Search Results (17)

Palatogenesis is a complex developmental process requiring temporospatially coordinated cellular and molecular events. The following review focuses on genetic, epigenetic, and environmental aspects directing palatal formation and their implication in orofacial clefting genesis. Essential for palatal shelf development and elevation (TGF-β, BMP, FGF, and WNT), the subsequent processes of fusion (SHH) and proliferation, migration, differentiation, and apoptosis of neural crest-derived cells are controlled through signaling pathways. Interruptions to these processes may result in the birth defect cleft lip and/or palate (CL/P), which happens in approximately 1 in every 700 live births worldwide. Recent progress has emphasized epigenetic regulations via the class of non-coding RNAs with microRNAs based on critically important biological processes, such as proliferation, apoptosis, and epithelial–mesenchymal transition. These environmental risks (maternal smoking, alcohol, retinoic acid, and folate deficiency) interact with genetic and epigenetic factors during palatogenesis, while teratogens like dexamethasone and TCDD inhibit palatal fusion. In orofacial cleft, genetic, epigenetic, and environmental impact on the complex epidemiology. This is an extensive review, offering current perspectives on gene-environment interactions, as well as non-coding RNAs, in palatogenesis and emphasizing open questions regarding these interactions in palatal development. Full article

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. Full article

Palatogenesis is a complex and intricate process involving the formation of the palate through various morphogenetic events highly dependent on the surrounding context. These events comprise outgrowth of palatal shelves from embryonic maxillary prominences, their elevation from a vertical to a horizontal position above the tongue, and their subsequent adhesion and fusion at the midline to separate oral and nasal cavities. Disruptions in any of these processes can result in cleft palate, a common congenital abnormality that significantly affects patient’s quality of life, despite surgical intervention. Although many genes involved in palatogenesis have been identified through studies on genetically modified mice and human genetics, the precise roles of these genes and their products in signaling networks that regulate palatogenesis remain elusive. Recent investigations have revealed that palatal shelf growth, patterning, adhesion, and fusion are intricately regulated by numerous transcription factors and signaling pathways, including Sonic hedgehog (Shh), bone morphogenetic protein (Bmp), fibroblast growth factor (Fgf), transforming growth factor beta (Tgf-β), Wnt signaling, and others. These studies have also identified a significant number of genes that are essential for palate development. Integrated information from these studies offers novel insights into gene regulatory networks and dynamic cellular processes underlying palatal shelf elevation, contact, and fusion, deepening our understanding of palatogenesis, and facilitating the development of more efficacious treatments for cleft palate. Full article

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; Fam20b^f/f mice, which exhibited complete cleft palate, malformed tongue, and micrognathia. In contrast, Osr2-Cre; Fam20b^f/f mice, in which Fam20b was deleted only in palatal mesenchyme, showed no abnormality, suggesting that failed palatal elevation in Wnt1-Cre; Fam20b^f/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. Full article

Background: Fgf9 mutation was found in cleft palate patients. Our previous study indicated that Fgf9 promotes timely elevation of palate by regulating hyaluronic acid (HA) accumulation at embryonic day 13.5 (E13.5). HA is synthesized by hyaluronic acid synthases (HAS) isoforms 1, 2, or 3. However, how FGF9 regulates HA in palatogenesis is still unclear. Methods: Using Ddx4-Cre mice, we generated the Fgf9^−/− mouse model (with exon 2 deletion). Immunohistochemistry was used to detect the location and expression of HAS2 in WT and the Fgf9^−/− palate at E13.5. We also predicted the association between Fgf9 and Has2 within the developing palate by performing a bioinformatics analysis. The expression of β-catenin, HAS2, and TCF7L2 were verified by Western blotting after knockout of Fgf9. Rescue experiments were performed by ELISA in vitro. Results: Fgf9^−/− mice exhibited 100% penetrance of the cleft palate. A knockout of Fgf9 confirmed that HAS2 and TCF7L2 expression was positively correlated with FGF9. TCF7L2 binds to the Has2 promoter, exhibiting the high specificity predicted by JASPAR. Additionally, increased HA expression by BML-284, TCF-dependent agonist, was blocked in Fgf9^−/− palate because of the significant decline in TCF7L2 expression. Conclusions: FGF9 promotes HAS2 expression via Wnt/β-catenin/TCF7L2 pathway with TCF7L2 activating transcription of Has2 in the palate. Full article

