferation was regulated by atRA and exogenous TGF-β3 could significantly antagonize the atRA-mediated suppression of MEPM cell proliferation, which is closely associated with the regulation of TGF-β/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been reported to activate TGF-β/Smad signaling, thereby regulating cellular proliferation, differentiation, and related processes. Here, we found that Meg3 expression increased significantly in atRA-treated MEPM cells while TGF-β3 treatment markedly inhibited Meg3 expression and antagonized the effect of atRA on Meg3. Moreover, Smad2 was found to interact directly with Meg3, and atRA treatment significantly enriched Meg3 in Smad2-immunoprecipitated samples. After Meg3 deletion, the effects of atRA on the proliferation of MEPM cells and TGF-β3-dependent protein expression were lost. Hence, we speculate that Meg3 has a role in the RA-induced suppression of MEPM cell proliferation by targeting Smad2 and thereby mediating TGF-β/Smad signaling inhibition.

Topics: Animals; Cell Proliferation; Cells, Cultured; Cleft Palate; Gene Expression Regulation, Developmental; Mesenchymal Stem Cells; Mice; Palate; Phosphorylation; RNA, Long Noncoding; Signal Transduction; Transforming Growth Factor beta3; Tretinoin

The molecular mechanisms of abnormal palatogenesis were investigated in this study. A key regulator, miR-106a-5p, and its target pathway were analyzed.. This research is trying to clarify the underlying mechanism of the modulation of miRNA transcription during the formation of cleft palate by 7T and 9.4T NMR metabolomic platforms.. Differentially expressed miRNAs and mRNAs were analyzed by microarray analysis and verified by qRT-PCR. The protein expression in TGFβ signaling pathways were analyzed by Western Blotting. The relationship between miR-106a-5p and TGFβ were analyzed by luciferase reporter assay. Cell apoptosis were analyzed by flow cytometer. And finally, the metabonomics were analyzed by NMR and multivariate data analysis models (MVDA).. The expression of miR-106a-5p increased in cleft palatal tissue and negatively correlated with the protein level of Tgfbr2. The luciferase assay further proved that the tgfbr2 was a direct target of miR-106a-5p. In another aspect, miR-106a-5p increased apoptosis level in palatal mesenchymal cells, possibly because its inhibition of TGFβ signaling pathway. Moreover, low cholesterol and choline levels with high citric acid and lipid levels were observed by 7T and 9.4T NMR metabonomic analysis, which inferred the disorder of cell membrane synthesis in cleft palate formation. Furthermore, transformation from choline to phosphatidylcholine regulated by miR-106a-5p was also disrupted, resulting in phosphatidic choline synthesis disorder and reduced cell membrane synthesis.. The regulatory mechanism of cleft palate was studied at transcriptional and metabolomics levels, which may provide important information in understanding the primary cause of this abnormality.

Topics: Animals; Apoptosis; Cell Membrane; Citric Acid; Cleft Palate; Disease Models, Animal; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Humans; Male; Membrane Lipids; Mesenchymal Stem Cells; Metabolome; Mice; MicroRNAs; Palate; Receptor, Transforming Growth Factor-beta Type II; Signal Transduction; Smad2 Protein; Smad3 Protein; Transcriptome; Transforming Growth Factor beta; Tretinoin

Recent studies have shown that lipid metabolism was abnormal during the formation of cleft palate. However, the composition of these lipid species remains unclear.. Aims of this study were to identify the lipid species components and reveal the key lipid metabolic disorders in cleft palate formation.. The pregnant mice were divided into experimental group exposed to all-trans retinoic acid (RA-treated group) (n = 12) and control group (n = 12) at embryonic gestation day 10.5 (E0.5). The component of the palatal tissue metabolome was analyzed using a LCMS-based nontargeted lipidomics approach. Multivariate statistical analysis was then carried out to assess the differences between the RA-treated group and the control group.. Twenty-nine lipid species were found to discriminate between RA-treated and control embryos. Among them, 28 lipid species increased and 1 lipid species decreased in the RA-treated group. Among these lipids, 13 were triglycerides, 9 were PEs, 3 were PCs, 2 were PSs, 2 were DGs. Further analysis of the number of carbons and unsaturated bond of triglycerides showed that TGs with high unsaturated bonds constituted a higher fraction in the RA-treated group. A higher amount of triglycerides containing 52, 54, 56, 58, 60 carbons, and 1 to 8 unsaturated bonds. Of note, under RA treatment, TG 50:1, 52:2, 56:6and 60:8 became the most prominent.. Lipid metabolism is significantly different in the formation of cleft palate induced by RA, and the unsaturated triglycerides increased in the RA-treated group may play an important role in the formation of cleft palate.

Topics: Animals; Cleft Palate; Female; Lipid Metabolism; Lipidomics; Lipids; Mice; Pregnancy; Tretinoin; Triglycerides

Cleft palate is a common congenital maxillofacial malformation in newborns. All-trans retinoic acid (atRA) is an ideal exogenous stimulus to construct a mouse cleft palate model. However, the precise pathogenic mechanism remains to be elucidated. In our study, to explore the toxicity of atRA on palatal shelves during different stages of palate development, a total of 100 mg/kg atRA was administered to C57BL/6 mice at embryonic day 10.5 (E10.5). Mouse embryonic palatal shelves at E13.5, E14.5, E15.5, and E16.5 were collected for RNA extraction and histological treatment. Changes in gene expression were tested through RNA-seq. Selected differentially expressed genes (DEGs) related to metabolic pathways, such as Ptgds, Ttr, Cyp2g1, Ugt2a1 and Mgst3, were validated and analyzed by Quantitative real-time PCR (qRT-PCR). In addition, Gene Oncology analysis showed that transcriptional changes of genes from extracellular matrix (ECM) components, such as Spp1, and crystallin family might play important role in palatal shelves elevation (E13.5-E14.5). Therefore, the protein expression level of Ttr and Spp1 from E13.5 to E16.5 were tested by immunohistochemistry (IHC). Besides, the mRNA level of Spp1, were down-regulated at E16.5 and the protein were down-regulated at E15.5 and E16.5 in all-trans retinoic acid group, suggesting that atRA may involve in palatal bone formation by regulating Spp1. Overall, gene transcriptional profiles were obviously different at each time point of palate development. Thus, this study summarized some pathways and genes that may be related to palatogenesis and cleft palate through RNA-seq, to provide a direction for subsequent studies on the mechanism and targeted therapy of cleft palate.

Topics: Animals; Cleft Palate; Female; Gene Expression Regulation, Developmental; Gene Ontology; Gestational Age; Mice; Mice, Inbred C57BL; Palate; Pregnancy; Prenatal Exposure Delayed Effects; Real-Time Polymerase Chain Reaction; RNA; RNA-Seq; Transcriptome; Tretinoin

Topics: Animals; Cleft Palate; Cytokines; Female; Male; Mice, Inbred C57BL; Placenta; Polychlorinated Dibenzodioxins; Pregnancy; Receptor for Advanced Glycation End Products; Teratogens; Tretinoin

Cleft palate(CP) is a widely studied congenital malformation. However, its etiology and pathogenesis still remain unclear. Proteins are fundamental molecules that participate in every biological process within cells. In this study, we established CP mouse models induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and retinoic acid (RA), using proteomics technology isobaric tags for relative and absolute quantitation (iTRAQ) to investigate the key proteins in the formation of CP. Pregnant mice were given a gavage of TCDD 28μg/kg or retinoic acid 80mg/kg of body weight or equivalent corn oil at gestational day 10.5(GD10.5) and sacrificed at GD 17.5. Foetal mice were recorded and collected for further detection. Western blot was performed to verify the iTRAQ results. Eventually, we obtained 18 common differentially expressed proteins in TCDD group and RA group compared with normal control, 17 up-regulated and 1 down-regulated. 14-3-3sigma and Annexin A1 were up-regulated in experimental groups at GD17.5, which was consistent with Western blot. We speculated that the common differentially expressed proteins might be one of the molecular mechanisms in the formation of cleft palate.

Topics: 14-3-3 Proteins; Animals; Annexin A1; Cleft Palate; Disease Models, Animal; Female; Male; Mice, Inbred C57BL; Polychlorinated Dibenzodioxins; Proteomics; Tretinoin

Cleft palate (CP) is one of the most common congenital craniofacial anomalies in humans and can be caused by either single or multiple genetic and environmental factor(s). With respect to environmental factors, excessive intake of vitamin A during early pregnancy is associated with increased incidence of CP in offspring both in humans and in animal models. Vitamin A is metabolized to retinoic acid (RA); however, the pathogenetic mechanism of CP caused by altered RA signaling during early embryogenesis is not fully understood. To investigate the detailed cellular and molecular mechanism of RA-induced CP, we administered all-trans RA to pregnant mice at embryonic day (E)8.5. In the RA-treated group, we observed altered expression of

Topics: Animals; Apoptosis; Cleft Palate; Embryo, Mammalian; Embryonic Development; Female; Hedgehog Proteins; Mice, Inbred ICR; Models, Biological; Neural Crest; Signal Transduction; Skull; SOXE Transcription Factors; Tretinoin

Epithelial-mesenchymal transformation of the medial edge epithelium is the most crucial process in embryonic palatal fusion. This study aimed to explore the relationship and potential mechanism between enhancer DNA methylation and mRNA expression of histone deacetylase 4 (HDAC4) during palatal fusion induced by maternal exposure to all-trans retinoic acid (ATRA). Pregnant mice were administered ATRA (70 mg/kg) by gavage at embryonic gestation day 10.5 (E10.5) to establish a cleft palate (CP) model in C57BL/6J mice. Control groups were given an equivalent volume of corn oil. Pregnant mice were dissected at E14.5 (n = 6) to obtain embryonic palates. HDAC4 enhancer DNA methylation data were obtained from a previous MethylRAD-seq. Methylation-specific polymerase chain reaction (MSP) and real-time quantitative PCR were used to quantify enhancer methylation and the mRNA expression level of HDAC4. Enhancer DNA methylation at a non-CpG site within the HDAC4 gene was hyper-methylated at E14.5 (P: 0.011, log

Topics: Animals; Cleft Palate; Disease Models, Animal; DNA Methylation; Enhancer Elements, Genetic; Epigenesis, Genetic; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Developmental; Histone Deacetylases; Maternal Exposure; Mice; Mice, Inbred C57BL; Palate; Pregnancy; RNA, Messenger; Tretinoin

Cleft palate is among the most common congenital disorders, and can be induced by exposure to all‑trans retinoic acid (atRA) during mice and human embryogenesis. However, the mechanism underlying the implication of atRA in the development of cleft palate has yet to be elucidated. In the present study, atRA administered by gavage resulted in formation of a cleft palate in 99% of treated C57BL/6 mice. Notch2 was revealed to be upregulated in mouse embryonic palate mesenchymal (MEPM) cells in the atRA‑treated group compared with untreated control mice between embryonic day (E)12.5 and E14.5. In addition, atRA was demonstrated to mediate Notch2 expression via the activation of RA receptors (RARs). Since Notch2 activation has previously been reported to inhibit the proliferation of MEPM cells, the expression levels of extracellular signal‑regulated kinase (ERK), p21, cyclin D1 and Ki‑67 were assessed in samples from atRA‑treated and control mouse embryos between E12.5 and E14.5. It was demonstrated that Notch2 silencing partially reversed the atRA‑induced inhibition of ERK phosphorylation in MEPM cells. In addition, the atRA‑induced cyclin D1 downregulation and p21 upregulation were partially reversed following Notch2 silencing, whereas the atRA‑induced inhibition of cellular proliferation was also attenuated. Furthermore, it was revealed that Notch2 expression was upregulated, whereas Ki‑67 expression was downregulated following atRA exposure, as assessed using resin bead‑released atRA in MEPM cells. The present findings suggested that during embryonic development, atRA may enhance the expression of Notch2, which may inhibit cellular proliferation, possibly through ERK signaling.

Topics: Animals; Cell Proliferation; Cells, Cultured; Cleft Palate; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Male; Mesenchymal Stem Cells; Mice, Inbred C57BL; Pregnancy; Prenatal Exposure Delayed Effects; Receptor, Notch2; Tretinoin; Up-Regulation

The current study was designed to elucidate the mechanism of retinol binding protein 4 (RBP4) in cleft palate induced by all‑trans retinoic acid (atRA). To establish a cleft palate model in C57BL/6J mice, pregnant mice were administered atRA (100 mg/kg) by gavage at the tenth embryonic stage (E10.0). Control groups were given the equivalent volume of corn oil. Pregnant mice were dissected at E12.5, E13.5 and E14.5 to obtain the embryonic palates. The expression levels of RBP4 in the embryonic palatal mesenchyme (EPM) were determined by immunohistochemistry, reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blotting. Human embryonic palatal mesenchymal cells were exposed to atRA to detect the variation in RBP4 induced by atRA in vitro. Small interfering RNA was used to suppress the expression of RBP4, and a plasmid overexpressing RBP4 was used to examine upregulated expression. The cell counting kit‑8 assay was used to evaluate the effect of RBP4 on cell proliferation. The expression levels of p27 and cyclin D1 were determined by RT‑qPCR and western blotting, while the expression levels of extracellular signal‑related kinase (ERK) 1/2 and protein kinase B (AKT) were assessed by western blotting. At E14.5, RBP4 was strongly expressed in the EPM, while it was downregulated following atRA treatment, which induced cleft palate in vivo. In vitro experiments indicated that atRA suppressed the expression of RBP4 and altered the expression of p27 and cyclin D1 to cause growth inhibition. Knockdown of RBP4 resulted in decreased expression of cyclin D1 and increased p27, and suppressed proliferation. Overexpression of RBP4 reversed the inhibitory effect of atRA and promoted proliferation via the ERK1/2 and AKT signaling pathways. These results suggested that RBP4 was involved in cleft palate induced by atRA and it can be suppressed by atRA to cause growth inhibition in the embryonic palate.

