Mouse Embryonic Palatal Mesenchymal Research Articles

Depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchymal cells.

Bone Morphogenetic Protein (BMP) signaling is essential for craniofacial development, though little is known about the mechanisms that govern BMP secretion. We show that depolarization induces calcium-dependent BMP4 release from mouse embryonic palate mesenchyme. We show endogenous transient changes in intracellular calcium occur in cranial neural crest cells, the cells from which embryonic palate mesenchyme derives. Waves of transient changes in intracellular calcium suggest that these cells are electrically coupled and may temporally coordinate BMP release. These transient changes in intracellular calcium persist in palate mesenchyme cells from embryonic day 9.5 to 13.5 mice. Disruption of a potassium channel called Kcnj2 significantly decreases the amplitude of calcium transients and the ability of cells to secrete BMP. Kcnj2 knockout mice have cleft palate and reduced BMP signaling. Our data suggest that temporal control of developmental cues is regulated by ion channels, depolarization, and intracellular calcium for mammalian craniofacial morphogenesis.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin Suppresses Mesenchymal Cell Proliferation and Migration Through miR-214-3p in Cleft Palate.

The aetiology of CL/P is complicated, with both genetic and environmental factors. This study aimed to investigate the association between TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) exposure and changes in the expression of miR-214-3p in the context of cleft palate. In this study, we established a fetal mouse cleft palate model using TCDD and differentially expressed miRNAs were analysed by microarray analysis and verified by qRT-PCR. Finally, we demonstrated the effects of TCDD and microRNAs on the proliferation and migration of mesenchymal cells by using CCK8, EDU, Transwell, and wound-healing assays. Our findings revealed significant upregulation of miRNAs such as miR-214-3p, miR-296-5p, and miR-33-5p in the TCDD intervention group, while miRNAs like miR-92a-3p, miR-126a-3p, and miR-411-5p were significantly downregulated. Notably, qRT-PCR testing confirmed a significant difference in miR-214-3P expression. Further investigations involved the overexpression of miR-214-3p, reducing cell proliferation and migration in primary mouse embryonic palatal mesenchymal (MEPM) cells. These results are consistent with the finding that TCDD suppresses palatal mesenchymal cell proliferation and migration through miR-214-3p. In conclusion, miR-214-3p probably plays a role in TCDD-induced cleft palates in mice.

Retinoic Acid Upregulates METTL14 Expression and the m6A Modification Level to Inhibit the Proliferation of Embryonic Palate Mesenchymal Cells in Cleft Palate Mice.

Cleft palate only (CPO) is one of the most common craniofacial birth defects. Environmental factors can induce cleft palate by affecting epigenetic modifications such as DNA methylation, histone acetylation, and non-coding RNA. However, there are few reports focusing on the RNA modifications. In this study, all-trans retinoic acid (atRA) was used to simulate environmental factors to induce a C57BL/6J fetal mouse cleft palate model. Techniques such as dot blotting and immunofluorescence were used to find the changes in m6A modification when cleft palate occurs. RNA-seq and KEGG analysis were used to screen for significantly differentially expressed pathways downstream. Primary mouse embryonic palate mesenchymal (MEPM) cells were successfully isolated and used for in vitro experimental verification. We found that an increased m6A methylation level was correlated with suppressed cell proliferation in the palatine process mesenchyme of cleft palate mice. This change is due to the abnormally high expression of m6A methyltransferase METTL14. When using siRNAs and the m6A methyltransferase complex inhibitor SAH to interfere with the expression or function of METTL14, the teratogenic effect of atRA on primary cells was partially alleviated. In conclusion, METTL14 regulates palatal mesenchymal cell proliferation and cycle-related protein expression relies on m6A methylation modification, affecting the occurrence of cleft palate.

Inhibition of Cxcr4 Disrupts Mouse Embryonic Palatal Mesenchymal Cell Migration and Induces Cleft Palate Occurrence.

