Epigenetic regulation of craniofacial development and disease.

BACKGROUND: The formation of the craniofacial complex relies on proper neural crest development. The gene regulatory networks (GRNs) and signaling pathways orchestrating this process have been extensively studied. These GRNs and signaling cascades are tightly regulated as alterations to any stage of neural crest development can lead to common congenital birth defects, including multiple syndromes affecting facial morphology as well as nonsyndromic facial defects, such as cleft lip with or without cleft palate. Epigenetic factors add a hierarchy to the regulation of transcriptional networks and influence the spatiotemporal activation or repression of specific gene regulatory cascades; however less is known about their exact mechanisms in controlling precise gene regulation. AIMS: In this review, we discuss the role of epigenetic factors during neural crest development, specifically during craniofacial development and how compromised activities of these regulators contribute to congenital defects that affect the craniofacial complex.

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.

Cleft palate and minor metabolic disturbances in a mouse global Arl15 gene knockout.

ARL15, a small GTPase protein, was linked to metabolic traits in association studies. We aimed to test the Arl15 gene as a functional candidate for metabolic traits in the mouse. CRISPR/Cas9 germline knockout (KO) of Arl15 showed that homozygotes were postnatal lethal and exhibited a complete cleft palate (CP). Also, decreased cell migration was observed from Arl15 KO mouse embryonic fibroblasts (MEFs). Metabolic phenotyping of heterozygotes showed that females had reduced fat mass on a chow diet from 14 weeks of age. Mild body composition phenotypes were also observed in heterozygous mice on a high-fat diet (HFD)/low-fat diet (LFD). Females on a HFD showed reduced body weight, gonadal fat depot weight and brown adipose tissue (BAT) weight. In contrast, in the LFD group, females showed increased bone mineral density (BMD), while males showed a trend toward reduced BMD. Clinical biochemistry analysis of plasma on HFD showed transient lower adiponectin at 20 weeks of age in females. Urinary and plasma Mg2+ concentrations were not significantly different. Our phenotyping data showed that Arl15 is essential for craniofacial development. Adult metabolic phenotyping revealed potential roles in brown adipose tissue and bone development.

[Regulatory role and mechanism of primary cilia in craniofacial and dental development].

Primary cilia protruding from cell surface are important cell receptors and exist in most types of vertebrate cells. Primary cilia can sense extracellular mechanical signals, chemical signals as well as optical signals, and transduce them into cells, which is crucial for embryonic development and maintenance of tissue homeostasis. Mutations of gene that are responsible for the structure or function of cilia can lead to abnormal cilia signal transport, which in turn leads to ciliopathies. About 30% of ciliopathies are characterized by craniofacial phenotype. The most common cilia-related craniofacial defects include micrognathia, cleft lip, cleft palate, orbital hypertelorism/hypotelorism, flat nasal bridge, prominent forehead, craniosynostosis, and so on, suggesting that primary cilia plays an important role in the normal development of craniofacial development. This review summarizes the key genes involved in the regulation of craniofacial development in primary cilia and the disease phenotypes caused by important cilia gene mutations, in order to provide a reference for understanding the etiology of primary cilia-related craniofacial congenital developmental defects.

Ezh2-dependent methylation in oral epithelia promotes secondary palatogenesis.