Apert syndrome is a rare genetic disorder characterized by craniosynostosis, midface retrusion, and limb anomalies. Cleft palate occurs in a subset of Apert syndrome patients. Although the genetic causes underlying Apert syndrome have been identified, the downstream signaling pathways and cellular mechanisms responsible for cleft palate are still elusive. To find clues for the pathogenic mechanisms of palatal defects in Apert syndrome, we review the clinical characteristics of the palate in cases of Apert syndrome, the palatal phenotypes in mouse models, and the potential signaling mechanisms involved in palatal defects. In Apert syndrome patients, cleft of the soft palate is more frequent than of the hard palate. The length of the hard palate is decreased. Cleft palate is associated most commonly with the S252W variant of FGFR2. In addition to cleft palate, high-arched palate, lateral palatal swelling, or bifid uvula are common in Apert syndrome patients. Mouse models of Apert syndrome display palatal defects, providing valuable tools to understand the underlying mechanisms. The mutations in FGFR2 causing Apert syndrome may change a signaling network in epithelial–mesenchymal interactions during palatogenesis. Understanding the pathogenic mechanisms of palatal defects in Apert syndrome may shed light on potential novel therapeutic solutions. Full article

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 Tgfβ3) 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. Full article

Morphogenesis requires a tight coordination between mechanical forces and biochemical signals to inform individual cellular behavior. For these developmental processes to happen correctly the organism requires precise spatial and temporal coordination of the adhesion, migration, growth, differentiation, and apoptosis of cells originating from the three key embryonic layers, namely the ectoderm, mesoderm, and endoderm. The cytoskeleton and its remodeling are essential to organize and amplify many of the signaling pathways required for proper morphogenesis. In particular, the interaction of the cell junctions with the cytoskeleton functions to amplify the behavior of individual cells into collective events that are critical for development. In this review we summarize the key morphogenic events that occur during the formation of the face and the palate, as well as the protein complexes required for cell-to-cell adhesions. We then integrate the current knowledge into a comprehensive review of how mutations in cell-to-cell adhesion genes lead to abnormal craniofacial development, with a particular focus on cleft lip with or without cleft palate. Full article

Craniofacial development including palatogenesis is a complex process which requires an orchestrated and spatiotemporal expression of various genes and factors for proper embryogenesis and organogenesis. One such group of genes essential for craniofacial development is the homeobox genes, transcriptional factors that are commonly associated with congenital abnormalities. Amongst these genes, DLX4, HOXB3, and MSX2 have been recently shown to be involved in the etiology of non-syndromic cleft lip and palate. Hence, we investigated the gene and protein expression of these genes in normal and cleft affected mucosal tissue obtained from 22 children, along with analyzing their role in promoting local-site inflammation using NF-κB. Additionally, we investigated the role of PTX3, which plays a critical role in tissue remodeling and wound repair. We found a residual gene and protein expression of DLX4 in cleft mucosa, although no differences in gene expression levels of HOXB3 and MSX2 were noted. However, a significant increase in protein expression for these genes was noted in the cleft mucosa (p < 0.05), indicating increased cellular proliferation. This was coupled with a significant increase in NF-κB protein expression in cleft mucosa (p < 0.05), highlighting the role of these genes in promotion of pro-inflammatory environment. Finally, no differences in gene expression of PTX3 were noted. Full article

