Palatal Shelves Research Articles

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.

Computer-Aided Design and Manufacture of Intraoral Splints: A Potential Role in Cleft Care

BackgroundNasoalveolar molding is a nonsurgical modality for the treatment of cleft lip and palate that uses an intraoral splint to align the palatal shelves. Repeated impressions are needed for splint modification, each carrying risk of airway obstruction. Computer-aided design and manufacturing (CAD/CAM) has the ability to simplify the process. As a precursor to CAD/CAM splint fabrication, a proof-of-concept study was conducted to compare three-dimensional splints printed from alginate impressions versus digital scans. We hypothesized that intraoral digital scanning would compare favorably to alginate impressions for palate registration and subsequent splint manufacture, with decreased production times. MethodsAlginate and digital impressions were taken from 25 healthy teenage volunteers. Digital impressions were performed with a commercially available intraoral scanner. Plaster casts made from alginate impressions were converted to Standard Triangle Language files. Patient-specific matched scans were evaluated for total surface area with the concordance correlation coefficient. Acrylic palatal splints were three-dimensionally printed from inverse digital molds. Subjective appliance fit was assessed using a five-point scale. ResultsA total of 23 participants were included. Most subjects preferred digital impression acquisition. Impression methods showed moderate agreement (concordance correlation coefficient 0.93). Subjects rated splints from digital impressions as having a more precise fit (4.4 versus 3.9). The digital approach decreased impression phase time by over 10-fold and overall production time by 28%. ConclusionsCAD/CAM has evolved extensively over the past two decades and is now commonplace in medicine. However, its utility in cleft patients has not been fully realized. This pilot study demonstrated that CAD/CAM technologies may prove useful in patients requiring intraoral splints.

Cleft Palate: Evaluation and Its Management Approach, Review Article

Cleft lip occurs when the frontonasal and maxillary processes fail to the lip. Moreover, cleft palate results when the fusion of the palatal shelves of the maxillary fail to complete, both soft and hard palate can be affected with or without cleft lip. In Saudi Arabia, the prevalence of cleft lip and palate (CLP) is at 0.65 per 1000 live births, with boys accounting for 63.7 percent and girls for 36.6 percent. We aimed to review the literature reviewing etiology of cleft lip and/or palate, clini

Cephalometric parameters in cleft palate patients with and without oronasal fistula

Introduction Failure in the fusion of medial nasal processes and maxillary prominences would result in the development of a cleft lip while failure in merging the palatal shelves would result in the development of a cleft palate (CP). At present, patients with a cleft palate (CP) have often undergone corrective surgery before 1 year of age. However, an oronasal fistula (ONF) often remains in the palate after surgery. Methodology In this historical cohort, 40 patients with CP, 6-10 years old, who had undergone corrective surgery at 1 year of age were selected by targeted sampling and assigned to two groups with ONF (N=20) and without ONF (n=20). The two groups were matched in terms of age and gender. All patients had an intraoral photograph of the maxilla and a lateral cephalogram. Twenty-three cephalometric parameters were measured prior to the orthodontic treatment and compared between the two groups using a t-test. Results CP patients with ONF had significantly higher Y-axis, gonial angle, Go1, Go2, GoGn-SN, U1-SN, and lower lip to E line compared with those without ONF (P<0.05). The Jarabak index was significantly lower in the CP patients with ONF compared with those without it (P<0.05). The difference in other cephalometric parameters was not significant between the two groups (P>0.05). Conclusion Patients with ONF had significantly greater growth in the vertical dimension compared with those without ONF. Yet, the difference in the anteroposterior dimension was not significant between the two groups.

Maxillary arch expansion along with fixed mechanotherapy using different expansion devices

Malocclusions can exist in all the three planes of space i.e in antero – posterior, vertical and transverse plane. Transverse discrepancies can manifest as dental, skeletal or functional discrepancies. Maxillary expansion is one of the most common treatment modality for arch length augmentation, posterior crossbite correction and removing cross arch interferences. Skeletal correction is done by orthopedic expansion or surgical expansion. Rapid Maxillary Expansion and Head Gears, produces orthopedic correction. Severe skeletal discrepancies require surgical correction of underlying skeletal deformity by using SARPE (Surgically Assisted Rapid Palatal Expansion) or surgical expansion of palatal shelves by midline osteotomies. Dental correction can be done using Rapid expansion in growing children and by slow expansion using Quad Helix, W Arches, Coffin Spring and certain modification of Jack screw appliances in other cases. In patients treated with fixed mechanotherapy expansions can not only be achieved by Arch wires, auxillary Jockey arches, cross elastics etc. but also by slow or rapid expansion devices that can be amalgamated with fixed orthodontic treatment. In this article, the use of slow maxillary expansion appliances in treatment of the three patients with transverse maxillary deficiency and undergoing fixed mechanotherapy will be discussed. Keywords: Expansion

Lithium-induced overexpression of β-catenin delays murine palatal shelf elevation by Cdc-42 mediated F-actin remodeling in mesenchymal cells.

