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BACKGROUND: 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. RESULTS: 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 Fgf18(c/c);Wnt1-Cre mutant mice. About 64% of Fgf18(c/c);Wnt1-Cre mice exhibited cleft palate. Whereas palatal shelf elevation was impaired in many Fgf18(c/c);Wnt1-Cre embryos, no significant difference in palatal cell proliferation was detected between Fgf18(c/c);Wnt1-Cre embryos and their control littermates. Embryonic maxillary explants from Fgf18(c/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. CONCLUSION: 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.

Tooth root development occurs through the interaction of multiple growth factors and transcription factors expressed in Hertwig's epithelial root sheath (HERS) and dental mesenchyme. Previously, we demonstrated that bobby sox homolog (Bbx) regulates odontoblast differentiation of human dental pulp stem cells. Here, we generated Bbx knockout (Bbx-/- ) mice to address the functional role of Bbx in tooth formation. During tooth development, Bbx was expressed in both dental epithelium and mesenchyme. However, molar and incisor morphology in Bbx-/- mice at postnatal Day 0 (P0) exhibited no prominent abnormalities compared with their wild-type (Bbx+/+ ) littermates. Until P28, the crown morphology in Bbx-/- mice was not distinctively different from Bbx+/+ littermates. Meanwhile, the length of the mandibular base in Bbx-/- mice was notably less at P28. Compared with Bbx+/+ mice, the mesial and distal root lengths of the first molar were reduced by 21.33% and 16.28% at P14 and 16.28% and 16.24% at P28, respectively, in Bbx-/- mice. The second molar of Bbx-/- mice also showed 10.16% and 6.4% reductions at P28 in the mesial and distal lengths, compared with Bbx+/+ mice, respectively. The gene expression analysis during early tooth root formation (P13) showed that the expression of dentin sialophosphoprotein (Dspp) was significantly decreased in Bbx-/- mice. Collectively, our data suggest that Bbx participates in tooth root formation and might be associated with the regulation of Dspp expression.

Tendons are fibrous connective tissues that transmit force between muscle and bone. Whereas the molecular and cellular mechanisms of bone and muscle development have been well studied, that of tendon development is poorly understood. Using the Scx-GFP transgenic mice, we isolated GFP(+) cells from the developing mouse limbs at E11.5, E13.5, and E15.5, respectively, and carried out whole transcriptome RNA-seq analysis. Comparing the gene expression profiles of GFP(+) and GFP(-) cells in the E13.5 limb isolated over 1,500 genes that exhibited enrichment of mRNA expression by at least 1.5-fold in the GFP(+) cells. Of these, 778 genes showed expression up-regulated by more than 1.5-fold from E11.5 to E13.5 and 516 genes showed expression up-regulated by more than 1.5-fold from E13.5 to E15.5 in the GFP(+) cell population. Interestingly, over 30 genes encoding transcription factors are among the early-activated genes in the GFP(+) cells. Whole mount and section in situ hybridization analyses showed that many of these transcription factor genes have distinct patterns of expression during limb development and identified Foxf2 expression as a specific marker for differentiated dorsal limb tendon cells. Together, these data provide a valuable resource for further investigation of the molecular mechanisms regulating tendon development.

