Fusion Of Palatal Shelves Research Articles

Palatal segment contributions to midfacial anterior-posterior growth.

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

Maxillary Sinus Volume in Patients With Unilateral Cleft Lip and Palate by CBCT.

Cleft lip and palate (CLP) is one of the most common congenital craniofacial anomalies, which can change the morphology of the skull, face, and maxillary sinus due to the disruption in the fusion of palatal shelves. This study aimed to investigate the volume and dimensions of the maxillary sinus in unilateral CLP patients along with comparing the healthy and affected sides. This cross-sectional study was conducted on 27 cone-beam computed tomography stereotypes (14 male and 13 female) of patients with unilateral CLP. Maxillary sinuses were traced separately on each side and analyzed by OnDemand3D software in a separate room with low light. The height and base area of the maxillary sinuses were measured on each side. After dividing each sinus into smaller pyramids, the volume of the sinus was obtained using the partial frustum model method and analyzed by paired t test. There was no significant difference between cleft and noncleft sides in terms of mean volume and height of the sinus ( P > 0.05). The average area of the sinus base on the cleft side was 32.77mm 2 more than the noncleft side, and this difference was statistically significant ( P = 0.027). The mean upper maxillary sinus volume on the cleft side was 541.62mm 3 larger than the noncleft side, but this difference was not statistically significant ( P = 0.075). Considering the age groups, the average upper sinus volume on the cleft side in patients under 20 years old was 4.66mm 3 less than the noncleft side. In the age group of more than 20 years, the average volume of the upper sinus on the cleft side was 978.66mm 3 more than the noncleft side. The mean volume of the lower sinus on the cleft side was 505.92mm 3 lower than the noncleft side, and this difference was statistically significant ( P = 0.010). The average area of the sinus base of the cleft side was significantly higher than the noncleft side. Also, the lower sinus volume of the cleft side was significantly lower than the noncleft side. However, no significant difference was observed in upper sinus volume between the cleft and noncleft sides.

Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos

Embryonic palate development involves bilateral vertical growth of palatal shelves – extensions from the maxillary processes – next to the tongue at embryonic day (E) 13.5. This vertical growth is followed by elevation and fusion above the tongue by E14.5. Recent models indicate that this process of elevation involves a complex vertical to horizontal remodeling of the palatal shelves. While earlier studies have implied that this is a rapid process, the precise timing has not been resolved. To further understand the dynamics of palatal shelf elevation, we employed time‐restricted pregnancies with a one‐hour resolution and magnetic resonance (MR) imaging of intermediate stages. Our data showed that in C57BL/6J mice, palatal shelves are unelevated in 100% of embryos at E14.0 and completely elevated in 80% of embryos at E14.25, which is only a 6‐hour timeframe. Interestingly, all E14.25 embryos with unelevated palatal shelves (20%) were female, suggesting a shift towards delayed elevation in female embryos. Even at E14.125, 25% of embryos had completely elevated palatal shelves suggesting that once initiated, the palatal shelves elevated rapidly with elevation completing in less than 3 hours in individual embryos. In contrast to C57BL/6J, in FVB/NJ mice, the elevation window was shifted earlier and was broader to E13.875‐E14.25 without any noticeable sex differences. In a proportion of embryos in both strains, we also captured an intermediate stage with unilateral palatal shelf elevation. Both right and left shelves were identified to be unilaterally elevated indicating that it was a random process. It remains to be determined whether palatal shelves elevate in a sequential manner, i.e., one‐at‐a‐time, with an obligate unilateral elevation stage. MR imaging of various stages showed a highly dynamic antero‐posterior remodeling of the shelves during elevation, beginning with the formation of bilateral bulges in the posterior palate. These bulges tapered anteriorly, however, palatal shelf fusion still began in the middle part of the palate. The bulge regions did not show changes in cell proliferation, consistent with the notion that rapid elevation was primarily due to cytoskeletal and extracellular matrix changes. We are using finite element analysis to identify the mechanical forces underlying these morphological changes. Thus, our data reveal critical novel insights into the rapid dynamic changes as well as strain and sex differences in palatal shelf elevation that lay the foundation for future studies of normal and abnormal palatogenesis.

Methods of Palate Culture in Later Palatogenesis: Elevation, Horizontal Outgrowth, and Fusion.

