Single-cell transcriptome and chromatin accessibility mapping of upper lip and primary palate fusion.

Cleft lip and/or primary palate (CL/P) represent a prevalent congenital malformation, the aetiology of which is highly intricate. Although it is generally accepted that the condition arises from failed fusion between the upper lip and primary palate, the precise mechanism underlying this fusion process remains enigmatic. In this study, we utilized transposase-accessible chromatin sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) to interrogate lambdoidal junction tissue derived from C57BL/6J mouse embryos at critical stages of embryogenesis (10.5, 11.5 and 12.5 embryonic days). We successfully identified distinct subgroups of mesenchymal and ectodermal cells involved in the fusion process and characterized their unique transcriptional profiles. Furthermore, we conducted cell differentiation trajectory analysis, revealing a dynamic repertoire of genes that are sequentially activated or repressed during pseudotime, facilitating the transition of relevant cell types. Additionally, we employed scATAC data to identify key genes associated with the fusion process and demonstrated differential chromatin accessibility across major cell types. Finally, we constructed a dynamic intercellular communication network and predicted upstream transcriptional regulators of critical genes involved in important signalling pathways. Our findings provide a valuable resource for future studies on upper lip and primary palate development, as well as congenital defects.

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.

An effective ultrasound fetal palate screening software based on the "sequential sector scan through the oral fissure" and three-dimensional ultrasound.

BACKGROUND: Orofacial clefts are one of the most common congenital malformations of the fetal face and ultrasound is mainly responsible for its diagnosis. It is difficult to view the fetal palate, so there is currently no unified standard for fetal palate screening, and the diagnosis of cleft palate is not included in the relevant prenatal ultrasound screening guidelines. Many prenatal diagnoses for cleft palate are missed due to the lack of effective screening methods. Therefore, it is imperative to increase the display rate of the fetal palate, which would improve the detection rate and diagnostic accuracy for cleft palate. We aim to introduce a fetal palate screening software based on the "sequential sector scan though the oral fissure", an effective method for fetal palate screening which was verified by our follow up results and three-dimensional ultrasound and to evaluate its feasibility and clinical practicability. METHODS: A software was designed and programmed based on "sequential sector scan through the oral fissure" and three-dimensional ultrasound. The three-dimensional ultrasound volume data of the fetal face were imported into the software. Then, the median sagittal plane was taken as the reference interface, the anterior upper margin of the mandibular alveolar bone was selected as the fulcrum, the interval angles, and the number of layers of the sector scan were set, after which the automatic scan was performed. Thus, the sector scan sequential planes of the mandibular alveolar bone, pharynx, soft palate, hard palate, and maxillary alveolar bone were obtained in sequence to display and evaluate the palate. In addition, the feasibility and accuracy of the software in fetal palate displaying and screening was evaluated by actual clinical cases. RESULTS: Full views of the normal fetal palates and the defective parts of the cleft palates were displayed, and relatively clear sequential tomographic images and continuous dynamic videos were formed after the three-dimensional volume data of 10 normal fetal palates and 10 cleft palates were imported into the software. CONCLUSIONS: The software can display fetal palates more directly which might allow for a new method of fetal palate screening and cleft palate diagnosis.

Protective effect of Sasa veitchii extract against all-trans-retinoic acid-induced inhibition of proliferation of cultured human palate cells.

Cleft palate is the most common facial birth defect worldwide. It is caused by environmental factors or genetic mutations. Environmental factors such as pharmaceutical exposure in women are known to induce cleft palate. The aim of the present study was to investigate the protective effect of Sasa veitchii extract against medicine-induced inhibition of proliferation of human embryonic palatal mesenchymal cells. We demonstrated that all-trans-retinoic acid inhibited human embryonic palatal mesenchymal cell proliferation in a dose-dependent manner, whereas dexamethasone treatment had no effect on cell proliferation. Cotreatment with Sasa veitchii extract repressed all-trans-retinoic acid-induced toxicity in human embryonic palatal mesenchymal cells. We found that cotreatment with Sasa veitchii extract protected all-trans-retinoic acid-induced cyclin D1 downregulation in human embryonic palatal mesenchymal cells. Furthermore, Sasa veitchii extract suppressed all-trans-retinoic acid-induced miR-4680-3p expression. Additionally, the expression levels of the genes that function downstream of the target genes ( ERBB2 and JADE1 ) of miR-4680-3p in signaling pathways were enhanced by cotreatment with Sasa veitchii extract and all-trans-retinoic acid compared to all-trans-retinoic acid treatment. These results suggest that Sasa veitchii extract suppresses all-trans-retinoic acid-induced inhibition of cell proliferation via modulation of miR-4680-3p expression.