BACKGROUND: In addition to genomic risk variants and environmental influences, increasing evidence suggests epigenetic modifications are important for orofacial development and their alterations can contribute to orofacial clefts. Ezh2 encodes a core catalytic component of the Polycomb repressive complex responsible for addition of methyl marks to Histone H3 as a mechanism of repressing target genes. The role of Ezh2 in orofacial clefts remains unknown. AIMS: To investigate the epithelial role of Ezh2-dependent methylation in secondary palatogenesis. METHODS: We used conditional gene-targeting methods to ablate Ezh2 in the surface ectoderm-derived oral epithelium of mouse embryos. We then performed single-cell RNA sequencing combined with immunofluorescence and RT-qPCR to investigate gene expression in conditional mutant palate. We also employed double knockout analyses of Ezh1 and Ezh2 to address if they have synergistic roles in palatogenesis. RESULTS: We found that conditional inactivation of Ezh2 in oral epithelia results in partially penetrant cleft palate. Double knockout analyses revealed that another family member Ezh1 is dispensable in orofacial development, and it does not have synergistic roles with Ezh2 in palatogenesis. Histochemistry and single-cell RNA-seq analyses revealed dysregulation of cell cycle regulators in the palatal epithelia of Ezh2 mutant mouse embryos disrupts palatogenesis. CONCLUSION: Ezh2-dependent histone H3K27 methylation represses expression of cell cycle regulator Cdkn1a and promotes proliferation in the epithelium of the developing palatal shelves. Loss of this regulation may perturb movement of the palatal shelves, causing a delay in palate elevation which may result in failure of the secondary palate to close altogether.

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.

Single cell sequencing of the mouse anterior palate reveals mesenchymal heterogeneity.

BACKGROUND: Cleft palate is one of the most prevalent birth defects. Mice are useful for studying palate development because of their morphological and genetic similarities to humans. In mice, palate development occurs between embryonic days (E)11.5 to 15.5. Single cell transcriptional profiles of palate cell populations have been a valuable resource for the craniofacial research community, but we lack a single cell transcriptional profile for anterior palate at E13.5, at the transition from proliferation to shelf elevation. RESULTS: A detailed single cell RNA sequencing analysis reveals heterogeneity in expression profiles of the cell populations of the E13.5 anterior palate. Hybridization chain reaction RNA fluorescent in situ hybridization (HCR RNA FISH) reveals epithelial populations segregate into layers. Mesenchymal populations spatially segregate into four domains. One of these mesenchymal populations expresses ligands and receptors distinct from the rest of the mesenchyme, suggesting that these cells have a unique function. RNA velocity analysis shows two terminal cell states that contribute to either the proximal or distal palatal regions emerge from a single progenitor pool. CONCLUSION: This single cell resolution expression data and detailed analysis from E13.5 anterior palate provides a powerful resource for mechanistic insight into secondary palate morphogenesis for the craniofacial research community.

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.

Allele-specific transcription factor binding in a cellular model of orofacial clefting.

Non-syndromic cleft lip with/without cleft palate (nsCL/P) is a frequent congenital malformation with multifactorial etiology. While recent genome-wide association studies (GWAS) have identified several nsCL/P risk loci, the functional effects of the associated non-coding variants are largely unknown. Furthermore, additional risk loci remain undetected due to lack of power. As genetic variants might alter binding of transcription factors (TF), we here hypothesized that the integration of data from TF binding sites, expression analyses and nsCL/P GWAS might help to (i) identify functionally relevant variants at GWAS loci, and (ii) highlight novel risk variants that have been previously undetected. Analysing the craniofacial TF TFAP2A in human embryonic palatal mesenchyme (HEPM) cells, we identified 2845 TFAP2A ChIP-seq peaks, several of which were located near nsCL/P candidate genes (e.g. MSX1 and SPRY2). Comparison with independent data suggest that 802 of them might be specific to craniofacial development, and genes near these peaks are enriched in processes relevant to nsCL/P. Integration with nsCL/P GWAS data, however, did not show robust evidence for co-localization of common nsCL/P risk variants with TFAP2A ChIP-seq peaks. This data set represents a new resource for the analyses of craniofacial processes, and similar approaches with additional cell lines and TFs could be applied to generate further insights into nsCL/P etiology.

Essential role of Msx1 in regulating anterior-posterior patterning of the secondary palate in mice.