Lithium chloride (LiCl) is widely used for the treatment of manic and other psychotic disorders, but the administration of lithium can result in several congenital defects in the fetus, including cleft palate (Meng, Wang, Torensma, Jw & Bian, 2015) (Szabo, 1970). However, the mechanism of Lithium's action as a developmental toxicant in palatogenesis is not well known. In this study, hematoxylin-eosin and immunofluorescence staining were employed to evaluate the phenotypes and the expression of related markers in the LiCl-treated mice model. The palatal mesenchymal cells were cultured in vitro, and stimulated with LiCl or SKL2000, and co-treated with CASIN. β-catenin protein and other cytoskeleton associated markers were evaluated by Western blotting. We found that Lithium disrupted palate elevation by increasing the expression of β-catenin in C57BL/6J mice with the high incidence of cleft palate (62.5%). LiCl disturbed the F-actin responsible for cytoskeletal remodeling in mesenchymal cells, which proved to be essential in generating the elevating force during palatal elevation. Additionally, our Western blotting analysis revealed that the overexpression of β-catenin resulted in up-regulation of Cdc42, which mediated the downstream F-actin synthesis. We concluded the LiCl-induced β-catenin overexpression delayed murine palatal shelf elevation by disturbing Cdc42 mediated F-actin cytoskeleton synthesis in the palatal mesenchyme.

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.

Med23 Regulates Sox9 Expression during Craniofacial Development

The etiology and pathogenesis of craniofacial birth defects are multifactorial and include both genetic and environmental factors. Despite the identification of numerous genes associated with congenital craniofacial anomalies, our understanding of their etiology remains incomplete, and many affected individuals have an unknown genetic diagnosis. Here, we show that conditional loss of a Mediator complex subunit protein, Med23 in mouse neural crest cells (Med23 fx/fx ;Wnt1-Cre), results in micrognathia, glossoptosis, and cleft palate, mimicking the phenotype of Pierre Robin sequence. Sox9 messenger RNA and protein levels are both upregulated in neural crest cell–derived mesenchyme surrounding Meckel’s cartilage and in the palatal shelves in Med23 fx/fx ;Wnt1-Cre mutant embryos compared to controls. Consistent with these observations, we demonstrate that Med23 binds to the promoter region of Sox9 and represses Sox9 expression in vitro. Interestingly, Sox9 binding to β-catenin is enhanced in Med23 fx/fx ;Wnt1-Cre mutant embryos, which, together with downregulation of Col2a1 and Wnt signaling target genes, results in decreased proliferation and altered jaw skeletal differentiation and cleft palate. Altogether, our data support a cell-autonomous requirement for Med23 in neural crest cells, potentially linking the global transcription machinery through Med23 to the etiology and pathogenesis of craniofacial anomalies such as micrognathia and cleft palate.

Load Transfer during Magnetic Mucoperiosteal Distraction in Newborns with Complete Unilateral and Bilateral Orofacial Clefts: A Three-Dimensional Finite Element Analysis