The ex vivo culture of the palate has provided a versatile model in which to study palatogenesis. Dysmorphias of the palate remain one of the most common birth defects globally, with great scope for future research in both normal and dysmorphic palatogenesis. This process can be studied in the mouse model using both the hyperoxic rolling culture of maxillary explants and Trowell-type static cultures, which are optimal for the study of different stages of palate development respectively. Here, we describe both methods: the former for the study of palatal shelf elevation and horizontal growth, and the latter for palatal shelf fusion . Both are applicable in murine embryos cultured at embryonic day 13.5 using nonspecialist equipment.

Three-dimensional reconstruction of systematic histological sections: application to observations on palatal shelf elevation

Normal mammalian secondary palate development undergoes a series of processes, including palatal shelf (PS) growth, elevation, adhesion and fusion, and palatal bone formation. It has been estimated that more than 90% of isolated cleft palate is caused by defects associated with the elevation process. However, because of the rapidly completed elevation process, the entire process of elevation will never be easy to clarify. In this article, we present a novel method for three-dimensional (3D) reconstruction of thick tissue blocks from two-dimensional (2D) histological sections. We established multiplanar sections of the palate and tongue in coronal and sagittal directions, and further performed 3D reconstruction to observe the morphological interaction and connection between the two components prior to and during elevation. The method completes an imaging system for simultaneous morphological analysis of thick tissue samples using both synthetic and real data. The new method will provide a comprehensive picture of reorientation morphology and gene expression pattern during the palatal elevation process.

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.

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.

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.

Genetic background influences the capacity for medial edge epithelium disintegration and phenotype of cleft palate in TGFβ3 knockout mice

ObjectivesCleft palate is a frequent congenital craniofacial malformation of unknown etiology. Transforming growth factor (TGF) β3 is required for palatal shelf fusion. Although TGFβ3 knockout (KO) mice are widely used mouse models for cleft palate, cleft palate phenotypes differ among these mice. This study aimed to determine the effects of genetic background on the cleft palate phenotype in mice. MethodsWe produced TGFβ3 KO congenic mouse strains with five different genetic backgrounds. The phenotypes of the congenic strains were determined by visual examination. The capacity for disintegration of the medial edge epithelium (MEE) and basement membrane (BM) of palatal shelves of all five mouse strains was analyzed by using immunofluorescence staining after single palatal shelf suspension culture. The relationship between phenotype and disappearance of the MEE and BM was analyzed. ResultsAlthough the five congenic strains carried the same defective Tgfb3 gene, the fetal palate phenotypes differed among strains. The loss of the MEE cells and BM also differed with the genetic background, and the degree of such loss correlated with the cleft palate phenotype. ConclusionsThe cleft palate phenotype in mice is influenced by the genetic background, which governs the capacity for MEE and BM disintegration.

Integrated analysis identifying long non-coding RNAs (lncRNAs) for competing endogenous RNAs (ceRNAs) network-regulated palatal shelf fusion in the development of mouse cleft palate.

Cleft palate results from the defective palatal fusion of the medial-edge epithelium after cells undergo epithelial-mesenchymal transition, a process that involves regulation by microRNAs (miRNAs). However, in palatal shelf fusion, miRNA regulation by long non-coding RNAs (lncRNAs) when acting as competing endogenous RNAs (ceRNAs) or miRNA sponges, remains unclear. We systematically analyzed the correlation between lncRNAs, miRNAs, and mRNAs from RNA sequencing profiling in embryonic gestation day 14.5 (E14.5) mouse embryos from control (n=3) and all-trans retinoic acid (ATRA)-treated (n=3) mice. We then constructed a lncRNA-associated ceRNA network. The expression profiles of mRNA, lncRNA, and miRNA were verified by quantitative polymerase chain reaction (qPCR). In total, 18 aberrantly expressed miRNAs, 861 mRNAs, and 583 lncRNAs were identified from palate samples of control and ATRA-treated samples. Bioinformatics data and integrative analysis identified 69 lncRNAs, 18 miRNAs, and 78 mRNAs that were aberrantly expressed, and a ceRNA network was then constructed. Finally, we identified a NONMMUT004850.2/NONMMUT024276.2-miR-741-3p/miR-465b-5p-Prkar1α ceRNA network associated with palatal shelf fusion at E14.5. The qPCR results showed that NONMMUT004850.2 (P=5E-05), NONMMUT024276.2 (P=0.0012), and Prkar1α (P=3E-05) were up-regulated, whereas miR-741-3p (P=0.006) and miR-465b-5p (P=1E-04) were down-regulated in ATRA-treated mice compared to the control samples. The qPCR results were in concordance with the RNA sequencing profiling. Our study demonstrated that NONMMUT004850.2/NONMMUT024276.2-miR-741-3p/miR-465b-5p-Prkar1α could potentially serve as an important regulatory mechanism of palatal fusion in the development of the cleft palate.

