Palatal Shelves Research Articles

Cleft Palate Induced by Augmented Fibroblast Growth Factor-9 Signaling in Cranial Neural Crest Cells in Mice.

Although enhanced fibroblast growth factor (FGF) signaling has been demonstrated to be crucial in many cases of syndromic cleft palate caused by tongue malposition in humans, animal models that recapitulate this phenotype are limited, and the precise mechanisms remain elusive. Mutations in FGF9 with the effect of either loss- or gain-of-function effects have been identified to be associated with cleft palate in humans. Here, we generated a mouse model with a transgenic Fgf9 allele specifically activated in cranial neural crest cells, aiming to elucidate the gain-of-function effects of Fgf9 in palatogenesis. We observed cleft palate with 100% penetrance in mutant mice. Further analysis demonstrated that no inherent defects in the morphogenic competence of palatal shelves could be found, but a passively lifted tongue prevented the elevation of palatal shelves, leading to the cleft palate. This tongue malposition was induced by posterior spatial confinement that was exerted by temporomandibular joint (TMJ) dysplasia characterized by a reduction in Sox9+ progenitors within the condyle and a structural decrease in the posterior dimension of the lower jaw. Our findings highlight the critical role of excessive FGF signaling in disrupting spatial coordination during palate development and suggest a potential association between palatal shelf elevation and early TMJ development.

Identification of novel genes regulating the development of the palate.

The International Mouse Phenotyping Consortium (IMPC) has generated thousands of knockout mouse lines, many of which exhibit embryonic or perinatal lethality. Using micro-computed tomography (micro-CT), the IMPC has created and publicly released 3D image datasets of embryos from these lethal and subviable lines. In this study, we leveraged this dataset to screen homozygous null mutants for anomalies in secondary palate development. We analyzed optical sections from 2,987 embryos at embryonic days E15.5 and E18.5, representing 484 homozygous mutant lines. Our analysis identified 45 novel genes implicated in palatogenesis. Gene set enrichment analysis highlighted biological processes and pathways relevant to palate development and uncovered 18 genes jointly regulating the development of the eye and the palate. These findings present a valuable resource for further research, offer novel insights into the molecular mechanisms underlying palatogenesis, and provide important context for understanding the etiology of rare human congenital disorders involving simultaneous malformations of the palate and other organs, including the eyes, ears, kidneys, and lungs.

Identification of competing endogenous RNA networks associated with circRNA and lncRNA in TCDD-induced cleft palate development

2,3,7,8 -tetrachlorodibenzo-p-dioxin (TCDD) is a teratogen that can induce cleft palate formation, a common birth defect. Competing endogenous RNAs (ceRNAs), including circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs), indirectly regulate gene expression via sharing microRNAs (miRNAs). Nevertheless, the mechanism by which they act as ceRNAs to regulate palatal development remains to be explored in greater detail. Here, the cleft palate model of C57BL/6 N pregnant mice was constructed by gavage of TCDD (64 ug/kg) on gestation day (GD) 10.5, and the palatal shelves were taken on gestation day (GD) 14.5 for whole-transcriptome sequencing to investigate the underlying mechanisms of the roles of circRNAs and lncRNAs as ceRNAs in cleft palate. Sequencing results revealed that 293 lncRNA, 589 circRNA, 47 miRNA, and 138 messenger RNA (mRNA) were significantly dysregulated, and the cytochrome P450 (CYP) enzymes and the aryl hydrocarbon receptor (AhR) pathway play key roles in the induction of cleft palate upon exposure to TCDD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed the function of TCDD function was mainly related to the metabolic processes of intracellular compounds, including the metabolic processes of cellular aromatic compounds and the metabolism of exogenous drugs by cytochrome P450, etc. Furthermore, quantitative reverse transcription polymerase chain reaction (qRT-PCR) indicated that the circRNA_1781/miR-30c-1–3p/PKIB and XR_380026.2/miR-1249–3p/DNAH10 ceRNA networks were hypothesized to be a hub involved in palatal development suggesting that the circRNA_1781/miR-30c-1–3p/PKIB and XR_380026.2/miR-1249–3p/DNAH10 ceRNA networks may be critical for palatogenesis, setting the foundation for the investigation of cleft palate.

