Palatal Mesenchymal Cells Research Articles

TCDD inhibited the osteogenic differentiation of human fetal palatal mesenchymal cells through AhR and BMP-2/TGF-β/Smad signaling

Exposure to environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes cleft palate at high rates, but little is known about the underlying biological mechanisms. In the present study, we cultured osteoblasts from human fetal palate mesenchymal cells (hFPMCs) to explore the effects of TCDD on osteogenic differentiation. The results showed that TCDD significantly decreased cell proliferation, alkaline phosphatase (ALP) activity and calcium deposition. RNA analyses and protein detection demonstrated that TCDD downregulated a wide array of pro-osteogenic biomarkers. Further investigation of the underlying molecular mechanisms revealed that exposure to TCDD activated aryl hydrocarbon receptor (AhR) signaling and inhibited BMP-2/TGF-β1/Smad pathway molecules. The inactivation of AhR signaling using CRISPR/Cas9-mediated AhR deletion or by genetic siRNA knockdown significantly blocked the effects induced by TCDD, suggesting a critical role of AhR activation in the TCDD-mediated inhibition of hFPMC osteogenic differentiation. The cotreatment with TGF-β1 or BMP-2 and TCDD significantly relieved the activation of AhR and rescued the impairment of osteogenesis caused by TCDD. Taken together, our findings indicated that TCDD inhibited the osteogenic differentiation of hFPMCs via crosstalk between AhR and BMP-2/TGF-β1/Smad signaling pathway.

MiR-106a-5p modulates apoptosis and metabonomics changes by TGF-β/Smad signaling pathway in cleft palate

BackgroundThe molecular mechanisms of abnormal palatogenesis were investigated in this study. A key regulator, miR-106a-5p, and its target pathway were analyzed. ObjectivesThis 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. MethodDifferentially 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). ResultsThe 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. ConclusionsThe 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.

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.

Mouse embryonic palatal mesenchymal cells maintain stemness through the PTEN-Akt-mTOR autophagic pathway

BackgroundBoth genetic and environmental factors are implicated in the pathogenesis of cleft palate. However, the molecular and cellular mechanisms that regulate the development of palatal shelves, which are composed of mesenchymal cells, have not yet been fully elucidated. This study aimed to determine the stemness and multilineage differentiation potential of mouse embryonic palatal mesenchyme (MEPM) cells in palatal shelves and to explore the underlying regulatory mechanism associated with cleft palate formation.MethodsPalatal shelves excised from mice models were cultured in vitro to ascertain whether MEPM are stem cells through immunofluorescence and flow cytometry. The osteogenic, adipogenic, and chondrogenic differentiation potential of MEPM cells were also determined to characterize MEPM stemness. In addition, the role of the PTEN-Akt-mTOR autophagic pathway was investigated using quantitative RT-PCR, Western blotting, and transmission electron microscopy.ResultsMEPM cells in culture exhibited cell surface marker expression profiles similar to that of mouse bone marrow stem cells and exhibited positive staining for vimentin (mesodermal marker), nestin (ectodermal marker), PDGFRα, Efnb1, Osr2, and Meox2 (MEPM cells markers). In addition, exposure to PDGFA stimulated chemotaxis of MEPM cells. MEPM cells exhibited stronger potential for osteogenic differentiation as compared to that for adipogenic and chondrogenic differentiation. Undifferentiated MEPM cells displayed a high concentration of autophagosomes, which disappeared after differentiation (at passage four), indicating the involvement of PTEN-Akt-mTOR signaling.ConclusionsOur findings suggest that MEPM cells are ectomesenchymal stem cells with a strong osteogenic differentiation potential and that maintenance of their stemness via PTEN/AKT/mTOR autophagic signaling prevents cleft palate development.

