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.

Assessing the fate and contribution of Foxd1-expressing embryonic precursors and their progeny in palatal development, homeostasis and excisional repair.

Oral mucosal tissues heal rapidly with minimal scarring, although palatal mucosa can be associated with excessive fibrosis in response to injury. Investigations on the balance between neovascularization and tissue repair suggests regulation of angiogenesis is an important determinant of repair versus scarring. Associated with pericyte mediated fibrosis in kidney injury, FoxD1 is implicated in growth centres during cranio-facial development, although which cell lineages are derived from these embryonic populations in development and in adult animals is unknown. Using a lineage tracing approach, we assessed the fate of embryonic Foxd1-expressing progenitor cells and their progeny in palatal development and during wound healing in adult mice. During palatal development as well as in post-natal tissues, Foxd1-lineage progeny were associated with the vasculature and the epineurium. Post-injury, de novo expression of FoxD1 was not detectable, although Foxd1-lineage progeny expanded while exhibiting low association with the fibroblast/myofibroblast markers PDGFα, PDGFß, vimentin, α-smooth muscle actin, as well as the neuronal associated markers S100ß and p75NTR. Foxd1-lineage progeny were primarily associated with CD146, CD31, and to a lesser extent CD105, remaining in close proximity to developing neovascular structures. Our findings demonstrate that FoxD1 derived cells are predominantly associated with the palatal vasculature and provide strong evidence that FoxD1 derived cells do not give rise to populations involved directly in the scarring of the palate.

MicroRNAs in Small Extracellular Vesicles from Amniotic Fluid and Maternal Plasma Associated with Fetal Palate Development in Mice.

Cleft palate (CP) is a common congenital birth defect. Cellular and morphological processes change dynamically during palatogenesis, and any disturbance in this process could result in CP. However, the molecular mechanisms steering this fundamental phase remain unclear. One study suggesting a role for miRNAs in palate development via maternal small extracellular vesicles (SEVs) drew our attention to their potential involvement in palatogenesis. In this study, we used an in vitro model to determine how SEVs derived from amniotic fluid (ASVs) and maternal plasma (MSVs) influence the biological behaviors of mouse embryonic palatal mesenchyme (MEPM) cells and medial edge epithelial (MEE) cells; we also compared time-dependent differential expression (DE) miRNAs in ASVs and MSVs with the DE mRNAs in palate tissue from E13.5 to E15.5 to study the dynamic co-regulation of miRNAs and mRNAs during palatogenesis in vivo. Our results demonstrate that some pivotal biological activities, such as MEPM proliferation, migration, osteogenesis, and MEE apoptosis, might be directed, in part, by stage-specific MSVs and ASVs. We further identified interconnected networks and key miRNAs such as miR-744-5p, miR-323-5p, and miR-3102-5p, offering a roadmap for mechanistic investigations and the identification of early CP biomarkers.

The roles of JAK2/STAT3 signaling in fusion of the secondary palate.

Cleft palate has a multifactorial etiology. In palatal fusion, the contacting medial edge epithelium (MEE) forms the epithelial seam, which is subsequently removed with the reduction of p63. Failure in this process results in a cleft palate. We herein report the involvement of janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling in palatal fusion and that folic acid rescues the fusing defect by reactivating JAK2/STAT3. In closure of bilateral palatal shelves, STAT3 phosphorylation was activated at the fusing MEE and mesenchyme underlying the MEE. JAK2 inhibition by AG490 inhibited STAT3 phosphorylation and resulted in palatal fusion failure without removal of the epithelial seam, in which p63 and keratin 17 (K17) periderm markers were retained. Folic acid application restored STAT3 phosphorylation in AG490-treated palatal explants and rescued the fusion defect, in which the p63- and K17-positive epithelial seam were removed. The AG490-induced palatal defect was also rescued in p63 haploinsufficient explants. These findings suggest that JAK2/STAT3 signaling is involved in palatal fusion by suppressing p63 expression in MEE and that folate restores the fusion defect by reactivating JAK2/STAT3.

Associations between the proliferation of palatal mesenchymal cells, Tgfß2 promoter methylation, Meg3 expression, and Smad signaling in atRA-induced cleft palate.