Topics: Animals; Cell Line; Cell Proliferation; Cleft Palate; Corn Oil; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Embryo, Mammalian; Excipients; Female; Gene Expression Regulation, Developmental; Humans; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Pregnancy; Proto-Oncogene Proteins c-akt; Retinol-Binding Proteins, Plasma; RNA, Small Interfering; Signal Transduction; Tretinoin

Long noncoding RNAs (lncRNAs) are the new class of transcripts and pervasively transcribed in the genome, which have been found to play important functional roles in many tissues and organs. LncRNAs can interact with target gene to exert their functions. However, the function and mechanism of lncRNA in cleft palate (CP) development remain elusive. Here, we investigated the role of lncRNA H19 and its target gene insulin-like growth factor 2 (IGF2) in CP of mice. All-trans retinoic acid (atRA) is a well-known teratogenic effecter of CP. After establishment of the CP mouse model using atRA in vivo, we found that the rate of CP in mice was 100%. The tail lengths of fetuses in atRA-treated mice were shorter than those of control mice from embryonic day (E)12 to E17. The expression of lncRNA H19 and IGF2 were embryo age-related differences between atRNA-treated and control mice. In addition, the the relationship between lncRNA H19 and IGF2 were negative correlation in the critical period of developmental palate. These findings suggest that lncRNA H19 mediate atRA-induced CP in mice.

Topics: Animals; Cleft Palate; Female; Insulin-Like Growth Factor II; Mice; Mice, Inbred C57BL; Pregnancy; RNA, Long Noncoding; Teratogens; Tretinoin

This study investigated the correlation between differentially expressed proteins in amniotic fluid (AF) and cleft palate induced by all-trans retinoic acid (atRA), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. Seven proteins were differentially expressed at embryonic day (E) 16.5 in atRA and control groups as revealed by label-based mouse antibody array. Enzyme-linked immunosorbent assay was further used to detect the expression levels of these proteins in AF from E13.5 to E16.5 in atRA, TCDD, and control groups. The cleft palate groups showed lower concentrations of receptor for advanced glycation end products (RAGE) and epiregulin at E16.5. RAGE immunostaining obviously decreased in palatal tissue sections obtained from E14.5 to E16.5 in the cleft palate groups as revealed by immunohistochemistry. These findings indicate that reduced levels of RAGE and epiregulin in AF are correlated to chemically induced cleft palate in mice.

Topics: Amniotic Fluid; Animals; Biomarkers; Cleft Palate; Disease Models, Animal; Epiregulin; Female; Gestational Age; Mice, Inbred C57BL; Palate; Polychlorinated Dibenzodioxins; Pregnancy; Receptor for Advanced Glycation End Products; Tretinoin

To investigate the effects of retinoic acid (RA) on the proliferation and apoptosis of mesenchyme and epithelium at the key stages of palatal development, and to determine the embryonic alterations associated with cleft palate.. 100 pregnant C57 females (E12) were equally divided into two groups. The experimental group was administered RA once (70 mg/kg) while the control group was administered corn oil only, and the heads of the embryos at E13.5-17.5 were collected and processed to paraffin. Sections were stained with hematoxylin and eosin for the morphological assessment of anterior,posterior, left and right palate, and BrdU and TUNEL assays for the detection of proliferation and survival of palatal mesenchyme and epithelium.. Simple treatment of RA at 70 mg/kg caused an incidence of 100％ cleft palate. During E13.5-15.5,cell proliferation was significantly promoted on the anterior palatal mesenchyme in the RA group, while no difference for the posterior palatal mesenchyme. Besides, the cell proliferation on anterior and posterior epithelium was comparable between the control and RA group. Less apoptotic cells were observed on epithelium during El3.5-14.5 in the RA group.. RA induces excessive cell proliferation of palatal mesenchyme, causing abnormal vertical palatal growth and failure of palatal shelves contact and fusion, which finally lead to cleft palate. RA also increases epithelium apoptosis.

Topics: Animals; Apoptosis; Cell Proliferation; Cleft Palate; Epithelium; Female; Keratolytic Agents; Mesoderm; Palate; Pregnancy; Tretinoin

Retinoic acid (RA)-induced cleft palate results from both extrinsic obstructions by the tongue and internal factors within the palatal shelves. Our previous study showed that the spatiotemporal expression of Rac1 regulates the fibronectin (FN) arrangement through cell density alterations that play an important role in palate development. In this study, we investigate the involvement of the Rac1 regulation of the FN arrangement in RA-induced cleft palate. Our results demonstrate that RA-induced intrinsic alterations in palatal shelves, including a delayed progress of cell condensation, delay palate development, even after the removal of the tongue. Further analysis shows that RA treatment diminishes the region-distinctive expression of Rac1 within the palatal shelves, which reversely alters the fibrillar arrangement of FN. Furthermore, RA treatment disrupts the formation of lamellipodia, which are indicative structures of cell migration that are regulated by Rac1. These results suggest that the Rac1 regulation of the FN arrangement is involved in RA-induced cleft palate through the regulation of cell migration, which delays the progress of cell condensation and subsequently influences the FN arrangement, inducing a delay in palate development. Our study provides new insights into the RA-induced impairment of palatal shelf elevation based on cell migration dynamics.

Topics: Animals; Cell Count; Cleft Palate; Fibronectins; Mesoderm; Mice, Inbred ICR; Palate; Pseudopodia; rac1 GTP-Binding Protein; Tongue; Tretinoin

Although numerous studies have confirmed that consumption of folic acid (FA) during early pregnancy reduces the risk of oral facial clefts in newborn infants, the optimal dose of FA for reducing this risk remains unknown. We evaluated various doses of FA for their ability to reduce the incidence of all-trans retinoic acid (ATRA)-induced cleft palate in mice.. Pregnant C57BL/6J mice were randomly assigned to eight groups dosed with corn oil (control group), ATRA (80 mg/kg), FA (40 mg/kg), or ATRA (80 mg/kg) + FA (2.5 mg, 5 mg, 10 mg, 20 mg, or 40 mg/kg body weight) on gestation day 11 (GD11), after which samples of maternal blood obtained on GD 11 were analyzed for serum folate levels. After receiving the doses, randomly selected mice in each dose group were sacrificed on GDs 13.5, 14.5, and 15.5, and the fetuses were removed for examination by light microscopy and scanning electron microscopy to detect the incidence of cleft palate.. Among the pregnant mice dosed with ATRA+FA, those dosed with 5 mg/kg FA had fetuses with the lowest incidence of cleft palate. In addition, the eight groups of pregnant mice had significantly different serum folate concentrations (P < .001).. When administered to pregnant mice at a specific dose and on the proper gestation day, FA showed an antiteratogenic effect by reducing the incidence of ATRA-induced cleft palate in fetal mice.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Female; Folic Acid; Maternal Exposure; Mice; Mice, Inbred C57BL; Pregnancy; Prenatal Care; Teratogens; Tretinoin

Mesenchymal cell proliferation is one of the processes in shelf outgrowth. Both all-trans retinoic acid (atRA) and transforming growth factor-β3 (TGF-β3) play an important role in mouse embryonic palate mesenchymal (MEPM) cell proliferation. The cellular effects of TGF-β are mediated by Smad-dependent or Smad-independent pathways. In the present study, we demonstrate that atRA promotes TGF-β3 promoter demethylation and protein expression, but can cause depression of mesenchymal cell proliferation, especially at embryonic day 14 (E14). Moreover, the inhibition of MEPM cell proliferation by atRA results in the downregulation of Smad signaling mediated by transforming growth interacting factor (TGIF). We speculate that the effects of atRA on MEPM cell proliferation may be mediated by Smad pathways, which are regulated by TGIF but are not related to TGF-β3 expression. Finally, the cellular effects of TGF-β3 on MEPM cell proliferation may be mediated by Smad-independent pathways.

Topics: Animals; Cell Proliferation; Cleft Palate; DNA Methylation; Embryo, Mammalian; Female; Mesenchymal Stem Cells; Mice, Inbred C57BL; Pregnancy; Signal Transduction; Smad Proteins; Transforming Growth Factor beta3; Tretinoin

Administration of all-trans retinoic acid (atRA) on E12.0 (embryonic day 12.0) leads to failure of medial edge epithelium (MEE) disappearance and cleft palate. However, the molecular mechanism underlying the relationship between atRA and MEE remains to be identified. In this study, atRA (200 mg/kg) administered by gavage induced a 75% incidence of cleft palate in C57BL/6 mice. Notch1 was up-regulated in MEE cells in the atRA-treated group compared with the controls at E15.0, together with reduced apoptosis and elevated proliferation. Next, we investigated the mechanisms underlying atRA, Notch1 and MEE degradation in palate organ culture. Our results revealed that down-regulation of Notch1 partially rescued the inhibition of atRA-induced palate fusion. Molecular analysis indicated that atRA increased the expression of Notch1 and Rbpj and decreased the expression of P21. In addition, depletion of Notch1 expression decreased the expression of Rbpj and increased the expression of P21. Moreover, inhibition of Rbpj expression partially reversed atRA-induced MEE persistence and increased P21 expression. These findings demonstrate that atRA inhibits MEE degradation, which in turn induces a cleft palate, possibly through the Notch1/RBPjk/P21 signaling pathway.

Topics: Animals; Apoptosis; Cleft Palate; Epithelium; Female; Gene Knockdown Techniques; Mice; Mice, Inbred C57BL; Pregnancy; Receptor, Notch1; Teratogens; Tretinoin

Cleft lip with or without palate (CL/P) is a common congenital anomaly in humans and is thought to be caused by genetic and environmental factors. However, the epigenetic mechanisms underlying orofacial clefts are not fully understood. Here, we investigate how the overdose of retinoic acid (RA), which can induce cleft palate in mice and humans, regulates histone methyltransferase, Wolf-Hirschhorn syndrome candidate 1 (WHSC1) during palatal development in mice. We treated mouse embryonic fibroblasts (MEFs) with 1 μM all-trans RA and discovered that the global level of H3K36me3 was downregulated and that expression of the H3K36 methyltransferase gene, Whsc1, was reduced. The expression level of WHSC1 in embryonic palatal shelves was reduced during palatogenesis, following maternal administration of 100 mg/kg body weight of RA by gastric intubation. Furthermore, the expression of WHSC1 in palatal shelves was observed in epithelial and mesenchymal cells at all stages, suggesting an important role for palatal development. Our results suggest that the pathogenesis of cleft palate observed after excessive RA exposure is likely to be associated with a reduction in the histone methyltransferase, WHSC1.

Topics: Animals; Cell Line; Cleft Palate; Down-Regulation; Drug Overdose; Female; Histone-Lysine N-Methyltransferase; Histones; Methylation; Mice; Mice, Inbred C57BL; Palate; RNA, Messenger; Tretinoin

Folate deficiency has been associated with numerous diseases and birth defects including orofacial defects. However, whether folate has a role in the face during early orofacial development has been unclear. The present study reveals that pharmacological and antisense oligonucleotide mediated inhibition of DHFR, an integral enzyme in the folate pathway, results in specific changes in the size and shape of the midface and embryonic mouth. Such defects are accompanied by a severe reduction in the muscle and cartilage jaw elements without significant change in neural crest pattern or global levels of methylation. We propose that the orofacial defects associated with DHFR deficient function are the result of decreased cell proliferation and increased cell death via DNA damage. In particular, localized apoptosis may also be depleting the cells of the face that express crucial genes for the differentiation of the jaw structures. Folate supplementation is widely known to reduce human risk for orofacial clefts. In the present study, we show that activating folate metabolism can reduce median oral clefts in the primary palate by increasing cell survival. Moreover, we demonstrate that a minor decrease in DHFR function exacerbates median facial clefts caused by RAR inhibition. This work suggests that folate deficiencies could be a major contributing factor to multifactorial orofacial defects.

Topics: Animals; Apoptosis; Biomarkers; Cartilage; Cell Cycle; Cell Proliferation; Cell Survival; Cleft Palate; DNA Damage; DNA Methylation; Embryo, Nonmammalian; Face; Folic Acid; Gene Expression Regulation, Developmental; Leucovorin; Methotrexate; Models, Biological; Morpholinos; Mouth; Muscles; Neural Crest; Oligonucleotides, Antisense; Receptors, Retinoic Acid; Signal Transduction; Tetrahydrofolate Dehydrogenase; Tretinoin; Xenopus laevis

To investigate the effects of all-trans retinoic acid (atRA) on the function of bone morphogenetic protein receptor 2 (BMPR2) expression in embryonic palate.. Cleft palate mice model was established by atRA. On gestation day (GD) 15 and GD 17, the pregnant mice were killed to obtain the embryos from the uteri. The embryonic palates were stained with hematoxylin-eosin, and the remaining sections were used for the immunohistochemistry of BMPR2 detection. Reverse transcription-polymerase chain reaction was performed to detect the expression levels of Bmpr2 mRNA.. In the atRA-treated group, short extensions and failure to fuse with each other were observed. The positive expression of BMPR2 was detected in developing palatal process from GD 15 to GD 17 in the control group. Compared with those of the control group, BMPR2 protein and Bmpr2 mRNA decreased in the atRA-treated group (P<0.05).. The treatment of pregnant mice with retinoic acid produces small palatal shelves in their fetuses and down-regulates BMPR2 expressions.

