Sprouty2 controls proliferation of palate mesenchymal cells via fibroblast growth factor signaling

Cleft palate is one of the most common craniofacial deformities. The fibroblast growth factor (FGF) plays a central role in reciprocal interactions between adjacent tissues during palatal development, and the FGF signaling pathway has been shown to be inhibited by members of the Sprouty protein family. In this study, we report the incidence of cleft palate, possibly caused by failure of palatal shelf elevation, in Sprouty2-deficient (KO) mice. Sprouty2-deficient palates fused completely in palatal organ culture. However, palate mesenchymal cell proliferation estimated by Ki-67 staining was increased in Sprouty2 KO mice compared with WT mice. Sprouty2-null palates expressed higher levels of FGF target genes, such as Msx1, Etv5, and Ptx1 than WT controls. Furthermore, proliferation and the extracellular signal-regulated kinase (Erk) activation in response to FGF was enhanced in palate mesenchymal cells transfected with Sprouty2 small interfering RNA. These results suggest that Sprouty2 regulates palate mesenchymal cell proliferation via FGF signaling and is involved in palatal shelf elevation.     

Augmented BMPRIA-Mediated BMP Signaling in Cranial Neural Crest Lineage Leads to Cleft Palate Formation and Delayed Tooth Differentiation

The importance of BMP receptor Ia (BMPRIa) mediated signaling in the development of craniofacial organs, including the tooth and palate, has been well illuminated in several mouse models of loss of function, and by its mutations associated with juvenile polyposis syndrome and facial defects in humans. In this study, we took a gain-of-function approach to further address the role of BMPR-IA-mediated signaling in the mesenchymal compartment during tooth and palate development. We generated transgenic mice expressing a constitutively active form of BmprIa (caBmprIa) in cranial neural crest (CNC) cells that contributes to the dental and palatal mesenchyme. Mice bearing enhanced BMPRIa-mediated signaling in CNC cells exhibit complete cleft palate and delayed odontogenic differentiation. We showed that the cleft palate defect in the transgenic animals is attributed to an altered cell proliferation rate in the anterior palatal mesenchyme and to the delayed palatal elevation in the posterior portion associated with ectopic cartilage formation. Despite enhanced activity of BMP signaling in the dental mesenchyme, tooth development and patterning in transgenic mice appeared normal except delayed odontogenic differentiation. These data support the hypothesis that a finely tuned level of BMPRIa-mediated signaling is essential for normal palate and tooth development.     

Intracellular dynamics of Smad-mediated TGFbeta signaling

The transforming growth factor-beta (TGFbeta) family represents a class of signaling molecules that plays a central role in morphogenesis, growth, and cell differentiation during normal embryonic development. Members of this growth factor family are particularly vital to development of the mammalian secondary palate where they regulate palate mesenchymal cell proliferation and extracellular matrix synthesis. Such regulation is particularly critical since perturbation of either cellular process results in a cleft of the palate. While the cellular and phenotypic effects of TGFbeta on embryonic craniofacial tissue have been extensively catalogued, the specific genes that function as downstream mediators of TGFbeta action in the embryo during palatal ontogenesis are poorly defined. Embryonic palatal tissue in vivo and murine embryonic palate mesenchymal (MEPM) cells in vitro secrete and respond to TGFbeta. In the current study, elements of the Smad component of the TGFbeta intracellular signaling system were identified and characterized in cells of the embryonic palate and functional activation of the Smad pathway by TGFbeta1, TGFbeta2, and TGFbeta3 was demonstrated. TGFbeta-initiated Smad signaling in cells of the embryonic palate was found to result in: (1) phosphorylation of Smad 2; (2) nuclear translocation of the Smads 2, 3, and 4 protein complex; (3) binding of Smads 3 and 4 to a consensus Smad binding element (SBE) oligonucleotide; (4) transactivation of transfected reporter constructs, containing TGFbeta-inducible Smad response elements; and (4) increased expression of gelatinases A and B (endogenous genes containing Smad response elements) whose expression is critical to matrix remodeling during palatal ontogenesis. Collectively, these data point to the presence of a functional Smad-mediated TGFbeta signaling system in cells of the developing murine palate.     