Potential teratogenic effect of prenatal dexamethasone administration on palate development: Experimental study in rats

BackgroundThe interaction of cell populations and synchronization of cell signaling pathways during craniofacial development can cause malformations such as facial clefts when interrupted by teratogenic agents including synthetic corticosteroids. Therefore, this study aimed to determine the potentially disturbing dexamethasone (Dex) effect on palatal shelf development in rat embryos and the possible reasons for this teratogenic potential in relation to fibroblast growth factor 10 (FGF10) as well as FGF10 receptor2 (FGFR2) signaling. MethodsThirty pregnant rats were used, which were then randomly categorized into three groups of ten animals each: the control group, where pregnant rats received no treatment, Sham group in which pregnant rats were injected subcutaneously with saline (0.4 mg/kg body weight) during the mid-pregnancy period at gestational day 9–14, and Dex-treated group, where pregnant rats received 0.4 mg/kg of Dex at mid-gestational period. Pregnant animals from all groups were sacrificed on day 20 of gestation before birth and the fetuses were removed for examination of the palatal shelves using a light microscope (LM). In addition, a real-time polymerase chain reaction and a reverse transcription-polymerase chain reaction (RT-PCR) were done to evaluate FGF10 and its receptor FGFR2 gene expression. ResultsThe cleft palate incidence rates in the groups of embryos were determined. Only the offspring in the Dex group showed a cleft palate. Moreover, the cleft palate incidence rate in the Dex group was significantly different compared to controls. Moreover, a significant decrease in FGF10 and FGFR2 expression levels was reported in the Dex group than in the controls. ConclusionsDex treatment in mid-gestation may increase the incidence of cleft palate development, which may be due to modulation of FGF signaling. This calls for caution when using this medication in the first half of pregnancy unless absolutely necessary and under close medical supervision.

Evaluation of palate morphology in patients treated with leaf expander and hyrax expander: A geometric morphometric analysis.

The aim of this study was to evaluate changes in shape of the palatal vault after maxillary expansion with hyrax expander (HE) and leaf expander (LE), using 3D Geometric Morphometric Analysis. Overall, 250 patients (110 M, 140 F) with maxillary transverse deficiency were selected for this study. In this study, 127 subjects were treated with HE, 123 with LE. Digital dental models were obtained pre-treatment (T0) and after 12 months from the cementation of the device (T1) and processed by means of a digital scanner. Linear and morphometric analyses were conducted to determine the effects of each appliance on dental measurements and palatal shape, and a multiple linear regression was performed to analyse the influence of anchorage and appliance type on final shape. Morphometric analysis showed that there was a lowering of the palatal vault in the HE group, while in the LE group it remained unchanged: the difference in palatal shape at time T0 and T1 was statistically significant in both treatments (HE vs. LE). In the HE group, the change in shape also included the upper part of the palatal vault in the vertical dimension, while in the LE group the change in shape interested mainly palatal shelves and the lower portion of the palate. Both LE and HE produce clinically significant changes in the morphology of the palatal vault.

Palatal segment contributions to midfacial anterior-posterior growth.