Topics: Animals; Bone Morphogenetic Protein 2; Cleft Palate; Disease Models, Animal; Down-Regulation; Female; Mice; Pregnancy; RNA, Messenger; Tretinoin

Retinoic acid (RA) is a key regulator of embryonic development and linked to several birth defects including cleft lip and palate (CLP). The aim was to investigate the effects of RA on proliferation and gene expression of human palatal keratinocytes (KCs) in vitro.. KCs from children with and without CLP were cultured with 2 and 5 μM RA. Proliferation was measured by quantification of DNA after 2, 4, 6, and 8 days. In addition, we analysed the effects of RA on messenger RNA expression of genes for proliferation, differentiation, apoptosis, and RA receptors.. RA similarly inhibited proliferation of palatal KC from cleft and non-cleft subjects. The proliferation of KCs from cleft subjects was reduced to 59.8±13.4% (2 μM) and 41.5±14.0% (5 μM, Day 6), while that of cells from age-matched non-cleft subjects was reduced to 66.9±12.1% (2 μM) and 33.9±10.1% (5 μM). RA treatment reduced the expression of several of the investigated genes; the proliferating cell nuclear antigen (PCNA) was reduced in CLP KCs only. Keratins 10 and 16 were downregulated in keratinocytes from both cleft and non-cleft subjects. P63, a master regulator for epithelial differentiation, was only downregulated in KCs from cleft subjects, as was the RXRa receptor. Two P63 target genes (GJB6 and DLX5) were strongly downregulated by RA in all cell lines. None of the apoptosis genes was affected.. Overall, RA similarly inhibits proliferation of palatal KCs from cleft and non-cleft subjects and reduces the expression of specific genes.

Topics: Case-Control Studies; Cell Differentiation; Cell Proliferation; Cells, Cultured; Child, Preschool; Cleft Palate; Down-Regulation; Gene Expression Regulation; Humans; Infant; Keratinocytes; RNA, Messenger; Tretinoin

To characterize the abnormal metabolic profile of all-trans-retinoic acid (ATRA)-induced craniofacial development in mouse embryos using proton magnetic resonance spectroscopy (1H-MRS).. Timed-pregnant mice were treated by oral gavage on the morning of embryonic gestation day 11 (E11) with all-trans-retinoic acid (ATRA). Dosing solutions were adjusted by maternal body weight to provide 30, 70, or 100 mg/kg RA. The control group was given an equivalent volume of the carrier alone. Using an Agilent 7.0 T MR system and a combination of surface coil coils, a 3 mm×3 mm×3 mm 1H-MRS voxel was selected along the embryonic craniofacial tissue. 1H-MRS was performed with a single-voxel method using PRESS sequence and analyzed using LCModel software. Hematoxylin and eosin was used to detect and confirm cleft palate.. 1H-MRS revealed elevated choline levels in embryonic craniofacial tissue in the RA70 and RA100 groups compared to controls (P<0.05). Increased choline levels were also found in the RA70 and RA100 groups compared with the RA30 group (P<0.01). High intra-myocellular lipids at 1.30 ppm (IMCL13) in the RA100 group compared to the RA30 group were found (P<0.01). There were no significant changes in taurine, intra-myocellular lipids at 2.10 ppm (IMCL21), and extra-myocellular lipids at 2.30 ppm (EMCL23). Cleft palate formation was observed in all fetuses carried by mice administered 70 and 100 mg/kg RA.. This novel study suggests that the elevated choline and lipid levels found by 1H-MRS may represent early biomarkers of craniofacial defects. Further studies will determine performance of this test and pathogenetic mechanisms of craniofacial malformation.

Topics: Abnormalities, Drug-Induced; Animals; Antineoplastic Agents; Choline; Cleft Palate; Craniofacial Abnormalities; Creatine; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Lipids; Male; Mice; Pregnancy; Proton Magnetic Resonance Spectroscopy; Taurine; Tretinoin

Although proper tongue development is relevant to other structures in the craniofacial region, the molecular details of muscle development in tongue remain poorly understood. Here, we report that pregnant mice treated with retinoic acid (+RA) produce embryos with tongue malformation and a cleft palate. Histological analyses revealed that at E14.5, the tongues of +RA fetuses failed to descend and flatten. Ultrastructural analysis showed that at perinatal stage E18.5, the myofilaments failed to form normal structures of sarcomeres, and arranged disorderly in the genioglossus. The proliferation and levels of myogenic determination markers (Myf5 and MyoD) and myosin in the genioglossus were profoundly reduced. Wnt5a and Camk2d expressions were down-regulated, while levels of Tbx1, Ror2, and PKCδ were up-regulated in the tongues of +RA fetuses. In mock- and Wnt5a-transfected C2C12 (Wnt5a-C2C12) cells, Wnt5a overexpression impaired proliferation, and maintained Myf5 at a relative high level after RA treatment. Furthermore, Wnt5a overexpression positively correlated with levels of Camk2d and Ror2 in C2C12 cells after RA exposure. These data support the hypothesis that the Wnt5a/CaMKII pathway is directly involved in RA-induced hypoplasia and disorder of tongue muscles.

Topics: Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line; Cell Proliferation; Cleft Palate; Embryo, Mammalian; Female; Mice; Mice, Inbred ICR; Muscle Development; MyoD Protein; Myogenic Regulatory Factor 5; Myosins; Pregnancy; Protein Kinase C-delta; Receptor Tyrosine Kinase-like Orphan Receptors; Sarcomeres; T-Box Domain Proteins; Tongue; Tretinoin; Wnt Proteins; Wnt-5a Protein

Little is known about oral clefts in developing countries. We aimed to identify micronutrient-related and environmental risk factors for oral clefts in Thailand. We tested hypotheses that maternal exposure during the periconceptional period to multivitamins or liver consumption would decrease cleft lip with or without cleft palate (CL ± P) risk and that menstrual regulation supplements would increase CL ± P risk. We conducted a multisite hospital-based case-control study in Thailand. We enrolled cases with CL ± P and 2 live births as controls at birth from the same hospital. Mothers completed a questionnaire. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Eighty-six cases and 172 controls were enrolled. Mothers who took a vitamin (adjusted OR, 0.39; 95% CI: 0.16, 0.94) or ate liver (adjusted OR, 0.26; 95% CI: 0.12, 0.57) were less likely than those who did not to have an affected child. Mothers who took a menstrual regulation supplement were more likely than mothers who did not to have an affected child. Findings did not differ for infants with a family history of other anomalies or with isolated CL ± P. If replicated, our finding that liver decreases CL ± P risk could offer a low-cost primary prevention strategy.

Topics: Animals; Calcium; Case-Control Studies; Cleft Lip; Cleft Palate; Common Cold; Contraceptive Agents, Female; Developing Countries; Diabetes Complications; Environment; Female; Folic Acid; Humans; Infant, Low Birth Weight; Infant, Newborn; Infant, Premature; Iron; Male; Meat; Micronutrients; Preconception Care; Risk Factors; Sex Factors; Swine; Thailand; Tretinoin; Vitamin A; Vitamin B Complex; Vitamins

Retinoic acid (RA) contributes to cleft palate; however, the cellular and molecular mechanisms responsible for the deleterious effects on the developing palate are unclear. Wnt signaling is a candidate pathway in the cleft palate and is associated with RA in organ development; thus, we aim to investigate whether RA-induced cleft palate also results from altered Wnt signaling. Administration of RA to mice altered cell proliferation and apoptosis in craniofacial tissues by regulating molecules controlling cell cycle and p38 MAPK signaling, respectively. This altered cell fate by RA is a crucial mechanism contributing to 100% incidence of cleft palate. Moreover, Wnt/β-catenin signaling was completely inhibited by RA in the early developing palate via its binding and activation with RA receptor (RAR) and is responsible for RA-induced cleft palate. Furthermore, PI3K/Akt signaling was also involved in actions of RA. Our findings help in elucidating the mechanisms of RA-induced cleft palate.

Topics: Animals; beta Catenin; Blotting, Western; Cell Proliferation; Cell Survival; Cells, Cultured; Cleft Palate; Epithelial Cells; Female; In Situ Nick-End Labeling; Mesoderm; Mice; Microscopy, Electron, Scanning; Pregnancy; Tretinoin; Wnt Proteins

Most previous studies focused on a small number of heat shock proteins (Hsps) and their relationships with embryogenesis, and the actual roles of these Hsps in normal and abnormal embryonic development remain unclear. It was found in the present systemic study that except for Grp170, whose expression was not detectable at GD18, all 19 Hsps of Hsp70, Hsp90 and Hsp110 families were expressed in the normal development of embryonic palate tissue in mice, but their expression patterns varied with different Hsps, presenting as a correlation with the developmental phases. In the treatment group by all-trans retinoic acid (atRA), the messenger RNA (mRNA) abundance of HspA1A, HspA1L, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, Hsp90AA1, Hsp90AB1, Grp94, Trap1, Hsp105, Hsp110 and Grp170 was higher in the palates at GD11 (the beginning of palate development), the mRNA abundance of HspA1A, HspA12A and HspA12B was higher at GD18 (before birth) and an mRNA expression peak of HspA1L, HspA8, HspA9, Hsp90AA1, Grp94, Hsp110 and Grp170 was observed at GD17. The mRNA abundance of most genes in atRA-induced cleft palates of the treatment group was different from that of the control group. Grp78, HspA14 and Hsp105 were closely associated with the normal palate development and cleft palate in mouse embryo, possibly as palate development-related genes. Except Grp170, the other genes may be closely associated with the development of mouse palates through participating in the stress response process and/or the antiapoptosis process.

Topics: Analysis of Variance; Animals; Cleft Palate; Embryonic Development; Endoplasmic Reticulum Chaperone BiP; Female; Heat-Shock Proteins; Male; Mice; Mice, Inbred ICR; Palate; RNA, Messenger; Tretinoin

To determine changes on craniofacial growth morphometrically in newborn mice with cleft palate induced by retinoic acid. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS: Gestation day 10 or 12 pregnant female C57BL/6N mice were given a single dose of all-trans retinoic acid (atRA) by gastric intubations via oral gavage. Sixty newborn mice with cleft palate (CP), 52 without CP from the experimental group, and 30 without CP from the control group were collected, and lateral cephalograms were taken of all of the mice.. Cephalometric analysis of the craniofacial skeleton was performed by means of a personal computer.. Inhibition of craniofacial growth was found in the experimental groups but not in the control groups. In the maxillary bone and mandible, the amount of growth was significantly reduced.. These results suggest that craniofacial growth is inhibited in newborn mice with cleft palate induced by retinoic acid.

Topics: Animals; Animals, Newborn; Cephalometry; Cleft Palate; Craniofacial Abnormalities; Embryo, Mammalian; Female; Fetus; Gestational Age; Mice; Mice, Inbred C57BL; Pregnancy; Radiography; Tretinoin

To screen the wnt and fibroblast growth factor (FGF) ligands involved in palatogenesis and cleft palate, and to study the dynamic expression of them in the different stages of palatal development and cleft palate formation.. Mouse model of retinoic acid (RA)-induced cleft palate was set up. At embryo day (ED) 14.5, the palatal tissues of RA-treated group and wild type were collected and prepared for gene-chip analysis. According to the gene-chip results, wnt3, wnt8a, fgf9 and fgf10 were selected and their expression level was detected at ED13.5-15.5 by using semi-quantitative reverse transcription-PCR (RT-PCR).. (1) Gene-chip analysis showed that in RA-induced cleft palate group wnt8a and fgf9 were down-regulated, wnt3 and fgf10 were up-regulated in conversely. (2)During the different stage of the control group palatogenesis, intense expression of wnt3, wnt8a, fgf9 and fgf10 were detected with a continuous dynamic pattern. (3)Compared with the control group, the expression level of wnt3, wnt8a, fgf9 and fgf10 in RA-induced cleft palate showed significant difference, respectively (P < 0.05).. wnt and FGF signaling molecules participate in the palatogenesis, and RA pathway may interact with wnt and FGF signaling pathway.

Topics: Animals; Cleft Palate; Fibroblast Growth Factor 10; Fibroblast Growth Factors; Genotype; Ligands; Mice; Tretinoin; Wnt3 Protein

Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF-β signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all-trans retinoic acid (ATRA)-induced cleft palate in organ culture were tested.. C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFβ3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis.. Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase-9 in MEPM cells were detected in the ATRA group than in the control or the ATRA+FA group. Compared with the control or the ATRA+FA group, ATRA had little effect on TGF-β3 in MEPM cells but significantly inhibited TGF-β receptor II.. Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFβ signaling in MEPM cells were involved in folic acid rescued ATRA-induced cleft palate.

Topics: Animals; Apoptosis; Caspase 9; Cells, Cultured; Cleft Palate; Folic Acid; Mice; Mice, Inbred C57BL; Organ Culture Techniques; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; Teratogens; Transforming Growth Factor beta3; Tretinoin; Vitamin B Complex

The effect of all-trans retinoic acid (atRA) on palatal fusion and the underlying mechanisms were investigated using organ culture. Compared with control group, the atRA-treated group (1 μM and 5 μM) had more medial edge epithelium (ME) remaining within the midline epithelial seam (MES). At 10 μM atRA, the opposing shelves were not in contact at the culture end (72 h). Cell death detection by TUNEL and laminin immunohistochemistry demonstrated that atRA (5 μM) induced apoptosis in mesenchyme and inhibited degradation of basal lamina within MES. Notably, migration and apoptosis of ME cells and degradation of basal lamina within MES markedly represented vehicle control palatal shelves in culture. Additionally, apoptosis was not detected in mesenchyme of control palatal shelves. Immunoblotting analysis revealed that Smad2 and Smad3 were endogenously activated and expression of Smad7 was inhibited during the fusion process. In contrast, atRA treatment abrogated phosphorylation of Smad2 and Smad3 and inducible expression of Smad7 in ME. From these data, it is assumed that inhibition of Smad pathway by atRA in ME may play a critical role in abrogation of the ME cell apoptosis and degradation of the basal laminin, which might contribute to failure of palatal fusion.