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 TGFß/Smad pathway are key in regulating embryonic development. Here, the impact of atRA on MEPM cell proliferation and associations between Tgfß2 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 Tgfß2 promoter expression. These analyses revealed that atRA suppressed MEPM cell proliferation, promoted the upregulation of Meg3, and reduced the levels of Smad2 and Tgfß2 expression phosphorylation, whereas Tgfß2 promoter methylation was unaffected. RNA immunoprecipitation experiments indicated that the TgfßI receptor is directly targeted by Meg3, suggesting that the ability of atRA to induce cleft palate may be mediated through the Tgfß/Smad signaling pathway.

Deficiency of Fam20b-Catalyzed Glycosaminoglycan Chain Synthesis in Neural Crest Leads to Cleft Palate.

Cleft palate is one of the most common birth defects. Previous studies revealed that multiple factors, including impaired intracellular or intercellular signals, and incoordination of oral organs led to cleft palate, but were little concerned about the contribution of the extracellular matrix (ECM) during palatogenesis. Proteoglycans (PGs) are one of the important macromolecules in the ECM. They exert biological functions through one or more glycosaminoglycan (GAG) chains attached to core proteins. The family with sequence similarity 20 member b (Fam20b) are newly identified kinase-phosphorylating xylose residues that promote the correct assembly of the tetrasaccharide linkage region by creating a premise for GAG chain elongation. In this study, we explored the function of GAG chains in palate development through Wnt1-Cre; Fam20bf/f mice, which exhibited complete cleft palate, malformed tongue, and micrognathia. In contrast, Osr2-Cre; Fam20bf/f mice, in which Fam20b was deleted only in palatal mesenchyme, showed no abnormality, suggesting that failed palatal elevation in Wnt1-Cre; Fam20bf/f mice was secondary to micrognathia. In addition, the reduced GAG chains promoted the apoptosis of palatal cells, primarily resulting in reduced cell density and decreased palatal volume. The suppressed BMP signaling and reduced mineralization indicated an impaired osteogenesis of palatine, which could be rescued partially by constitutively active Bmpr1a. Together, our study highlighted the key role of GAG chains in palate morphogenesis.

High throughput miRNA sequencing and bioinformatics analysis identify the mesenchymal cell proliferation and apoptosis related miRNAs during fetal mice palate development.

BACKGROUND: Palatogenesis requires a precise spatiotemporal regulation of gene expression. Recent studies indicate that microRNAs (miRNAs) are key factors in normal palatogenesis. The present study aimed to explain the regulatory mechanisms of miRNAs during palate development. METHODS: Pregnant ICR mice were choose at embryonic day 10.5 (E10.5). Hemotoxylin and eosin (H&E) staining was used to observe the morphological changes during the development of palatal process at embryonic day (E)13.5, E14.0, E14.5, E15.0 and E15.5. The fetal palatal tissues were collected at E13.5, E14.0, E14.5 and E15.0 to explore miRNA expression and function by high throughput sequencing and bioinformatic analysis. Mfuzz cluster analysis was used to look for miRNAs related to the fetal mice palate formation. The target genes of miRNAs were predicted by miRWalk. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed base on target genes. The mesenchymal cell proliferation and apoptosis related miRNAs-genes networks were predicted and constructed using miRWalk and Cytoscape software. The expression of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was detected by a quantitative real-time PCR (RT-qPCR) assay. RESULTS: H&E staining found that the palatal process grows vertically along the sides of the tongue at E13.5, the position of the tongue begins to descend and the bilateral palatal processes rise above the tongue at E14.0, the palatal process grows horizontally at E14.5, there is palatal contact fusion at E15.0, and the palatal suture disappeared at E15.5. Nine clusters of miRNA expression changes were identified in the fetal mice palate formation progression, including two reducing trends, two rising trends and five disordered trends. Next, the heatmap showed the miRNA expression from Clusters 4, 6, 9, 12 in the E13.5, E14.0, E14.5 and E15.0 groups. GO functional and KEGG pathway enrichment analysis found target genes of miRNAs in clusters involved in regulation of mesenchymal phenotype and the mitogen-activated protein kinase (MAPK) signaling pathway. Next, mesenchymal phenotype related miRNA-genes networks were constructed. The heatmap showing that the mesenchymal phenotype related miRNA expression of Clusters 4, 6, 9 and 12 at E13.5, E14.0, E14.5 and E15.0. Furthermore, the mesenchymal cell proliferation and apoptosis related miRNA-gene networks were identified in Clusters 6 and 12, including mmu-miR-504-3p-Hnf1b, etc. The expression level of mesenchymal cell proliferation and apoptosis related miRNAs at the E13.5, E14.0, E14.5, and E15.0 was verified by a RT-qPCR assay. CONCLUSIONS: For the first time, we identified that clear dynamic miRNA expression during palate development. Furthermore, we demonstrated that mesenchymal cell proliferation and apoptosis related miRNAs, genes and the MAPK signaling pathway are important during fetal mice palate development.

