Palate In Mice Research Articles

Runx1-Stat3-Tgfb3 signaling network regulating the anterior palatal development

Runx1 deficiency results in an anteriorly specific cleft palate at the boundary between the primary and secondary palates and in the first rugae area of the secondary palate in mice. However, the cellular and molecular pathogenesis underlying such regional specificity remain unknown. In this study, Runx1 epithelial-specific deletion led to the failed disintegration of the contacting palatal epithelium and markedly downregulated Tgfb3 expression in the primary palate and nasal septum. In culture, TGFB3 protein rescued the clefting of the mutant. Furthermore, Stat3 phosphorylation was disturbed in the corresponding cleft regions in Runx1 mutants. The Stat3 function was manifested by palatal fusion defects in culture following Stat3 inhibitor treatment with significant downregulation of Tgfb3. Tgfb3 is therefore a critical target of Runx1 signaling, and this signaling axis could be mediated by Stat3 activation. Interestingly, the expression of Socs3, an inhibitor of Stat3, was specific in the primary palate and upregulated by Runx1 deficiency. Thus, the involvement of Socs3 in Runx1-Tgfb3 signaling might explain, at least in part, the anteriorly specific downregulation of Tgfb3 expression and Stat3 activity in Runx1 mutants. This is the first study to show that the novel Runx1-Stat3-Tgfb3 axis is essential in anterior palatogenesis.

Development of Normal and Cleft Palate: A Central Role for Connective Tissue Growth Factor (CTGF)/CCN2

Development of the palate is the result of an organized series of events that require exquisite spatial and temporal regulation at the cellular level. There are a myriad of growth factors, receptors and signaling pathways that have been shown to play an important role in growth, elevation and/or fusion of the palatal shelves. Altered expression or activation of a number of these factors, receptors and signaling pathways have been shown to cause cleft palate in humans or mice with varying degrees of penetrance. This review will focus on connective tissue growth factor (CTGF) or CCN2, which was recently shown to play an essential role in formation of the secondary palate. Specifically, the absence of CCN2 in KO mice results in defective cellular processes that contribute to failure of palatal shelf growth, elevation and/or fusion. CCN2 is unique in that it has been shown to interact with a number of other factors important for palate development, including bone morphogenetic proteins (BMPs), fibroblast growth factors (FGFs), epidermal growth factor (EGF), Wnt proteins and transforming growth factor-βs (TGF-βs), thereby influencing their ability to bind to their receptors and mediate intracellular signaling. The role that these factors play in palate development and their specific interactions with CCN2 will also be reviewed. Future studies to elucidate the precise mechanisms of action for CCN2 and its interactions with other regulatory proteins during palatogenesis are expected to provide novel information with the potential for development of new pharmacologic or genetic treatment strategies for clinical intervention of cleft palate during development.

Constitutive activation of hedgehog signaling adversely affects epithelial cell fate during palatal fusion

Cleft palate is one of the most common craniofacial congenital defects in humans. It is associated with multiple genetic and environmental risk factors, including mutations in the genes encoding signaling molecules in the sonic hedgehog (Shh) pathway, which are risk factors for cleft palate in both humans and mice. However, the function of Shh signaling in the palatal epithelium during palatal fusion remains largely unknown. Although components of the Shh pathway are localized in the palatal epithelium, specific inhibition of Shh signaling in palatal epithelium does not affect palatogenesis. We therefore utilized a hedgehog (Hh) signaling gain-of-function mouse model, K14-Cre;R26SmoM2, to uncover the role of Shh signaling in the palatal epithelium during palatal fusion. In this study, we discovered that constitutive activation of Hh signaling in the palatal epithelium results in submucous cleft palate and persistence of the medial edge epithelium (MEE). Further investigation revealed that precise downregulation of Shh signaling is required at a specific time point in the MEE during palatal fusion. Upregulation of Hh signaling in the palatal epithelium maintains the proliferation of MEE cells. This may be due to a dysfunctional p63/Irf6 regulatory loop. The resistance of MEE cells to apoptosis is likely conferred by enhancement of a cell adhesion network through the maintenance of p63 expression. Collectively, our data illustrate that persistent Hh signaling in the palatal epithelium contributes to the etiology and pathogenesis of submucous cleft palate through its interaction with a p63/Irf6-dependent biological regulatory loop and through a p63-induced cell adhesion network.