Topics: Animals; Apoptosis; Basement Membrane; Cleft Palate; Epithelium; Female; Mice; Mice, Inbred ICR; Models, Animal; Organ Culture Techniques; Palate; Phosphorylation; Pregnancy; Signal Transduction; Smad Proteins; Smad2 Protein; Smad3 Protein; Smad7 Protein; Tretinoin

Palate development depends on complex events and is very sensitive to disruption. Accordingly, clefts are the most common congenital malformations worldwide, and a connection is proposed with fetal exposure to toxic factors or environmental contaminants, such as dioxins. There is increasing evidence that dioxin interferes with all-trans-retinoic acid (atRA), a hormone-like signal derived from vitamin A, which plays an essential role during embryonic development. Although similarities have been described between dioxin-induced toxicity and the outcome of altered atRA signaling during palate development, their relationship needs to be clarified.. We used a genetic approach to understand the interaction between atRA and dioxin and to identify the cell type targeted by dioxin toxicity during secondary palate formation in mice.. We analyzed the phenotype of mouse embryos harboring an atRA-sensitive reporter transgene or bearing null mutations for atRA-synthesizing enzymes (RALDH) or atRA receptors (RAR) and maternally exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at gestation day 10.5.. We found that an intact atRA signal was required to enable TCDD to induce cleft palate. This mandatory atRA signal was generated through the activity of RALDH3 in the nasal epithelium and was transduced by RARγ (RARG) in the nasal mesenchyme, where it notably controlled aryl hydrocarbon receptor (Ahr) transcript levels. TCDD also did not alter the developmental pattern of atRA signaling during palate formation.. TCDD-induced alteration of secondary palate development in the mouse appears to depend on atRA signaling, which controls AHR expression. This mechanism is likely conserved throughout vertebrate evolution and may therefore be relevant in humans.

Topics: Animals; Cleft Palate; Mesoderm; Mice; Mice, Transgenic; Models, Animal; Palate; Polychlorinated Dibenzodioxins; Real-Time Polymerase Chain Reaction; Receptors, Aryl Hydrocarbon; Receptors, Retinoic Acid; Retinal Dehydrogenase; Retinoic Acid Receptor alpha; Retinoic Acid Receptor gamma; Tretinoin

Although the mechanism by which retinoic acid (RA) induces cleft palate has been intensely investigated, some controversies remain. Some researchers argue that RA inhibits apoptosis, resulting in a failure of palatal shelves to fuse, whereas others propose that RA disrupts elevation or retards the growth of palatal shelves. This study investigated the mechanism underlying RA-induced formation of cleft palate in the rat, focusing mainly on the role of apoptosis.. Using an RA-induced cleft palate model of Sprague-Dawley rats described in our previous study, we examined a total of 92 embryos. Retinoic acid was injected intraperitoneally in experimental group animals on embryonic day 11 (D11), a time when our previous study indicated that RA-induced cleft palate was maximally developed. Control animals were treated with normal saline mixed with sesame oil. Timed pregnant rats were killed by an overdose of ether on D13, D14, D15, D16, and D17, and 8 sections were prepared from the anterior to the posterior of the palate. Growth of palatal shelves was evaluated histologically by examining sections stained with hematoxylin-eosin, periodic acid-Schiff, trichrome, and cresyl violet. Differences in apoptosis were monitored using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays.. Histologic examinations revealed underdevelopment of palatal shelves in the experimental group compared with the control group. In the RA-treated group, the overall process of palatal shelf development was delayed 1 day, and palatal elevation was observed. In hard-palate areas of both groups, apoptosis was maximal immediately after the fusion of palatal shelves. In the soft-palate areas, the saline-treated group showed fusion of palatal shelves, whereas the RA-treated group showed retarded growth of palatal shelves that resulted in failure of palatal fusion. Moreover, apoptosis occurred before palatal contact.. Apoptotic manifestations did not differ between RA-induced cleft palates and control palates, suggesting that apoptosis makes a minimal contribution to the cleft palate formed in response to RA. Instead, growth retardation of the palatal shelves appears to play a major role in RA-induced cleft palate.

Topics: Animals; Apoptosis; Cleft Palate; Disease Models, Animal; Female; In Situ Nick-End Labeling; Pregnancy; Rats; Rats, Sprague-Dawley; Staining and Labeling; Tretinoin

Topics: Animals; Apoptosis; Cleft Palate; Female; Pregnancy; Tretinoin

Cleft palate is one of the major malformations induced by retinoic acid in both rodents and humans. The purpose of the present study was to elucidate the mechanism by which all-trans retinoic acid (atRA) induces the cleft palate.. The cell cycle distribution of mouse embryonic palate mesenchymal (MEPM) cells under atRA (100 mg/kg) treatment on gestation day (GD) 10 or GD 12 were measured by immunohistochemistry and flow cytometry. The p21, phospho-Rb, Smad2/3, phospho-Smad2 and phospho-Smad3 protein expression levels were detected by western blot, respectively. Quantitative real-time PCR was performed for p21, Smad2, and Smad3 gene expression in each group under both conditions. Small interfering RNA (siRNA) was applied to inhibit Smad2/3 expression in MEPM cells and the effect was detected by western blot and flow cytometry.. The G(0)/G(1) arrest in MEPM cells in vivo was induced by atRA on GD 10. The protein expression levels of p21, Smad2/3, phospho-Smad2, and phospho-Smad3 were increased, while phospho-Rb was decreased in MEPM after atRA treatment on GD 10. These changes were not observed on the GD 12 group. Moreover, the mRNA expression levels of p21, Smad2, and Smad3 detected by quantitative real-time PCR were almost consistent with their protein expression trends. Furthermore, p21 was partially decreased and G(0)/G(1) arrest was partially released following Smad2/3 siRNA knockdown.. The induction of G(0)/G(1) block by atRA in MEPM cells varied with the development stage of exposure. Our study demonstrated that Smad2/3 regulation of p21 was partly required for atRA-induced cell cycle perturbations in MEPM cells.

Topics: Animals; Cell Cycle; Cleft Palate; Cyclin-Dependent Kinase Inhibitor p21; Disease Models, Animal; Gene Expression Regulation, Developmental; Gene Silencing; Mesoderm; Mice; Palate; RNA, Small Interfering; Smad2 Protein; Smad3 Protein; Teratogens; Tretinoin

To investigate the effects of excessive all-trans retinoic acid (atRA) on palatogenesis in mouse fetuses and level of Smad2 phophorylation (pSmad2).. atRA (80 mg/kg x d) was orally given to mice on embryonic day 11 (E11). Palatal fusion was examined by Hemagglutinin Esterase (HE). The level of Smad2 phophorylation (pSmad2) in medial edge epithelial (MEE) was detected by immunohistochemistry and Western Blot, respectively.. The incidence of cleft palate (CP) was 90% of total fetus in atRA-treated group. Compared with vehicle control group, no pSmad2 was identified in MEE in atRA-treated palatal shelves, which was correlated with the persistence of the MEE and failure of palatal fusion.. Inhibition of Smad phosphorylation might be involved in cleft palate induced by atRA.

Topics: Animals; Cleft Palate; Female; Maternal Exposure; Mice; Mice, Inbred ICR; Phosphorylation; Pregnancy; Random Allocation; Signal Transduction; Smad2 Protein; Tretinoin

The aim of this study was to analyze epigenetic (specifically, DNA methylation) participation in the mechanisms of cleft palate only induced by maternal exposure to all-trans retinoic acid in mice.. Cleft palate only was induced in fetuses by maternal exposure to all-trans retinoic acid. Their secondary palates were excised for analysis. Cytosine extension assay and restriction landmark genomic scanning were performed to analyze DNA methylation status. The expression levels of the DNA methyltransferases were examined by real-time reverse transcriptase-polymerase chain reaction.. Using cytosine extension assay, on gestation day 14.5, the status of DNA methylation within CpG islands and in global DNA was decreased significantly in all-trans retinoic acid-treated groups compared with the controls (p < .01 and p < .05). In the controls, the status within CpG islands on gestation day 14.5 was significantly increased compared with gestation days 13.5 and 18.5 (p < .01). Using real-time reverse transcriptase-polymerase chain reaction, there was no significant change in the expression of DNA methyltransferases, except on gestation day 18.5. Using restriction landmark genomic scanning on gestation day 18.5, five spots (0.49%) in the controls and one spot (0.1%) in all-trans retinoic acid-treated groups were specifically detected.. These results indicate that changes in DNA methylation may play an important role in the manifestation of cleft palate only caused by environmental factors such as maternal exposure to all-trans retinoic acid.

Topics: Animals; Cleft Palate; CpG Islands; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; Epigenesis, Genetic; Female; Gestational Age; Maternal Exposure; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Palate; Restriction Mapping; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

To study the effect of retinoic acid (RA) on cell proliferation and apoptosis of palatal shelves by 5-bromo-2-deoxyuridine (BrdU) and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL).. C57BL/6J mice were used to establish the RA induced cleft palate animal model, in which the pregnant mice were given a single dose of RA at 100 mg/kg body weight on gestation day 10 (GD10) and GD12, respectively. Specimens were prepared for immunohistochemical staining by using BrdU and TUNEL monoclonal antibodies.. The percentages of BrdU positive cells of embryonic palatal mesenchyme (EPM) and medial edge epithelium (MEE) in GD10 RA group were both obviously lower than that of control group. There was no difference between GD12 RA group and control group. Abnormally intense staining of TUNEL was detected in the palatal mesenchymal cells of GD10 day RA group but not in control group and GD12 day RA group.. After exposure of embryonic mice to RA on GD10, the proliferation and apoptosis of palatal mesenchymal cells are increased, this causes the smaller size of shelves and failure of fusion. The MEE cells keep a bilayer midline epithelial seam after exposure on GD12 with normal apoptosis, this indicates that cell apoptosis in MEE cells be not the only process required for palatal fusion.

Topics: Animals; Apoptosis; Cell Proliferation; Cleft Palate; Disease Models, Animal; Epithelial Cells; Epithelium; Female; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Pregnancy; Tretinoin

To evaluate the effects of retinoic acid (RA) on expression of bone morphogenetic protein 7 (BMP-7) in rat fetus with cleft palate, and the effects of RA on proliferation and apoptosis of osteoblasts.. All-trans RA (ATRA) was used to induce congenital cleft palate in Wistar rat BMP-7 mRNA expression in maxillary bone tissue of fetal rats was measured by Northern blotting analysis. Flow cytometry and MTT assay were used to measure the apoptosis and proliferation of ATRA-treated MC-3T3-E1 cells. BMP-7 mRNA and protein expressions in ATRA-treated MC-3T3-E1 cells were detected by RT-PCR and Western blotting analysis.. ATRA could induce cleft palate of rat fetus. The incidence rate of cleft palate induced by 100 mg/kg AT-RA (45.5%) was significantly higher than 50 mg/kg ATRA (12.5%, P < 0.05). BMP-7 mRNA expression decreased in maxillary bone tissue of rat fetus with cleft palate. MC-3T3-E1 cells proliferation treated with 1 x 10(-6) mol/L ATRA decreased by 60%, the cell apoptosis increased by 2 times. BMP-7 mRNA and protein levels in MC-3T3-E1 cells treated with 1 x 10(-6) mol/L ATRA decreased by 60% and 80%, respectively, compared with ATRA-untreated cells (P < 0.05).. BMP-7 may play an important role in embryonic palate development RA may possess the ability to down-regulate cell proliferation through regulation of BMP-7 gene expression.

Topics: 3T3 Cells; Animals; Apoptosis; Base Sequence; Blotting, Northern; Bone Morphogenetic Protein 7; Cell Proliferation; Cleft Palate; DNA Primers; Down-Regulation; Female; Mice; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; Tretinoin

Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior to palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G(1)/S progression of palatal mesenchymal cells through upregulation of p21(Cip1), leading to Rb hypophospholylation. Thus, RA appears to cause G(1) arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.

Topics: Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Cycle Proteins; Cleft Palate; Embryo, Mammalian; Epithelial Cells; Female; Fetal Development; G1 Phase; In Situ Nick-End Labeling; Intubation, Gastrointestinal; Jaw; Male; Maxilla; Mice; Mice, Inbred ICR; Microscopy, Electron, Scanning; Movement; Palate; Pregnancy; S Phase; Tongue; Tretinoin

To investigate the effect of all-trans retinoic acid (atRA) on the development of mouse palates and its possible mechanism, a model of fetal mouse rolling plate organ culture was establishted.. Mouse embryonic palates were explanted on GD12 and cultured in a roller device for 72h induced by atRA in different concentrations from 10(-5) micromol/L to 10(-1) micromol/L.. Similar to development in vivo, palates in control normal fused. Compared with the control, palates development and fusion were promoted in group of 10(-15) micromol/L, while were inhibited in groups of 10(-4) micromol/L or greater, resulting in cleft palate. The proportion of fusion was reduced in a significant dose-response pattern.. In the model, the cultured palates continued developing and fusing. Palatal fusion was promoted by normally atRA in 10(-5) micromol/L level and were inhibited by atRA in 10(-4) micromol/L level or greater, which resulting in cleft palate. These mentioned confirm the validity and reliability of this model.