The primary correction of congenital complete unilateral cleft lip and palate (UCLP) and bilateral cleft lip and palate (BCLP) is challenging due to inherent lack of palatal tissue and small extent of the palatal shelves at birth. The tissue deficiency affects the nasal mucosa, maxillary bone and palatal mucosa. This condition has driven the evolution of several surgical and non-surgical techniques for mitigating the inherent problem of anatomical deficits. These techniques share the common principle of altering the neighboring tissues around the defect area in order to form a functional seal between the oral and nasal cavity. However, there is currently no option for rectifying the tissue deficiency itself. Investigations have repeatedly shown that despite the structural tissue deficiency of the cleft, craniofacial growth proceeds normal if the clefts remain untreated, but the cleft remains wide. Conversely, craniofacial growth is reduced after surgical repair and the related alteration of the tissues. Therefore, numerous attempts have been made to change the surgical technique and timing so as to reduce the effects of surgical repairs on craniofacial growth, but they have been only minimally effective so far. We have determined whether the intrinsic structural soft and hard tissue deficiency can be ameliorated before surgical repair using the principles of periosteal distraction by means of magnetic traction. Two three-dimensional maxillary finite element models, with cleft patterns of UCLP and BCLP, respectively, were created from computed tomography slice data using dedicated image analysis software. A virtual dental magnet was positioned on either side of the cleft at the mucoperiosteal borders, and an incremental magnetic attraction force of up to 5 N was applied to simulate periosteal distraction. The stresses and strains in the periosteal tissue induced by the magnet were calculated using finite element analysis. For a 1 N attraction force the maximum strains did not exceed 1500 µstrain suggesting that adaptive remodeling will not take place for attraction forces lower than 1 N. At 5 N the regions subject to remodeling differed between the UCLP and BCLP models. Stresses and strains at the periosteum of the palatal shelf ridges in the absence of compressive forces at the alveolar borders were greater in the UCLP model than the BCLP model. The findings suggest that in newborns with UCLP and BCLP, periosteal distraction by means of a magnetic 5 N attraction force can promote the generation of soft and hard tissues along the cleft edges and rectify the tissue deficiency associated with the malformation.

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

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. 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. 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.

Observation of the Epithelial Cell Behavior in the Nasal Septum During Primary Palate Closure in Mice.

Epithelial fusion is critical in palatogenesis, and incomplete fusion results in various type of facial cleft, depending on the region that fails to fuse. In mammalian palatogenesis, the bilateral secondary palatal processes fuse in the middle of the face to form the secondary palate. Later, the dorsal side of the secondary palatal shelves fuses with the nasal septum to complete palatogenesis. Importantly, the anterior border of the secondary palatal shelf fuses with the primary palate, which is located at the anterior and ventral border of the nasal septum. While numerous studies have investigated the mechanism of fusion between secondary palatal shelves, very little is known about how the primary palate touches and fuses with the secondary palatal shelves. In this study, we investigate the possible epithelial cell behaviors on the surface of the primary palate using palatal explant cultures of K14-GFP mice. A time-lapse observation of the GFP-labeled epithelium and an SEM analysis revealed that the extrusion epithelium appeared at the region corresponding to the fusing area and expanded rostrally on the nasal septum surface in the absence of the secondary palatal processes. Unlike on the secondary palate surface, cellular migration and subsequent autonomous mesenchymal exposure were not evident on the nasal septum or the primary palate. TUNEL staining revealed that these extrusion epithelia were undergoing apoptosis. These findings indicated that extrusion with apoptosis was autonomously initiated at the presumptive region of the fusion without contact with the opposing secondary palate.

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

BackgroundCleft 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. MethodsWhole 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. ResultsWES 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. ConclusionsThese findings demonstrate that nuclear-localized Bag6 and p300 co-operatively enhance FoxO1 acetylation to promote FasL-mediated MEE apoptosis during palate fusion.

Innovative Molecular and Cellular Therapeutics in Cleft Palate Tissue Engineering.

Clefts of the lip and/or palate are the most prevalent orofacial birth defects occurring in about 1:700 live human births worldwide. Early postnatal surgical interventions are extensive and staged to bring about optimal growth and fusion of palatal shelves. Severe cleft defects pose a challenge to correct with surgery alone, resulting in complications and sequelae requiring life-long, multidisciplinary care. Advances made in materials science innovation, including scaffold-based delivery systems for precision tissue engineering, now offer new avenues for stimulating bone formation at the site of surgical correction for palatal clefts. In this study, we review the present scientific literature on key developmental events that can go awry in palate development and the common surgical practices and challenges faced in correcting cleft defects. How key osteoinductive pathways implicated in palatogenesis inform the design and optimization of constructs for cleft palate correction is discussed within the context of translation to humans. Finally, we highlight new osteogenic agents and innovative delivery systems with the potential to be adopted in engineering-based therapeutic approaches for the correction of palatal defects. Impact statement Tissue-engineered scaffolds supplemented with osteogenic growth factors have attractive, largely unexplored possibilities to modulate molecular signaling networks relevant to driving palatogenesis in the context of congenital anomalies (e.g., cleft palate). Constructs that address this need may obviate current use of autologous bone grafts, thereby avoiding donor-site morbidity and other regenerative challenges in patients afflicted with palatal clefts. Combinations of biomaterials and drug delivery of diverse regenerative cues and biologics are currently transforming strategies exploited by engineers, scientists, and clinicians for palatal cleft repair.