Increased miR-200c levels disrupt palatal fusion by affecting apoptosis, cell proliferation, and cell migration.

The mammalian palate separates the oral and nasal cavities, facilitating proper feeding, respiration, and speech. Palatal shelves, composed of neural crest-derived mesenchyme and surrounding epithelium, are a pair of maxillary prominences contributing to this structure. Palatogenesis reaches completion upon the fusion of the midline epithelial seam (MES) following contact between medial edge epithelium (MEE) cells in the palatal shelves. This process entails numerous cellular and molecular occurrences, including apoptosis, cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT). MicroRNAs (miRs) are small, endogenous, non-coding RNAs derived from double-stranded hairpin precursors that regulate gene expression by binding to target mRNA sequences. Although miR-200c is a positive regulator of E-cadherin, its role in palatogenesis remains unclear. This study aims to explore the role of miR-200c in palate development. Before contact with palatal shelves, mir-200c was expressed in the MEE along with E-cadherin. After palatal shelf contact, miR-200c was present in the palatal epithelial lining and epithelial islands surrounding the fusion region but absent in the mesenchyme. The function of miR-200c was investigated by utilizing a lentiviral vector to facilitate overexpression. Ectopic expression of miR-200c resulted in E-cadherin upregulation, impaired dissolution of the MES, and reduced cell migration for palatal fusion. The findings imply that miR-200c is essential in palatal fusion as it governs E-cadherin expression, cell death, and cell migration, acting as a non-coding RNA. This study may contribute to clarifying the underlying molecular mechanisms in palate formation and provides insights into potential gene therapies for cleft palate.

Periderm Fate during Palatogenesis: TGF-ß and Periderm Dedifferentiation.

Failure of palatogenesis results in cleft palate, one of the most common congenital disabilities in humans. During the final phases of palatogenesis, the protective function of the peridermal cell layer must be eliminated for the medial edge epithelia to adhere properly, which is a prerequisite for the successful fusion of the secondary palate. However, a deeper understanding of the role and fate of the periderm in palatal adherence and fusion has been hampered due to a lack of appropriate periderm-specific genetic tools to examine this cell type in vivo. Here we used the cytokeratin-6A (Krt-6a) locus to develop both constitutive (Krt6ai-Cre) and inducible (Krt6ai-CreERT2) periderm-specific Cre driver mouse lines. These novel lines allowed us to achieve both the spatial and temporal control needed to dissect the periderm fate on a cellular resolution during palatogenesis. Our studies suggest that, already before the opposing palatal shelves contact each other, at least some palatal periderm cells start to gradually lose their squamous periderm-like phenotype and dedifferentiate into cuboidal cells, reminiscent of the basal epithelial cells seen in the palatal midline seam. Moreover, we show that transforming growth factor-ß (TGF-ß) signaling plays a critical periderm-specific role in palatogenesis. Thirty-three percent of embryos lacking a gene encoding the TGF-ß type I receptor (Tgfbr1) in the periderm display a complete cleft of the secondary palate. Our subsequent experiments demonstrated that Tgfbr1-deficient periderm fails to undergo appropriate dedifferentiation. These studies define the periderm cell fate during palatogenesis and reveal a novel, critical role for TGF-ß signaling in periderm dedifferentiation, which is a prerequisite for appropriate palatal epithelial adhesion and fusion.