Identification of circular RNA-associated competing endogenous RNA network in the development of cleft palate.

Circular RNAs (circRNAs) serve as competing endogenous RNAs (ceRNAs) and indirectly regulate gene expression through shared microRNAs (miRNAs). However, the regulatory mechanisms of circRNA as ceRNA associated with the fusion of palatal shelves in palatogenesis are yet unclear. This study aimed to explore the potential mechanism underlying the role of circRNA as ceRNA in cleft palate (CP). First, we systematically analyzed RNA-seq and miRNA-seq data after high-throughput sequencing for embryonic palatal shelf tissues from a mouse CP model induced by maternal exposure to all-trans retinoic acid on embryonic gestation day 14.5 (E14.5). Thirty-nine circRNAs, 18 miRNAs, and 936 messenger RNAs (mRNAs) were significantly dysregulated (log2 [fold change {FC}] > 1; P < 0.05). Thereafter, we constructed a circRNA-associated ceRNA network. Finally, we determined the circRNA_0954-miRNA-881-3p-PRKAR1α ceRNA network as a hub involved in palatogenesis. Gene Ontology analysis revealed that ceRNA-related genes were associated with facial morphogenesis and developmental gene silencing. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that ceRNA-related genes are involved in apoptosis (P < 0.05, fold enrichment >1). Quantitative reverse transcription polymerase chain reaction was performed to verify the results of ceRNA analysis. We found that the circRNA-miRNA-mRNA ceRNA network is involved in palatogenesis. The present results imply that circRNA_0954-miRNA-881-3p-PRKAR1α ceRNA network may cause dysfunctional palatal fusion and might facilitate the development of novel epigenetic biomarkers to treat CP in the future.

Position-dependent correlation between TBX22 exon 5 methylation and palatal shelf fusion in the development of cleft palate.

DNA methylation is essential for spatiotemporally-regulated gene expression in embryonic development. TBX22 (Chr X: 107667964-107688978) functioning as a transcriptional repressor affects DNA binding, sumoylation, and transcriptional repression associated with X-linked cleft palate. This study aimed to explore the relationship and potential mechanism between TBX22 exon 5 methylation and palatal shelf fusion induced by all-trans retinoic acid (ATRA). We performed DNA methylation profiling, using MethylRAD-seq, after high throughput sequencing of mouse embryos from control (n=9) and ATRA-treated (to induce cleft palate, n=9) C57BL/6J mice at embryonic gestation days(E) 13.5, 14.5 and 16.5. TBX22 exon 5 was hyper-methylated at the CpG site at E13.5 (P=0.025, log2FC=1.5) and E14.5 (P=0.011, log2FC:1.5) in ATRA-treated, whereas methylation TBX22 exon 5 at the CpG site was not significantly different at E16.5 (P=0.808, log2FC=-0.2) between control and ATRA-treated. MSP results showed a similar trend consistent with the MethylRAD-seq results. qPCR showed the change in TBX22 exon 5 expression level negatively correlated with its TBX22 exon 5 methylation level. These results indicate that changes in TBX22 exon 5 methylation might play an important regulatory role during palatal shelf fusion, and may enlighten the development of novel epigenetic biomarkers in the treatment of CP in the future.