Topics: Animals; Cleft Palate; Dose-Response Relationship, Drug; Embryo, Mammalian; Female; Male; Mice; Mice, Inbred ICR; Models, Animal; Organ Culture Techniques; Palate; Pregnancy; Tretinoin

We cloned a novel mouse cDNA, Mcpr1 (mouse cleft palate-related gene 1), between retinoic acid (RA)-treated murine embryonic palatal and control shelves by improved subtractive hybridization. Its transcript was identified by Northern blotting. The open reading frame encodes 132 amino acids and shows almost no identity to other genetic products. Mcpr1 expression could be detected extensively in adult mouse tissues and during murine embryonic development. It was identified to be significantly stimulated by RA in murine palatal shelves at embryonic day 12 and in palatal mesenchymal cells in vitro. We demonstrate that MCPR1 protein was localized primarily in the cytoplasm and could be synthesized and secreted by transfected COS-7 cells. Both the secretory and recombinant proteins of Mcpr1 inhibited proliferation of murine embryonic palatal mesenchymal cells and impeded the progression from the G1 to S phase in the cell cycle. The cells were prone to apoptosis after exposure to glutathione S-transferase-MCPR1. Furthermore, knockdown of MCPR1 protein levels by antisense oligodeoxynucleotides promoted progression of cells from the G1 to S phase and completely abolished the RA-induced block of the cell cycle from the G1 to S phase. These findings suggest that Mcpr1 might function as one of the RA-up-regulated genes involved in inhibiting cell proliferation during palatogenesis and RA-induced cleft palate by regulating proliferation and apoptosis of embryonic palatal mesenchymal cells and might even play a role in the development of many other organs.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Blotting, Northern; Cell Proliferation; Cleft Palate; Cloning, Molecular; Female; Gene Library; Glutathione Transferase; Male; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Oligodeoxyribonucleotides, Antisense; Palate; Recombinant Fusion Proteins; Subtraction Technique; Tretinoin

Epidermal growth factor (EGF) and transforming growth factor-alpha (TGFalpha) regulate cell proliferation and differentiation in the embryo. The induction of cleft palate (CP) by all trans-retinoic acid (RA) was associated with altered expression of TGFalpha, EGF receptor, and binding of EGF. This study uses knockout (KO) mice to examine the roles of EGF and TGFalpha in teratogenic responses of embryos exposed to RA.. Pregnant wild-type (WT) mice of mixed genetic background, EGF KO, C57BL/6J, and TGFalpha KO mice were given a single oral dose of RA (100 mg/kg, 10 ml/kg) or corn oil on GD 10 at 12 PM, GD 11 at 12 PM or 4 PM, or GD 12 at 8 AM or 12 PM (plug day = GD 0). GD 18 fetuses were examined for external, visceral, and skeletal effects.. After exposure to RA on GD 12, the incidence of CP in EGF KO was significantly reduced relative to WT. In TGFalpha KO fetuses, RA exposure on GD 10 increased the incidence of CP versus C57BL/6J. The incidence of skeletal defects in the limbs, vertebrae, sternebrae, and ribs were also affected by lack of expression of EGF or TGFalpha with region-specific amelioration or exacerbation of the effects of RA. In TGFalpha KO fetuses, incidences of forelimb long bone and digit defects increased relative to C57BL/6J. In EGF KO fetuses, relative to WT, the incidence of hindlimb oligodactyly was increased. In EGF KO, but not WT, RA produced short, bent radius, humerus, and ulna. Both TGFalpha and EGF KO mice had increased incidences of dilation of the renal pelvis and this was reduced by RA.. RA exposure produced skeletal and visceral defects in all genotypes; however, EGF or TGFalpha KO influenced the incidence and severity of defects. This study supports a role for EGF and TGFalpha in the response to RA.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Cleft Palate; Epidermal Growth Factor; Female; Fetus; Kidney Pelvis; Mice; Mice, Knockout; Phenotype; Pregnancy; Prenatal Injuries; Transforming Growth Factor alpha; Tretinoin

To study the expression of sHsps in normal palate and cleft palate during mouse embryogenesis.. At GD10, gestational mice of the treatment and the control were administered with 80 mg/kg retinoic acid and the same volume vegetable oil separately, and the normal palate and cleft palate of embryos were harvested in GD15-GD17. The relative abundance of sHsps of all samples was measured by reverse transcript polymerase chain reaction (RT-PCR).. In the normal limbs, except that there is no expression of Hspb10 at GD17, all the other Hsps expressed obviously in GD15-GD17. To the normal palates, the expressional abundance of Hsp20, Hsp25, Hsp27, Hsp32, Hspb2, Hspb3, Hspb7 was stable, that of Hsp30, Hspb5 was increased following the embryos aging, and the expressional peak of Hsp10, Hsp22, Hspb4 occurred at GD16. In GD15-GD17, the expressional abundance of Hsp30, Hsp32, Hspb4, Hspb10 of the cleft palates was higher than that of the normal palates, but the expressional abundance of Hsp60, Hspb5, Hspb9 of the cleft palates was lower than that of the normal palates. The expressional models of Hspb9, Hspb 10 of the normal palates were different from those of the cleft palates obviously.. Except that there is no expression of HspblO at GD17, all the other Hsps expressed obviously in GD15-GD17 during normal clefts' development. To different Hsps, there is different expressional characteristic. Hsp30, Hsp32, Hspb4, Hspb10 maybe play a protective role in the stress action of cleft palate, and Hsp10, Hsp60, Hspb5, Hspb9, Hspb10 were maybe relative to cleft palate.

Topics: Animals; Cleft Palate; Embryo, Mammalian; Female; Gene Expression Regulation, Developmental; Heat-Shock Proteins; Male; Mice; Mice, Inbred ICR; Tretinoin

To investigate the effect of all-trans retinoic acid (atRA) on proliferation activity and cell cycle distribution in mouse embryonic palatal mesenchymal (MEPM) cells and the underlying molecular mechanisms.. MEPM cells were prepared from palate shelves of mouse fetal on gestation day 13. Cell viability was determined by MTI assay. Cell cycle distribution and subdiploid population were analyzed by cytometry. The expression of cyclin D and E and phosphorylation of retinoblastoma protein was examined using Western-blot.. atRA remarkably inhibited the growth of MEPM cells in a dose-dependent manner. atRA also caused an increase in the proportion of cells in G0/G1 and a decrease in the proportion of cells in S phase. atRA inhibited expression of cyclins D and E at protein level. Furthermore, atRA treatment reduced phosphorylated Rb.. These data suggested that atRA had antiproliferative activity by modulating G1/S cell cycle regulators and by inhibition of Rb phosphorylation in MEPM cells, which might account for the pathogenesis of cleft palate induced by retinoic acid.

Topics: Animals; Apoptosis; Cell Cycle; Cells, Cultured; Cleft Palate; Embryo, Mammalian; Female; Mesoderm; Mice; Palate; Retinoblastoma Protein; Teratogens; Tretinoin

The present work studied the induction of cleft palate formation in embryos developed from pregnant BALB/c mice treated orally with retinoic acid (RA). Previous studies on mature somatic cell types showed that RA exerted inhibitory effects on inducible nitric oxide synthase (iNOS) production. For the first time, our study has shown that RA actually stimulates significant expression of iNOS at specific zones of the affected embryonic palatal tissues at three consecutive stages, from gestation day 13 (GD13) to day 16 (GD16). Enzymatically, iNOS facilitates intracellular nitric oxide (NO) synthesis from L-arginine. When NO reacts with reactive superoxides it may result in irreparable cell injury. NO was also reported to induce apoptosis in some mammalian cell systems. Based on our findings, we propose that such an increase in NO production might be associated with apoptosis in the embryonic palatal tissues in the RA-treated mice. The detrimental effects of NO resulted in a reduction in proliferating palatal cells and therefore disturbed the normal plasticity of the palatal shelves. With iNOS overexpression, our findings also showed that there was significant concomitant down-regulation in the expressions of Bone Morphogenetic Proteins (BMPs) -2, 4, and 7 with regional variations particularly in the palatal mesenchymal cells for those embryos developing cleft palate. Since specific spatial and temporal expressions of BMPs -2, 4, and 7 are critical during normal palatal morphogenesis, any deficiency in the epithelial-mesenchymal interaction may result in retarding growth at the embryonic palatal shelves. Taken together, our study has demonstrated cleft palate formation in the BALB/c embryos involved overexpression of iNOS and down-regulation of BMPs-2, 4 and 7.

Topics: Animals; Bone Morphogenetic Protein 4; Bone Morphogenetic Proteins; Cleft Palate; Down-Regulation; Gene Expression Regulation, Developmental; Immunohistochemistry; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Teratogens; Time Factors; Tissue Distribution; Tretinoin

The objectives of this study were to (1) compare two techniques (computerized image analysis and visual morphological evaluation) for the assessment of fetal forelimb malformations and (2) increase the robustness of the dose-response curve for forelimb and cleft palate malformations resulting from all-trans retinoic acid (RA) exposure in GD 11 mice.. Pregnant CD-1 mice were administered a single oral dose of all-trans RA (0, 2.5, 10, 30, 60, or 100 mg/kg) on GD 11. GD 18 fetuses were examined for malformations using visual morphological scoring and computerized image analysis.. Dose-dependent changes occurred in the size and shape of the humerus, radius, and ulna based on both assessment methodologies. The most sensitive indicators for the lowest effect level (10 mg/kg) on forelimbs were roundness, a shape measurement determined by image analysis, and visual morphological scoring. For all other bone measurements (proximal and distal width, area, length, and perimeter), the lowest effect level was 30 mg/kg. The maximum effect for limb defects and total malformed fetuses was seen at 60 mg/kg and higher. Incidence of cleft palate increased over the entire range of administered doses reaching a maximum of 74% (100 mg/kg).. Overall, results indicate that computerized image analysis was no more sensitive in detecting changes in the humerus, radius, and ulna than gross visual examination. Dose-response modeling of developmental endpoints yielded comparable benchmark dose levels for long bones and cleft palate that ranged from 0.24 to 7.6 mg/kg all-trans RA. Birth Defects Res B 71:289-295, 2004.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Dose-Response Relationship, Drug; Female; Fetus; Forelimb; Image Processing, Computer-Assisted; Mice; Pregnancy; Tretinoin

To explore the molecular mechanism of cleft palate induced by chemicals.. Retinoic acid was used as a known teratogen to induce cleft palate in ICR mice and a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes that related to cleft palate of ICR mice.. 14 reverse differently and 9 forward differentially expressed clones were obtained. Some clones were selected to be sequenced and aligned to GenBank.. In this study, suppressed Gpc3 and Insulin-Induced protein 1 could affect growth of palate shelves and resulted in cleft palate by reducing the size of the palate shelves. Down-regulation of Ptprs interfered with a cell signal pathway and down-regulation of Tn C inhibited the cell de-adhesion and expression of Egfr, then suppressed Egfr prevented the normal expression of MMPs that influenced the medial edge epithelium disruption and caused cleft palate. Tn C could bind to Ptprs and Gpcs, and HSPGs were ligands for Ptrps. Up-regulate of Rps25 might play a role in cleft palate by excessively apoptosis.

Topics: Animals; Apoptosis; Cleft Palate; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Developmental; Glypicans; Heparan Sulfate Proteoglycans; Male; Membrane Proteins; Mice; Mice, Inbred ICR; Nucleic Acid Hybridization; Tenascin; Tretinoin; Up-Regulation

Proteoglycans are involved in secondary palate formation. In the present study, we focused on two small leucine-rich proteoglycans, decorin and biglycan, because they assembled extracellular matrix molecules such as collagens and modulated signaling pathway of transforming growth factor-beta. To investigate the functions of decorin and biglycan in palatogenesis, we compared their mRNA expression patterns between normal palate and retinoic acid-induced cleft palate in mice by using in situ hybridization analysis during the period of embryonic day 13.5 (E13.5) to E15.5. On E13.5, decorin mRNA was expressed in the epithelia and mesenchyme on the nasal side of the developing secondary palate. During the period the palate shelves were fusing (E14.5), decorin mRNA was strongly expressed in the mesenchyme but its expression pattern was asymmetric; decorin mRNA expression area in the nasal side was broader than that in the oral side. The expression of decorin mRNA was hardly detected in the mesenchyme on either side of the medial edge epithelium. After fusion (E15.5), its expression converged to the mesenchyme just around the palatine bone. Biglycan mRNA was ubiquitously distributed throughout the palatal mesenchyme for the mid-gestation period. Its expression area became limited to the ossification area within the palate after the late gestation period. In the retinoic acid-treated mice, the area of the decorin gene expression expanded to the core region of the palate primordium where little signal was observed in control mice. On the other hand, biglycan in the retinoic acid-treated mice did not show remarkable change in its distribution patterns compared with that in the control mice. These findings suggest that decorin and biglycan play distinct roles in palatogenesis, and decorin was more actively involved in the process of secondary palate formation than biglycan. Up-regulation of decorin gene expression in the retinoic acid-treated mice might influence the pathogenesis of cleft palate.