Craniosynostosis, the premature fusion of one or more sutures between the calvarial bones, is a common birth defect. Mutations in genes encoding receptors for the transforming growth factor-beta (TGFβ) family of signaling molecules have been associated with craniosynostosis, but how TGFβ signaling is regulated during suture development is not known. In the present study, we found that expression of Smad2 and Smad3, intracellular mediators of canonical TGFβ signaling, gradually increases during early postnatal suture development in rat in both the coronal suture (CS), which remains patent throughout life, and the posterior frontal suture (PFS), which undergoes programmed closure by postnatal day 22. The amounts of phosphorylated Smad2 and Smad3 proteins showed a similar gradual increase in the PFS and CS, but in the CS, Smad2/3 activation was suppressed after neonatal day 10. The suppression of Smad2/3 activation in the CS correlated with upregulation of Smad7 expression. We demonstrate that siRNA-mediated knockdown of Smad7 caused increased phosphorylation of Smad2 and Smad3 and induced osseous obliteration of the CS from postnatal days 10 to 22. The Smad7 siRNA-induced CS closure was associated with significantly increased levels of Fgf10 and phosphorylated ERK1/2 in the suture mesenchyme. Moreover, addition of the Erk1/2 inhibitor U0126 partially blocked Smad7-siRNA-induced CS closure. These findings suggest that canonical TGFβ signaling induces suture closure at least in part through activation of FGF and ERK signaling and that Smad7 plays an important role in maintaining suture patency by suppressing canonical TGFβ signaling during suture development.

Background and objective Wingless-type MMTV integration site family (Wnt)/β-catenin signaling plays an essential role in cellular differentiation and matrix formation during skeletal development. However, little is known about its role in tooth-root formation. In a previous study, we found excessive formation of dentin and cementum in mice with constitutive β-catenin stabilization in the dental mesenchyme. In the present study we analyzed the molar roots of these mice to investigate the role of Wnt/β-catenin signaling in root formation in more detail. Material and methods We generated OC-Cre:Catnb(+/lox(ex3)) mice by intercrossing Catnb(+/lox(ex3)) and OC-Cre mice, and we analyzed their mandibular molars using radiography, histomorphometry and immunohistochemistry. Results OC-Cre:Catnb(+/lox(ex3)) mice showed impaired root formation. At the beginning of root formation in mutant molars, dental papilla cells did not show normal differentiation into odontoblasts; rather, they were prematurely differentiated and had a disorganized arrangement. Interestingly, SMAD family member 4 was upregulated in premature odontoblasts. In 4-wk-old mutant mice, molar roots were about half the length of those in their wild-type littermates. In contrast to excessively formed dentin in crown, root dentin was thin and hypomineralized in mutant mice. Biglycan and dentin sialophosphoprotein were downregulated in root dentin of mutant mice, whereas dentin matrix protein 1 and Dickkopf-related protein 1 were upregulated. Additionally, ectonucleotide pyrophosphatase/phosphodiesterase 1 was significantly downregulated in the cementoblasts of mutant molars. Finally, in the cementum of mutant mice, bone sialoprotein was downregulated but Dickkopf-related protein 2 was upregulated. Conclusion These results suggest that temporospatial regulation of Wnt/β-catenin signaling plays an important role in cell differentiation and matrix formation during root and cementum formation.

Axin1 is a critical negative regulator of the canonical Wnt-signaling pathway. It is a concentration-limiting factor in the β-catenin degradation complex. Axin1 null mutant mouse embryos died at embryonic day 9.5, precluding direct genetic analysis of the roles of Axin1 in many developmental and physiological processes using these mutant mice. In this study, we have generated mice carrying two directly repeated loxP sites flanking the exon 2 region of the Axin1 gene. We show that floxed-allele-carrying mice (Axin1( fx/fx) ) mice appear normal and fertile. Upon crossing the Axin1( fx/fx) mice to the CMV-Cre transgenic mice, the loxP-flanked exon 2 region that encodes the N-terminus and the conserved regulation of G-protein signaling domain was efficiently deleted by Cre-mediated excision in vivo. Moreover, we show that mouse embryos homozygous for the Cre/loxP-mediated deletion of exon 2 of the Axin1 gene display embryonic lethality and developmental defects similar to those reported for Axin1(-/-) mice. Thus, this Axin1(fx/fx) mouse model will be valuable for systematic tissue-specific dissection of the roles of Axin1 in embryonic and postnatal development and diseases.