We analyzed the immunohistochemical expression of Ki-67, pRb, Bax, and MMP-9 during the human secondary palate formation (7th to 12th developmental weeks (DWs). The most significant proliferation was observed in the seventh DW with 32% of Ki-67-positive cells in the epithelium, while loose ectomesenchyme condensations (lec) and loose non-condensing ectomesenchyme (lnc) had only 18 and 11%, respectively (Kruskal–Wallis, p < 0.001), and diminished afterwards. Contrarily, pRb-positive cells were mostly located in the lnc (67%), with significant difference in comparison to epithelium and lec in all investigated periods (Kruskal–Wallis, p < 0.001). Ki-67- and pRb-positive cells co-expressed occasionally in all investigated periods. MMP-9 displayed a strong expression pattern with the highest number of positive cells during the seventh DW in the epithelium, with significant difference in comparison to lec and lnc (Kruskal–Wallis, p < 0.0001). The ninth DW is particularly important for the Bax expression, especially in the epithelium (84%), in comparison to lec (58%) and lnc (47%) (Kruskal–Wallis, p < 0.001). The co-expression of Bax and MMP-9 was seen only in the epithelium during seventh and ninth DWs. Our study indicates the parallel persistence of proliferation (Ki-67, pRb) and remodeling (MMP-9) that enables growth and apoptotic activity (Bax) that enable the removal of the epithelial cells at the fusion point during secondary palate formation. Full article

In the present study, we examined the bone healing capacity of Meox2, a homeobox gene that plays essential roles in the differentiation of a range of developing tissues, and identified its putative function in palatogenesis. We applied the knocking down of Meox2 in human periodontal ligament fibroblasts to examine the osteogenic potential of Meox2. Additionally, we applied in vivo periodontitis induced experiment to reveal the possible application of Meox2 knockdown for 1 and 2 weeks in bone healing processes. We examined the detailed histomorphological changes using Masson’s trichrome staining and micro-computed tomography evaluation. Moreover, we observed the localization patterns of various signaling molecules, including α-SMA, CK14, IL-1β, and MPO to examine the altered bone healing processes. Furthermore, we investigated the process of bone formation using immunohistochemistry of Osteocalcin and Runx2. On the basis of the results, we suggest that the knocking down of Meox2 via the activation of osteoblast and modulation of inflammation would be a plausible answer for bone regeneration as a gene therapy. Additionally, we propose that the purpose-dependent selection and application of developmental regulation genes are important for the functional regeneration of specific tissues and organs, where the pathological condition of tooth loss lesion would be. Full article

Both infectious as non-infectious inflammation can cause placental dysfunction and pregnancy complications. During the first trimester of human gestation, when palatogenesis takes place, intrauterine hematoma and hemorrhage are common phenomena, causing the release of large amounts of heme, a well-known alarmin. We postulated that exposure of pregnant mice to heme during palatogenesis would initiate oxidative and inflammatory stress, leading to pathological pregnancy, increasing the incidence of palatal clefting and abortion. Both heme oxygenase isoforms (HO-1 and HO-2) break down heme, thereby generating anti-oxidative and -inflammatory products. HO may thus counteract these heme-induced injurious stresses. To test this hypothesis, we administered heme to pregnant CD1 outbred mice at Day E12 by intraperitoneal injection in increasing doses: 30, 75 or 150 μmol/kg body weight (30H, 75H or 150H) in the presence or absence of HO-activity inhibitor SnMP from Day E11. Exposure to heme resulted in a dose-dependent increase in abortion. At 75H half of the fetuses where resorbed, while at 150H all fetuses were aborted. HO-activity protected against heme-induced abortion since inhibition of HO-activity aggravated heme-induced detrimental effects. The fetuses surviving heme administration demonstrated normal palatal fusion. Immunostainings at Day E16 demonstrated higher numbers of ICAM-1 positive blood vessels, macrophages and HO-1 positive cells in placenta after administration of 75H or SnMP + 30H. Summarizing, heme acts as an endogenous “alarmin” during pregnancy in a dose-dependent fashion, while HO-activity protects against heme-induced placental vascular inflammation and abortion. Full article