Anterior-posterior (A-P) elongation of the palate is a critical aspect of integrated midfacial morphogenesis. Reciprocal epithelial-mesenchymal interactions drive secondary palate elongation that is coupled to the periodic formation of signaling centers within the rugae growth zone (RGZ). However, the relationship between RGZ driven morphogenetic processes, the differentiative dynamics of underlying palatal bone mesenchymal precursors, and the segmental organization of the upper jaw has remained enigmatic. A detailed ontogenetic study of these relationships is important, because palatal segment growth is a critical aspect of normal midfacial growth, can be modified to produce dysmorphology, and is a likely basis for evolutionary differences in upper jaw morphology. Variation in palatal-segment specific growth may also underlie known differences in palatal segment proportions between inbred mouse strains. We completed a combined whole mount gene expression and morphometric analysis of normal murine palatal growth dynamics and their association with palatal segment elongation and resulting upper jaw morphology. Our results demonstrated that the first formed palatal ruga (ruga 1), found just posterior to the RGZ, maintained an association with important nasal, neurovascular and palatal structures throughout early midfacial development; suggesting that these features are positioned at a proximal source of embryonic midfacial directional growth. Our detailed characterization of midfacial morphogenesis revealed a one-to-one relationship between palatal segments and upper jaw bones during the earliest stages of palatal elongation. Growth of the maxillary anlage within the anterior secondary palate is uniquely coupled to RGZ-driven morphogenesis that more than doubles the length of this palatal segment prior to palatal shelf fusion. Our results also demonstrate that the future maxillary-palatine suture, approximated by the position ruga 1 and consistently associated with the palatine anlage, forms predominantly via the posterior differentiation of the maxilla within the expanding anterior secondary palate. Our complementary ontogenetic comparison of three inbred mouse strains identified small but significant strain-specific differences in early embryonic palatal segment contributions to the upper jaw. Although early palatal segment specific growth is not primarily responsible for adult differences in upper jaw morphology between these strains, our ontogenetic series of measurements provide a useful foundation for understanding the impact of background genetic effects on facial shape and elongation. In combination, our results provide a novel and particularly detailed picture of the earliest spatiotemporal dynamics of intramembranous midfacial skeletal specification and differentiation within the context of the surrounding palatal segment A-P elongation and associated rugae formation.

Early embryogenesis in CHDFIDD mouse model reveals facial clefts and altered cranial neurogenesis.

CDK13-related disorder, also known as congenital heart defects, dysmorphic facial features and intellectual developmental disorder (CHDFIDD) is associated with mutations in the CDK13 gene encoding transcription-regulating cyclin-dependent kinase 13 (CDK13). Here, we focused on the development of craniofacial structures and analyzed early embryonic stages in CHDFIDD mouse models, with one model comprising a hypomorphic mutation in Cdk13 and exhibiting cleft lip/palate, and another model comprising knockout of Cdk13, featuring a stronger phenotype including midfacial cleft. Cdk13 was found to be physiologically expressed at high levels in the mouse embryonic craniofacial structures, namely in the forebrain, nasal epithelium and maxillary mesenchyme. We also uncovered that Cdk13 deficiency leads to development of hypoplastic branches of the trigeminal nerve including the maxillary branch. Additionally, we detected significant changes in the expression levels of genes involved in neurogenesis (Ache, Dcx, Mef2c, Neurog1, Ntn1, Pou4f1) within the developing palatal shelves. These results, together with changes in the expression pattern of other key face-specific genes (Fgf8, Foxd1, Msx1, Meis2 and Shh) at early stages in Cdk13 mutant embryos, demonstrate a key role of CDK13 in the regulation of craniofacial morphogenesis.

Spatiotemporal Evolution of Developing Palate in Mice

The intricate formation of the palate involves a series of complex events, yet its mechanistic basis remains uncertain. To explore major cell populations in the palate and their roles during development, we constructed a spatiotemporal transcription landscape of palatal cells. Palate samples from C57BL/6 J mice at embryonic days 12.5 (E12.5), 14.5 (E14.5), and 16.5 (E16.5) underwent single-cell RNA sequencing (scRNA-seq) to identify distinct cell subsets. In addition, spatial enhanced resolution omics-sequencing (stereo-seq) was used to characterize the spatial distribution of these subsets. Integrating scRNA-seq and stereo-seq with CellTrek annotated mesenchymal and epithelial cellular components of the palate during development. Furthermore, cellular communication networks between these cell subpopulations were analyzed to discover intercellular signaling during palate development. From the analysis of the middle palate, both mesenchymal and epithelial populations were spatially segregated into 3 domains. The middle palate mesenchymal subpopulations were associated with tooth formation, ossification, and tissue remodeling, with initial state cell populations located proximal to the dental lamina. The nasal epithelium of the palatal shelf exhibited richer humoral immune responses than the oral side. Specific enrichment of Tgfβ3 and Pthlh signals in the midline epithelial seam at E14.5 suggested a role in epithelial–mesenchymal transition. In summary, this study provides high-resolution transcriptomic information, contributing to a deeper mechanistic understanding of palate biology and pathophysiology.