Many processes take place during embryogenesis, and the development of the palate mainly involves proliferation, migration, osteogenesis, and epithelial-mesenchymal transition. Abnormalities in any of these processes can be the cause of cleft palate (CP). There have been few reports on whether C-X-C motif chemokine receptor 4 (CXCR4), which is involved in embryonic development, participates in these processes. In our study, the knockdown of Cxcr4 inhibited the migration of mouse embryonic palatal mesenchymal (MEPM) cells similarly to the use of its inhibitor plerixafor, and the inhibition of cell migration in the Cxcr4 knockdown group was partially reversed by supplementation with C-X-C motif chemokine ligand 12 (CXCL12). In combination with low-dose retinoic acid (RA), plerixafor increased the incidence of cleft palates in mice by decreasing the expression of Cxcr4 and its downstream migration-regulating gene Rac family small GTPase 1 (RAC1) mediating actin cytoskeleton to affect lamellipodia formation and focal complex assembly and ras homolog family member A (RHOA) regulating the actin cytoskeleton to affect stress fiber formation and focal complex maturation into focal adhesions. Our results indicate that the disruption of cell migration and impaired normal palatal development by inhibition of Cxcr4 expression might be mediated through Rac1 with RhoA. The combination of retinoic acid and plerixafor might increase the incidence of cleft palate, which also provided a rationale to guide the use of the drug during conception.

Transcriptome sequencing analysis of the role of β-catenin in F-actin reorganization in embryonic palatal mesenchymal cells.

Palatogenesis is a highly regulated and coordinated developmental process that is coordinated by multiple transcription factors and signaling pathways. Our previous studies identified that defective palatal shelf reorientation due to aberrant mesenchymal β-catenin signaling is associated with Filamentous actin (F-actin) dysregulation. Herein, the underlying mechanism of mesenchymal β-catenin in regulating F-actin cytoskeleton reorganization is further investigated. Firstly, β-catenin silenced and overexpressed mouse embryonic palatal mesenchymal (MEPM) cells were established by adenovirus-mediated transduction. Subsequently, we compared transcriptomes of negative control (NC) group, β-catenin knockdown (KD) group, or β-catenin overexpression group respectively using RNA-sequencing (RNA-seq), and differentially expressed genes (DEGs) screened were further identified by quantitative real-time polymerase chain reaction (qRT-PCR). Finally, in vivo experiments further verified the expression change of critical molecules. We discovered 184 and 522 DEGs in the knockdown and overexpression groups compared to the NC group, respectively (adjusted P<0.05; |fold change| >2.0). Among these, 106 DEGs were altered in both groups. Gene Ontology (GO) enrichment analysis relating to biological functions identified cytokine-cytokine receptor interaction, and positive modulation of cellular migration. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment assessment indicated that these DEGs were chiefly linked by the regulation of signaling receptor activity and chemokine signaling pathways. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that the similar expression trend of serum amyloid A3 (Saa3) and CXC-chemokine ligand 5 (Cxcl5) possibly involved in cytoskeletal rearrangement with RNA-seq data. Experiments in vivo displayed that no significant expression change of Saa3 and Cxcl5 was observed in β-catenin knockout and overexpressed mouse models. Our study provides an expression landscape of DEGs in β-catenin silenced and overexpressed MEPM cells, which emphasizes the important role of processes such as chemotactic factor and cell migration. Our data gain deeper insight into genes associated with F-actin reorganization that is regulated by β-catenin either directly or by another route, which will contribute to further investigation of the exact mechanism of mesenchymal β-catenin/F-actin in palatal shelf reorientation.