Orofacial clefts (OFCs) are the most frequent craniofacial birth defects. An orofacial cleft (OFC) occurs as a result of deviations in palatogenesis. Cell proliferation, differentiation, adhesion, migration and apoptosis are crucial in palatogenesis. We hypothesized that deregulation of these processes in oral keratinocytes contributes to OFC. We performed microarray expression analysis on palatal keratinocytes from OFC and non-OFC individuals. Principal component analysis showed a clear difference in gene expression with 24% and 17% for the first and second component, respectively. In OFC cells, 228 genes were differentially expressed (p < 0.001). Gene ontology analysis showed enrichment of genes involved in β1 integrin-mediated adhesion and migration, as well as in P-cadherin expression. A scratch assay demonstrated reduced migration of OFC keratinocytes (343.6 ± 29.62 μm) vs. non-OFC keratinocytes (503.4 ± 41.81 μm, p < 0.05). Our results indicate that adhesion and migration are deregulated in OFC keratinocytes, which might contribute to OFC pathogenesis. Full article

Normal mammalian palatogenesis is a complex process that requires the occurrence of a tightly regulated series of specific and sequentially regulated cellular events. Cleft lip/palate (CLP), the most frequent craniofacial malformation birth defects, may occur if any of these events undergo abnormal interference. Such defects not only affect the patients, but also pose a financial risk for the families. In our recent study, the miniature pig was shown to be a valuable alternative large animal model for exploring human palate development by histology. However, few reports exist in the literature to document gene expression and function during swine palatogenesis. To better understand the genetic regulation of palate development, an mRNA expression profiling analysis was performed on miniature pigs, Sus scrofa. Five key developmental stages of miniature pigs from embryonic days (E) 30–50 were selected for transcriptome sequencing. Gene expression profiles in different palate development stages of miniature pigs were identified. Nine hundred twenty significant differentially expressed genes were identified, and the functional characteristics of these genes were determined by gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Some of these genes were associated with HH (hedgehog), WNT (wingless-type mouse mammary tumor virus integration site family), and MAPK (mitogen-activated protein kinase) signaling, etc., which were shown in the literature to affect palate development, while some genes, such as HIP (hedgehog interacting protein), WNT16, MAPK10, and LAMC2 (laminin subunit gamma 2), were additions to the current understanding of palate development. The present study provided a comprehensive analysis for understanding the dynamic gene regulation during palate development and provided potential ideas and resources to further study normal palate development and the etiology of cleft palate. Full article

Dioxins and related compounds induce morphological abnormalities in developing animals in an aryl hydrocarbon receptor (AhR)-dependent manner. Here we review the studies in which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is used as a prototypical compound to elucidate the pathogenesis of morphological abnormalities. TCDD-induced cleft palate in fetal mice involves a delay in palatogenesis and dissociation of fused palate shelves. TCDD-induced hydronephrosis, once considered to be caused by the anatomical obstruction of the ureter, is now separated into TCDD-induced obstructive and non-obstructive hydronephrosis, which develops during fetal and neonatal periods, respectively. In the latter, a prostaglandin E₂ synthesis pathway and urine concentration system are involved. TCDD-induced abnormal development of prostate involves agenesis of the ventral lobe. A suggested mechanism is that AhR activation in the urogenital sinus mesenchyme by TCDD modulates the wingless-type MMTV integration site family (WNT)/β-catenin signaling cascade to interfere with budding from urogenital sinus epithelium. TCDD exposure to zebrafish embryos induces loss of epicardium progenitor cells and heart malformation. AHR2-dependent downregulation of Sox9b expression in cardiomyocytes is a suggested underlying mechanism. TCDD-induced craniofacial malformation in zebrafish is considered to result from the AHR2-dependent reduction in SRY-box 9b (SOX9b), probably partly via the noncoding RNA slincR, resulting in the underdevelopment of chondrocytes and cartilage. Full article