Topics: Animals; Antineoplastic Agents; Biglycan; Cell Division; Cleft Palate; Decorin; Extracellular Matrix; Extracellular Matrix Proteins; Female; Gene Expression Regulation, Developmental; Male; Mesoderm; Mice; Mice, Inbred ICR; Palate; Pregnancy; Proteoglycans; RNA, Messenger; Signal Transduction; Transforming Growth Factor beta; Tretinoin

During formation of the secondary palate, clefting may result when critical developmental events are altered. The purpose of this study was to reduce the incidence of retinoic acid (RA)-induced cleft palate (CP) in mice by the co-administration of folic acid (FA), methionine (ME) or a combination of both.. Four groups of time-pregnant Swiss Webster mice were injected intraperitoneally with 50 mg/kg RA on gestational day (GD) 10. Likewise, 4.0 mg/kg FA and 187 mg/kg ME were administered on GD 8-11. The experiment included a control group (RA plus H2O) and three experimental groups, (RA plus therapeutic doses of FA, ME, or FA + ME). Necropsies were carried out on GD 18 and pups were analyzed for teratogenic effects.. Litters that received no therapy exhibited 100% CP with individual pups showing 76% susceptibility. Each therapy administered separately reduced the frequency of CP to approximately 6%, and the combination of FA and ME completely prevented the occurrence of RA-induced cleft palate (0%). A second experiment was conducted in which therapy levels were decreased by 25%. Litters that did not receive therapy experienced 100% clefting and individual pups exhibited CP at 86%. These therapies administered separately did not alter significantly the frequency of cleft palate. The combined doses of FA and ME, however, lowered significantly the frequency of cleft palate to 46%. Decreases in limb and tail defects with FA + ME therapy were also observed in both experiments.. Although FA and ME, at appropriate levels, can reduce individually the frequency of RA-induced cleft palate and other defects in mice, the results from the present study suggest that there is an additive interaction between the two therapeutic agents that can reduce further the teratogenic impact of RA. Further studies are needed to assess the mechanism of action of concomitant doses of FA and ME in the reduction of drug-induced birth defects.

Topics: Abnormalities, Drug-Induced; Animals; Bone and Bones; Cleft Palate; Disease Models, Animal; Drug Synergism; Drug Therapy, Combination; Female; Folic Acid; Injections, Intraperitoneal; Methionine; Mice; Pregnancy; Teratogens; Tretinoin

To investigate the morphologic changes of embryonic palatal development exposed to retinoic acid (RA) in mouse, and to detect the significance of the expression of TGFbeta1, TGFbeta3, EGF and BCL2.. The stage of palatal development was examined by light microscopy. S-P immunohistochemistry and in-situ hybridization was used to detect spatio-temporal patterns of expression of TGFbeta1, TGFbeta3, EGF and BCL2 in embryonic palate.. The fetus exposed to RA resulted in formation of small palatal shelves without contact and fusion of each other to form and intact palate. RA can regulate the embryonic palatal expression of genes involved in RA-induced cleft palate.. RA can inhibit the proliferation of MEPM cell to form small palatal shelves and induce abnormal differentiation of MEE cell causing the bi-palatal shelves no contact and fuse with each other, then induce the formation of cleft palate. RA can regulate the spatio-temporal patterns of expression of TGFbeta1, TGFbeta3 and EGF in embryonic palatal processes and the change of special expression of these genes in embryonic palatal processes are involved in RA-induced cleft palate.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Embryo, Mammalian; Epidermal Growth Factor; Female; Mice; Mice, Inbred C57BL; Palate; Transforming Growth Factor beta; Tretinoin

The actual role of programmed cell death (PCD) in embryonic processes and the extrinsic signals that define the death fate in developing cells are still poorly understood. Here, we show that during secondary palate shelf fusion in the mouse, PCD appeared in the medial edge epithelia (MEE) of the anterior region only after shelf contact. Contact was necessary for efficient cell death activation in the MEE. However, exogenous all-trans-retinoic acid (RA) increased cell death independently of contact. Competence to induce cell death by contact or by RA exposure was obtained when the MEE were close to touch. Endogenous RA is a relevant regulator of the secondary palate PCD since this was reduced by a retinol dehydrogenase inhibitor and an RAR specific antagonist. Bmp-7 expression was positively regulated by RA. However, BMP-7 was unable to activate cell death within the palate tissue and NOGGIN, a natural BMP antagonist, did not block PCD. Reduction of PCD at the MEE directly with a caspase inhibitor or by inhibiting retinol dehydrogenase resulted in unfused palate shelves, but adhesion was not affected. In contrast, exogenous RA also blocked fusion, but in this situation the increased cell death within the MEE appeared to affect adhesion, thereby causing cleft palate in vivo.

Topics: Animals; Apoptosis; Bone Morphogenetic Protein 7; Bone Morphogenetic Proteins; Cleft Palate; Female; Gene Expression Regulation, Developmental; In Situ Hybridization; Mice; Palate; Pregnancy; Transforming Growth Factor beta; Tretinoin

It has been shown that apoptosis is involved in normal embryonic development. The aim of the present study was to elucidate the role and alteration of apoptosis in the pathogenesis of cleft palate induced by retinoic acid (RA) and the ectomesenchymal stem (EMS) cells influenced by RA. RA was administered by gavage to pregnant C57BL/6N strain mice in the experimental group, and the control group received oil alone. Pregnant mice were killed at set periods of time thereafter and histologically analyzed. EMS cells explanted from the palatal shelves of embryonic mice were cultured and characterized by immunohistochemistry, growth curves and population-doubling time. The alterations of apoptosis of EMS cells and developing palatal shelves influenced by RA were evaluated by the terminal deoxynucleotidyl transferase-mediated UTP-biotin nick end-labeling (TUNEL) method. RA-treated mice showed formation of cleft palates resulted from the small size of the palatal shelves and their failure to lift. TUNEL staining showed that the number of apoptotic mesenchymal cells in palatal shelves in the RA-treated mice was increased significantly when compared with the control group. The primary culture of EMS cells proceeded successfully. The population-doubling time of RA-treated cells was much longer compared with non-treated EMS cells. RA also dramatically increased the number of apoptotic cells in EMS cells in vitro. We concluded that EMS cells are the crucial cells in palate development. RA could inhibit the proliferation and induced the apoptosis of EMS cells. The inhibition of growth and excess apoptosis of EMS cells may contribute to the formation of cleft palate and other orofacial congenital malformations.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cleft Palate; Female; Male; Mesoderm; Mice; Mice, Inbred C57BL; Palate; Pregnancy; Stem Cells; Tretinoin

To identify the function of these bone morphogenetic proteins (BMP) during pathogenesis of cleft palate, an experimental model was established in BALB/c mice. Cleft palate was induced by exposure to retinoic acid on embryonic day (E)12. The expression of BMP-2,3,4,5 mRNA in normal and abnormal embryonic palatal shelves was then examined from E13 to E16 by in situ hybridization. The results showed that BMP-4 mRNA was expressed strongly and uniformly in normal epithelial cells and dispersed mesenchymal cells on E13. BMP-2,5 mRNA expression appeared only in dispersed mesenchymal cells. With the development of shelves, the staining density of BMP-2,4,5 decreased gradually in mesenchymal cells outside of the condensation and increased inside the condensation. After shelves had fused on E16, no positive signals for BMP-2,4,5 were detected in dispersed mesenchymal cells, but their expression persisted in the condensation. Exposure to retinoic acid delayed the formation of the condensation and decreased BMP-2,4,5 mRNA dramatically in mesenchyme from E13 to E15. BMP-3 mRNA expression were almost negative in either control or retinoic acid-treated groups during all stages. It was concluded that spatial and temporal expression of BMP-2,4,5 was required during normal palatogenesis, and that a deficiency of their mRNA expression may contribute to the pathogenesis of cleft palate.

Topics: Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 3; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein 5; Bone Morphogenetic Proteins; Cleft Palate; Coloring Agents; Disease Models, Animal; Epithelial Cells; Gene Expression Regulation; Growth Substances; In Situ Hybridization; Mesoderm; Mice; Mice, Inbred BALB C; Palate; RNA, Messenger; Transforming Growth Factor beta; Tretinoin

Malformations of the palate were induced in white rat embryos following maternal exposure to retinoic acid (tretinoin). Five experimental groups and the controls were treated by the following protocol: Group 1: pregnant rats received 100 mg retinoic acid (RA)/kg b.w. suspended in corn oil on gestational day (GD) 11.5; Group 2: 20 mg RA/kg b.w. from GD 8-12; Group 3: 20 mg RA/kg b.w. from GD 7.5-11.5; Group 4: 100 mg RA/kg b.w. on GD 10-11; Group 5: 100 mg RA/kg b.w. on GD 10 and 12; Group 6 received corn oil vehicle from GD 7-14.5; and Group 6: served as non-injected controls. In all retinoic acid treated groups, varying degrees of clefts with occasional attempts of fusion were noted. The severity and frequency of the malformations were dependent on dosage or gestational day of drug treatment. Our results indicate that RA, even at the lowest dose tested (20 mg/kg b.w.) severely affects the various tissues constituting the embryonic palatal shelves by altering cell interaction and possibly programmed cell death. These events would then result in lack of or inadequate differentiation with subsequent formation of aberrant craniofacial architecture.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Female; Palate; Pregnancy; Rats; Rats, Wistar; Tretinoin

To observe the changes of programmed cell death (PCD) in C57BL/6N strain mouse cleft palate formation and study related gene of PCD in future.. 16 mice (gestation of 10 days, GD10) were divided into the experimental and control groups randomly, then the mice of the experimental group were dosed with retinoic acid(RA) 80 mg/kg, and the tissue slices of the embryo mouse were prepared for TdT-mediated dUTP nick end labeling (TUNEL) staining at GD13(14)(the 14th hour of the 13th day gestation), GD13(22), GD14(8), GD14(14), GD14(22), GD15(8), GD15(22) and GD16(8) respectively.. In the early development of palate process, the positive index of PCD was significantly higher in the experimental group than in the control group (P < 0.05).. The effects of RA on mesenchymal cells of the early development of palate process in the experimental group induce PCD, which result in the formation of small palate process and failure of fusing of the shelves.

Topics: Animals; Apoptosis; Cleft Palate; Embryo, Mammalian; Female; In Situ Nick-End Labeling; Male; Mice; Mice, Inbred C57BL; Palate; Pregnancy; Tretinoin

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.

Topics: Animals; Cleft Palate; Fetus; Mice; Mice, Inbred ICR; Mice, Knockout; Receptors, Retinoic Acid; Retinoid X Receptors; Teratogens; Transcription Factors; Tretinoin

Retinoic acid (RA) plays an important role in embryogenesis, by regulating morphogenesis, cell proliferation, differentiation, and extracellular matrix production. RA exposure on gestational day (GD) 12 in CD-1 mice results in delayed palatal shelf elevation and subsequent clefts in the secondary palate. Given the dynamic and complex nature of palate development, it is not surprising that this system is susceptible to changes in retinoid levels. There is evidence that experimental manipulation of retinoid status during development alters normal transforming growth factor-beta (TGF-beta) status. To study the role of perturbation in TGF-beta levels in RA-induced cleft palate, gravid CD-1 mice were treated with 70 mg/kg RA on GD 12. We examined changes in TGF-beta proteins and the steady-state level of TGF-beta mRNA within the first 24 hr after exposure. The interactions between RA and TGF-beta s were very complex. RA differentially regulated the mRNA and protein levels of TGF-beta 1. Changes in mRNA steady-state levels were rapid and transient in nature, indicating a direct mediation by RA. Differential regulation was evident, because RA treatment resulted in an increase in TGF-beta 1 mRNA steady levels followed by a decrease in the intracellular and extracellular forms of TGF-beta 1 protein. Moreover, the patterns of localization and levels of TGF-beta 2 and TGF-beta 3 proteins were not dramatically affected, although there was an increase in TGF-beta 3 mRNA steady-state levels. The increases in mRNA steady-state levels for TGF-beta 2 and TGF-beta 3, as for TGF-beta 1, were rapid and transient in nature, again arguing for direct mediation by RA. These data provide evidence for interactions between RA and TGF-beta s, and indicate that RA is capable of differentially regulating TGF-beta isoforms through processes involving different stages of TGF-beta synthesis and secretion. Further, changes in TGF-beta isoforms were observed prior to changes in mesenchyme morphology and must be considered as mediators of RA's effects on mesenchyme development.

Topics: Animals; Cleft Palate; Gestational Age; Immunohistochemistry; Mice; Palate; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Transforming Growth Factor beta; Tretinoin

The growth and differentiation factor transforming growth factor-beta2 (TGFbeta2) is thought to play important roles in multiple developmental processes. Targeted disruption of the TGFbeta2 gene was undertaken to determine its essential role in vivo. TGFbeta2-null mice exhibit perinatal mortality and a wide range of developmental defects for a single gene disruption. These include cardiac, lung, craniofacial, limb, spinal column, eye, inner ear and urogenital defects. The developmental processes most commonly involved in the affected tissues include epithelial-mesenchymal interactions, cell growth, extracellular matrix production and tissue remodeling. In addition, many affected tissues have neural crest-derived components and simulate neural crest deficiencies. There is no phenotypic overlap with TGFbeta1- and TGFbeta3-null mice indicating numerous non-compensated functions between the TGFbeta isoforms.

Topics: Abnormalities, Multiple; Animals; Bone and Bones; Cleft Palate; Craniofacial Abnormalities; Cyanosis; Ear, Inner; Embryonic Induction; Epithelium; Eye Abnormalities; Genes, Homeobox; Heart Defects, Congenital; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Transforming Growth Factor beta; Tretinoin; Urogenital Abnormalities

The face is one of the most intricately patterned structures in human and yet little is known of the mechanisms by which the tissues are instructed to grow, fuse, and differentiate. We undertook a study to determine if the craniofacial primordia used the same molecular cues that mediate growth and patterning in other embryonic tissues such as the neural tube and the limb. Here we provide evidence for the presence of organizer-like tissues in the craniofacial primordia. These candidate organizers express the polarizing signal sonic hedghog (shh) and its putative receptor, patched, as well as fibroblast growth factor 8 and bone morphogeneic protein 2. Shh-expressing epithelial grafts functioned as organizing tissues in a limb bud assay system, where they evoked duplications of the digit pattern. High doses of retinoic acid, which are known to truncate the growth of the frontonasal and maxillary processes and thus produce bilateral clefting of the lip and palate, inhibited the expression of shh and patched but not fgf8, in the craniofacial primordia, and abolished polarizing activity of these tissues. From these studies we conclude that the embryonic face contains signaling centers in the epithelium that participate in craniofacial growth and patterning. In addition, we discuss a novel mechanism whereby retinoids can exert a teratogenic effect on craniofacial morphogenesis independent of its effects on Hox gene expression or neural crest cell migration.