Axin1 is a critical negative regulator of the canonical Wnt-signaling pathway. It is a concentration-limiting factor in the β-catenin degradation complex. Axin1 null mutant mouse embryos died at embryonic day 9.5, precluding direct genetic analysis of the roles of Axin1 in many developmental and physiological processes using these mutant mice. In this study, we have generated mice carrying two directly repeated loxP sites flanking the exon 2 region of the Axin1 gene. We show that floxed-allele-carrying mice (Axin1( fx/fx) ) mice appear normal and fertile. Upon crossing the Axin1( fx/fx) mice to the CMV-Cre transgenic mice, the loxP-flanked exon 2 region that encodes the N-terminus and the conserved regulation of G-protein signaling domain was efficiently deleted by Cre-mediated excision in vivo. Moreover, we show that mouse embryos homozygous for the Cre/loxP-mediated deletion of exon 2 of the Axin1 gene display embryonic lethality and developmental defects similar to those reported for Axin1(-/-) mice. Thus, this Axin1(fx/fx) mouse model will be valuable for systematic tissue-specific dissection of the roles of Axin1 in embryonic and postnatal development and diseases.

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 Osr2IresCre knockin allele activated β-galactosidase expression specifically throughout the developing palatal mesenchyme from the onset of palatal shelf outgrowth. In addition, the Osr2IresCre 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 Osr2IresCre knockin mice provide a unique tool for tissue-specific studies of the molecular mechanisms regulating palate and kidney development. genesis 45:618–624, 2007. © 2007 Wiley-Liss, Inc.

The vertebrate upper lip forms from initially freely projecting maxillary, medial nasal, and lateral nasal prominences at the rostral and lateral boundaries of the primitive oral cavity. These facial prominences arise during early embryogenesis from ventrally migrating neural crest cells in combination with the head ectoderm and mesoderm and undergo directed growth and expansion around the nasal pits to actively fuse with each other. Initial fusion is between lateral and medial nasal processes and is followed by fusion between maxillary and medial nasal processes. Fusion between these prominences involves active epithelial filopodial and adhering interactions as well as programmed cell death. Slight defects in growth and patterning of the facial mesenchyme or epithelial fusion result in cleft lip with or without cleft palate, the most common and disfiguring craniofacial birth defect. Recent studies of craniofacial development in animal models have identified components of several major signaling pathways, including Bmp, Fgf, Shh, and Wnt signaling, that are critical for proper midfacial morphogenesis and/or lip fusion. There is also accumulating evidence that these signaling pathways cross-regulate genetically as well as crosstalk intracellularly to control cell proliferation and tissue patterning. This review will summarize the current understanding of the basic morphogenetic processes and molecular mechanisms underlying upper lip development and discuss the complex interactions of the various signaling pathways and challenges for understanding cleft lip pathogenesis.

During mammalian palatogenesis, palatal shelves initially grow vertically from the medial sides of the paired maxillary processes flanking the developing tongue and subsequently elevate and fuse with each other above the tongue to form the intact secondary palate. Pathological palate-mandible or palate-tongue fusions have been reported in humans and other mammals, but the molecular and cellular mechanisms that prevent such aberrant adhesions during normal palate development are unknown. We previously reported that mice deficient in Jag2, which encodes a cell surface ligand for the Notch family receptors, have cleft palate associated with palate-tongue fusions. In this report, we show that Jag2 is expressed throughout the oral epithelium and is required for Notch1 activation during oral epithelial differentiation. We show that Notch1 is normally highly activated in the differentiating oral periderm cells covering the developing tongue and the lateral oral surfaces of the mandibular and maxillary processes during palate development. Oral periderm activation of Notch1 is significantly attenuated during palate development in the Jag2 mutants. Further molecular and ultrastructural analyses indicate that oral epithelial organization and periderm differentiation are disrupted in the Jag2 mutants. Moreover, we show that the Jag2 mutant tongue fused to wild-type palatal shelves in recombinant explant cultures. These data indicate that Jag2-Notch1 signaling is spatiotemporally regulated in the oral epithelia during palate development to prevent premature palatal shelf adhesion to other oral tissues and to facilitate normal adhesion between the elevated palatal shelves.