Uji Efek Teratogenik dari Yoghurt Terhadap Fetus Mencit Putih (Mus musculus)

Yoghurt merupakan salah satu olahan susu yang dibuat dengan fermentasi asam laktat yaitu bakteri Lactobacillus bulgaricus dan Streptococcus thermophilus.Pada penelitian ini dilakukan uji efek teratogen dari yoghurt terhadap fetus mencit putih betina (Mus musculus). Induk mencit putih betina sebanyak 20 ekor yang dibagi menjadi 4 kelompok perlakuan. Kelompok kontrol, D1, D2, dan D3 berturut-turut yaitu 0,52 gram, 1,04 gram, dan 2,08 gram yoghurt. Data hasil penelitian diolah menggunakan ANOVA Satu Arah dan uji wilayah berganda Duncan. Hasil Penelitian menunjukan bahwa pemberian yoghurt selama kehamilan dapat mempengaruhi berat badan induk mencit (P < 0,05). Pemberian yoghurt selama kehamilan tidak mempengaruhi jumlah fetus, dan berat badan fetus secara bermakna (P > 0,05). Pengamatan dengan larutan Alizarin tidak ditemukan cacat skeletal setelah dibandingkan dengan kelompok kontrol. Pengamatan dengan larutan Bouin’s ditemukan cacat visceralcleft palate fetus mencit pada kelompok yoghurt D3. Dapat disimpulkan yoghurt aman dikonsumsi pada kelompok D1 dan D2. Yoghurt berpotensi menyebabkan teratogen pada beberapa fetus pada kelompok D3

Patient‐derived MAFB Missense Mutation Is Associated with Cleft Palate in Mice

Non‐syndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology. Amongst the numerous genes involved, mutations in MAFB (MAF BZIP Transcription Factor B) were recently associated with increased risk for NSCL/P. MAFB, a single‐exon gene of 3.3kb, encodes the protein MAFB, which is a transcription factor expressed in the craniofacial region at time points critical for palatal development. Previous sequencing of MAFB in cases revealed a novel missense mutation which was significantly associated with an increased risk of NSCL/P. This patient‐derived MAFB mutation, leading to the amino acid change H131Q, was knocked‐into the mouse Mafb, resulting in the allele MafbH131Q. In contrast to Mafb nulls, which die shortly after birth, MafbH131Q mice survived into adulthood at Mendelian ratios. MafbH131Q embryos were harvested for serial sectioning and histological characterization at time points critical for craniofacial development. One MafbH131Q/H131Q animal at embryonic day (e) 18.5 (N = 12) exhibited highly abnormal craniofacial morphology that included a cleft palate and shortened snout. At e13.5, our preliminary data indicate the presence of mild oral adhesions in MafbH131Q/+ and MafbH131Q/H131Q mice that were absent in wild type animals. However, immunofluorescence for periderm markers did not indicate altered expression of keratin 6 and keratin 17. These data demonstrate functional significance of the patient‐derived H131Q mutation and its role in the etiology of NSCL/P.Support or Funding InformationMD ‐ NIH/NIAMS AR067739. Additional partial financial support was provided by a grant from the NIH R37DE08559 and by the FaceBase consortium (grants DE020052 and DE020057)CP ‐ Iowa Biosciences Academy, funded by NIH/NIGMS R25GM058939, the University of Iowa (UI) Office of the Vice President for Research, and the UI Chief Diversity Office.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Palatal Epithelial Cells Migrate Independently of Ephrin Signaling during Fusion