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.

Pax9's dual roles in modulating Wnt signaling during murine palatogenesis.

Despite the strides made in understanding the complex network of key regulatory genes and cellular processes that drive palate morphogenesis, patients suffering from these conditions face treatment options that are limited to complex surgeries and multidisciplinary care throughout life. Hence, a better understanding of how molecular interactions drive palatal growth and fusion is critical for the development of treatment and preventive strategies for cleft palates in humans. Our previous work demonstrated that Pax9-dependent Wnt signaling is critical for the growth and fusion of palatal shelves. We showed that controlled intravenous delivery of small molecule Wnt agonists specifically blocks the action of Dkks (inhibitors of Wnt signaling) and corrects secondary palatal clefts in Pax9-/- mice. While these data underscore the importance of the functional upstream relationship of Pax9 to the Wnt pathway, not much is known about how the genetic nature of Pax9's interactions in vivo and how it modulates the actions of these downstream effectors during palate formation. Here, we show that the genetic reduction of Dkk1 during palatogenesis corrected secondary palatal clefts in Pax9-/- mice with restoration of Wnt signaling activities. In contrast, genetically induced overexpression of Dkk1 mice phenocopied the defects in tooth and palate development visible in Pax9-/- strains. Results of ChIP-qPCR assays showed that Pax9 can bind to regions near the transcription start sites of Dkk1 and Dkk2 as well as the intergenic region of Wnt9b and Wnt3 ligands that are downregulated in Pax9-/- palates. Taken together, these data suggest that the molecular mechanisms underlying Pax9's role in modulating Wnt signaling activity likely involve the inhibition of Dkk expression and the control of Wnt ligands during palatogenesis.

Polarized Sonic Hedgehog Protein Localization and a Shift in the Expression of Region-Specific Molecules Is Associated With the Secondary Palate Development in the Veiled Chameleon.

Secondary palate development is characterized by the formation of two palatal shelves on the maxillary prominences, which fuse in the midline in mammalian embryos. However, in reptilian species, such as turtles, crocodilians, and lizards, the palatal shelves of the secondary palate develop to a variable extent and morphology. While in most Squamates, the palate is widely open, crocodilians develop a fully closed secondary palate. Here, we analyzed developmental processes that underlie secondary palate formation in chameleons, where large palatal shelves extend horizontally toward the midline. The growth of the palatal shelves continued during post-hatching stages and closure of the secondary palate can be observed in several adult animals. The massive proliferation of a multilayered oral epithelium and mesenchymal cells in the dorsal part of the palatal shelves underlined the initiation of their horizontal outgrowth, and was decreased later in development. The polarized cellular localization of primary cilia and Sonic hedgehog protein was associated with horizontal growth of the palatal shelves. Moreover, the development of large palatal shelves, supported by the pterygoid and palatine bones, was coupled with the shift in Meox2, Msx1, and Pax9 gene expression along the rostro-caudal axis. In conclusion, our results revealed distinctive developmental processes that contribute to the expansion and closure of the secondary palate in chameleons and highlighted divergences in palate formation across amniote species.

ROS Regulate Caspase-Dependent Cell Delamination without Apoptosis in the Drosophila Pupal Notum.

SummaryThorax fusion occurs in the midline of the Drosophila pupal notum and involves epithelial cell delamination requiring apoptotic signaling. By genetic screening, we found that NADPH oxidases (Nox and Duox) associated with superoxide anion (O˙-2) are responsible for caspase-3 activation and delamination. We observed that Nox is upregulated in cells that undergo delamination and that delamination depends on caspase activation. However, the cell morphology and the almost complete lack of propidium iodide incorporation suggested little membrane disruption and signified apoptotic modulation. These results demonstrate that most delaminating cells undergo caspase activation, but this activation is not sufficient for apoptosis. We showed that the expression of Catalase, encoding an H2O2 scavenger in the cytosol, increases delamination and induces apoptotic nuclear fragmentation in caspase-3-activated cells. These findings suggest that the roles of O˙-2 and intracellular H2O2 for delamination differs before and after caspase-3 activation, which involves live cell delamination.