Development of the lip and palate involves a complex series of events that are frequently disturbed resulting in the congenital anomalies cleft lip and cleft palate. Van der Woude syndrome (VWS) is an autosomal dominant disorder that is characterised by cleft lip, cleft palate, lower lip pits, and hypodontia. VWS arises as the result of mutations in the gene encoding interferon regulatory factor 6 (IRF6). To provide insights into the role of IRF6 during embryogenesis, we have analysed the expression of this molecule during mouse and chick facial development. Irf6 was expressed in the ectoderm covering the facial processes during their fusion to form the upper lip and primary palate in both mouse and chick. However, while Irf6 was expressed in the medial edge epithelia of the developing secondary palate of the mouse, which fuses as in man, Irf6 was not expressed in the medial edge epithelia of the naturally cleft chick secondary palate. Similarly, Irf6 was found to be down-regulated in the medial edge epithelia of transforming growth factor beta3-null mice, which also exhibit cleft palate. Together, these results support a role for IRF6 during the fusion events that occur during development of the lip and palate. Developmental Dynamics 235:1441–1447, 2006. © 2005 Wiley-Liss, Inc.

Cleft lip with or without cleft palate (CLP) is the most common craniofacial birth defect in humans. Recently, mutations in the WNT3 and Wnt9b genes, encoding two members of the Wnt family of signaling molecules, were found associated with CLP in human and mice, respectively. To investigate whether Wnt3 and Wnt9b directly regulate facial development, we analyzed their developmental expression patterns and found that both Wnt3 and Wnt9b are expressed in the facial ectoderm at critical stages of midfacial morphogenesis during mouse embryogenesis. Whereas Wnt3 mRNA is mainly expressed in the maxillary and medial nasal ectoderm, Wnt9b mRNA is expressed in maxillary, medial nasal, and lateral nasal ectoderm. During lip fusion, Wnt9b, but not Wnt3, is expressed in the epithelial seam between the fusing medial and lateral nasal processes. Furthermore, we found that expression of TOPGAL, a transgenic reporter of activation of canonical Wnt signaling pathway, is specifically activated in the distal regions of the medial nasal, lateral nasal, and maxillary processes prior to lip fusion. During lip fusion, the epithelial seam between the medial and lateral nasal processes as well as the facial mesenchyme directly beneath the fusing epithelia strongly expresses TOPGAL. These data, together with the CLP lip phenotype in WNT3-/- humans and Wnt9b-/- mutant mice, indicate that Wnt3 and Wnt9b signal through the canonical Wnt signaling pathway to regulate midfacial development and lip fusion.

Mutations in the TBX22 gene have been identified recently in patients with the X-linked cleft palate and ankyloglossia syndrome, suggesting that the TBX22 transcription factor plays an important role in palate development. However, because ankyloglossia has been reported in the majority of patients with TBX22 mutations, it has been speculated that the cleft palate phenotype is secondary to defective fetal tongue movement. To understand the role of TBX22 in disease pathogenesis and in normal development, it is necessary to carry out a detailed temporal and spatial gene expression analysis. We report here the isolation and developmental expression analysis of the mouse homolog Tbx22. The mouse Tbx22 gene encodes a putative protein of 517 amino acid residues, which shares 72% overall amino acid sequence identity with the human TBX22 protein. By using interspecific backcross analysis, we have localized the Tbx22 gene to mouse chromosome X, in a region syntenic to human chromosome Xq21, where the TBX22 gene resides, indicating that Tbx22 is the ortholog of human TBX22. Our in situ hybridization analysis shows that Tbx22 is expressed in a temporally and spatially highly restricted pattern during mouse palate and tongue development. Together with the mutant phenotypes in human patients, our data indicate a primary role for Tbx22 in both palate and tongue development. © 2002 Wiley-Liss, Inc.