Development of the secondary palate displays molecular heterogeneity along the anterior-posterior axis; however, the underlying molecular mechanism remains largely unknown. MSX1 is an anteriorly expressed transcription repressor required for palate development. Here, we investigate the role of Msx1 in regional patterning of the secondary palate. The Wnt1-Cre-mediated expression of Msx1 (RosaMsx1Wnt1-Cre) throughout the palatal mesenchyme leads to cleft palate in mice, associated with aberrant cell proliferation and cell death. Osteogenic patterning of the hard palate in RosaMsx1Wnt1-Cre mice is severely impaired, as revealed by a marked reduction in palatine bone formation and decreased expression of the osteogenic regulator Sp7. Overexpression and knockout of Msx1 in mice show that the transcription repressor promotes the expression of the anterior palate-specific Alx1 but represses the expression of the medial-posterior palate genes Barx1, Meox2, and Tbx22. Furthermore, Tbx22 constitutes a direct Msx1 target gene in the secondary palate, suggesting that Msx1 can directly repress the expression of medial-posterior specific genes. Finally, we determine that Sp7 is downstream of Tbx22 in palatal mesenchymal cells, suggesting that a Msx1/Tbx22/Sp7 axis participates in the regulation of palate development. Our findings unveil a novel role for Msx1 in regulating the anterior-posterior growth and patterning of the secondary palate.

Ras signaling and RREB1 are required for the dissociation of medial edge epithelial cells in murine palatogenesis.

Cleft palate is one of the major congenital craniofacial birth defects. The etiology underlying the pathogenesis of cleft palate has yet to be fully elucidated. Dissociation of the medial edge epithelium (MEE) at the contacting region of palatal shelves and subsequent migration or apoptosis of MEE cells is required for proper MEE removal. Ras-responsive element-binding protein 1 (RREB1), a RAS transcriptional effector, has recently been shown to play a crucial role in developmental epithelial-mesenchymal transition (EMT), in which loss of epithelial characteristics is an initial step, during mid-gastrulation of embryonic development. Interestingly, the involvement of RREB1 in cleft palate has been indicated in humans. Here, we demonstrated that pan-Ras inhibitor prevents the dissociation of MEE during murine palatal fusion. Rreb1 is expressed in the palatal epithelium during palatal fusion, and knockdown of Rreb1 in palatal organ culture resulted in palatal fusion defects by inhibiting the dissociation of MEE cells. Our present findings provide evidence that RREB1-mediated Ras signaling is required during palatal fusion. Aberrant RREB1-mediated Ras signaling might be involved in the pathogenesis of cleft palate.

Dexamethasone Suppresses Palatal Cell Proliferation through miR-130a-3p.

Cleft lip with or without cleft palate (CL/P) is one of the most common congenital birth defects. This study aims to identify novel pathogenic microRNAs associated with cleft palate (CP). Through data analyses of miRNA-sequencing for developing palatal shelves of C57BL/6J mice, we found that miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p were significantly upregulated, and that miR-19a-3p, miR-130a-3p, miR-301a-3p, and miR-486b-5p were significantly downregulated, at embryonic day E14.5 compared to E13.5. Among them, overexpression of the miR-449 family (miR-449a-3p, miR-449a-5p, miR-449b, miR-449c-3p, and miR-449c-5p) and miR-486b-5p resulted in reduced cell proliferation in primary mouse embryonic palatal mesenchymal (MEPM) cells and mouse cranial neural crest cell line O9-1. On the other hand, inhibitors of miR-130a-3p and miR-301a-3p significantly reduced cell proliferation in MEPM and O9-1 cells. Notably, we found that treatment with dexamethasone, a glucocorticoid known to induce CP in mice, suppressed miR-130a-3p expression in both MEPM and O9-1 cells. Moreover, a miR-130a-3p mimic could ameliorate the cell proliferation defect induced by dexamethasone through normalization of Slc24a2 expression. Taken together, our results suggest that miR-130-3p plays a crucial role in dexamethasone-induced CP in mice.

FAM20C Overview: Classic and Novel Targets, Pathogenic Variants and Raine Syndrome Phenotypes.