A unique mouse strain expressing Cre recombinase for tissue-specific analysis of gene function in palate and kidney development

Mammalian palate development is a multistep process, involving initial bilateral downward outgrowth of the palatal shelves from the oral side of the maxillary processes, followed by stage-specific palatal shelf elevation to the horizontal position above the developing tongue and subsequent fusion of the bilateral palatal shelves at the midline to form the intact roof of the oral cavity. While mutations in many genes have been associated with cleft palate pathogenesis, the molecular mechanisms regulating palatal shelf growth, patterning, and elevation are not well understood. Genetic studies of the molecular mechanisms controlling palate development in mutant mouse models are often complicated by early embryonic lethality or gross craniofacial malformation. We report here the development of a mouse strain for tissue-specific analysis of gene function in palate development. We inserted an IresCre bicistronic expression cassette into the 3' untranslated region of the mouse Osr2 gene through gene targeting. We show, upon crossing to the R26R reporter mice, that Cre expression from the Osr2(IresCre) knockin allele activated beta-galactosidase expression specifically throughout the developing palatal mesenchyme from the onset of palatal shelf outgrowth. In addition, the Osr2(IresCre) mice display exclusive Cre-mediated recombination in the glomeruli tissues derived from the metanephric mesenchyme and complete absence of Cre activity in other epithelial and mesenchymal tissues in the developing metanephric kidney. These data indicate that the Osr2(IresCre) knockin mice provide a unique tool for tissue-specific studies of the molecular mechanisms regulating palate and kidney development.     

Quantification and localization of ornithine decarboxylase in the embryonic palate.

Ornithine decarboxylase (ODC; EC4.1.1.17), the key enzyme in polyamine biosynthesis, and intracellular polyamines increase rapidly and markedly in tissues and cells that are actively proliferating as well as differentiating and decrease as these processes cease. ODC activity has also been implicated as playing a role in the proliferation and differentiation of cells derived from the developing palate. Ornithine decarboxylase activity was thus quantified and ODC localized in the developing murine palate in vivo. Levels of ODC activity showed little variation during the ontogeny of the palate, averaging 126 pmol CO2/mg protein/hr. When difluoromethylornithine (DFMO), an irreversible inhibitor of ODC activity, was administered to pregnant mice throughout the period of palate development (days 11-14), palatal tissue ODC activity was reduced by 85%. No craniofacial malformations were observed, however. The lack of a teratogenic effect by DFMO treatment could be due to sufficient remaining ODC activity in craniofacial tissue and/or maintenance of intracellular polyamine levels by the activity of a polyamine transport system. The activity of this system was demonstrated by the ability of palatal tissue in vivo to take up radiolabeled putrescine. The presence of a polyamine transport system was previously suggested by the demonstration of such a system in palate mesenchymal cells in vitro. Dramatic temporal and spatial shifts in tissue patterns of immunolocalization for ODC in developing palatal tissue were also seen. Immunostaining for ODC was evenly distributed in oral, nasal, and medial edge palate epithelial cells on day 12 of gestation. The basal aspects of epithelial cells were, however, more intensely stained. Mesenchymal cells exhibited a peri-nuclear immunostaining pattern. On days 12 and 13 of gestation, the staining patterns for ODC in palate epithelial and mesenchymal cells were comparable. On day 14 of gestation, all regions of the palate epithelium, particularly the medial edge epithelia, were immunostained for ODC, whereas the intensity of staining in the mesenchymal cells was significantly reduced. This study represents essential initial observations toward understanding the role that ODC may play in normal craniofacial development.     

Msx-1 gene expression and regulation in embryonic palatal tissue

Summary The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.     

In vitro development of the hamster and chick secondary palate

A series of experiments were undertaken to compare the in vitro behaviour of the medial edge epithelium (MEE) of hamster, in which palatal shelves normally fuse, and chick, in which they do not fuse. Homotypic pairs of hamster and chick embryo palatal processes, single palatal processes, and heterotypic palatal shelves of both animals were grown in vitro. The results indicated that contact between palatal shelves may not be crucial for MEE differentiation in mammals. The ability to acquire pre-fusion characteristics may be present in mammalian palatal tissue from their early development and may be expressed by cessation of DNA synthesis in the MEE, elevation of cAMP, and MEE cell death. Isolated chick palatal shelf cultured under identical conditions did not express these mammalian pre-fusion characteristics. When MEE of hamster and chick palatal shelves were placed in contact with one another, the intervening epithelia underwent cytolysis. This could be due to either the destruction of chick MEE by lysosomal enzymes liberated from adjacent degenerating hamster MEE cells, or by induction of cell death in chick MEE by hamster mesenchyme. Heterotypic palatal tissue combinations also suggest that release of lysosomal enzymes in the hamster MEE, which leads to its dissolution, may be the terminal event in epithelial differentiation prior to the establishment of mesenchymal continuity. It is suggested that an inverse relationship exists between DNA synthesis and cAMP levels during palatogenesis: when palate closes (as in mammals) the MEE is eliminated by increasing cAMP levels, whereas when palate remains open (as in birds) low level of cAMP preserve the integrity of MEE by supporting DNA synthesis.     