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 Tgfß3 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.

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.

The role of the histone methyltransferase SET domain bifurcated 1 during palatal development.

The histone methyltransferase SET domain bifurcated 1 (SETDB1) catalyzes the trimethylation of lysine 9 of histone H3, thereby regulating gene expression. In this study, we used conditional knockout mice, where Setdb1 was deleted only in neural crest cells (Setdb1fl/fl,Wnt1-Cre + mice), to clarify the role of SETDB1 in palatal development. Setdb1fl/fl,Wnt1-Cre + mice died shortly after birth due to a cleft palate with full penetration. Reduced palatal mesenchyme proliferation was seen in Setdb1fl/fl,Wnt1-Cre + mice, which might be a possible mechanism of cleft palate development. Quantitative RT-PCR and in situ hybridization showed that expression of the Pax9, Bmp4, Bmpr1a, Wnt5a, and Fgf10 genes, known to be important for palatal development, were markedly decreased in the palatal mesenchyme of Setdb1fl/fl,Wnt1-Cre + mice. Along with these phenomena, SMAD1/5/9 phosphorylation was decreased by the loss of Setdb1. Our results demonstrated that SETDB1 is indispensable for palatal development partially through its proliferative effect. Taken together with previous reports that PAX9 regulates BMP signaling during palatal development which implies that loss of Setdb1 may be involved in the cleft palate development by decreasing SMAD-dependent BMP signaling through Pax9.

Impact of GAD65 and/or GAD67 deficiency on perinatal development in rats.

GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1-/- ; Gad2-/- rats died at postnatal day 0 and showed little alveolar space in their lungs, suggesting that the cause of their death was respiratory failure. All Gad1-/- ; Gad2-/- rats and 18% of Gad1-/- ; Gad2+/- rats showed cleft palate. In contrast, none of the Gad mutant rats including Gad1-/- ; Gad2-/- rats, showed omphalocele. These results suggest that both rat GAD65 and GAD67 are involved in palate formation, while neither isoform is critical for abdominal wall formation. The GABA content in Gad1-/- ; Gad2-/- rat forebrains and retinas at embryonic day 20 was extremely low, indicating that almost all GABA was synthesized from glutamate by GADs in the perinatal period. The present study shows that Gad mutant rats are a good model for further defining the role of GABA during development.

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.

The Mafb cleft-associated variant H131Q is not required for palatogenesis in the mouse.

BACKGROUND: Orofacial clefts (OFCs) are common birth defects with complex etiology. Genome wide association studies for OFC have identified SNPs in and near MAFB. MAFB is a transcription factor critical for structural development of digits, kidneys, skin, and brain. MAFB is also expressed in the craniofacial region. Previous sequencing of MAFB in a Filipino population revealed a novel missense variant significantly associated with an increased risk for OFC. This MAFB variant, leading to the amino acid change H131Q, was knocked into the mouse Mafb, resulting in the MafbH131Q allele. The MafbH131Q construct was engineered to allow for deletion of Mafb ("Mafbdel "). RESULTS: Mafbdel/del animals died shortly after birth. Conversely, MafbH131Q/H131Q mice survived into adulthood at Mendelian ratios. Mafbdel/del and MafbH131Q/H131Q heads exhibited normal macroscopic and histological appearance at all embryonic time points evaluated. The periderm was intact based on expression of keratin 6, p63, and E-cadherin. Despite no effect on craniofacial morphogenesis, H131Q inhibited the Mafb-dependent promoter activation of Arhgap29 in palatal mesenchymal, but not ectodermal-derived epithelial cells in a luciferase assay. CONCLUSIONS: Mafb is dispensable for murine palatogenesis in vivo, and the cleft-associated variant H131Q, despite its lack of morphogenic effect, altered the expression of Arhgap29 in a cell-dependent context.

Anti-epileptic drug topiramate upregulates TGFß1 and SOX9 expression in primary embryonic palatal mesenchyme cells: Implications for teratogenicity.