Expression of Gene Runx2, Wnt and OPG in Palate Cleft Reconstruction Material

Abstract Introduction. Facial morphogenesis occurs from the fourth to the twelfth gestation week, when the cells from nerve crest migrate to the region of face, forming the primary palate. The cleft palate is an abnormality in embryogenesis period, which is characterized by the absence of fusion of palatal shelves. The incidence of cleft lip and palate is one in 700 live births. In recent years the effect of different genes and signaling molecules, including Runx2, Wnt3 and OPG have been studied in the development of cleft palate, because these substances are considered to be regulators of pathogenesis responsible for formation of bone and cartilage tissue and particularly the bone. Aim of the Study. The aim of the work was to evaluate the expression of Runx2, Wnt3 and OPG in palate bone and nasal cartilage for children with cleft palate. Material and methods.Eleven bone and cartilage samples were obtained from 21 children of the lip, soft and hard palate correction surgery. All the patients were diagnosed with clefts of the lip, alveolar process of maxilla, and palate. In the tissue sections using the immunohistochemistry method (IMH), were determined Runx2 (code: AB192256, 1: 250, Abcam GB, rabbit), Wnt3 (code: AB1992, 1: 800, Abcam GB, rabbit), and OPG (code: A0611, 1: 100, The Orbit USA, rabbit) local expression. We used a semi-quantitative census method for quantifying the positive structures. Results. Runx2 expression was observed in five patient bone tissue samples and six patient cartilage tissue samples. Of the Runx2 positive bone tissue, in one case we observed occasional, in two cases- few, in one case- moderate to numerous and in one case numerous positive osteocytes while in tissue of cartilage in two cases we observed few, in one case- few to moderate, in two casesmoderate, and in one case numerous positive chondrocytes. A significant difference in Wnt3 expiation was observed between bone and cartilage tissues. Wnt3 expressing chondrocytes were observed in all samples, where in one case- occasional, in three cases few, in one case-moderate, and in six cases-numerous positive cartilage cells were observed. The expression of the gene in the bone was observed in nine cases, which contained mostly occasional or few positive structures, except in three cases where in one Wnt3 was marked by few to moderate and in two cases numerous positive osteocytes. OPG expression was observed in all samples, but in the cartilage, the expression was more pronounced. In the cartilage in seven cases, there were numerous positive chondrocytes, in one case- few to moderate, in two cases moderate to numerous and in one case few to moderate number of chondrocytes. OPG showed variable expression. In four cases, we observed occasional to few, in one case few to moderate, in one case- moderate, in one case moderate to numerous and in four cases numerous positive bone cells. Conclusion. Cartilage tissue expresses significantly more Runx2, Wnt3 genes and OPG proteins, indicating a greater compensatory tissue capacity. In the case of palate clefts, the high expression of Wnt3 and OPG and lower expression of Runx2 could indicate a significant tissue proliferation which predominates over mineralization and ossification processes.

Live Imaging of Mouse Secondary Palate Fusion.

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Small-molecule Wnt agonists correct cleft palates in Pax9 mutant mice in utero.

Clefts of the palate and/or lip are among the most common human craniofacial malformations and involve multiple genetic and environmental factors. Defects can only be corrected surgically and require complex life-long treatments. Our studies utilized the well-characterized Pax9-/- mouse model with a consistent cleft palate phenotype to test small-molecule Wnt agonist therapies. We show that the absence of Pax9 alters the expression of Wnt pathway genes including Dkk1 and Dkk2, proven antagonists of Wnt signaling. The functional interactions between Pax9 and Dkk1 are shown by the genetic rescue of secondary palate clefts in Pax9-/-Dkk1f/+;Wnt1Cre embryos. The controlled intravenous delivery of small-molecule Wnt agonists (Dkk inhibitors) into pregnant Pax9+/- mice restored Wnt signaling and led to the growth and fusion of palatal shelves, as marked by an increase in cell proliferation and osteogenesis in utero, while other organ defects were not corrected. This work underscores the importance of Pax9-dependent Wnt signaling in palatogenesis and suggests that this functional upstream molecular relationship can be exploited for the development of therapies for human cleft palates that arise from single-gene disorders.

The A-kinase Anchoring Protein GSKIP Regulates GSK3β Activity and Controls Palatal Shelf Fusion in Mice

A-kinase anchoring proteins (AKAPs) represent a family of structurally diverse proteins, all of which bind PKA. A member of this family is glycogen synthase kinase 3β (GSK3β) interaction protein (GSKIP). GSKIP interacts with PKA and also directly interacts with GSK3β. The physiological function of the GSKIP protein in vivo is unknown. We developed and characterized a conditional knock-out mouse model and found that GSKIP deficiency caused lethality at birth. Embryos obtained through Caesarean section at embryonic day 18.5 were cyanotic, suffered from respiratory distress, and failed to initiate breathing properly. Additionally, all GSKIP-deficient embryos showed an incomplete closure of the palatal shelves accompanied by a delay in ossification along the fusion area of secondary palatal bones. On the molecular level, GSKIP deficiency resulted in decreased phosphorylation of GSK3β at Ser-9 starting early in development (embryonic day 10.5), leading to enhanced GSK3β activity. At embryonic day 18.5, GSK3β activity decreased to levels close to that of wild type. Our findings reveal a novel, crucial role for GSKIP in the coordination of GSK3β signaling in palatal shelf fusion.