MicroRNA-374a, -4680, and -133b suppress cell proliferation through the regulation of genes associated with human cleft palate in cultured human palate cells

BackgroundCleft palate (CP) is the second most common congenital birth defect; however, the relationship between CP-associated genes and epigenetic regulation remains largely unknown. In this study, we investigated the contribution of microRNAs (miRNAs) to cell proliferation and regulation of genes involved in CP development.MethodsIn order to identify all genes for which mutations or association/linkage have been found in individuals with CP, we conducted a systematic literature search, followed by bioinformatics analyses for these genes. We validated the bioinformatics results experimentally by conducting cell proliferation assays and miRNA-gene regulatory analyses in cultured human palatal mesenchymal cells treated with each miRNA mimic.ResultsWe identified 131 CP-associated genes in the systematic review. The bioinformatics analysis indicated that the CP genes were associated with signaling pathways, microRNAs (miRNAs), metabolic pathways, and cell proliferation. A total 17 miRNAs were recognized as potential modifiers of human CP genes. To validate miRNA function in cell proliferation, a main cause of CP, we conducted cell proliferation/viability assays for the top 11 candidate miRNAs from our bioinformatics analysis. Overexpression of miR-133b, miR-374a-5p, and miR-4680-3p resulted in a more than 30% reduction in cell proliferation activity in human palatal mesenchymal cell cultures. We found that several downstream target CP genes predicted by the bioinformatics analyses were significantly downregulated through induction of these miRNAs (FGFR1, GCH1, PAX7, SMC2, and SUMO1 by miR-133b; ARNT, BMP2, CRISPLD1, FGFR2, JARID2, MSX1, NOG, RHPN2, RUNX2, WNT5A and ZNF236 by miR-374a-5p; and ERBB2, JADE1, MTHFD1 and WNT5A by miR-4680-3p) in cultured cells.ConclusionsOur results indicate that miR-374a-5p, miR-4680-3p, and miR-133b regulate expression of genes that are involved in the etiology of human CP, providing insight into the association between CP-associated genes and potential targets of miRNAs in palate development.

SPECC1L‐Deficient Cells Show Impaired Collective Cell Migration Attributes that are Rescued by Upregulation of PI3K‐AKT Pathway

Clefts of the lip and/or palate (CL/P) are common anomalies that occur in 1/700 live births. Pathogenic SPECC1L variants identified in patients with rare atypical clefts and syndromic CL/P suggest the gene plays a primary role in face and palate development. Null mutants are lethal at embryonic day 9.5 with defective neural tube closure and cranial neural crest cell delamination. Hypomorphic Specc1lΔ200 mutants die perinatally, but do not show cleft palate. Specc1lnull/Δ200 compound mutants resulted in transient oral adhesions, subepidermal blebbing, and a delay in palate elevation. Palate development requires extensive mesenchymal remodeling especially during palatal shelf elevation. We posit that this remodeling involves collective cell migration (CCM) of neural crest derived palate mesenchymal cells. Live time‐lapse microscopy was performed to visualize in vitro wound‐repair assays with SPECC1L‐deficient U2OS osteosarcoma and primary mouse embryo palatal mesenchyme (MEPM) cells. SPECC1L‐deficient cells consistently showed delayed wound closure. Directional movement of neighboring cells is a hallmark of CCM. In wound‐repair assays, an increase in movement parallel to the direction of wound front propagation results can be measured as an attribute of CCM. Tracking of individual trajectories of SPECC1L deficient cells during wound‐repair indeed shows an increase in cell movement perpendicular to the direction of wound closure. This diminished directionality causes delayed wound closure even though cells are adequately motile. We previously demonstrated SPECC1L to be a novel regulator of PI3K‐AKT signaling, with in vitro and in vivo SPECC1L deficiency exhibiting reduced PI3K‐AKT signaling. Indeed, wound closure delay, and the underlying CCM defect, in SPECC1L‐deficient U2OS and MEPM cells can be rescued by activation of the PI3K‐AKT signaling pathway using 740 Y‐P small molecule activator. Our data are the first to show CCM attributes in MEPM cells, and propose that MEPM cells can be used to model mesenchymal remodeling during palatal shelf elevation. We also show a novel role for SPECC1L in CCM through modulation of P13K‐AKT signaling.Support or Funding InformationThis project was supported in part by the National Institutes of Health grants DE026172 (I.S.). Core support and pilot funding was also provided by National Institutes of Health Center of Biomedical Research Excellence (COBRE) grant (P20 GM104936), Kansas IDeA Network for Biomedical Research Excellence (INBRE) grant (P20 GM103418), and Kansas Intellectual and Developmental Disabilities Research Center (IDDRC) grant (P30 HD 002528).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