Topiramate is an anti-epileptic drug that is commonly prescribed not just to prevent seizures but also migraine headaches, with over 8 million prescriptions dispensed annually. Topiramate use during pregnancy has been linked to significantly increased risk of babies born with orofacial clefts (OFCs). However, the exact molecular mechanism of topiramate teratogenicity is unknown. In this study, we first used an unbiased antibody array analysis to test the effect of topiramate on human embryonic palatal mesenchyme (HEPM) cells. This analysis identified 40 differentially expressed proteins, showing strong connectivity to known genes associated with orofacial clefts. However, among known OFC genes, only TGFß1 was significantly upregulated in the antibody array analysis. Next, we validated that topiramate could increase expression of TGFß1 and of downstream target phospho-SMAD2 in primary mouse embryonic palatal mesenchyme (MEPM) cells. Furthermore, we showed that topiramate treatment of primary MEPM cells increased expression of SOX9. SOX9 overexpression in chondrocytes is known to cause cleft palate in mouse. We propose that topiramate mediates upregulation of TGFß1 signaling through activation of γ-aminobutyric acid (GABA) receptors in the palate. TGFß1 and SOX9 play critical roles in orofacial morphogenesis, and their abnormal overexpression provides a plausible etiologic molecular mechanism for the teratogenic effects of topiramate.

SPECC1L-deficient primary mouse embryonic palatal mesenchyme cells show speed and directionality defects.

Cleft lip and/or palate (CL/P) are common anomalies occurring in 1/800 live-births. Pathogenic SPECC1L variants have been identified in patients with CL/P, which signifies a primary role for SPECC1L in craniofacial development. Specc1l mutant mouse embryos exhibit delayed palatal shelf elevation accompanied by epithelial defects. We now posit that the process of palate elevation is itself abnormal in Specc1l mutants, due to defective remodeling of palatal mesenchyme. To characterize the underlying cellular defect, we studied the movement of primary mouse embryonic palatal mesenchyme (MEPM) cells using live-imaging of wound-repair assays. SPECC1L-deficient MEPM cells exhibited delayed wound-repair, however, reduced cell speed only partially accounted for this delay. Interestingly, mutant MEPM cells were also defective in coordinated cell movement. Therefore, we used open-field 2D cultures of wildtype MEPM cells to show that they indeed formed cell streams at high density, which is an important attribute of collective movement. Furthermore, activation of the PI3K-AKT pathway rescued both cell speed and guidance defects in Specc1l mutant MEPM cells. Thus, we show that live-imaging of primary MEPM cells can be used to assess mesenchymal remodeling defects during palatal shelf elevation, and identify a novel role for SPECC1L in collective movement through modulation of PI3K-AKT signaling.

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

Palatal mesenchymal cell proliferation is essential to the process of palatogenesis, and the proliferation of mouse embryonic palate mesenchymal (MEPM) cells is impacted by both all-trans retinoic acid (atRA) and the TGF-ß/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been shown to activate TGF-ß/Smad signaling and to thereby regulate cell proliferation, differentiation, and related processes. Herein, we found that atRA treatment (100 mg/kg) promoted Meg3 upregulation in MEPM cells, and that such upregulation was linked to the suppression of MEPM cell proliferation in the context of secondary palate fusion on gestational day (GD) 13 and 14. Moreover, the demethylation of specific CpG sites within the lncRNA Meg3 promoter was detected in atRA-treated MEPM cells, likely explaining the observed upregulation of this lncRNA. Smad signaling was also suppressed by atRA treatment in these cells, and RNA immunoprecipitation analyses revealed that Smad2 can directly interact with Meg3 in MEPM cells following atRA treatment. Therefore, we propose a model wherein Meg3 is involved in the suppression of MEPM cell proliferation, functioning at least in part via interacting with the Smad2 protein and thereby suppressing Smad signaling in the context of atRA-induced cleft palate.

Maternal Folic Acid Deficiency Is Associated to Developing Nasal and Palate Malformations in Mice.

Craniofacial development requires extremely fine-tuned developmental coordination of multiple specialized tissues. It has been evidenced that a folate deficiency (vitamin B9), or its synthetic form, folic acid (FA), in maternal diet could trigger multiple craniofacial malformations as oral clefts, tongue, or mandible abnormalities. In this study, a folic acid-deficient (FAD) diet was administered to eight-week-old C57/BL/6J female mouse for 2-16 weeks. The head symmetry, palate and nasal region were studied in 24 control and 260 experimental fetuses. Our results showed a significant reduction in the mean number of fetuses per litter according to maternal weeks on FAD diet (p < 0.01). Fetuses were affected by cleft palate (3.8%) as well as other severe congenital abnormalities, for the first time related to maternal FAD diet, as head asymmetries (4.6%), high arched palate (3.5%), nasal septum malformed (7.3%), nasopharynx duct shape (15%), and cilia and epithelium abnormalities (11.2% and 5.8%). Dysmorphologies of the nasal region were the most frequent, appearing at just four weeks following a maternal FAD diet. This is the first time that nasal region development is experimentally related to this vitamin deficiency. In conclusion, our report offers novel discoveries about the importance of maternal folate intake on midface craniofacial development of the embryos. Moreover, the longer the deficit lasts, the more serious the consequent effects appear to be.