Tamoxifen exposure induces cleft palate in mice

Cleft palate is a common birth defect in mammals, which can be caused by genetic or environmental factors, or both. Decades have witnessed that many environmental exposures during gestation extremely increase the incidence of cleft palate. Tamoxifen (TAM), a widely-used drug in treating breast cancer, has been reported to be associated with craniofacial defects including micrognathia and cleft palate in humans. However, its exact effects on the developing palate remain unclear. Here we took advantage of a mouse model to explore how TAM affects palatal development at the molecular level. We showed that excess exposure of TAM in the early embryonic stages indeed leads to cleft palate in mice. RNA-sequencing results strongly suggest the involvement of mitogen-activated protein kinase (MAPK) signalling in TAM-induced cleft palate. Interestingly, in the anterior portion of the TAM-treated palatal shelf, phosphorylated (p)-AKT and p-ERK1/2 were activated but p-p38 was inhibited, while in the posterior palate, the p-AKT increased but the levels of p-p38 and p-JNK decreased. We conclude that excess TAM exposure causes cleft palate defects in mice by regulating MAPK pathways, which implicates the importance of tightly-regulated MAPK signalling in palatal development. This study provides a basis for further exploration of the molecular aetiology of cleft palate defects caused by environmental factors and, based on our results, we would give a serious warning regarding prescription of TAM and potential cleft palate defects in animal models involving the inducible Cre-LoxP system.

Secondary Palate Development in the Dog (Canis lupus familiaris).

To investigate the gestational timing of morphologic events in normal canine secondary palate development as a baseline for studies in dog models of isolated cleft palate (CP). Beagle and beagle/cocker spaniel-hybrid fetal dogs were obtained by cesarean-section on various days of gestation, timed from the initial rise of serum progesterone concentration. Morphology of fetal heads was determined by examining serial coronal sections. On gestational day 35 (d35), the palatal shelves pointed ventrally alongside the tongue. On d36, palatal shelves were elongated and elevated to a horizontal position above the tongue but were not touching. On d37, palatine shelves and vomer were touching, but the medial epithelial seam (MES) between the apposed shelves remained. Immunostaining with epithelial protein markers showed that the MES gradually dissolved and was replaced by mesenchyme during d37-d44, and palate fusion was complete by d44. Examination of remnant MES suggested that fusion of palatal shelves began in mid-palate and moved rostrally and caudally. Palate development occurs in dogs in the steps described in humans and mice, but palate closure occurs at an intermediate time in gestation. These normative data will form the basis of future studies to determine pathophysiologic mechanisms in dog models of CP. Added clinical significance is the enhancement of dogs as a large animal model to test new approaches for palate repair, with the obvious advantage of achieving full maturity within 2 years rather than 2 decades.

Sox2 Controls Periderm and Rugae Development to Inhibit Oral Adhesions

In humans, ankyloglossia and cleft palate are common congenital craniofacial anomalies, and these are regulated by a complex gene regulatory network. Understanding the genetic underpinnings of ankyloglossia and cleft palate will be an important step toward rational treatment of these complex anomalies. We inactivated the Sry (sex-determining region Y)–box 2 (Sox2) gene in the developing oral epithelium, including the periderm, a transient structure that prevents abnormal oral adhesions during development. This resulted in ankyloglossia and cleft palate with 100% penetrance in embryos examined after embryonic day 14.5. In Sox2 conditional knockout embryos, the oral epithelium failed to differentiate, as demonstrated by the lack of keratin 6, a marker of the periderm. Further examination revealed that the adhesion of the tongue and mandible expressed the epithelial markers E-Cad and P63. The expanded epithelia are Sox9-, Pitx2-, and Tbx1-positive cells, which are markers of the dental epithelium; thus, the dental epithelium contributes to the development of oral adhesions. Furthermore, we found that Sox2 is required for palatal shelf extension, as well as for the formation of palatal rugae, which are signaling centers that regulate palatogenesis. In conclusion, the deletion of Sox2 in oral epithelium disrupts palatal shelf extension, palatal rugae formation, tooth development, and periderm formation. The periderm is required to inhibit oral adhesions and ankyloglossia, which is regulated by Sox2. In addition, oral adhesions occur through an expanded dental epithelial layer that inhibits epithelial invagination and incisor development. This process may contribute to dental anomalies due to ankyloglossia.