Single-cell multiomics decodes regulatory programs for mouse secondary palate development

Perturbations in gene regulation during palatogenesis can lead to cleft palate, which is among the most common congenital birth defects. Here, we perform single-cell multiome sequencing and profile chromatin accessibility and gene expression simultaneously within the same cells (n = 36,154) isolated from mouse secondary palate across embryonic days (E) 12.5, E13.5, E14.0, and E14.5. We construct five trajectories representing continuous differentiation of cranial neural crest-derived multipotent cells into distinct lineages. By linking open chromatin signals to gene expression changes, we characterize the underlying lineage-determining transcription factors. In silico perturbation analysis identifies transcription factors SHOX2 and MEOX2 as important regulators of the development of the anterior and posterior palate, respectively. In conclusion, our study charts epigenetic and transcriptional dynamics in palatogenesis, serving as a valuable resource for further cleft palate research.

Unveiling dysregulated lncRNAs and networks in non-syndromic cleft lip with or without cleft palate pathogenesis

Non-syndromic cleft lip with or without cleft palate (NSCL/P) is a common congenital facial malformation with a complex, incompletely understood origin. Long noncoding RNAs (lncRNAs) have emerged as pivotal regulators of gene expression, potentially shedding light on NSCL/P's etiology. This study aimed to identify critical lncRNAs and construct regulatory networks to unveil NSCL/P's underlying molecular mechanisms. Integrating gene expression profiles from the Gene Expression Omnibus (GEO) database, we pinpointed 30 dysregulated NSCL/P-associated lncRNAs. Subsequent analyses enabled the creation of competing endogenous RNA (ceRNA) networks, lncRNA-RNA binding protein (RBP) interaction networks, and lncRNA cis and trans regulation networks. RT-qPCR was used to examine the regulatory networks of lncRNA in vivo and in vitro. Furthermore, protein levels of lncRNA target genes were validated in human NSCL/P tissue samples and murine palatal shelves. Consequently, two lncRNAs and three mRNAs: FENDRR (log2FC = − 0.671, P = 0.040), TPT1-AS1 (log2FC = 0.854, P = 0.003), EIF3H (log2FC = − 1.081, P = 0.041), RBBP6 (log2FC = 0.914, P = 0.037), and SRSF1 (log2FC = 0.763, P = 0.026) emerged as potential contributors to NSCL/P pathogenesis. Functional enrichment analyses illuminated the biological functions and pathways associated with these lncRNA-related networks in NSCL/P. In summary, this study comprehensively delineates the dysregulated transcriptional landscape, identifies associated lncRNAs, and reveals pivotal sub-networks relevant to NSCL/P development, aiding our understanding of its molecular progression and setting the stage for further exploration of lncRNA and mRNA regulation in NSCL/P.

Disruption of DNA methylation-mediated cranial neural crest proliferation and differentiation causes orofacial clefts in mice.