Novel insights into the development of the avian nasal cavity.

In embryonic amniotes, patterning of the oral and nasal cavities requires bilateral fusion between craniofacial prominences, ensuring an intact primary palate and upper jaw. After fusion has taken place, the embryonic nasal cavities open anteriorly through paired external nares positioned directly above the fusion zones and bordered by the medial nasal and lateral nasal prominences. In this study, we show that in the chicken embryo, the external nares initially form as patent openings but only remain so for a short period of time. Soon after the nasal cavities form, the medial nasal and lateral nasal prominences fuse together in stage 29 embryos, entirely closing off the external nares for a substantial portion of embryonic and fetal development. The epithelium between the fused prominences is then retained and eventually develops into a nasal plug that obstructs the nasal vestibule through the majority of the fetal period. At stage 40, the nasal plug begins to break down through a combination of cellular remodeling, apoptosis, as well as non-apoptotic necrosis, leading to completely patent nasal cavities at hatching. These findings place chickens in a category with several species of nonavian reptiles and mammals (including humans) that have been found to develop a transient embryonic nasal plug. Our findings are discussed in the context of previously reported cases of nasal plugs as part of normal embryonic development and provide novel insight into the craniofacial development of a key model organism in developmental biology.

Tissue-specific analysis of Fgf18 gene function in palate development.

BACKGROUND: Previous studies showed that mice lacking Fgf18 function had cleft palate defects and that the FGF18 locus was associated with cleft lip and palate in humans, but what specific roles Fgf18 plays during palatogenesis are unclear. RESULTS: We show that Fgf18 exhibits regionally restricted expression in developing palatal shelves, mandible, and tongue, during palatal outgrowth and fusion in mouse embryos. Tissue-specific inactivation of Fgf18 throughout neural crest-derived craniofacial mesenchyme caused shortened mandible and reduction in ossification of the frontal, nasal, and anterior cranial base skeletal elements in Fgf18c/c ;Wnt1-Cre mutant mice. About 64% of Fgf18c/c ;Wnt1-Cre mice exhibited cleft palate. Whereas palatal shelf elevation was impaired in many Fgf18c/c ;Wnt1-Cre embryos, no significant difference in palatal cell proliferation was detected between Fgf18c/c ;Wnt1-Cre embryos and their control littermates. Embryonic maxillary explants from Fgf18c/c ;Wnt1-Cre embryos showed successful palatal shelf elevation and fusion in organ culture similar to the maxillary explants from control embryos. Furthermore, tissue-specific inactivation of Fgf18 in the early palatal mesenchyme did not cause cleft palate. CONCLUSION: These results demonstrate a critical role for Fgf18 expression in the neural crest-derived mesenchyme for the development of the mandible and multiple craniofacial bones but Fgf18 expression in the palatal mesenchyme is dispensable for palatogenesis.

Embryology of the naso-palatal complex in Gekkota based on detailed 3D analysis in Lepidodactylus lugubris and Eublepharis macularius.

The vomeronasal organ (VNO), nasal cavity, lacrimal duct, choanal groove, and associated parts of the superficial (soft tissue) palate are called the naso-palatal complex. Despite the morphological diversity of the squamate noses, little is known about the embryological basis of this variation. Moreover, developmental data might be especially interesting in light of the morpho-molecular discordance of squamate phylogeny, since a 'molecular scenario' implies an occurrence of unexpected scale of homoplasy also in olfactory systems. In this study, we used X-ray microtomography and light microscopy to describe morphogenesis of the naso-palatal complex in two gekkotans: Lepidodactylus lugubris (Gekkonidae) and Eublepharis macularius (Eublepharidae). Our embryological data confirmed recent findings about the nature of some developmental processes in squamates, for example, involvement of the lateral nasal prominence in the formation of the choanal groove. Moreover, our study revealed previously unknown differences between the studied gekkotans and allows us to propose redefinition of the anterior concha of Sphenodon. Interpretation of some described conditions might be problematic in the phylogenetic context, since they represent unknown: squamate, nonophidian squamate, or gekkotan features.

Altered BMP-Smad4 signaling causes complete cleft palate by disturbing osteogenesis in palatal mesenchyme.