Topics: Animals; Chick Embryo; Cleft Lip; Cleft Palate; Craniofacial Abnormalities; Ectoderm; Embryonic Induction; Endoderm; Epithelium; Face; Gene Expression Regulation, Developmental; Hedgehog Proteins; Humans; Limb Buds; Maxilla; Morphogenesis; Protein Biosynthesis; Proteins; Skull; Teratogens; Trans-Activators; Transcription, Genetic; Tretinoin

A major challenge for biomedical research is the reduction and/or prevention of congenital craniofacial abnormalities which can be induced by some extrinsic toxicants such as retinoic acids (e.g. isotretinoin, Accutane) and glucocorticoids (corticosteroid hormones) during embryonic craniofacial morphogenesis. Our present studies using a genetically susceptible mouse strain (B10.A) indicate that the teratogenic actions of exogenous retinoic acid or glucocorticoid in secondary cleft palate induction can be largely reduced or even completely rescued by subsequent administration of methionine. The greatest reduction in frequency of all-trans retinoic acid- or triamcinolone-induced secondary cleft palate was obtained by a single-dose IP administration of methionine at 187 mg/kg to pregnant mice on E13 21 hr. It appears that detrimental toxic effects were not observed in mice treated with this therapeutic level of methionine. Our present findings support the need for further research into the role of exogenous methionine in cleft palate reduction, that will provide a biological rationale for considering methionine as a therapeutic agent.

Topics: Animals; Cleft Palate; Female; Male; Methionine; Mice; Mice, Inbred Strains; Teratogens; Tretinoin; Triamcinolone

The polychlorinated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin, commonly referred to as dioxin or TCDD, has been shown to cause cleft palate in mice. TCDD displays an interesting interaction with another cleft palate teratogen, retinoic acid (RA): when mice are treated with TCDD and RA simultaneously, palatal clefts can be observed in 100% of offspring of mothers at dose levels far below those required for either agent to produce clefting if given singly. This synergy strongly suggests that the pathways controlled by these agents converge at one or more points in cells of the developing palate. In this study, we examined the effects of TCDD on induction of the type II cellular retinoic acid binding protein (CRABP-II) and the retinoic acid receptor beta (RAR beta) by RA in murine embryonic palate mesenchyme (MEPM) cells. While TCDD alone had no effect on basal levels of expression of either gene, the induction of both genes by RA was strongly inhibited by TCDD. These results represent the first evidence for a direct molecular interaction between the RA and TCDD-mediated signaling pathways.

Topics: Animals; Cells, Cultured; Cleft Palate; Embryo, Mammalian; Female; Gene Expression; Mice; Mice, Inbred ICR; Palate; Polychlorinated Dibenzodioxins; Pregnancy; Receptors, Retinoic Acid; Tretinoin

The diverse effects of retinoids on the development, growth, and homeostasis of vertebrate organisms are mediated in part by three distinct isoforms of retinoic acid receptors (RARs). These proteins, which are structurally and functionally closely related to thyroid hormone receptors and the oncogene product v-ErbA, regulate patterns of gene expression in target tissues. One approach to study the distinct effects of retinoic acid in cells is to subvert this activity of endogenous receptors by expression of dominant negative receptor derivatives. We demonstrate here that RAR alpha, RAR beta, and RAR gamma can be converted into potent negative transcriptional regulators that block wild-type RAR function. Furthermore, these mutant RARs, but not the wild-type receptors, actively repress the basal transcription level of target promoters. When expressed in transgenic mice, the most potent of these inhibitory receptor mutants is apparently able to disturb developmental processes by inducing a cleft palate in transgenic offspring.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Carrier Proteins; Cell Line; Chloramphenicol O-Acetyltransferase; Cleft Palate; Congenital Abnormalities; Genes, Dominant; Humans; Kinetics; Mice; Mice, Transgenic; Molecular Sequence Data; Oncogene Proteins v-erbA; Promoter Regions, Genetic; Protein-Tyrosine Kinases; Receptors, Retinoic Acid; Receptors, Thyroid Hormone; Recombinant Fusion Proteins; Retroviridae Proteins, Oncogenic; Sequence Homology, Amino Acid; Transfection; Tretinoin

The first association study of cleft lip with or without cleft palate (CL/P), with candidate genes, found an association with the transforming growth-factor alpha (TGFA) locus. This finding has since been replicated, in whole or in part, in three independent studies. Here we extend our original analysis of the TGFA TaqI RFLP to two other TGFA RFLPs and seven other RFLPs at five candidate genes in 117 nonsyndromic cases of CL/P and 113 controls. The other candidate genes were the retinoic acid receptor (RARA), the bcl-2 oncogene, and the homeobox genes 2F, 2G, and EN2. Significant associations with the TGFA TaqI and BamHI RFLPs were confirmed, although associations of clefting with previously reported haplotypes did not reach significance. Of particular interest, in view of the known teratogenic role of retinoic acid, was a significant association with the RARA PstI RFLP (P = .016; not corrected for multiple testing). The effect on risk of the A2 allele appears to be additive, and although the A2A2 homozygote only has an odds ratio of about 2 and recurrence risk to first-degree relatives (lambda 1) of 1.06, because it is so common it may account for as much as a third of the attributable risk of clefting. There is no evidence of interaction between the TGFA and RARA polymorphisms on risk, and jointly they appear to account for almost half the attributable risk of clefting.

Topics: Blotting, Southern; Carrier Proteins; Cleft Lip; Cleft Palate; Female; Genes, Homeobox; Haplotypes; Humans; Male; Polymorphism, Restriction Fragment Length; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Retinoic Acid; Transforming Growth Factor alpha; Tretinoin

Retinoic acid (RA) is teratogenic in many species, producing multiple malformations, including cleft palate. The effects of RA which lead to cleft palate vary depending on the stage of development exposed. After exposure of embryonic mice to RA on gestation day (GD) 10, abnormally small palatal shelves form. After exposure on GD 12 shelves of normal size form, but fail to fuse, as the medial cells proliferate and differentiate into a nasal-like epithelium. Growth factors and their receptors play an important role in regulating development, and the expression of EGF receptors, EGF, TGF-alpha, TFG-beta 1, and TGF-beta 2 has been reported in the mouse embryo. In a variety of cell types in culture, these growth factors are capable of regulating proliferation, differentiation, expression of matrix proteins, and other cellular events including epithelial-mesenchymal transformations. The present study examines immunohistochemically the expression of EGF, TGF-alpha, TGF-beta 1, and TGF-beta 2 in the control embryonic palatal shelves from GD 12 to 15 and the effects of RA treatment on GD 10 or 12 on their expression on GD 14 and 16. These growth factors were shown to have specific temporal and spatial expression in the palatal shelf. With advancing development the levels of TGF-alpha decreased while the expression of EGF increased. TGF-beta 2 localization became regional by GD 14-15, with higher levels found in epithelial cells and chondrogenic mesenchyme. TGF-beta 1 occurred in epithelial and mesenchymal cells and distribution did not change substantially with advancing development. RA exposure altered the expression of TFG-alpha, TGF-beta 1, and TGF-beta 2, but significant effects on EGF were not found. The effects on TGF-alpha and TGF-beta 1 expression were dependent on the gestational age exposed. Levels of TGF-alpha on GD 14 decreased after RA exposure on GD 10, but increased after GD 12 exposure. TGF-beta 1 expression in the mesenchyme was increased after exposure on GD 12, but was unaffected by RA on GD 10. After exposure on either day, the levels of TGF-beta 2 increased in GD 14 nasal epithelial cells. Acting in concert, growth factors could regulate events critical to formation of the secondary palate, including cessation of medial epithelial cell proliferation, synthesis of extracellular matrix proteins in the mesenchyme, programmed cell death of medial epithelial peridermal cells, and transformation of basal epithelial medial cells to mesenchymal cells.

Topics: Abnormalities, Drug-Induced; Animals; Cell Differentiation; Cleft Palate; Epidermal Growth Factor; Female; Gene Expression Regulation; Male; Mice; Mice, Inbred C57BL; Morphogenesis; Palate; Pregnancy; Transforming Growth Factors; Tretinoin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is teratogenic in mice, inducing cleft palate and hydronephrosis at doses which are not overtly maternally toxic or embryotoxic. After TCDD exposure the palatal shelves of normal size come into contact, but fail to fuse due to altered differentiation of the medial epithelial cells. These cells continue to express EGF receptors, proliferate, and differentiate into an oral-like stratified squamous epithelium. The present study examines the effect of TCDD on the expression of growth factors which are believed to regulate differentiation and proliferation in the palate. This study also examined the combined effect of TCDD and retinoic acid (RA), since in teratology studies coadministration of these agents results in an enhancement of cleft palate incidence. Embryos were exposed in vivo on Gestation Day (GD) 10 or 12 to TCDD ot TCDD + RA and the palatal shelves were dissected on GD 14-16. Growth factor expression was determined immunohistochemically using antibodies to TGF-alpha, EGF, TGF-beta 1, or TGF-beta 2. The growth factors displayed specific spatial and temporal expression in the palatal shelves. TCDD reduced the expression of TGF-alpha, EGF, and TGF-beta 1 in epithelial and mesenchymal cells. The degree of reduction was generally greater after exposure on GD 10 to TCDD alone or in combination with RA when compared to that on GD 12. The abnormal proliferation and differentiation of TCDD-exposed medial cells may be a response to reduced expression of EGF and TGF-alpha. Low levels of these factors may be related to the previously observed elevated levels of EGF receptors in medial cells. In other systems, low levels of ligand have resulted in upregulation of the EGF receptor. Continued proliferation and altered differentiation could also be attributable to decreased levels of TGF-beta 1, a factor inhibitory to epithelial proliferation. Since TGF-beta 1 stimulates mesenchymal growth and TGF-alpha and EGF stimulate epithelial proliferation, the formation of small shelves after exposure to TCDD + RA on GD 10 may be due to the severe reduction in these factors. Only a slight to moderate reduction in growth factor expression occurs after exposure to TCDD + RA on GD 12 and in this case shelves of normal size form. Since TCDD and RA appear to act in part through pathways that involve TGF-beta 1, in vitro experiments were designed to examine the involvement of TGF-beta 1 in TCDD teratogenicity.(ABSTRACT TRUNCATED AT 400

Topics: Animals; Cleft Palate; Drug Synergism; Epidermal Growth Factor; Female; Immunohistochemistry; Male; Mice; Mice, Inbred C57BL; Polychlorinated Dibenzodioxins; Teratogens; Transforming Growth Factors; Tretinoin

Retinoic acid (RA) has been shown to be teratogenic in many species, and 13-cis-RA is teratogenic in humans. Exposure to RA during embryonic morphogenesis produced a variety of malformations including limb defects and cleft palate. The type and severity of malformation depended on the stage of development exposed. The purpose of this study was to compare the effects of RA exposure in vivo on different stages of palate development. These results were compared to effects observed after exposure in organ culture. The vehicle used in RA dosing was also shown to be a major factor in the incidence of RA-induced cleft palate. For the in vivo studies, RA (100 mg/kg) in 10 ml corn oil/kg was given p.o. on gestation day (GD) 10 or 12, and the embryos were examined on GD 14 and 16. Exposure to RA in an oil:DMSO vehicle resulted in much higher incidences of cleft palate than were observed after dosing with RA in oil only. After exposure on GD 10, to RA, small palatal shelves formed which did not make contact and fuse on GD 14. The medial cells did not undergo programmed cell death. Instead, the medial cells differentiated into a stratified, squamous, oral-like epithelium. The RA-exposed medial cells did not incorporate 3H-TdR on GD 14 or 16, but the cells expressed EGF receptors and bound 125I-EGF. In contrast, RA-induced clefting after exposure on GD 12 did not involve growth inhibition. Shelves of normal size formed and made contact, but because of altered medial cell differentiation did not fuse. Medial cells differentiated into a pseudostratified, ciliated, nasal-like epithelium. This response was produced in vivo at exposure levels which produced cleft palate, and after exposure of palatal shelves to RA in vitro from GD 12-15. The medial cells exposed on GD 12 incorporated 3H-TdR on GD 14, expressed EGF receptors, and bound 125I-EGF. The responses to RA which lead to cleft palate differed after exposure on GD 10 or 12, and the pathways of differentiation which the medial cells followed depended on the developmental stage exposed.