Dhcr7 Regulates Palatal Shelf Fusion through Regulation of Shh and Bmp2 Expression.

The aim of this study was to investigate the effect of the 7-dehydrocholesterol reductase (Dhcr7) gene and identify signaling pathways involved in regulation of embryonic palatogenesis. The expression of Dhcr7 and its protein product were examined during murine normal embryonic palatogenesis via a reverse transcription polymerase chain reaction (RT-PCR) and Western blot (WB). RNA interference (RNAi) technology was used to inhibit Dhcr7 expression in a palatal shelf culture in vitro. The effects of Dhcr7 on palatogenesis and palatal fusion were examined by scanning electron microscopy (SEM). The expression changes of Dhcr7, Sonic Hedgehog (Shh), and bone morphogenetic protein-2 (Bmp2) were measured by RT-PCR and WB after Dhcr7 gene silencing and the addition of exogenous cholesterol. The results showed that the palatal shelf failed to complete normal development and fusion when Dhcr7 expression was inhibited. The inhibitory effect study of RNAi on the development of the palatal shelf supported that cholesterol supplementation did not alter the silencing of Dhcr7. Shh and Bmp2 expressions were reduced after Dhcr7 gene silencing, and administration of exogenous cholesterol did not affect Dhcr7 expression; however Shh and Bmp2 expressions increased. We conclude that Dhcr7 plays a role in growth of the palatal shelf and can regulate palatogenesis through alterations in the levels of Shh and Bmp2.

A Comprehensive Study of Soft Palate Development in Mice.

Cleft palate is one of the most common congenital birth defects. Tremendous efforts have been made over the last decades towards understanding hard palate development. However, little is known about soft palate morphogenesis and myogenesis. Finding an appropriate surgical repair to restore physiological functions of the soft palate in patients with cleft palate is a major challenge for surgeons, and complete restoration is not always achievable. Here, we first analyzed the morphology, orientation and attachments of the four muscles of the murine soft palate and found that they are very similar to their counterparts in humans, validating the use of mus musculus as a model for future studies. Our data suggests that muscle differentiation extends from the lateral region to the midline following palatal fusion. We also detected an epithelial seam in the fusing soft palatal shelves, consistent with the process of fusion of the posterior palatal shelves, followed by degradation of the epithelial remnants. Innervation and vascularization are present mainly in the oral side of the soft palate, complementing the differentiated muscles. Cell lineage tracing using Wnt1-Cre;Zsgreen fl/fl mice indicated that all the tendons and mesenchyme embedding the soft palate muscles are neural crest-derived. We propose that the posterior attachment of the soft palate to the pharyngeal wall is an interface between the neural crest- and mesoderm-derived mesenchyme in the craniofacial region, and thus can serve as a potential model for the study of boundaries during development. Taken together, our study provides a comprehensive view of the development and morphology of the murine soft palate and serves as a reference for further molecular analyses.

Convergence and extrusion driven by non‐muscle myosin II are required for normal fusion of the mammalian secondary palate.

Fusion of two distinct embryonic prominences to form a single continuous structure occurs in numerous developmental contexts and typically requires integration of two epithelia and subsequent removal of that intervening epithelium. During fusion of the mammalian secondary palatal shelves, the medial epithelial seam (MES) is removed to form a continuous secondary palate with mesenchymal confluence, but the cellular mechanisms remain incompletely understood. Three main hypotheses for how this occurs have been proposed. Whereas considerable investigation has been made into epithelial to mesenchymal transition (EMT) and programmed cell death of the intervening epithelium as potential cellular mechanisms, the contribution of other cellular dynamics of the intervening epithelium has not been directly evaluated. Using confocal live imaging, we have now directly observed the cellular processes underlying tissue fusion in the palatal shelves. We find that convergence of a multi‐layered epithelium into a single‐layer epithelium is an essential early step driven by cell intercalation and is concurrent to orthogonal displacement of epithelial cells. Surprisingly, we find that epithelial cell extrusion, a process in which cells are surrounded and squeezed from an epithelium in an actomyosin contractility‐dependent manner functions in the removal of the MES during palate fusion. Functional studies in mice indicate that these processes require an actomyosin contractility pathway involving Rho kinase (ROCK) and myosin light chain kinase (MLCK), culminating in the activation of non‐muscle myosin IIA (NMIIA). Together, these data indicate that actomyosin contractility drives cell intercalation and cell extrusion during palate fusion and suggest a general mechanism for tissue fusion in development.