Orofacial clefts of the lip and palate are widely recognized to result from complex gene-environment interactions, but inadequate understanding of environmental risk factors has stymied development of prevention strategies. We interrogated the role of DNA methylation, an environmentally malleable epigenetic mechanism, in orofacial development. Expression of the key DNA methyltransferase enzyme DNMT1 was detected throughout palate morphogenesis in the epithelium and underlying cranial neural crest cell (cNCC) mesenchyme, a highly proliferative multipotent stem cell population that forms orofacial connective tissue. Genetic and pharmacologic manipulations of DNMT activity were then applied to define the tissue- and timing-dependent requirement of DNA methylation in orofacial development. cNCC-specific Dnmt1 inactivation targeting initial palate outgrowth resulted in OFCs, while later targeting during palatal shelf elevation and elongation did not. Conditional Dnmt1 deletion reduced cNCC proliferation and subsequent differentiation trajectory, resulting in attenuated outgrowth of the palatal shelves and altered development of cNCC-derived skeletal elements. Finally, we found that the cellular mechanisms of cleft pathogenesis observed in vivo can be recapitulated by pharmacologically reducing DNA methylation in multipotent cNCCs cultured in vitro. These findings demonstrate that DNA methylation is a crucial epigenetic regulator of cNCC biology, define a critical period of development in which its disruption directly causes OFCs, and provide opportunities to identify environmental influences that contribute to OFC risk.

Research progress in molecular mechanism of palatal development

Cleft palate is one of the most common maxillofacial birth defects, which can occur alone or accompany with many known deformities. Palatal selves need to complete the process of vertical growth, elevation, adhesion and fusion in a specific time window of embryo development. Any abnormality in this process will lead to cleft palate. Although previous studies have identified many molecular networks that regulate the growth, location and fusion of palatal selves, there are still many unknown mechanisms for palatal development. The pathogenesis of cleft palate has not been clarified so far. In recent years, the molecular research on palate development has been deepened continuously. Here we summarize major recent advances and integrate the genes and molecular pathways with the cellular and morphogenetic processes of palatal shelf growth, patterning, elevation, adhesion, and fusion, in order to comprehensively understand the genotype-phenotype functional relationship and provide assistance in formulating effective prevention strategies for cleft palate disease.

The influence of zinc on apoptosis and cell proliferation in palatal shelves during the fusion phase in mice and identification of a special protein family based on gene expression in cleft palate

Objective: To investigate the effect of the trace element zinc (Zn) on apoptosis and cell proliferation in palate shelvesduring the fusion phase, and to screen candidate genes of the Zn-finger special protein (Sp) family that were differentially expressed between the cleft palate and the normal palate to explore the mechanism of Zn in the development of cleft palate. Methods: Zn-rich, normal-Zn, low-Zn, and Zn-deficient diets were fed to female mice and, for the resultant fetuses, paraffin slices of their heads were made at embryonicdays 14.5 and 16.5. Using terminaldeoxynucleotidyltransferase-mediated dUTP nick-end labeling, the number of apoptotic cells in the palatal shelves was counted, and cell proliferation activity was detected using proliferating cell nuclear antigen staining. Total RNA from the palatal shelves of fetal mice was extracted from the Zn-rich diet, normal Zn-diet, and Zn-deficient-diet groups. We used microarray analysis to examine the expression of genes to identify intergroup differential gene expression and polymerase chain reaction tests to validate the results. Results: At ED14.5, the incidence of cleft palate in the regular zinc group, zinc rich group, low zinc group, and zinc deficient group was 8% (3/36), 2% (1/39), 29% (12/41), and 39% (15/38), respectively. The HE staining results at ED14.5 showed that both the left and right palatal processes in the zinc group had been lifted up and were in contact and connected with each other. In the zinc deficiency group, the left and right palatine processes remained vertically downwards on both sides of the tongue, ultimately forming cleft palate; In the low zinc group, the left and right palatine processes were raised but not in contact, ultimately resulting in cleft palate. There is no significant difference between the zinc rich group and the regular zinc group. At ED14.5, the positive rates of proliferative cells in the palatal process of fetal mice in the regular zinc group (80.29% ± 7.39%) and the zinc rich group (87.69% ± 6.62%) were significantly higher than those in the zinc deficient group (56.05% ± 16.13%) and the low zinc group (56.22% ± 9.61%) (t=4.32, P<0.05). The apoptosis index of fetal rat palatal process cells in the zinc deficient group (38.80% ± 3.10%) and the low zinc group (28.80% ± 6.19%) were significantly higher than those in the regular zinc group (16.80% ± 1.82%) (t=19.35, P<0.001; t=5.81, P<0.001). There were 663 differentially expressed genes in the zinc rich group and the zinc deficient group, with 513 up-regulated genes and 150 down-regulated genes, among which Sp5 was found to be located. The real time PCR results showed that compared with the regular zinc group (2.22 ± 0.36), the expression level of Sp5mRNA in the palatal process tissue of the zinc deficient group (1.23 ± 0.38) significantly increased (P<0.05), while the zinc rich group (3.68 ± 0.90) significantly decreased (P<0.05). Conclusions: Trace element Zn content was found to be closely related to the occurrence of cleft palate in mice offspring, with a lack of Zn leading to cleft palate.