The mammalian secondary palate forms from two shelves of mesenchyme sheathed in a single layered epithelium. Failure of the midline epithelial seam (MES) to degrade when these shelves meet at midline prevents mesenchymal confluence and results in a cleft palate. Our previous studies showed that cultured MES cells undergo features of epithelial‐to‐mesenchymal transition (EMT) when ephrin reverse signaling is activated. We therefore hypothesized that midline epithelial cells are removed during fusion by migration away from the seam in response to ephrin action. In order to observe this migration, we performed real time imaging of fusing embryonic mouse palates in culture using two‐photon confocal microscopy. The palates came from mice expressing an ephrin‐B2‐driven green fluorescent protein (GFP) that specifically labels the nuclei of epithelial cells in the palate. Immediately after 48 h of imaging, tissues were fixed, sectioned, and examined for fusion. The motion of individual MES cells was tracked over the time course of culture using Imaris software. MES cells migrated in a surprisingly unidirectional fashion toward the oral surface over 48 h of imaging, regardless of tissue orientation on the culture dish, and maintained their GFP expression for the duration of the experiment. Fusion of the imaged palates was confirmed histologically, thus demonstrating that our culture time frame encompassed the period required for palatal fusion. Addition of unclustered EphA4/Fc recombinant protein during the culture period to block ephrin reverse signaling inhibited fusion, as we previously established, but did not change the observed migration of GFP‐labeled cells. Because these cells maintained GFP expression, we conclude that they remained alive and that MES cell death by apoptosis is therefore not required for fusion, as was previously thought. Although ephrin reverse signaling is required for palate fusion, we found that it is not necessary for MES cell migration.Support or Funding InformationNIH/NIDCR R01DE022804This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of cleft palate in C57BL/6N mice induced by retinoic acid

Objective: To investigate the mechanism of cleft palate in mice induced by excessive all-trans retinoic acid (atRA). Methods: The pregnant mice were randomly divided into atRA-treated group (n=27) and control group (n=27). atRA-treated group was given by gavage a single dose of atRA (100 mg/kg) at 8: 00 AM on gestation day 10 (GD10) and the control group was given by gavage the isopyknic corn oil. At GD13-GD15, the fetal mice palate development was observed by HE staining. The mouse embryonic palatal mesenchymal cell proliferation was detected by 5-bromo-2-deoxyuridine (BrdU) immunohistochemistry. The expressions of Smad2, phospho-Smad2 (p-Smad2), Smad4 and Smad7 in mouse embryonic palatal mesenchyme were analyzed by Western blotting. Results: At GD13-GD15, compared with the control, the ratio of BrdU-positive cells in the palatal mesenchyme of atRA-treated fetuses decreased significantly (P<0.05), especially at GD14, atRA inhibition rate was (65.4±1.7)%. Moreover, atRA decreased the levels of p-Smad2 and Smad4 in embryonic palate mesenchymal cells, whereas the expression of Smad7 was significantly increased at GD14 and GD15. Conclusions: atRA may lead to cleft palate by inhibiting the activation of Smad signaling pathway and affecting the proliferation of palatal mesenchymal cells.

Inhibition of periderm removal in all-trans retinoic acid-induced cleft palate in mice.

Cleft palate is a common craniofacial birth defect. The aim of the present study was to investigate the effect of excess all-trans retinoic acid (atRA) on periderm removal and the disappearance of basal medial edge epithelial (MEE) cells during palatogenesis, particularly during the stage prior to contact. atRA (200 mg/kg) was administered to C57BL/6N mice at embryonic day (E) 12.0 by gavage. Fetal palates were processed and analyzed by histology and electron microscopy. Single palate shelf peridermal cells were removed and cultured in the presence of atRA (3 µM) only or in the presence of or the caspase inhibitor, Z-VAD (100 µM) only, for 48 h. Once cultured, morphological changes were analyzed by histological staining and electron microscopy. A TUNEL assay was used to detect apoptotic neurons. Paired palatal shelves with periderm removal were cultured in the presence of atRA (3 µM) only or in the presence of Z-VAD (100 µM) only for 48 h and analyzed by hematoxylin and eosin staining. At E14.5, medial edge epithelium periderm was retained in the atRA-treated palates but had been shed prior to contact in the control groups. In addition, atRA was revealed to disrupt the cell cycle in the periderm by downregulating p21. Furthermore, atRA inhibited apoptosis in the periderm and basal MEE cells; however, atRA exhibited no effect on basement membrane degradation in single palatal organ culture. Additionally, once paired palates were cultured for 48 h, all of the groups in which the periderm had been removed exhibited confluence of the embryonic palatal mesenchyme. The present results suggest that periderm removal is inhibited in atRA-induced cleft palate in mice and that removal of the periderm contributes to EPM confluence in vitro.