Topics: Animals; Cleft Palate; Corn Oil; Dimethyl Sulfoxide; Epidermal Growth Factor; Gestational Age; Mice; Mice, Inbred C57BL; Microscopy, Electron; Organ Culture Techniques; Palate; Pharmaceutical Vehicles; Tretinoin

TCDD is one of the most toxic man-made compounds and an extremely potent teratogen in mice. Many of its toxic symptoms resemble those seen during vitamin A deficiency. Vitamin A and its derivatives, such as alltrans-retinoic acid (RA), are also teratogenic in mice, as well as many other species. Both TCDD and RA produce cleft palate in susceptible strains of mice. However, while TCDD produces hydronephrosis, RA does not, and TCDD does not produce limb bud defects while RA does. To determine whether TCDD and RA would enhance or antagonize the teratogenic effects of the other compound, C57BL/6N dams were treated po on Gestation Day (gd) 10 or 12 with 10 ml corn oil/kg containing TCDD (0-18 micrograms/kg), RA (0-200 mg/kg), or combinations of the two chemicals. Dams were killed on gd 18 and toxicity and teratogenicity assessed. Coadministration of TCDD and RA had no effect on maternal or fetal toxicity beyond what would be expected by either compound alone. Cleft palate was induced by RA at lower doses on gd 10 than on gd 12, but by TCDD at lower doses on gd 12 than on gd 10. Sensitivity to TCDD-induced hydronephrosis was similar on both gd 10 and 12. The limb bud defects were only observed when RA was administered on gd 10, not when given on gd 12. No other soft tissue or skeletal malformations were related to administration of TCDD or RA. No effect of TCDD was observed on the incidence or severity of limb bud defects induced by RA, nor did RA influence the incidence or severity of hydronephrosis induced by TCDD. However, the incidence of cleft palate was dramatically enhanced by coadministration of the xenobiotic and vitamin. On both gd 10 and 12, the dose-response curves for cleft palate induction were parallel, suggesting some similarities in mechanism between the two compounds. However, combination treatment resulted in a synergistic response that varied with the stage of development and was tissue specific.

Topics: Abnormalities, Drug-Induced; Animals; Body Weight; Bone and Bones; Cleft Palate; Dioxins; Dose-Response Relationship, Drug; Drug Synergism; Female; Hydronephrosis; Mice; Mice, Inbred C57BL; Polychlorinated Dibenzodioxins; Pregnancy; Tretinoin

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and retinoic acid (RA) are both teratogenic in mice. TCDD is a highly toxic, stable environmental contaminant, while RA is a naturally occurring form of vitamin A. Exposure to TCDD induces hydronephrosis and cleft palate, and exposure to RA induces limb defects and cleft palate. Teratology studies previously have shown that the incidence of clefting is higher after exposure to RA + TCDD than would be observed for the same doses of either compound given alone. This study examines the cellular effects which result in cleft palate, after po administration on gestation Day (GD) 10 or 12 of RA + TCDD in corn oil (10 ml/kg total volume). Exposure on GD 10 to 6 micrograms TCDD + 40 mg RA/kg inhibited early growth of the shelves and clefting was due to a failure of shelves to meet and fuse. This effect on mesenchyme was observed in previous studies to occur after exposure on GD 10 to 40 mg/kg RA alone, but not after TCDD alone. After exposure on GD 12 to 6 micrograms TCDD + 80 mg RA/kg, clefting was due to a failure of shelves to fuse after making contact, because the medial cells differentiated into an oral-like epithelium. This response was observed in previous studies to occur after exposure to TCDD alone, but RA alone on GD 12 resulted in differentiation toward nasal-like cells. The interaction between TCDD and RA results in RA-like clefting after exposure on GD 10 and TCDD-like clefting after exposure on GD 12, and this clefting occurs at higher incidences than would occur after the same levels of either agent alone. After exposure on either GD 10 or 12 to RA + TCDD, the programmed cell death of the medial cells does not occur, and these cells continue to express EGF receptors and to bind 125I-EGF. The effects of RA and TCDD may involve modulation of the cells responses to embryonic growth and differentiation factors.

Topics: Administration, Oral; Animals; Autoradiography; Cleft Palate; Dioxins; Drug Synergism; ErbB Receptors; Female; Iodine Radioisotopes; Mice; Mice, Inbred C57BL; Polychlorinated Dibenzodioxins; Pregnancy; Tretinoin; Tritium

Previous observations have indicated that isotretinoin (IT), a drug in common use for therapy of cystic acne, is teratogenic in humans but possesses low embryotoxicity in pregnant mice, probably because of its shorter half-life and limited placental transfer in rodents. In human volunteers and patients, one major blood metabolite of IT is 4-oxo-isotretinoin (4-oxo-IT) which undergoes slower elimination than IT and may itself be a participant in teratogenesis. To investigate the problem of species differences displayed by IT and the role of its metabolism, embryotoxic effects of 4-oxo-IT were examined after its single or repeated intubations into pregnant ICR mice and compared with the effects of a similar regimen of IT. The two compounds were also tested for their relative ability to suppress chrondrogenesis in the in vitro cell and organ culture assays. We found that a single dose of 4-oxo-IT, 100 mg/kg, given on day 11 of gestation (plug day = day 0 of gestation) produced a moderate incidence of limb reduction defects and cleft palate (39% and 27% of surviving fetuses, respectively), while a dose of 150 mg/kg affected virtually every fetus. IT, on the other hand, produced no defects in fetuses exposed to similar dose levels. Repeated intubations with IT, however, resulted in increasing the frequencies of limb reduction defects and cleft palate to levels obtained after 4-oxo-IT administration. We found that a 3-hour interval between IT intubations was more effective in this regard than an 8-hour interval. Repeated IT intubations also uncovered sharper stage-dependency of limb and palatal defects than obtained otherwise.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Abnormalities, Drug-Induced; Animals; Cartilage; Cleft Palate; Female; In Vitro Techniques; Isotretinoin; Limb Deformities, Congenital; Male; Mice; Mice, Inbred ICR; Pregnancy; Teratogens; Tretinoin

In cultured fetal rat bones, cyclohexanetriones that stimulate prostaglandin synthesis inhibited retinoic acid-induced cartilage degradation in a dose-dependent manner. The inhibition by the cyclohexanetrione Ro 31-0521 was reversible, indicating that the effect was not due to cytotoxicity. Excess retinoic acid is teratogenic in rats and adversely affects the normal differentiation of various morphogenetic systems, depending on the time of administration. The following retinoic acid-induced malformations were suppressed by Ro 31-0521: malformations of long bones and of apical phalanges induced on days 13 and 15 of gestation, respectively; spina bifida and tail malformations induced on day 11 of gestation and cleft palate induced on day 15 of gestation. However, cleft palate and other head malformations including exencephaly induced by retinoic acid on day 11 of gestation were not suppressed but even increased by Ro 31-0521. At a high dose, Ro 31-0521 given alone on day 11 of gestation was embryolethal and teratogenic but was not on the tested other days, indicating that the cyclohexanetrione at specific stages and doses also interfered with normal morphogenesis like retinoic acid. Assuming that stimulation of prostaglandin synthesis is the main biological effect of the cyclohexanetriones, our findings suggest that prostaglandins may be involved in mediating retinoid action.

Topics: Abnormalities, Drug-Induced; Animals; Bone Resorption; Cartilage; Cleft Palate; Cyclohexanes; Cyclohexanones; Gestational Age; Limb Deformities, Congenital; Male; Organ Culture Techniques; Prostaglandins; Proteoglycans; Rats; Reproduction; Tretinoin

Chronokinetic synergism, a holistic and extremely sensitive experimental design, has shown in the mouse embryo that site-specific epigenetic forces differentially regulate genesis of the palate (cleft palate) and limb bud organogenesis (shortened stature). Acute exposures of 11-day pregnant mice to minimally effective doses of thymidine or ethanol followed 5 or 8 hr later by minimal exposure to retinoic acid have enabled quantitative and qualitative assay for genomic-epigenetic interactions. These site-specific morphogenetic regulations occurred during palatal genesis from the maxillary prominence of the first pharyngeal arch and during limb bud prechondrogenesis. Thymidine is presumed to induce its response by inhibition of DNA polymerase and hence by transitory cytostatic block. (Embryo size was not detectably changed). Ethanol is interpreted, guilt by associated response, indirectly to interfere with histone regulation of transcription. Two central findings have demonstrated the coordinated regulation of genomic and epigenetic positional information. First, thymidine or ethanol as epigenetic probes for limb prechondrogenesis and palatal precursor cells have activated distinctive site-specific responses. Second, responses to chronokinetic synergisms have indicated that epigenetic regulators for limb and palate dysmorphogenesis may affect distinctly different phases of the cell division cycle and hence induce differential DNA expressions. Although each of palate and limb is concurrently susceptible to epigenetic regulation, their differential intrinsic genomic capabilities appear to have been uncoupled. The putative homeostatic balance of genomic expressions in the palate precursor and the prechondrogenic limb bud cells of the 11-day mouse embryo has been characterized as epigenetically regulated, alternatively expressed, and positionally restricted. We propose that the chronokinetic synergisms have disclosed the existence of distinctive palate-determining genes and stature-determining genes.

Topics: Abnormalities, Drug-Induced; Animals; Cell Cycle; Cell Division; Cleft Palate; Embryo, Mammalian; Ethanol; Genes; Heterozygote; Limb Deformities, Congenital; Mice; Thymidine; Tretinoin

Two closely related retinoids, all-trans and 13-cis retinoic acids, were assessed for their relative activities as teratogens in ICR mice by monitoring the frequency with which either isomer produced discrete dysmorphogenesis of the embryonic limb and the secondary palate. A single oral dose of all-trans retinoic acid at 100 mg/kg on either day 11.5 or 12.0 of gestation (plug day = day one) was maximally effective; more than 90% of the treated embryos developed reduction defects of the limb bones and an equally high percentage also had cleft palate. The limb development was most sensitive on day 11.5 of gestation while the peak susceptibility for palatal clefts began on day 12.0. Under identical experimental conditions, treatment with 100 mg/kg 13-cis retinoic acid produced no apparent teratogenic effects. By assessing the relative incidence of readily identifiable malformations of the limb and palate associated with various doses of the two isomers, we found that 13-cis retinoic acid was four to eight times less embryopathic than all-trans retinoic acid. Since the mechanism of teratogenic action of retinoids is still far from clear, it is suggested that further studies on causative factors will be greatly assisted by the use of these two closely related retinoids, which substantially differ from each other in their teratogenic potency.

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Dose-Response Relationship, Drug; Female; Isomerism; Limb Deformities, Congenital; Mice; Mice, Inbred ICR; Palate; Teratogens; Tretinoin

C57B1 mice were given orally one, two , or three doses of ritinyl palmitate or retinoic acid on day 11, 12, 13, or 14 or pregnancy to determine 1) if it was possible to produce cleft palate at any time during palatal development of the mouse embryo, and 2) the maternal and fetal levels of vitamin A associated with the production of clefts. Whereas a single dose of either substance was sufficient to cause clefts on day 11 or day 12, three doses at 3-hour intervals were necessary to cause clefts on day 14. Most of these clefts were incomplete. Measurement of vitamin A levels in maternal serum and fetuses of dams after administration of retinyl palmitate or retinoic acid on day 11 and day 14 of pregnancy showed that transplacental passage of retinyl palmitate, retinol, and retinoic acid had taken place. Analysis of fetal serum on day 14 showed increase in levels of retinyl palmitate, retinol, and retinoic acid after dosing with retinyl palmitate, and in retinoic acid alone after dosing with retinoic acid. In both cases peak levels were observed within 3 hours. Our findings suggest that the effect of vitamin A on the developing mouse palate is direct, occurring soon after its administration, and that duration of exposure is as important as its concentration in vivo. The apparent change in sensitivity of mouse palate between day 11 and day 14 of pregnancy is discussed.

Topics: Animals; Cleft Palate; Dose-Response Relationship, Drug; Female; Gestational Age; Mice; Mice, Inbred C57BL; Palate; Pregnancy; Tretinoin; Vitamin A

A fixed-exposure culture technique is described using palatal shelves removed from mouse fetuses on day 14 of pregnancy. In this technique growth is eliminated as a possible variable. The addition of retinyl palmitate, retinol, or retinoic acid to culture medium at concentrations similar to those found associated with cleft palates produced in vivo after dosing dams with vitamin A on day 14 prevented fusion of explanted shelves in vitro after a 24-hour exposure period. Retinoic acid prevented fusion of explants after as little as 4 hours exposure. Retinoic acid was more active in vitro than retinol, which was itself eight to ten times more active than retinyl palmitate. An explanation of the effect of vitamin A on the fusing mouse palate is give based on altered glycoprotein synthesis.

Topics: Animals; Cleft Palate; Culture Media; Culture Techniques; Female; Germ Layers; Mice; Mice, Inbred C57BL; Palate; Pregnancy; Tretinoin; Vitamin A

Both retinoic acid and retinyl acetate, administered in high doses on days 13--15 of gestation, are capable of causing a 90 per cent incidence of cleft palate in Charles River rats. However, an attempt to develop as in vivo rabbit model system for the induction of clefts via hypervitaminosis A was unsuccessful. In the rat, the retinoic acid form of vitamin A is the more potent teratogen, inducing clefts at less than half the dose required to produce them with retinyl acetate. Histologic examination of fetal rat heads confirmed the biochemical evidence that retinoic acid is the more potent teratogen. Both forms of vitamin A prevented palatal shelf reorientation from occurring at the correct gestational age. The retinyl acetate treatment delayed the rotation for approximately 12 hours, the retinoic acid for at least 48 hours.

Topics: Animals; Cleft Palate; Disease Models, Animal; Diterpenes; Palate; Rabbits; Rats; Retinaldehyde; Retinyl Esters; Teratogens; Tretinoin; Vitamin A

Topics: Abnormalities, Drug-Induced; Animals; Cleft Palate; Ectromelia; Female; Forelimb; Hindlimb; Limb Deformities, Congenital; Mice; Mice, Inbred DBA; Pregnancy; Teratogens; Time Factors; Tretinoin; Vitamin A