Fabrication of obturators for veau’s class III cleft lip and palate patients - A case series

A fissured palate is a genetic condition where there is a gap or opening in the roof of the mouth (palate). This opening occurs due to the inability of the palatal shelves, which are responsible for forming the palate, to properly fuse during fetal development. This results in oronasal communication, or an opening between the palate and the base of the nose. Cleft palates can result from a combination of genetic factors, chromosomal disorders, heredity, and environmental factors. Possible environmental causes include nutritional deficiencies, maternal alcohol or cigarette consumption, exposure to radiation during pregnancy, hormonal imbalances, the ingestion of teratogenic substances by the mother, and infections. Newborns with cleft palates face difficulties in feeding and swallowing due to the gap in the palate. This can lead to problems such as stunted growth and being underweight. As these children grow, they can experience additional challenges including difficulties in speech, dental problems, altered facial appearance, and psychological issues. One immediate solution for infants with cleft palate is the fabrication of a feeding plate, often called an obturator. This device helps close the oronasal opening, enabling improved feeding, and reducing the risk of infections and regurgitation. This intervention can also aid in achieving proper weight gain, which is essential for future reconstructive surgery to repair the cleft palate. This paper presents a case series of infants with cleft palate who received feeding plates. The feeding plates were created using putty impressions to customize them for each patient's needs. This approach aimed to help the infants feed effectively, maintain their weight, and prepare for future reconstructive surgery to address the cleft palate.

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.

Retinoic acid delays murine palatal shelf elevation by inhibiting Wnt5a-mediated noncanonical Wnt signaling and downstream Cdc-42/F-actin remodeling in mesenchymal cells.

Mammalian palatal shelves erupted from maxillary prominences undergo vertical extention, transient elevation, and horizontal growth to fuse. Previous studies in mice reported that the retinoic acid (RA) contributed to cleft palate in high incidence by delaying the elevating procedure, but little was known about the underlying biological mechanisms. In this study, hematoxylin-eosin and immunofluorescence staining were employed to evaluate the phenotypes and the expression of related markers in the RA-treated mice model. In situ hybridization and RT-qPCR were used to detect the expression of genes involved in Wnt signaling pathway. The palatal mesenchymal cells were cultured in vitro, and stimulated with RA or CASIN, and co-treated with Foxy5. Wnt5a and Ccd42 expression were evaluated by immunofluorescence staining. Phalloidin was used to label the microfilament cytoskeleton (F-actin) in cultured cells. We revealed that RA resulted in 100% incidence of cleft palate in mouse embryos, and the expression of genes responsible for Wnt5a-mediated noncanonical Wnt signal transduction were specifically downregulated in mesenchymal palatal shelves. The in vitro study of palatal mesenchymal cells indicated that RA treatment disrupted the organized remodeling of cytoskeleton, an indicative structure of cell migration regulated by the small Rho GTPase Cdc42. Moreover, we showed that the suppression of cytoskeleton and cell migration induced by RA was partially restored using the small molecule Foxy-5-mediated activation of Wnt5A, and this restoration was attenuated by CASIN (a selective GTPase Cdc42 inhibitor) again. These data identified a crucial mechanism for Wnt5a-mediated noncanonical Wnt signaling in acting downstream of Rho GTPase Cdc42 to regulate cytoskeletal remodeling and cell migration during the process of palate elevation. Our study provided a new explanation for the cause of cleft palate induced by RA.