Spatiotemporal MicroRNA-Gene Expression Network Related to Orofacial Clefts

Craniofacial structures change dynamically in morphology during development through the coordinated regulation of various cellular molecules. However, it remains unclear how these complex mechanisms are regulated in a spatiotemporal manner. Here we applied natural cubic splines to model gene and microRNA (miRNA) expression from embryonic day (E) 10.5 to E14.5 in the proximal and distal regions of the maxillary processes to identify spatiotemporal patterns of gene and miRNA expression, followed by constructing corresponding regulatory networks. Three major groups of differentially expressed genes (DEGs) were identified, including 3,927 temporal, 314 spatial, and 494 spatiotemporal DEGs. Unsupervised clustering further resolved these spatiotemporal DEGs into 8 clusters with distinct expression patterns. Interestingly, we found 2 clusters of differentially expressed miRNAs: 1 had 80 miRNAs monotonically decreasing and the other had 97 increasing across developmental stages. To evaluate the phenotypic relevance of these DEGs during craniofacial development, we integrated data from the CleftGeneDB database and constructed the regulatory networks of genes related to orofacial clefts. Our analysis revealed 2 hub miRNAs, mmu-miR-325-3p and mmu-miR-384-5p, that repressed cleft-related genes Adamts3, Runx2, Fgfr2, Acvr1, and Edn2, while their expression increased over time. On the contrary, 2 hub miRNAs, mmu-miR-218-5p and mmu-miR-338-5p, repressed cleft-related genes Pbx2, Ermp1, Snai1, Tbx2, and Bmi1, while their expression decreased over time. Our experiments indicated that these miRNA mimics significantly inhibited cell proliferation in mouse embryonic palatal mesenchymal (MEPM) cells and O9-1 cells through the regulation of genes associated with cleft palate and validated the role of our regulatory networks in orofacial clefts. To facilitate interactive exploration of these data, we developed a user-friendly web tool to visualize the gene and miRNA expression patterns across developmental stages, as well as the regulatory networks (https://fyan.shinyapps.io/facebase_shiny/). Taken together, our results provide a valuable resource that serves as a reference map for future research in craniofacial development.

Mechanism of cleft palate in C57BL/6N mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin

Objective: To explore the molecular mechanism of cleft palate in mice induced by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). Methods: The pregnant mice were randomly divided into TCDD-treated group (n=42) and control group (n=42). TCDD-treated group was given by gavage a single dose of TCDD (64 μg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunofluorescence. The localization and expression of maternally expressed gene3 (MEG3) in mouse embryonic palatal mesenchymal cells was detected by situ hybridization and real-time PCR (RT-PCR). The key protein expressions of transforming growth factor-β (TGF-β)/Smad signaling pathway in mouse embryonic palatal mesenchyme were analyzed by Western blotting. The interaction of MEG3 and TGF-β receptor Ⅰ (TGF-βRⅠ) was examined by RNA binding protein immunoprecipitation (RIP). Results: At GD13 and GD14, compared with the control group, the ratio of BrdU-positive cells in the palatal mesenchyme of TCDD-treated fetuses decreased significantly (GD13, t=6.66, P=0.003; GD14, t=6.56, P=0.003). However, at GD15, the ratio of BrdU-positive cells was significantly increased (t=-5.98, P=0.004). MEG3 was mainly expressed in the nuclei of fetal mouse palatal mesenchymal cells, and the expression of MEG3 in TCDD group was significantly increased at GD13, GD14 and GD15(GD13, t=39.28, P=0.012; GD14, t=18.75, P=0.042; GD15, t=28.36, P=0.045). At GD14, TCDD decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells (p-Smad2, t=9.48, P=0.001;Smad4, t=63.10, P=0.001), whereas the expression of Smad7 was significantly increased at GD14 (t=30.77, P<0.001). The results of the RIP experiment showed that the amount of TGF-βRⅠ-bound MEG3 in mouse embryonic palatal mesenchymal cells in the TCDD group (23.940±1.301) was higher than that in the control group (8.537±1.523)(t=24.55, P<0.001). Conclusions: MEG3 is involved in the suppression of mouse embryonic palatal mesenchymal cell proliferation, functioning at least in part via interacting with the TGF-βRⅠ protein and thereby suppressing Smad signaling in the context of TCDD induced cleft palate.