255-nm Light-emitting Diode Kills Enterococcus faecalis and Induces the Production of Cellular Biomarkers in Human Embryonic Palatal Mesenchyme Cells and Gingival Fibroblasts

IntroductionThe successful treatment of infected or inflamed endodontic tissues requires chemomechanical debridement of the canal spaces and proper sealing of the coronal and apical canal openings. Only a few methods are available to further disinfect areas or initiate regeneration of local tissues. In this study, we assessed the ability of 255-nm and 405-nm light-emitting diode (LED) treatment to kill planktonic cultures of Enterococcus faecalis and induce the production of cellular biomarkers related to endodontic tissue regeneration. MethodsWe determined the antimicrobial effects of 255-nm and 405-nm LED treatment on E. faecalis and the effects of 255-nm and 405-nm LED treatment on the production of osteoinductive, angiogenic, proliferative, and proinflammatory biomarkers from human embryonic palatal mesenchyme (HEPM) cells and gingival fibroblasts. ResultsWe showed that 255-nm LED but not 405-nm LED treatment killed E. faecalis; the 255-nm LED and sodium hypochlorite more efficiently killed E. faecalis; neither 255-nm nor 405-nm LED treatment affected the viability of HEPM cells and gingival fibroblasts; and 255-nm LED treatment, alone or in combination with 405-nm LED treatment, of HEPM cells and gingival fibroblasts induced the production of biomarkers related to endodontic tissue regeneration. ConclusionsThe results of this study suggest a new treatment modality using short periods of 255-nm LED treatment as an adjunct to chemomechanical debridement for the disinfection of inflamed sites and the production of biomarkers related to endodontic tissue regeneration.

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.

Embryonic Midfacial Palatal Organ Culture Methods in Developmental Toxicology.

The morphogenesis of the secondary palate provides an interesting model for many of the processes involved in embryonic development. A number of in vitro models have been used to study craniofacial development, including whole embryo culture, palatal mesenchymal and micromass cell cultures, and Trowell-like palatal cultures in which dissected palates are cultured individually or as pairs in contact on a support above medium. This chapter presents a detailed protocol for the culture of maxillary midfacial tissues, including the palatal shelves, in suspension culture. This method involves isolation of the midfacial tissues (maxillary arch and palatal shelves) and suspension of the tissues in medium in flasks. On a rocker in an incubator, the palatal shelves elevate, grow, make contact, and fuse in a time span analogous to that occurring in the intact embryo in utero.

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.

Protein Arginine Methyltransferase PRMT1 Is Essential for Palatogenesis

Cleft palate is among the most common birth defects. Currently, only 30% of cases have identified genetic causes, whereas the etiology of the majority remains to be discovered. We identified a new regulator of palate development, protein arginine methyltransferase 1 (PRMT1), and demonstrated that disruption of PRMT1 function in neural crest cells caused complete cleft palate and craniofacial malformations. PRMT1 is the most highly expressed of the protein arginine methyltransferases, enzymes responsible for methylation of arginine motifs on histone and nonhistone proteins. PRMT1 regulates signal transduction and transcriptional activity that affect multiple signal pathways crucial in craniofacial development, such as the BMP, TGFβ, and WNT pathways. We demonstrated that Wnt1-Cre;Prmt1 fl/fl mice displayed a decrease in palatal mesenchymal cell proliferation and failure of palatal shelves to reach the midline. Further analysis in signal pathways revealed that loss of Prmt1 in mutant mice decreased BMP signaling activation and reduced the deposition of H4R3me2a mark. Collectively, our study demonstrates that Prmt1 is crucial in palate development. Our study may facilitate the development of a better strategy to interrupt the formation of cleft palate through manipulation of PRMT1 activity.