Trio-based GWAS identifies novel associations and subtype-specific risk factors for cleft palate

Cleft palate (CP) is one of the most common craniofacial birth defects; however, there are relatively few established genetic risk factors associated with its occurrence despite high heritability. Historically, CP has been studied as a single phenotype, although it manifests across a spectrum of defects involving the hard and/or soft palate. We performed a genome-wide association study using transmission disequilibrium tests of 435 case-parent trios to evaluate broad risks for any cleft palate (ACP) (n= 435), and subtype-specific risks for any cleft soft palate (CSP), (n= 259) and any cleft hard palate (CHP) (n= 125). We identified a single genome-wide significant locus at 9q33.3 (lead SNP rs7035976, p= 4.24×10-8) associated with CHP. One gene at this locus, angiopoietin-like 2 (ANGPTL2), plays a role in osteoblast differentiation. It is expressed both in craniofacial tissue of human embryos and developing mouse palatal shelves. We found 19 additional loci reaching suggestive significance (p<5×10-6), of which only one overlapped between groups (chromosome 17q24.2, ACP and CSP). Odds ratios for the 20 loci were most similar across all 3 groups for SNPs associated with the ACP group, but more distinct when comparing SNPs associated with either subtype. We also found nominal evidence of replication (p<0.05) for 22 SNPs previously associated with orofacial clefts. Our study to evaluate CP risks in the context of its subtypes and we provide newly reported associations affecting the broad risk for CP as well as evidence of subtype-specific risks.

RA-induced prominence-specific response resulted in distinctive regulation of Wnt and osteogenesis.

Proper retinoic acid (RA) signaling is essential for normal craniofacial development. Both excessive RA and RA deficiency in early embryonic stage may lead to a variety of craniofacial malformations, for example, cleft palate, which have been investigated extensively. Dysregulated Wnt and Shh signaling were shown to underlie the pathogenesis of RA-induced craniofacial defects. In our present study, we showed a spatiotemporal-specific effect of RA signaling in regulating early development of facial prominences. Although inhibited Wnt activities was observed in E12.5/E13.5 mouse palatal shelves, early exposure of excessive RA induced Wnt signaling and Wnt-related gene expression in E11.5/E12.5 mouse embryonic frontonasal/maxillary processes. A conserved regulatory network of miR-484-Fzd5 was identified to play critical roles in RA-regulated craniofacial development using RNA-seq. In addition, subsequent osteogenic/chondrogenic differentiation were differentially regulated in discrete mouse embryonic facial prominences in response to early RA induction, demonstrated using both in vitro and in vivo analyses.

An improved multicellular human organoid model for the study of chemical effects on palatal fusion.

Tissue fusion is a mechanism involved in the development of the heart, iris, genital tubercle, neural tube, and palate during embryogenesis. Failed fusion of the palatal shelves could result in cleft palate (CP), a common birth defect. Organotypic models constructed of human cells offer an opportunity to investigate developmental processes in the human. Previously, our laboratory developed an organoid model of the human palate that contains human mesenchyme and epithelial progenitor cells to study the effects of chemicals on fusion. Here, we developed an organoid model more representative of the embryonic palate that includes three cell types: mesenchyme, endothelial, and epithelial cells. We measured fusion by a decrease in epithelial cells at the contact point between the organoids and compared the effects of CP teratogens on fusion and toxicity in the previous and current organoid models. We further tested additional suspect teratogens in our new model. We found that the three-cell-type model is more sensitive to fusion inhibition by valproic acid and inhibitors of FGF, BMP, and TGFβRI/II. In this new model, we tested other suspect CP teratogens and found that nocodazole, topiramate, and Y27632 inhibit fusion at concentrations that do not induce toxicity. This sensitive human three-cell-type organotypic model accurately evaluates chemicals for cleft palate teratogenicity.

Gene Regulatory Networks and Signaling Pathways in Palatogenesis and Cleft Palate: A Comprehensive Review.

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.