Comparative analysis of mouse embryonic palatal mesenchymal cells isolated by two primary culture methods

Cell culture plays a vital role in mechanism research, as a supplement to experiments in vivo. At present, there are two main methods to obtain mouse embryonic palatal mesenchymal (MEPM) cells, while no systematic investigation about characteristics of cells using these two methods to get a clear application. In this study, using the traditional two-step primary culture method as the control group, we found that the MEPM cells of the simplified one-step method and the control group were both consistent with the surface markers of mesenchymal stem cells. The vimentin (mesenchymal marker) was positive, cytokeratin 14 (CK14, epithelium marker) was negative. In addition, along with the increasing passages, the cells cultured by two methods had similar changes with a decreasing proliferation activity, increasing apoptosis rate, declining migration, diminishing stemness, and obvious senescence. Moreover, the senescence of MEPM cells in the one-step group was lower than that in the control group. Our findings indicate that there are no significant differences between the two methods and one-step method can be a good method in investigating palatal development mechanisms by shortening the processing of the MEPM cell culture and may reduce the damage to the cells done by more enzymes and processing.

Dexamethasone Suppresses Palatal Cell Proliferation through miR-130a-3p

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.

The role of lncRNA Meg3 in the proliferation of all-trans retinoic acid-treated mouse embryonic palate mesenchymal cells involves the Smad pathway

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.

MicroRNA-124-3p Plays a Crucial Role in Cleft Palate Induced by Retinoic Acid.

Cleft lip with/without cleft palate (CL/P) is one of the most common congenital birth defects, showing the complexity of both genetic and environmental contributions [e.g., maternal exposure to alcohol, cigarette, and retinoic acid (RA)] in humans. Recent studies suggest that epigenetic factors, including microRNAs (miRs), are altered by various environmental factors. In this study, to investigate whether and how miRs are involved in cleft palate (CP) induced by excessive intake of all-trans RA (atRA), we evaluated top 10 candidate miRs, which were selected through our bioinformatic analyses, in mouse embryonic palatal mesenchymal (MEPM) cells as well as in mouse embryos treated with atRA. Among them, overexpression of miR-27a-3p, miR-27b-3p, and miR-124-3p resulted in the significant reduction of cell proliferation in MEPM cells through the downregulation of CP-associated genes. Notably, we found that excessive atRA upregulated the expression of miR-124-3p, but not of miR-27a-3p and miR-27b-3p, in both in vivo and in vitro. Importantly, treatment with a specific inhibitor for miR-124-3p restored decreased cell proliferation through the normalization of target gene expression in atRA-treated MEPM cells and atRA-exposed mouse embryos, resulting in the rescue of CP in mice. Taken together, our results indicate that atRA causes CP through the induction of miR-124-3p in mice.

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes.

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

LncRNA Meg3-mediated regulation of the Smad pathway in atRA-induced cleft palate

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.

Down-regulation of miR-381-3p inhibits osteogenic differentiation of mouse embryonic palatal mesenchymal cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin-induced cleft palate of fetal mice