Inactivation of Fgfr2 gene in mouse secondary palate mesenchymal cells leads to cleft palate

Numerous studies have been conducted to understand the molecular mechanisms controlling mammalian secondary palate development such as growth, reorientation and fusion. However, little is known about the signaling factors regulating palate initiation. Mouse fibroblast growth factor (FGF) receptor 2 gene (Fgfr2) is expressed on E11.5 in the palate outgrowth within the maxillary process, in a region that is responsible for palate cell specification and shelf initiation. Fgfr2 continues to express in palate on E12.5 and E13.5 in both epithelial and mesenchymal cells, and inactivation of Fgfr2 expression in mesenchymal cells using floxed Fgfr2 allele and Osr2-Cre leads to cleft palate at various stages including reorientation, horizontal growth and fusion. Notably, some mutant embryos displayed no sign of palate shelf formation suggesting that FGF receptor 2 mediated FGF signaling may play an important role in palate initiation.

Diverse effect of BMP-2 homodimer on mesenchymal progenitors of different origin

Bone morphogenetic protein-2 (BMP-2), is a potential factor to enhance osseointegration of dental implants. However, the appropriate cellular system to investigate the osteogenic effect of BMP-2 in vitro in a standardized manner still needs to be defined. The aim of this study was to examine the effect of BMP-2 on the cell proliferation and osteogenic differentiation of human osteogenic progenitors of various origins: dental pulp stem cells (DPSC), human osteosarcoma cell line (Saos-2) and human embryonic palatal mesenchymal cell line (HEPM). For induction of osteogenic differentiation, cell culture medium was supplemented with BMP-2 homodimer alone or in combination with conventionally used differentiation inducing agents. Differentiation was monitored for 6–18 days. To assess differentiation, proliferation rate, alkaline phosphatase activity, calcium deposition and the expression level of osteogenic differentiation marker genes (Runx2, BMP-2) were measured. BMP-2 inhibited cell proliferation in a concentration and time-dependent manner. In a concentration which caused maximal cell proliferation, BMP-2 did not induce osteogenic differentiation in any of the tested systems. However, it had a synergistic effect with the osteoinductive medium in both DPSC and Saos-2, but not in HEPM cells. We also found that the differentiation process was faster in Saos-2 than in DPSCs. Osteogenic differentiation could not be induced in the osteoblast progenitor HEPM cells. Our data suggest that in a concentration that inhibits proliferation the differentiation inducing effect of BMP-2 is evident only in the presence of permissive osteoinductive components. β-glycerophosphate, was identified interacting with BMP-2 in a synergistic manner.

FGF8 Signaling Alters the Osteogenic Cell Fate in the Hard Palate

Fibroblast growth factor (FGF) signaling has been implicated in the regulation of osteogenesis in both intramembranous and endochondral ossifications. In the developing palate, the anterior bony palate forms by direct differentiation of cranial neural crest (CNC)–derived mesenchymal cells, but the signals that regulate the osteogenic cell fate in the developing palate remain unclear. In the present study, we investigated the potential role of FGF signaling in osteogenic fate determination of the palatal mesenchymal cells. We showed that locally activated FGF8 signaling in the anterior palate using a Shox2Cre knock-in allele and an R26RFgf8 allele leads to a unique palatal defect: a complete loss of the palatine process of the maxilla as well as formation of ectopic cartilaginous tissues in the anterior palate. This aberrant developmental process was accompanied by a significantly elevated level of cell proliferation, which contributes to an abnormally thickened palatal tissue, where the palatine process of the maxilla would normally form, and by a complete inhibition of Osterix expression, which accounts for the lack of bone formation. The coexpression of Runx2 initially with Sox9 and subsequently with Col II in the ectopic cartilaginous tissues indicates a conversion of osteogenic fate to a chondrogenic one. Consistent with the unique palatal phenotype, RNA-Sequencing analysis revealed that the augmented FGF8 signaling downregulated genes involved in ossification, biomineral tissue development, and bone mineralization but upregulated genes involved in cell proliferation, cartilage development, and cell fate commitment, which was further supported by quantitative real-time reverse transcription polymerase chain reaction validation of selected genes. Our results demonstrate that FGF8 signaling functions as a negative regulator of osteogenic fate and is sufficient to convert a subset of CNC cell-derived mesenchymal cells into cartilage in the anterior hard palate, which will have implications in future directed differentiation of CNC-derived precursor cells for clinical application.

TGF-β signaling inhibits canonical BMP signaling pathway during palate development.