An E2F1/MiR-17-92 Negative Feedback Loop mediates proliferation of Mouse Palatal Mesenchymal Cells

Normal cell cycle progression and proliferation of palatal mesenchymal cells are important for palatal development. As targets of miR-17-92, E2F transcription factors family has been suggested to induce the transcription of miR-17-92 in several cell types. In the present study, we sought to investigate whether this negative feedback loop exists in mouse PMCs and what the function of this negative feedback loop would be in palatal mesenchymal cells. Using GeneMANIA, we revealed that the most important function of experimentally verified targets of miR-17-92 is cell cycle regulation. E2F1 and E2F3, but not E2F2, were extensively expressed in mouse palate. Over-expression of E2F1 significantly increased the expression of all the members of miR-17-92. After increased by E2F1, miR-17 and miR-20a may negatively target E2F1, and thereby prevent the cells from excessive proliferation. We suggest that the negative feedback loop between E2F1 and miR-17-92 may contribute to palatal development by regulating the proliferation and cell cycle of palatal mesenchymal cells.

Modulating Wnt Signaling Rescues Palate Morphogenesis in Pax9 Mutant Mice

Cleft palate is a common birth defect caused by disruption of palatogenesis during embryonic development. Although mutations disrupting components of the Wnt signaling pathway have been associated with cleft lip and palate in humans and mice, the mechanisms involving canonical Wnt signaling and its regulation in secondary palate development are not well understood. Here, we report that canonical Wnt signaling plays an important role in Pax9-mediated regulation of secondary palate development. We found that cleft palate pathogenesis in Pax9-deficient embryos is accompanied by significantly reduced expression of Axin2, an endogenous target of canonical Wnt signaling, in the developing palatal mesenchyme, particularly in the posterior regions of the palatal shelves. We found that expression of Dkk2, encoding a secreted Wnt antagonist, is significantly increased whereas the levels of active β-catenin protein, the essential transcriptional coactivator of canonical Wnt signaling, is significantly decreased in the posterior regions of the palatal shelves in embryonic day 13.5 Pax9-deficent embryos in comparison with control littermates. We show that small molecule–mediated inhibition of Dickkopf (DKK) activity in utero during palatal shelf morphogenesis partly rescued secondary palate development in Pax9-deficient embryos. Moreover, we found that genetic inactivation of Wise, which is expressed in the developing palatal shelves and encodes another secreted antagonist of canonical Wnt signaling, also rescued palate morphogenesis in Pax9-deficient mice. Furthermore, whereas Pax9del/del embryos exhibit defects in palatal shelf elevation/reorientation and significant reduction in accumulation of hyaluronic acid—a high molecular extracellular matrix glycosaminoglycan implicated in playing an important role in palatal shelf elevation—80% of Pax9del/del;Wise-/- double-mutant mouse embryos exhibit rescued palatal shelf elevation/reorientation, accompanied by restored hyaluronic acid accumulation in the palatal mesenchyme. Together, these data identify a crucial role for canonical Wnt signaling in acting downstream of Pax9 to regulate palate morphogenesis.

Early Craniofacial Defects in Zebrafish That Have Reduced Function of a Wnt-Interacting Extracellular Matrix Protein, Tinagl1.

Tinagl1 has a weak genetic association with craniosynostosis, but its functions in cartilage and bone development are unknown. Knockdown of Tinagl1 in zebrafish embryos allowed an initial characterization of its potential effects on craniofacial cartilage development and a test of whether these effects could involve Wnt signaling. Tinagl1 knockdown resulted in dose-dependent reductions and defects in ventral pharyngeal arch cartilages as well as the ethmoid plate, a zebrafish correlate to the palate. These defects could be correlated to reduced numbers of cranial neural crest cells in the pharyngeal arches and could be reproduced with comanipulation of Tinagl1 and Wnt3a by morpholino-based knockdown. These results suggest that Tinagl1 is required early in the proliferation or migration of cranial neural crest cells and that its effects are mediated via Wnt3a signaling. Because Wnt3a is among the Wnts that contribute to nonsyndromic cleft lip and cleft palate in mouse and man, further investigation of Tinagl1 may help to elucidate mechanisms underlying these disorders.