Modulation of BMP signaling by Noggin is required for the maintenance of palatal epithelial integrity during palatogenesis

BMP signaling plays many important roles during organ development, including palatogenesis. Loss of BMP signaling leads to cleft palate formation. During development, BMP activities are finely tuned by a number of modulators at the extracellular and intracellular levels. Among the extracellular BMP antagonists is Noggin, which preferentialy binds to BMP2, BMP4 and BMP7, all of which are expressed in the developing palatal shelves. Here we use targeted Noggin mutant mice as a model for gain of BMP signaling function to investigate the role of BMP signaling in palate development. We find prominent Noggin expression in the palatal epithelium along the anterior-posterior axis during early palate development. Loss of Noggin function leads to overactive BMP signaling, particularly in the palatal epithelium. This results in disregulation of cell proliferation, excessive cell death, and changes in gene expression, leading to formation of complete palatal cleft. The excessive cell death in the epithelium disrupts the palatal epithelium integrity, which in turn leads to an abnormal palate-mandible fusion and prevents palatal shelf elevation. This phenotype is recapitulated by ectopic expression of a constitutively active form of BMPR-IA but not BMPR-IB in the epithelium of the developing palate; this suggests a role for BMPR-IA in mediating overactive BMP signaling in the absence of Noggin. Together with the evidence that overexpression of Noggin in the palatal epithelium does not cause a cleft palate defect, we conclude from our results that Noggin mediated modulation of BMP signaling is essential for palatal epithelium integrity and for normal palate development.     

A possible explanation of what's happening at the moment of palatal shelf elevation

Cleft lip and palate is multifactorial in aetiology. The elevation of palatal shelves is a key point of palatogenesis. However, there were many different opinions on the explanation of the elevation. In this article, we offered a new explanation. Before sixth week of gestation in humans, Palatal mesenchymal proliferation was along the horizontal direction. Because of the block of the tongue, the palatal shelves had to grow first vertically in the oral cavity. In the process of cells migration, much horizontal stress accumulated in the palatal shelves, meanwhile increased the collagen secretion of the palatal mesenchymal cells in order to strengthen the elasticity of palatal shelf and maintain the integrity to make palatal shelf look like an elastic palate. The intrinsic elevating force and the block of tongue made the palatal shelf curved. After seventh week facial structures grew predominantly in the sagittal plane. The activity of the geniohyoid and genioglossus muscles caused the mandibular retraction and the widening of the angulation between the bilateral hemimandibles. These changes provided the space for palatal shelf elevation. At some moment of the eighth to tenth weeks, the elastic stress center of the palatal shelf was above the horizontal surface because of the drop of the tongue. The palatal shelves might bounce up and elevate in a horizontal position when enough horizontal stress accumulated, and then adhered and fused.     

Cytokeratin, vimentin and E-cadherin immunodetection in the embryonic palate in two strains of mice with different susceptibility to glucocorticoid-induced clefting

An immunohistochemical study analyzing the pattern of distribution of some intermediate filament proteins, keratin and vimentin and, one adhesion molecule, cadherin in different stages of developing secondary palate in two strains of mice with different H-2 backgrounds was undertaken to investigate differences between a strain that is susceptible to glucocorticoid-induced cleft palate (A/Sn) and one that is resistant to glucocorticoid-induced cleft palate (C57/BL). The heads of embryos were processed by standard immunohistochemistry with antipancytokeratin (KAE1), antikeratins 18 (K18) and 19 (K19), antivimentin, and anti E-cadherin antibodies. Immunostaining with KAE1 antibody showed differences between the strains. The reaction was stronger in the medial edge epithelia of palatal processes in the A/Sn strain at all stages of palatogenesis. The C57/BL strain showed a weak immunostain to KAE1. Antivimentin antibody stained the mesenchymal cells of palatal processes and K18 and K19 showed no reaction in either strain of mice. Anti E-cadherin antibody was detected in the medial palatal epithelium of both strains of mice and in all stages of palate development. No differences were observed in E-cadherin and vimentin immunostain in palatal epithelium between the strains. The different expression of some cytokeratins in the embryonic palatal epithelium suggests that these intermediate filament proteins may be involved in different susceptibility to glucocorticoid-induced cleft palate in the mouse. The decreased immunoreaction of cytokeratins observed in the resistant strain would facilitate the disappearance of this molecule during the transformation from an epithelial to a mesenchymal phenotype that takes place during the development of the palate. These results may be related to the loss of cytokeratin expression observed during epithelial-mesenchymal transformation in the embryonic palate.     