During early palate development, gene expression and regulation exhibit heterogeneity along the anterior-posterior axis. Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) signaling pathways play essential roles in secondary palatal formation but the exact relationship between the TGF-β and BMP pathways in palate development remains unknown. Here, we demonstrate that, during early secondary palate development, phospho-(p)Smad1/5/8 is highly expressed in the anterior palate but relatively lowly expressed in the posterior palate. Conversely, pSmad2/3 has a lower expression level in the anterior palate than in the posterior palate. With the BRE-Gal reporter, we found that the canonical BMP signaling pathway was not activated in the anterior palate but exhibited a moderate level in the posterior palate. Co-immunoprecipitation revealed that Smad4 bound to pSmad1/5/8 only in the posterior palate and not in the anterior palate. Knocking-out of Tgfbr2 (Wnt1-Cre;Tgfbr2 f/f;BRE) in the palatal mesenchyme enhanced canonical BMP activity in the posterior palate but not in the anterior palate, because of decreased pSmad2/3. pSmad1/5/8-Smad4 complexes were found to be dramatically increased in posterior palatal mesenchymal cells at embryonic day 13.5 cultured in the presence of SB525334. Proximity ligation assay also showed pSmad1/5/8-Smad4 complexes were increased in the posterior palate of Wnt1-Cre;Tgfbr2 f/f mice. Therefore, the reduction of pSmad2/3 level in the palatal mesenchyme of Wnt1-Cre;Tgfbr2 f/f;BRE mice contributes primarily to the increase of pSmad1/5/8-Smad4 complexes leading to enhanced canonical BMP activity in the posterior palate. Moreover, during early development, canonical BMP signaling operates in the posterior palate but is completely absent in the anterior palate. Canonical TGF-β signaling suppresses canonical BMP signaling activity in the posterior palate by competing limited Smad4.

Six2 Plays an Intrinsic Role in Regulating Proliferation of Mesenchymal Cells in the Developing Palate.

Cleft palate is a common congenital abnormality that results from defective secondary palate (SP) formation. The Sine oculis-related homeobox 2 (Six2) gene has been linked to abnormalities of craniofacial and kidney development. Our current study examined, for the first time, the specific role of Six2 in embryonic mouse SP development. Six2 mRNA and protein expression were identified in the palatal shelves from embryonic days (E)12.5 to E15.5, with peak levels during early stages of palatal shelf outgrowth. Immunohistochemical staining (IHC) showed that Six2 protein is abundant throughout the mesenchyme in the oral half of each palatal shelf, whereas there is a pronounced decline in Six2 expression by mesenchyme cells in the nasal half of the palatal shelf by stages E14.5–15.5. An opposite pattern was observed in the surface epithelium of the palatal shelf. Six2 expression was prominent at all stages in the epithelial cell layer located on the nasal side of each palatal shelf but absent from the epithelium located on the oral side of the palatal shelf. Six2 is a putative downstream target of transcription factor Hoxa2 and we previously demonstrated that Hoxa2 plays an intrinsic role in embryonic palate formation. We therefore investigated whether Six2 expression was altered in the developing SP of Hoxa2 null mice. Reverse transcriptase PCR and Western blot analyses revealed that Six2 mRNA and protein levels were upregulated in Hoxa2−/− palatal shelves at stages E12.5–14.5. Moreover, the domain of Six2 protein expression in the palatal mesenchyme of Hoxa2−/− embryos was expanded to include the entire nasal half of the palatal shelf in addition to the oral half. The palatal shelves of Hoxa2−/− embryos displayed a higher density of proliferating, Ki-67 positive palatal mesenchyme cells, as well as a higher density of Six2/Ki-67 double-positive cells. Furthermore, Hoxa2−/− palatal mesenchyme cells in culture displayed both increased proliferation and elevated Cyclin D1 expression relative to wild-type cultures. Conversely, siRNA-mediated Six2 knockdown restored proliferation and Cyclin D1 expression in Hoxa2−/− palatal mesenchyme cultures to near wild-type levels. Our findings demonstrate that Six2 functions downstream of Hoxa2 as a positive regulator of mesenchymal cell proliferation during SP development.

Controlled JAGGED1 delivery induces human embryonic palate mesenchymal cells to form osteoblasts.