Spatiotemporal Gene Expression Regions along the Anterior-Posterior Axis in Mouse Embryos before and after Palatal Elevation.

The mammalian secondary palate is formed through complex developmental processes: growth, elevation, and fusion. Although it is known that the palatal elevation pattern changes along the anterior-posterior axis, it is unclear what molecules are expressed and whether their locations change before and after elevation. We examined the expression regions of molecules associated with palatal shelf elevation (Pax9, Osr2, and Tgfß3) and tissue deformation (F-actin, E-cadherin, and Ki67) using immunohistochemistry and RT-PCR in mouse embryos at E13.5 (before elevation) and E14.5 (after elevation). Pax9 was expressed at significantly higher levels in the lingual/nasal region in the anterior and middle parts, as well as in the buccal/oral region in the posterior part at E13.5. At E14.5, Pax9 was expressed at significantly higher levels in both the lingual/nasal and buccal/oral regions in the anterior and middle parts and the buccal/oral regions in the posterior part. Osr2 was expressed at significantly higher levels in the buccal/oral region in all parts at E13.5 and was more strongly expressed at E13.5 than at E14.5 in all regions. No spatiotemporal changes were found in the other molecules. These results suggested that Pax9 and Osr2 are critical molecules leading to differences in the elevation pattern in palatogenesis.

A unique form of collective epithelial migration is crucial for tissue fusion in the secondary palate and can overcome loss of epithelial apoptosis.

Tissue fusion frequently requires the removal of an epithelium that intervenes distinct primordia to form one continuous structure. In the mammalian secondary palate, a midline epithelial seam (MES) forms between two palatal shelves and must be removed to allow mesenchymal confluence. Abundant apoptosis and cell extrusion support their importance in MES removal. However, genetically disrupting the intrinsic apoptotic regulators BAX and BAK within the MES results in complete loss of cell death and cell extrusion, but successful removal of the MES. Novel static- and live-imaging approaches reveal that the MES is removed through streaming migration of epithelial trails and islands to reach the oral and nasal epithelial surfaces. Epithelial trail cells that express the basal epithelial marker ΔNp63 begin to express periderm markers, suggesting that migration is concomitant with differentiation. Live imaging reveals anisotropic actomyosin contractility within epithelial trails, and genetic ablation of actomyosin contractility results in dispersion of epithelial collectives and failure of normal MES migration. These findings demonstrate redundancy between cellular mechanisms of morphogenesis, and reveal a crucial and unique form of collective epithelial migration during tissue fusion.

[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-ß (TGF-ß)/Smad signaling pathway in mouse embryonic palatal mesenchyme were analyzed by Western blotting. The interaction of MEG3 and TGF-ß receptor Ⅰ (TGF-ßRⅠ) 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-ßRⅠ-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-ßRⅠ protein and thereby suppressing Smad signaling in the context of TCDD induced cleft palate.

Resistant starch-An accessible fiber ingredient acceptable to the Western palate.

Dietary fiber intakes in Western societies are concerningly low and do not reflect global recommended dietary fiber intakes for chronic disease prevention. Resistant starch (RS) is a fermentable dietary fiber that has attracted research interest. As an isolated ingredient, its fine particle size, relatively bland flavor, and white appearance may offer an appealing fiber source to the Western palate, accustomed to highly refined, processed grains. This review aims to provide a comprehensive insight into the current knowledge (classification, production methods, and characterization methods), health benefits, applications, and acceptability of RS. It further discusses the present market for commercially available RS ingredients and products containing ingredients high in RS. The literature currently highlights beneficial effects for dietary RS supplementation with respect to glucose metabolism, satiety, blood lipid profiles, and colonic health. An exploration of the market for commercial RS ingredients indicates a diverse range of products (from isolated RS2, RS3, and RS4) with numerous potential applications as partial or whole substitutes for traditional flour sources. They may increase the nutritional profile of a food product (e.g., by increasing the fiber content and lowering energy values) without significantly compromising its sensory and functional properties. Incorporating RS ingredients into staple food products (such as bread, pasta, and sweet baked goods) may thus offer an array of nutritional benefits to the consumer and a highly accessible functional ingredient to be greater exploited by the food industry.