Ein In-vitro-Mausmodell für die Palatogenese und die Entstehung von Gaumenspalten

AbstractThe mouse is an excellent model to study palatogenesis since, like humans, it is a mammal, it can easily be bred and a wide array of molecular tools are available. Moreover, the isolated embryonic mouse palate can be cultured in vitro, which allows us to study the actual fusion process in detail.Just prior to the actual fusion at E13, the maxilla with the 2 palatal shelves can be dissected from the embryo head. The maxilla is cultured on a filter paper on top of a stainless-steel grid at the interface of the culture medium and air. After 24 h in culture, the palatal shelves are in contact and the MES is clearly visible. At 48 h, the MES has nearly completely disintegrated with small remnants at the nasal and oral side of the palate. Finally, at 72 h, the palatal shelves are completely fused and osteoid tissue is beginning to form in the lateral areas.The exact downstream molecular mechanisms that lead to clefting are far from being unraveled. The model for palate fusion presented here offers the possibility to analyze the molecular and cellular mechanisms that lead to clefting as a result of specific genetic and environmental factors. This will pave the way to pharmacological intervention for the prevention of cleft lip and/or palate in susceptible individuals.

Lower concentrations of receptor for advanced glycation end products and epiregulin in amniotic fluid correlate to chemically induced cleft palate in mice.

This study investigated the correlation between differentially expressed proteins in amniotic fluid (AF) and cleft palate induced by all-trans retinoic acid (atRA), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice. Seven proteins were differentially expressed at embryonic day (E) 16.5 in atRA and control groups as revealed by label-based mouse antibody array. Enzyme-linked immunosorbent assay was further used to detect the expression levels of these proteins in AF from E13.5 to E16.5 in atRA, TCDD, and control groups. The cleft palate groups showed lower concentrations of receptor for advanced glycation end products (RAGE) and epiregulin at E16.5. RAGE immunostaining obviously decreased in palatal tissue sections obtained from E14.5 to E16.5 in the cleft palate groups as revealed by immunohistochemistry. These findings indicate that reduced levels of RAGE and epiregulin in AF are correlated to chemically induced cleft palate in mice.

RNAi of Grp78 may disturb the fusion of ICR mouse palate cultured in vitro.

RNA interference (RNAi) is a powerful tool to silence or minimize gene expression, and palate culture in vitro is an important technique for study of the palate development. Our previous study demonstrated that the gene expression of glucose-regulated protein-78 (Grp78) was downregulation in the all-trans retinoic acid-induced mouse models of cleft palate (CP) during embryogenesis. To find the role of Grp78, the small interfering RNA (siRNA) of this gene carried by fluorescent vector was injected with a microinjector, through which about 30 pmol siRNA was injected into the Institute of Cancer Research (ICR) mouse palate explants. After 6, 12, 24, 48, and 72 h, these palate explants were removed from culture to observe their fluorescent and Alcian blue-staining phenotypes, and the expression of the unfolded protein response (UPR) key members (Grp78, Inositol-responsive enzyme 1, protein kinase RNA-like endoplasmic reticulum kinase, activating transcription factor-6 and X-box binding protein-1) was measured. After cultured for 72 h, the partially or completely fused bilateral palates were observed in the control siRNA group, while CPs were found in the Grp78 siRNA group. In the Grp78 siRNA group, the relatively mRNA abundance of the key genes belonged to UPR at each time point was lower than that of the control siRNA group, and their protein expression also displayed the same change. By the system of RNAi strategies with mouse palate culture, we found the siRNA of Grp78 disturbed the fusion of mouse palate cultured in vitro.