Osteoblast commitment and differentiation are controlled by multiple growth factors including members of the Notch signaling pathway. JAGGED1 is a cell surface ligand of the Notch pathway that is necessary for murine bone formation. The delivery of JAGGED1 to induce bone formation is complicated by its need to be presented in a bound form to allow for proper Notch receptor signaling. In this study, we investigate whether the sustained release of JAGGED1 stimulates human mesenchymal cells to commit to osteoblast cell fate using polyethylene glycol malemeide (PEG-MAL) hydrogel delivery system. Our data demonstrated that PEG-MAL hydrogel constructs are stable in culture for at least three weeks and maintain human mesenchymal cell viability with little cytotoxicity in vitro. JAGGED1 loaded on PEG-MAL hydrogel (JAGGED1-PEG-MAL) showed continuous release from the gel for up to three weeks, with induction of Notch signaling using a CHO cell line with a Notch1 reporter construct, and qPCR gene expression analysis in vitro. Importantly, JAGGED1-PEG-MAL hydrogel induced mesenchymal cells towards osteogenic differentiation based on increased Alkaline phosphatase activity and osteoblast genes expression including RUNX2, ALP, COL1, and BSP. These results thus indicated that JAGGED1 delivery in vitro using PEG-MAL hydrogel induced osteoblast commitment, suggesting that this may be a viable in vivo approach to bone regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 552-560, 2018.

Hoxa2 Inhibits Bone Morphogenetic Protein Signaling during Osteogenic Differentiation of the Palatal Mesenchyme.

Cleft palate is one of the most common congenital birth defects worldwide. The homeobox (Hox) family of genes are key regulators of embryogenesis, with Hoxa2 having a direct role in secondary palate development. Hoxa2−/− mice exhibit cleft palate; however, the cellular and molecular mechanisms leading to cleft palate in Hoxa2−/− mice is largely unknown. Addressing this issue, we found that Hoxa2 regulates spatial and temporal programs of osteogenic differentiation in the developing palate by inhibiting bone morphogenetic protein (BMP) signaling dependent osteoblast markers. Expression of osteoblast markers, including Runx2, Sp7, and AlpI were increased in Hoxa2−/− palatal shelves at embryonic day (E) 13.5 and E15.5. Hoxa2−/− mouse embryonic palatal mesenchyme (MEPM) cells exhibited increased bone matrix deposition and mineralization in vitro. Moreover, loss of Hoxa2 resulted in increased osteoprogenitor cell proliferation and osteogenic commitment during early stages of palate development at E13.5. Consistent with upregulation of osteoblast markers, Hoxa2−/− palatal shelves displayed higher expression of canonical BMP signaling in vivo. Blocking BMP signaling in Hoxa2−/− primary MEPM cells using dorsomorphin restored cell proliferation and osteogenic differentiation to wild-type levels. Collectively, these data demonstrate for the first time that Hoxa2 may regulate palate development by inhibiting osteogenic differentiation of palatal mesenchyme via modulating BMP signaling.

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.

Wnt6 influences the viability of mouse embryonic palatal mesenchymal cells via the β-catenin pathway.

The embryological stages of palatal shelf elongation and elevation, mainly induced by the proliferation and extracellular matrix secretion of embryonic palatal mesenchymal (MEPM) cells, are essential for normal palatal development. Wingless-related MMTV integration site gene family (Wnt) signaling pathways serve key roles in craniofacial development and palate formation. Recent studies have indicated that Wnt6 participates in embryonic development of the palate, though its exact role in palate development remains unclear. In the present study, to investigate the role of Wnt6 during the stages of palatal shelves elongation and elevation, mouse MEPM cells were cultured from dissected palatal shelves at embryonic day 13.5. Results of an MTT assay and flow cytometric analysis demonstrated that treatment with recombinant Wnt6 increased the viability of MEPM cells (P<0.01) and the proportion of cells in the S and G2/M phases (P<0.01). Meanwhile, Wnt6 activated the β-catenin signaling pathway as indicated by the dual luciferase assay result, and blockade of the WNT/β-catenin pathway reduced the cytoactivity of Wnt6 in MEPM cells (P<0.01). Collectively, these findings indicate that Wnt6 promotes the vitality of MEPM cells by increasing the S + G2/M-phase cell population, potentially through activation of the β-catenin pathway during palatal shelf elongation and elevation.