FAM20C is a gene coding for a protein kinase that targets S-X-E/pS motifs on different phosphoproteins belonging to diverse tissues. Pathogenic variants of FAM20C are responsible for Raine syndrome (RS), initially described as a lethal and congenital osteosclerotic dysplasia characterized by generalized atherosclerosis with periosteal bone formation, characteristic facial dysmorphisms and intracerebral calcifications. The aim of this review is to give an overview of targets and variants of FAM20C as well as RS aspects. We performed a wide phenotypic review focusing on clinical aspects and differences between all lethal (LRS) and non-lethal (NLRS) reported cases, besides the FAM20C pathogenic variant description for each. As new targets of FAM20C kinase have been identified, we reviewed FAM20C targets and their functions in bone and other tissues, with emphasis on novel targets not previously considered. We found the classic lethal and milder non-lethal phenotypes. The milder phenotype is defined by a large spectrum ranging from osteonecrosis to osteosclerosis with additional congenital defects or intellectual disability in some cases. We discuss our current understanding of FAM20C deficiency, its mechanism in RS through classic FAM20C targets in bone tissue and its potential biological relevance through novel targets in non-bone tissues.

In-frame deletion of SPECC1L microtubule association domain results in gain-of-function phenotypes affecting embryonic tissue movement and fusion events.

Patients with autosomal dominant SPECC1L variants show syndromic malformations, including hypertelorism, cleft palate and omphalocele. These SPECC1L variants largely cluster in the second coiled-coil domain (CCD2), which facilitates association with microtubules. To study SPECC1L function in mice, we first generated a null allele (Specc1lΔEx4) lacking the entire SPECC1L protein. Homozygous mutants for these truncations died perinatally without cleft palate or omphalocele. Given the clustering of human variants in CCD2, we hypothesized that targeted perturbation of CCD2 may be required. Indeed, homozygotes for in-frame deletions involving CCD2 (Specc1lΔCCD2) resulted in exencephaly, cleft palate and ventral body wall closure defects (omphalocele). Interestingly, exencephaly and cleft palate were never observed in the same embryo. Further examination revealed a narrower oral cavity in exencephalic embryos, which allowed palatal shelves to elevate and fuse despite their defect. In the cell, wild-type SPECC1L was evenly distributed throughout the cytoplasm and colocalized with both microtubules and filamentous actin. In contrast, mutant SPECC1L-ΔCCD2 protein showed abnormal perinuclear accumulation with diminished overlap with microtubules, indicating that SPECC1L used microtubule association for trafficking in the cell. The perinuclear accumulation in the mutant also resulted in abnormally increased actin and non-muscle myosin II bundles dislocated to the cell periphery. Disrupted actomyosin cytoskeletal organization in SPECC1L CCD2 mutants would affect cell alignment and coordinated movement during neural tube, palate and ventral body wall closure. Thus, we show that perturbation of CCD2 in the context of full SPECC1L protein affects tissue fusion dynamics, indicating that human SPECC1L CCD2 variants are gain-of-function.

Susceptibility to DNA damage caused by abrogation of Rad54 homolog B: A putative mechanism for chemically induced cleft palate.