Excessive retinoic acid inhibit mouse embryonic palate mesenchymal cell growth through involvement of Smad signaling

ABSTRACTAll-trans retinoic acid (atRA), the oxidative metabolite of retinoic acid (RA), is essential for palatogenesis. Overdose RA is capable of inducing cleft palate in mice and humans. Normal embryonic palatal mesenchymal (EPM) cell growth is crucial for shelf growth. Smad signaling is involved in many biological processes. However, it is not much clear if atRA could affect Smad signaling during EPM cells growth. In this study, the timed pregnant mice with maternal administration of 100 mg/kg body weight of RA by gastric intubation were cervical dislocation executed to evaluate growth changes of palatal shelves by hematoxylin and eosin (H&E) staining. At the same time, a primary mouse EPM (MEPM) cell culture model was also established. MEPM cells were treated with atRA (0.1, 0.5, 1, 5 and 10 μM) for 24, 48 and 72 h. The results indicated that the sizes of the shelves were smaller than those in control. AtRA inhibited MEPM cell growth with both increasing concentration and increasing incubation time, especially at 72 h in vitro. Moreover, atRA significantly increased the mRNA and protein expression levels of Smad7 (P < .05), but the mRNA and protein expression levels of PCNA were reduced (P < .05). We also found atRA inhibited phosphorylation of Smad2 compared with untreated group (P < .05). However, the protein and mRNA levels of Smad2 did not change both in atRA-treated and untreated group (P > .05). We demonstrated that RA induced inhibition of MEPM cell growth that could cause cleft palate partly by down-regulation of Smad pathway.

Dose-Dependent Antiteratogenic Effects of Folic Acid on All-Trans Retinoic Acid-Induced Cleft Palate in Fetal Mice.

Although numerous studies have confirmed that consumption of folic acid (FA) during early pregnancy reduces the risk of oral facial clefts in newborn infants, the optimal dose of FA for reducing this risk remains unknown. We evaluated various doses of FA for their ability to reduce the incidence of all-trans retinoic acid (ATRA)-induced cleft palate in mice. Pregnant C57BL/6J mice were randomly assigned to eight groups dosed with corn oil (control group), ATRA (80 mg/kg), FA (40 mg/kg), or ATRA (80 mg/kg) + FA (2.5 mg, 5 mg, 10 mg, 20 mg, or 40 mg/kg body weight) on gestation day 11 (GD11), after which samples of maternal blood obtained on GD 11 were analyzed for serum folate levels. After receiving the doses, randomly selected mice in each dose group were sacrificed on GDs 13.5, 14.5, and 15.5, and the fetuses were removed for examination by light microscopy and scanning electron microscopy to detect the incidence of cleft palate. Among the pregnant mice dosed with ATRA+FA, those dosed with 5 mg/kg FA had fetuses with the lowest incidence of cleft palate. In addition, the eight groups of pregnant mice had significantly different serum folate concentrations (P < .001). When administered to pregnant mice at a specific dose and on the proper gestation day, FA showed an antiteratogenic effect by reducing the incidence of ATRA-induced cleft palate in fetal mice.

Activation of Notch1 inhibits medial edge epithelium apoptosis in all-trans retinoic acid-induced cleft palate in mice

Administration of all-trans retinoic acid (atRA) on E12.0 (embryonic day 12.0) leads to failure of medial edge epithelium (MEE) disappearance and cleft palate. However, the molecular mechanism underlying the relationship between atRA and MEE remains to be identified. In this study, atRA (200 mg/kg) administered by gavage induced a 75% incidence of cleft palate in C57BL/6 mice. Notch1 was up-regulated in MEE cells in the atRA-treated group compared with the controls at E15.0, together with reduced apoptosis and elevated proliferation. Next, we investigated the mechanisms underlying atRA, Notch1 and MEE degradation in palate organ culture. Our results revealed that down-regulation of Notch1 partially rescued the inhibition of atRA-induced palate fusion. Molecular analysis indicated that atRA increased the expression of Notch1 and Rbpj and decreased the expression of P21. In addition, depletion of Notch1 expression decreased the expression of Rbpj and increased the expression of P21. Moreover, inhibition of Rbpj expression partially reversed atRA-induced MEE persistence and increased P21 expression. These findings demonstrate that atRA inhibits MEE degradation, which in turn induces a cleft palate, possibly through the Notch1/RBPjk/P21 signaling pathway.