Histological and Immunohistochemical Studies to Determine the Mechanism of Cleft Palate Induction after Palatal Fusion in Mice Exposed to TCDD.

Rupture of the basement membrane in fused palate tissue can cause the palate to separate after fusion in mice, leading to the development of cleft palate. Here, we further elucidate the mechanism of palatal separation after palatal fusion in 8-10-week-old ICR female mice. On day 12 of gestation, 40 µg/kg of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), sufficient to cause cleft palate in 100% of mice, was dissolved in 0.4 mL of olive oil containing toluene and administered as a single dose via a gastric tube. Fetal palatine frontal sections were observed by H&E staining, and epithelial cell adhesion factors, apoptosis, and cell proliferation were observed from the anterior to posterior palate. TUNEL-positive cells and Ki67-positive cells were observed around the posterior palatal dissection area of the TCDD-treated group. Moreover, in fetal mice exposed to TCDD, some fetuses exhibited cleft palate dehiscence during fusion. The results suggest that palatal dehiscence may be caused by abnormal cell proliferation in epithelial tissues, decreased intercellular adhesion, and inhibition of mesenchymal cell proliferation. By elucidating the mechanism of cleavage after palatal fusion, this research can contribute to establishing methods for the prevention of cleft palate development.

Candidate positive targets of LHX6 and LHX8 transcription factors in the developing upper jaw.

Craniofacial development is controlled by a large number of genes, which interact with one another to form a complex gene regulatory network (GRN). Key components of GRN are signaling molecules and transcription factors. Therefore, identifying targets of core transcription factors is an important part of the overall efforts toward building a comprehensive and accurate model of GRN. LHX6 and LHX8 are transcription factors expressed in the oral mesenchyme of the first pharyngeal arch (PA1), and they are crucial regulators of palate and tooth development. Previously, we performed genome-wide transcriptional profiling and chromatin immunoprecipitation to identify target genes of LHX6 and LHX8 in PA1, and described a set of genes repressed by LHX. However, there has not been any discussion of the genes positively regulated by LHX6 and LHX8. In this paper, we revisited the above datasets to identify candidate positive targets of LHX in PA1. Focusing on those with known connections to craniofacial development, we performed RNA in situ hybridization to confirm the changes in expression in Lhx6;Lhx8 mutant. We also confirmed the binding of LHX6 to several putative enhancers near the candidate target genes. Together, we have uncovered novel connections between Lhx and other important regulators of craniofacial development, including Eya1, Barx1, Rspo2, Rspo3, and Wnt11.

The involvement of hormone-sensitive lipase in all-trans retinoic acid induced cleft palate.

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 in vivo. In the atRA group, the proliferation of HSL siRNA-transfected cells was remarkably promoted, and the migration rate significantly increased in the HSL siRNA-transfected MEPM cells. These results suggested that HSL may be involved in cleft palate induced by atRA and that atRA enhances HSL levels to inhibit embryonic palate growth.

Sox9CreER-mediated deletion of ß-catenin in palatal mesenchyme results in delayed palatal elevation accompanied with repressed canonical Wnt signaling and reduced actin polymerization.

Cleft palate is a good model to pushing us toward a deeper understanding of the molecular mechanisms of spatiotemporal patterns in tissues and organisms because of the multiple-step processes such as elevation and fusion. Previous studies have shown that the epithelial ß-catenin is crucial for palatal fusion, however, the function of the mesenchymal ß-catenin remains elusive. We investigate the role of mesenchymal ß-catenin in palatal development by generating a ß-catenin conditional knockout mouse (CKO) (Sox9CreER; Ctnnb1F/F ). We found that the CKO mice exhibited delayed palatal elevation, leading to cleft palate in both in vivo and ex vivo. Abnormal cell proliferation and repressed mesenchymal canonical Wnt signaling were found in the CKO palate. Interestingly, Filamentous actin (F-actin) polymerization was significantly reduced in the palatal mesenchyme of mutant embryos. Furthermore, overexpression of adenovirus-mediated transfection with Acta1 in the mutant could help to elevate the palatal shelves but could not prevent cleft palate in ex vivo. Our results suggest that conditionally knock out ß-catenin in the palatal mesenchyme by Sox9CreER leading to delayed palatal elevation, which results in repressed mesenchymal canonical Wnt signaling, decreased cell proliferation, and reduced actin polymerization, finally causes cleft palate.