To investigate the correlation between down-regulation of miR-381-3p and inhibition of osteogenic differentiation of mouse embryonic palatal mesenchymal (MEPM) cells in 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD)-induced cleft palate of fetal mice. Thirty-two pregnant mice were randomly divided into TCDD group and control group, 16 in each group. On embryonic day 10.5 (E10.5), the pregnant mice in TCDD group were orally administrated with TCDD at dosage of 28 μg/kg, while the pregnant mice in control group received equivalent corn oil. The pregnant mice in each group were sacrificed on E13.5 and E14.5, fetal palates were collected for analysis. The expression of miR-381-3p was detected by real-time fluorescent quantitative PCR and the protein expressions of runt- related transcription factor 2 (RUNX2) and osteopontin (OPN) were detected by Western blot. MEPM cells were extracted from fetal palates on E14.5 in control group and passaged. The 3rd passage cells were cultured with TCDD at dosage of 10 nmol/L for 0, 0.5, 1, 2, and 3 days. The expression of miR-381-3p was detected after 0, 0.5, 1, 2, and 3 days and the protein expressions of RUNX2 and OPN were detected after 0, 1, 2, and 3 days. Then, the 3rd passage cells were divided into 4 groups. The MEPM cells were transfected with miR-381-3p inhibitor (inhibitor group), NC inhibitor (NC inhibitor group) and miR-381-3p mimics (mimics group), NC mimics (NC mimics group) for 48 hours, respectively. And the expressions of miR-381-3p and the protein expressions of RUNX2 and OPN were detected. On E13.5 and E14.5, 96 fetal mice in control group and 92 in TCDD group were obtained. The bilateral palates contacted in control group on E14.5, and a gap between the bilateral palates existed in TCDD group. On E13.5 and E14.5, the relative expressions of miR-381-3p and RUNX2 and OPN proteins were significant lower in TCDD group than in control group ( P<0.05). The relative expression of miR-381-3p at 0.5 and 1 day after TCDD treatment of MEPM cells were significantly lower than that at 0 day ( P<0.05); then, the relative expressions at 2 and 3 days significantly increased, showing no significant difference when compared with that at 0 day ( P>0.05). The relative expressions of RUNX2 and OPN proteins at 1, 2, and 3 days were significantly lower than that at 0 day ( P<0.05). The relative expressions of miR-381-3p and RUNX2 and OPN proteins significantly lower in inhibitor group than in NC inhibitor group ( P<0.05) and higher in mimics group than in NC mimics group ( P<0.05). Down-regulation of miR-381-3p expression may be associated with inhibition of osteogenic differentiation of MEPM cells in TCDD-induced cleft palate of fetal mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and TGFβ3-Mediated Mouse Embryonic Palatal Mesenchymal Cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental teratogenic effector for cleft palate. Transforming growth factor 3 (TGF-β3) is an essential growth factor for palatogenesis. The objective of this study is to clarify the effects of TCDD and TGF-β3 in mouse embryonic palatal mesenchymal (MEPM) cells. The effects of 10 nM TCDD, 10 ng/mL TGF-β3, or a combination of 10 nM TCDD and 10 ng/mL TGF-β3 on MEPM cells were revealed by cell and biological methods. With the increase in TCDD (0.5-10 nM), the expression of TGF-β3 increased, but at TCDD concentrations greater than 10 nM, the expression of TGF-β3 reduced. The viabilities of MEPM cells decreased in the 10 nM TCDD-treated group. But the viabilities increased in the 10 ng/mL TGF-β3-treated group, and the viabilities were intermediate in the group treated with a combination of 10 nM TCDD and 10 ng/mL TGF-β3. This phenomenon was the same as that of the motilities. In addition, we found that the expression of p-Smad2, p-Smad3,and Smad7 were increased by TCDD, TGF-β3, combination of TCDD and TGF-β3, but the expression of Smad4 were decreased by TCDD, TGF-β3, combination of TCDD and TGF-β3. These data revealed that TCDD and TGF-β3 interacted and affected MEPM cells.

2,3,7,8-Tetrachlorodibenzo-p-Dioxin and TGF-β3 Mediated-Mouse Embryonic Palatal Mesenchymal Cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a well-known environmental teratogenic agent for cleft palate. But transforming growth factor β3 (TGF-β3) is an essential growth factor for palatogenesis. This study is to clarify effects of TCDD and TGF-β3 in mouse embryonic palatal mesenchymal (MEPM) cells. The result showed that with increase of TCDD (0.5 nM-10 nM), the expression of TGF-β3 increased, but after 10 nM TCDD, the expression of TGF-β3 reduced. The viabilities of MEPM cells decreased in 10 nM TCDD-treated group. But the viabilities increased in 10 ng/mL TGF-β3-treated group, or the viabilities were between that of them in combination of 10 nM TCDD and 10 ng/mL TGF-β3-treated group. This phenomenon was the same as the motilities. In addition, we found that the expression of phosphorylated Smad2/3 and Smad7 was increased by 10 nM TCDD, 10 ng/mL TGF-β3, or combination of 10 nM TCDD and 10 ng/mL TGF-β3 induced, but the expression of Smad4 was decreased. These data revealed that the TGF-β/Smad signaling pathway affected TCDD and TGF-β3 in MEPM cells.