Histone acetylation is involved in TCDD-induced cleft palate formation in fetal mice

The aim of the present was to evaluate the effects of DNA methylation and histone acetylation on 2,3,7,8-tetrachlo-rodibenzo-p-dioxin (TCDD)-induced cleft palate in fetal mice. Pregnant mice (n=10) were randomly divided into two groups: i) TCDD group, mice were treated with 28 µg/kg TCDD on gestation day (GD) 10 by oral gavage; ii) control group, mice were treated with an equal volume of corn oil. On GD 16.5, the fetal mice were evaluated for the presence of a cleft palate. An additional 36 pregnant mice were divided into the control and TCDD groups, and palate samples were collected on GD 13.5, GD 14.5 and GD 15.5, respectively. Transforming growth factor-β3 (TGF-β3) mRNA expression, TGF-β3 promoter methylation, histone acetyltransferase (HAT) activity and histone H3 (H3) acetylation in the palates were evaluated in the two groups. The incidence of a cleft palate in the TCDD group was 93.55%, and no cases of cleft palate were identified in the control group. On GD 13.5 and GD 14.5, TGF-β3 mRNA expression, HAT activity and acetylated H3 levels were significantly increased in the TCDD group compared with the control. Methylated bands were not observed in the TCDD or control groups. In conclusion, at the critical period of palate fusion (GD 13.5–14.5), TCDD significantly increased TGF-β3 gene expression, HAT activity and H3 acetylation. Therefore, histone acetylation may be involved in TCDD-induced cleft palate formation in fetal mice.

G-Protein α-Subunit Gsα Is Required for Craniofacial Morphogenesis

The heterotrimeric G protein subunit Gsα couples receptors to activate adenylyl cyclase and is required for the intracellular cAMP response and protein kinase A (PKA) activation. Gsα is ubiquitously expressed in many cell types; however, the role of Gsα in neural crest cells (NCCs) remains unclear. Here we report that NCCs-specific Gsα knockout mice die within hours after birth and exhibit dramatic craniofacial malformations, including hypoplastic maxilla and mandible, cleft palate and craniofacial skeleton defects. Histological and anatomical analysis reveal that the cleft palate in Gsα knockout mice is a secondary defect resulting from craniofacial skeleton deficiencies. In Gsα knockout mice, the morphologies of NCCs-derived cranial nerves are normal, but the development of dorsal root and sympathetic ganglia are impaired. Furthermore, loss of Gsα in NCCs does not affect cranial NCCs migration or cell proliferation, but significantly accelerate osteochondrogenic differentiation. Taken together, our study suggests that Gsα is required for neural crest cells-derived craniofacial development.

Linking suckling biomechanics to the development of the palate.

Skulls are amongst the most informative documents of evolutionary history but a complex geometry, coupled with composite material properties and complicated biomechanics, have made it particularly challenging to identify mechanical principles guiding the skull’s morphogenesis. Despite this challenge, multiple lines of evidence, for example the relationship between masticatory function and the evolution of jaw shape, nonetheless suggest that mechanobiology plays a major role in skull morphogenesis. To begin to tackle this persistent challenge, cellular, molecular and tissue-level analyses of the developing mouse palate were coupled with finite element modeling to demonstrate that patterns of strain created by mammalian-specific oral behaviors produce complementary patterns of chondrogenic gene expression in an initially homogeneous population of cranial neural crest cells. Neural crest cells change from an osteogenic to a chondrogenic fate, leading to the materialization of cartilaginous growth plate-like structures in the palatal midline. These growth plates contribute to lateral expansion of the head but are transient structures; when the strain patterns associated with suckling dissipate at weaning, the growth plates disappear and the palate ossifies. Thus, mechanical cues such as strain appear to co-regulate cell fate specification and ultimately, help drive large-scale morphogenetic changes in head shape.