Intraperitoneal injection of PDTC on the NF-kB signaling pathway and osteogenesis indexes of young adult rats with anterior palatal suture expansion model.

In recent years, many studies have found that mechanical tension can activiate NF-kB signal pathway and NF-kB plays an important role in the process of osteogenesis. However, it is still unclear whether this process exists in the anterior palatal suture expansion. In this paper, we mainly studied the effect of intraperitoneal injection of PDTC on the NF-kB signaling pathway and osteogenesis index of the anterior palatal suture expansion model in young adult rats. The expansion model is grouped and established: 45 male 8-week-old Sprague-Dawley rats were randomly divided into three groups, an expansion only (EO) group, an expansion plus PDTC (PE) group, and a control group. The results revealed that PDTC inhibited the activity of NF-kB signaling pathway and promote one morphogenetic protein 2 (BMP-2), steocalatin (OCN) expression. Compared with the control group, the optical density (OD) value of BMP in the EO group and PE group rats increased significantly from the first day to the seventh day, and the difference was statistically significant (P<0.05). After 6.0Gy irradiation, PDTC administration group could slightly increase the total SOD level in the liver and serum of rats, and reduce the MDA level in the liver and serum, especially the effect of 60mg/kg and 90mg/kg was the most obvious.

Identification and profiles of microRNAs in different development stages of miniature pig secondary palate.

Cleft palate is one of the most frequent craniofacial malformation birth defects. Miniature pigs (Sus scrofa) are a valuable alternative large animal model to explore human palate development. Presently, the microRNA (miRNA) expression profiles in miniature pigs during palatogenesis from embryonic day (E) 30 to 50 were identified. A total of 2044 known miRNAs and 192 novel miRNAs were identified. The functional characteristics of their potential target genes were identified using Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway analysis. MiRNAs displayed diverse expression levels among the different stages. Using Short Time-series Expression Miner software to investigate the expression patterns of miRNAs from E30-50, all miRNAs were clustered into 20 profiles. The profiles showing miRNAs expression decreased (profile 0)/increased (profile 19) from E30-50 were the main patterns during palatogenesis. Hub genes of four significant modules were identified by weighted correlation network analysis, including ssc-miR-98, ssc-miR-27a_R + 1, and ssc-miR-150, etc. which might be novel potential targets for regulating palate development. The data are expected to improve the understanding of palate development and the etiology of cleft palate in further studies.

Functional identification of a rare vascular endothelial growth factor a (VEGFA) variant associating with the nonsyndromic cleft lip with/without cleft palate.

Vascular endothelial growth factor A (VEGFA) is a crucial growth factor, which participates in multiple processes of human growth and development, such as angiogenesis and osteogenesis and is also necessary for development of palate. The purpose of this study was to investigate the effect of a rare VEGFA mutation (NM_001025366.2 773 T > C p.Val258Ala) on the cell functions and osteogenesis. Here, we found that the VEGFA mutation has adverse effects on the function of human embryonic palatal plate mesenchymal (HEPM) cells, and may affect the development of palate. The VEGFA mutation has adverse effects on promoting cell proliferation and migration and inhibiting apoptosis in HEPM and HEK-293 cells. In addition, the mutant VEGFA allele has a negative influence on osteogenesis. Taken together, the rare variant of the VEGFA gene had an adverse effect on cell functions and osteogenesis, which may impact the development of the palate. And these findings suggested that VEGFA mutation (c.773 T > C) may lead to nonsyndromic cleft lip with or without cleft palate and also provide a new insight into the mechanism of VEGFA gene in osteogenesis and palatogenesis.