Overexpression of iNOS and down-regulation of BMPs-2, 4 and 7 in retinoic acid induced cleft palate formation

The present work studied the induction of cleft palate formation in embryos developed from pregnant BALB/c mice treated orally with retinoic acid (RA). Previous studies on mature somatic cell types showed that RA exerted inhibitory effects on inducible nitric oxide synthase (iNOS) production. For the first time, our study has shown that RA actually stimulates significant expression of iNOS at specific zones of the affected embryonic palatal tissues at three consecutive stages, from gestation day 13 (GD13) to day 16 (GD16). Enzymatically, iNOS facilitates intracellular nitric oxide (NO) synthesis from L-arginine. When NO reacts with reactive superoxides it may result in irreparable cell injury. NO was also reported to induce apoptosis in some mammalian cell systems. Based on our findings, we propose that such an increase in NO production might be associated with apoptosis in the embryonic palatal tissues in the RA-treated mice. The detrimental effects of NO resulted in a reduction in proliferating palatal cells and therefore disturbed the normal plasticity of the palatal shelves. With iNOS overexpression, our findings also showed that there was significant concomitant down-regulation in the expressions of Bone Morphogenetic Proteins (BMPs) -2, 4, and 7 with regional variations particularly in the palatal mesenchymal cells for those embryos developing cleft palate. Since specific spatial and temporal expressions of BMPs -2, 4, and 7 are critical during normal palatal morphogenesis, any deficiency in the epithelial-mesenchymal interaction may result in retarding growth at the embryonic palatal shelves. Taken together, our study has demonstrated cleft palate formation in the BALB/c embryos involved overexpression of iNOS and down-regulation of BMPs-2, 4 and 7.     

Morphological observations in normal primary palate and cleft lip embryos in the Kyoto collection

Normal developmental events during human primary palate formation and alterations associated with cleft lip remain poorly defined. The purpose of this study was to analyze serially sectioned human embryos to identify morphological changes during normal palatal closure and alterations associated with failure of palatal formation. Normal and cleft embryos from the histological collection at the Congenital Anomaly Research Center at the University of Kyoto were studied and photographed for detailed evaluation. Seven serially sectioned cleft lip embryos of stages shortly after primary palate formation (Streeter-O'Rahilly stages 19, 20, and 22) with unilateral or bilateral clefts with varying degrees of clefting were studied. In the normal Kyoto embryos, initial nasal fin (epithelial seam) formation was observed between the medial nasal process and the lateral nasal and maxillary processes at stage 17. During stages 18 and 19, the nasal fin epithelium was replaced by an enlarging mesenchymal bridge, as the maxillary processes united with the medial nasal processes to form the primary palate. The most prominent features observed in the cleft embryos were a reduced thickness of mesenchymal bridging between the medial nasal and maxillary processes, with an excessive amount of epithelium at the junctions between these processes. With ingrowth of the maxillary processes, greater cell dispersion and apparent extracellular matrix accumulation were observed in the medial nasal region. During closure of the primary palate, terminal branches of the maxillary nerve crossed the mesenchymal bridge to the medial nasal region. The partial clefts had reduced maxillary ingrowth and smaller union areas with the medial nasal process. Detailed studies of experimental animal models are required to identify regional growth required for contact between the facial prominences, to clarify the mechanisms of mesenchymal ingrowth and epithelial displacement during palatal formation, and to identify local and/or general factors causing alterations that lead to primary palatal clefting.     