Exposure to environmental toxicants such as all-trans retinoic acid (atRA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may cause cleft palate (CP), which process is related to DNA damage. Rad54B, an important DNA damage repaired protein, has been proved to be associated with non-syndromic cleft lip with palate (NSCLP). In the present study, we sought to clarify the role of Rad54B in palatal development and environment-induced CP. atRA (100 mg/kg) and TCDD (40 µg/kg) were used to induce CP in mice (C57BL/6 J mice). In this study, mouse embryonic heads were collected on embryonic day (E) 13.5∼16.5. The expression level of DNA repair protein Rad54 homolog B (Rad54B) was significantly decreased while those of the DNA double-strand breaks (DSBs) marker γ-H2A.X, apoptosis marker caspase-3 and p53 were significantly increased in the palatal shelves upon exposure to atRA and TCDD relative to the control. Primary mouse embryonic palatal mesenchymal cells (MEPMs) were cultured and transfected with siRNA or adenovirus in vitro to knock down or increase the level of Rad54B. Rad54B knockdown resulted in increased cellular S-phase arrest and apoptosis as well as decreased cell proliferation. Rad54B overexpression also increased apoptosis and reduced cell proliferation. Western blotting was used to detect the level of γ-H2A.X in transfected cells stimulated with etoposide (ETO, a DSBs inducer), and after 5 µM ETO stimulation of transfected MEPMs, the expression of γ-H2A.X was increased in Rad54B-knockdown cells. The expression of Mdm2, Mdmx and p53 with changes in Rad54B was also detected and coimmunoprecipitation was performed to analyze the combination of Mdm2 and p53 when Rad54B was changed in MEPMs. Knockdown of Rad54B inhibited the expression of Mdm2 and Mdmx, while the level of p53 increased. The coimmunoprecipitation results showed a decreased combination of Mdm2 and p53 when Rad54B was knocked down. Therefore, Rad54B can regulate the cell cycle, proliferation, and apoptosis of MEPMs. The loss of Rad54B increased the sensitivity of MEPMs to DSBs inducers, promoted apoptosis, and suppressed the proliferation of MEPMs by inhibiting the degradation of p53. Taken together, these findings suggest that Rad54B may play a key regulatory role in environment-induced CP.

The role of MEG3 in the proliferation of palatal mesenchymal cells is related to the TGFß/Smad pathway in TCDD inducing cleft palate.

Cleft palate (CP) is a common birth defect with a high incidence of occurrence in humans. The 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic halogenated aromatic hydrocarbon, with a strong CP effect on mice. Increasing recent evidences have shown that long-noncoding RNAs (lncRNAs) play an important role in several diseases, including CP. However, there is a paucity of studies on the role of lncRNA MEG3 in the occurrence and development of TCDD-induced CP. In this study, the relationship between MEG3 and the proliferation of palatal mesenchymal cells and the underlying molecular mechanism were studied by establishing fetal CP with TCDD (64 µg/kg) in C57BL/6N mice. The results revealed that MEG3 was highly expressed during the critical period of CP formation and that the fetal mesenchymal proliferation was significantly inhibited at certain critical periods in the mice receiving TCDD. In addition, we noted a possibility of a crosstalk between MEG3 and the TGF-ß/Smad pathway, such that the inhibition of the TGF-ß/Smad pathway was induced by TCDD. Cumulatively, our study suggests that TCDD-induced CP may be caused by MEG3 inhibition of the proliferation of palatal mesenchymal cells involving the TGFß/Smad pathway, which may provide a novel perspective to understand the pathogenesis of CP.

RERE deficiency contributes to the development of orofacial clefts in humans and mice.

Deletions of chromosome 1p36 are the most common telomeric deletions in humans and are associated with an increased risk of orofacial clefting. Deletion/phenotype mapping, combined with data from human and mouse studies, suggests the existence of multiple 1p36 genes associated with orofacial clefting including SKI, PRDM16, PAX7 and GRHL3. The arginine-glutamic acid dipeptide (RE) repeats gene (RERE) is located in the proximal critical region for 1p36 deletion syndrome and encodes a nuclear receptor co-regulator. Pathogenic RERE variants have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye or heart (NEDBEH). Cleft lip has previously been described in one individual with NEDBEH. Here we report the first individual with NEDBEH to have a cleft palate. We confirm that RERE is broadly expressed in the palate during mouse embryonic development, and we demonstrate that the majority of RERE-deficient mouse embryos on C57BL/6 background have cleft palate. We go on to show that ablation of Rere in cranial neural crest (CNC) cells, mediated by a Wnt1-Cre, leads to delayed elevation of the palatal shelves and cleft palate and that proliferation of mesenchymal cells in the palatal shelves is significantly reduced in Rereflox/flox; Wnt1-Cre embryos. We conclude that loss of RERE function contributes to the development of orofacial clefts in individuals with proximal 1p36 deletions and NEDBEH and that RERE expression in CNC cells and their derivatives is required for normal palatal development.

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.

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.