Mechanism of cleft palate in C57BL/6N mice induced by retinoic acid

Objective: To investigate the mechanism of cleft palate in mice induced by excessive all-trans retinoic acid (atRA). Methods: The pregnant mice were randomly divided into atRA-treated group (n=27) and control group (n=27). atRA-treated group was given by gavage a single dose of atRA (100 mg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The expressions of Smad2, phospho-Smad2 (p-Smad2), Smad4 and Smad7 in mouse embryonic palatal mesenchyme were analyzed by Western blotting. Results: At GD13-GD15, compared with the control, the ratio of BrdU-positive cells in the palatal mesenchyme of atRA-treated fetuses decreased significantly (P<0.05), especially at GD14, atRA inhibition rate was (65.4±1.7)%. Moreover, atRA decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells, whereas the expression of Smad7 was significantly increased at GD14 and GD15. Conclusions: atRA may lead to cleft palate by inhibiting the activation of Smad signaling pathway and affecting the proliferation of palatal mesenchymal cells.

Involvement of Notch2 in all‑trans retinoic acid‑induced inhibition of mouse embryonic palate mesenchymal cell proliferation.

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.

Excessive retinoic acid inhibit mouse embryonic palate mesenchymal cell growth through involvement of Smad signaling

ABSTRACTAll-trans retinoic acid (atRA), the oxidative metabolite of retinoic acid (RA), is essential for palatogenesis. Overdose RA is capable of inducing cleft palate in mice and humans. Normal embryonic palatal mesenchymal (EPM) cell growth is crucial for shelf growth. Smad signaling is involved in many biological processes. However, it is not much clear if atRA could affect Smad signaling during EPM cells growth. In this study, the timed pregnant mice with maternal administration of 100 mg/kg body weight of RA by gastric intubation were cervical dislocation executed to evaluate growth changes of palatal shelves by hematoxylin and eosin (H&E) staining. At the same time, a primary mouse EPM (MEPM) cell culture model was also established. MEPM cells were treated with atRA (0.1, 0.5, 1, 5 and 10 μM) for 24, 48 and 72 h. The results indicated that the sizes of the shelves were smaller than those in control. AtRA inhibited MEPM cell growth with both increasing concentration and increasing incubation time, especially at 72 h in vitro. Moreover, atRA significantly increased the mRNA and protein expression levels of Smad7 (P < .05), but the mRNA and protein expression levels of PCNA were reduced (P < .05). We also found atRA inhibited phosphorylation of Smad2 compared with untreated group (P < .05). However, the protein and mRNA levels of Smad2 did not change both in atRA-treated and untreated group (P > .05). We demonstrated that RA induced inhibition of MEPM cell growth that could cause cleft palate partly by down-regulation of Smad pathway.

2,3,7,8-Tetrachlorodibenzo-p-dioxin Mediated Cleft palate by Mouse Embryonic Palate Mesenchymal Cells

ObjectiveTo evaluate the effects of mouse embryonic palatal mesenchymal (MEPM) cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced. Study designAn experimental study at Xinxiang Medical University. MethodsPrimary MEPM cells were derived from palatal tissue on 30 pregnant C57BL/6 female mice (embryonic day13, GD13). The MEPM cells were placed in a humidified incubator at 37°C with 5% CO2 atmosphere with media replaced every other day. The third passage cells were seeded, and one part of cells were treated with 10nM TCDD (TCDD group). And others were treated with DMSO (≤0.05%, as control group). After 72h, MTT assay was analysed cell viability. Scratch wound-healing was analysed cell motility. Flow cytometry was analysed cell apoptosis and cycle. Western blot was analysed the expression of cyclinE, CDK2, P16 and P21. ResultsTCDD inhibited the growth and migration of MEPM cells, while increased cell apoptosis. TCDD exposure inhibited the progression of cells from G1 to S phase and tended to reduce the number of cells entering the G2/M phase. TCDD inhibited expression of cyclinE and CDK2 at the protein level, instead increased the expression of P16 and p21 proteins. ConclusionsTCDD might induce cleft palate by altering MEPM cells.