Cooperation of two ADAMTS metalloproteases in closure of the mouse palate identifies a requirement for versican proteolysis in regulating palatal mesenchyme proliferation

We have identified a role for two evolutionarily related, secreted metalloproteases of the ADAMTS family, ADAMTS20 and ADAMTS9, in palatogenesis. Adamts20 mutations cause the mouse white-spotting mutant belted (bt), whereas Adamts9 is essential for survival beyond 7.5 days gestation (E7.5). Functional overlap of Adamts9 with Adamts20 was identified using Adamts9(+/-);bt/bt mice, which have a fully penetrant cleft palate. Palate closure was delayed, although eventually completed, in both Adamts9(+/-);bt/+ and bt/bt mice, demonstrating cooperation of these genes. Adamts20 is expressed in palatal mesenchyme, whereas Adamts9 is expressed exclusively in palate microvascular endothelium. Palatal shelves isolated from Adamts9(+/-);bt/bt mice fused in culture, suggesting an intact epithelial TGFβ3 signaling pathway. Cleft palate resulted from a temporally specific delay in palatal shelf elevation and growth towards the midline. Mesenchyme of Adamts9(+/-);bt/bt palatal shelves had reduced cell proliferation, a lower cell density and decreased processing of versican (VCAN), an extracellular matrix (ECM) proteoglycan and ADAMTS9/20 substrate, from E13.5 to E14.5. Vcan haploinsufficiency led to greater penetrance of cleft palate in bt mice, with a similar defect in palatal shelf extension as Adamts9(+/-);bt/bt mice. Cell density was normal in bt/bt;Vcan(hdf)(/+) mice, consistent with reduced total intact versican in ECM, but impaired proliferation persisted in palate mesenchyme, suggesting that ADAMTS-cleaved versican is required for cell proliferation. These findings support a model in which cooperative versican proteolysis by ADAMTS9 in vascular endothelium and by ADAMTS20 in palate mesenchyme drives palatal shelf sculpting and extension.     

Shox2-deficient mice exhibit a rare type of incomplete clefting of the secondary palate

The short stature homeobox gene SHOX is associated with idiopathic short stature in humans, as seen in Turner syndrome and Leri-Weill dyschondrosteosis, while little is known about its close relative SHOX2. We report the restricted expression of Shox2 in the anterior domain of the secondary palate in mice and humans. Shox2-/- mice develop an incomplete cleft that is confined to the anterior region of the palate, an extremely rare type of clefting in humans. The Shox2-/- palatal shelves initiate, grow and elevate normally, but the anterior region fails to contact and fuse at the midline, owing to altered cell proliferation and apoptosis, leading to incomplete clefting within the presumptive hard palate. Accompanied with these cellular alterations is an ectopic expression of Fgf10 and Fgfr2c in the anterior palatal mesenchyme of the mutants. Tissue recombination and bead implantation experiments revealed that signals from the anterior palatal epithelium are responsible for the restricted mesenchymal Shox2 expression. BMP activity is necessary but not sufficient for the induction of palatal Shox2 expression. Our results demonstrate an intrinsic requirement for Shox2 in palatogenesis, and support the idea that palatogenesis is differentially regulated along the anteroposterior axis. Furthermore, our results demonstrate that fusion of the posterior palate can occur independently of fusion in the anterior palate.     

Calmodulin quantification and immunolocalization in developing embryonic orofacial tissue

Temporally and quantitatively coordinated synthesis of cyclic adenosine monophosphate appears to be critical for normal development of the mammalian secondary palate. Calmodulin has been implicated as being involved in mediating the activity of a number of fundamental calcium-regulated intracellular enzyme systems including phosphodiesterases, adenylate cyclase, and a variety of kinases, all of which may regulate or be regulated by intracellular cAMP. Calmodulin levels were thus quantified, and endogenous calmodulin was immunolocalized in developing palatal tissue in vivo and in embryonic palatal mesenchymal cells in vitro. Endogenous palatal calmodulin levels, determined by radioimmunoassay, showed little variation during the period of murine palatal ontogenesis and averaged 0.23 ng/micrograms protein. Murine palate mesenchymal cells in monolayer, either in logarithmic growth or at confluency, contained similar levels of calmodulin. In palate mesenchymal cells in primary culture, specific anti-calmodulin staining was confined to the cell cytoplasm and was concentrated in the perinuclear region. Since immunostaining for calmodulin appeared to be associated with discrete cytoplasmic filaments, distribution of actin and tubulin were investigated. Immunostaining for tubulin in these cells was also localized to the perinuclear region, while immunolocalization of actin showed staining patterns, reflective of stress fibers, which were quite different from those seen for calmodulin. Immunostaining was seen in vivo in all regions of the palatal epithelium with superficial peridermal cells staining most intensely. Specific immunostaining was also evident in palatal mesenchyme, where a pericellular distribution was seen. Staining patterns were similar throughout the period of palatal ontogeny. In addition, a sharply defined localization of calmodulin to cartilagenous extracellular matrix was noted. This study provides a useful initial approach toward understanding the role calmodulin may play in embryonic orofacial development.