As the major receptor mediated BMP signaling in craniofacial development, Bmpr1a expression was detected in the anterior palatal shelves from E13.5 and the posterior palatal shelves from E14.5. However, inactivating BMP receptor in the mesenchyme only leads to anterior cleft palate or submucous cleft palate. The role of BMP signaling in posterior palatal mesenchyme and palatal osteogenesis is still unknown. In this study, a secreted BMP antagonist, Noggin was over-expressed by Osr2-creKI to suppress BMP signaling intensively in mouse palatal mesenchyme, which made the newborn mouse displaying complete cleft palate, a phenotype much severer than the anterior or submucous cleft palate. Immunohistochemical analysis indicated that in the anterior and posterior palatal mesenchyme, the canonical BMP-Smad4 signaling was dramatically down-regulated, while the non-canonical BMP signaling pathways were altered little. Although cell proliferation was reduced only in the anterior palatal mesenchyme, the osteogenic condensation and Osterix distribution were remarkably repressed in the posterior palatal mesenchyme by Noggin over-expression. These findings suggested that BMP-Smad4 signaling was essential for the cell proliferation in the anterior palatal mesenchyme, and for the osteogenesis in the posterior palatal mesenchyme. Interestingly, the constitutive activation of Bmpr1a in palatal mesenchyme also caused the complete cleft palate, in which the enhanced BMP-Smad4 signaling resulted in the premature osteogenic differentiation in palatal mesenchyme. Moreover, neither the Noggin over-expression nor Bmpr1a activation disrupted the elevation of palatal shelves. Our study not only suggested that BMP signaling played the differential roles in the anterior and posterior palatal mesenchyme, but also indicated that BMP-Smad4 signaling was required to be finely tuned for the osteogenesis of palatal mesenchyme.

The cleft palate candidate gene BAG6 supports FoxO1 acetylation to promote FasL-mediated apoptosis during palate fusion.

BACKGROUND: Cleft palate is a common craniofacial defect, which occurs when the palate fails to fuse during development. During fusion, the palatal shelves migrate towards the embryonic midline to form a seam. Apoptotic elimination of medial edge epithelium (MEE) cells along this seam is required for the completion of palate fusion. METHODS: Whole exome sequencing (WES) of six Chinese cleft palate families was applied to identify novel cleft palate-associated gene variants. Palatal fusion and immunofluorescence studies were performed in a murine palatal shelf organ culture model. Gene and protein expression were analyzed by qPCR and immunoblotting in murine MEE cells during seam formation in vivo. Mechanistic immunoprecipitation studies were performed in murine MEE cells in vitro. RESULTS: WES identified Bcl-2 associated anthanogene 6 (BAG6) as a novel cleft palate-associated gene. In murine MEE cells, we discovered upregulation of Bag6 and the transcription factor forkhead box protein O1 (FoxO1) during seam formation in vivo. Using a palatal shelf organ culture model, we demonstrate that nuclear-localized Bag6 enhances MEE cell apoptosis by promoting p300's acetylation of FoxO1, thereby promoting transcription of the pro-apoptotic Fas ligand (FasL). Subsequent gain- and loss-of-function studies in the organ culture model demonstrated that FasL is required for Bag6/acFoxO1-mediated activation of pro-apoptotic Bax/caspase-3 signaling, MEE apoptosis, and palate fusion. Palatal shelf contact was shown to enhance Bag6 nuclear localization and upregulate nuclear acFoxO1 in MEE cells. CONCLUSIONS: These findings demonstrate that nuclear-localized Bag6 and p300 co-operatively enhance FoxO1 acetylation to promote FasL-mediated MEE apoptosis during palate fusion.

Comparison of the biological behaviors of palatal mesenchymal and epithelial cells induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro.

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) effectively induces cleft palate at increased doses, but its mechanism of involvement is unclear, and arguments have examined palatal shelf contact and/or fusion failure. The role of different types of cells constituting palatal skulls remains elusive regarding TCDD dosage. No reports have simultaneously compared the biological behaviors of TCDD- induced mesenchymal and epithelial cells in vitro. This study employed primary epithelial and mesenchymal cells as models in vitro to explore proliferation, migration, apoptosis and epithelial-to-mesenchymal transition with two different doses of TCDD (10 nmol/L, 100 nmol/L), contrasted with a control group without TCDD. Interestingly, we found the EMT process of primary palatal epithelial cells occurred automatically in vitro without helping bilateral palatal contact. The results showed that, with the low dose of TCDD, transformation of epithelial cells to mesenchymal cells was inhibited, and mesenchymal cell proliferation and migration were promoted. At high doses, mesenchymal cells decreased, preventing palate development, uprising and contact, while the EMT of epithelial cells decreased. Regardless of dose of TCDD, no impact on migration and apoptosis of epithelial cells was noted, but there was increased apoptosis of mesenchymal cell in a dose-dependent manner.