Cleft palate is a congenital disorder arising from a failure in the multistep process of palate development. In its mildest form the cleft affects only the posterior soft palate. In more severe cases the cleft includes the soft (

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.

Mammalian palatogenesis depends on interactions between the stomodium-derived epithelium and the cranial neural crest-derived ectomesenchyme. Fibroblast growth factor 10 (FGF10) is a mesenchymal signaling factor that guides the morphogenesis of multiple organs through tissue-tissue interactions. This is consistent with widespread agenesis and dysgenesis of organs observed in Fgf10-/- mice. In this study, we report the presence of a wide-open cleft secondary palate in Fgf10 homozygous null mutant mice. Fgf10 transcripts were detected in the palatal mesenchyme from E11.5 to E13.5 during normal palatogenesis and were enriched in the anterior and middle portions of the palatal shelves. In Fgf10-/- embryos, histological analyses revealed aberrant adhesion of the palatal shelves with the tongue in the anterior and fusion with the mandible in the middle and posterior beginning at E13.5, which could prevent normal elevation of the palatal shelves leading to a cleft palate. TUNEL and BrdU assays demonstrated significant levels of apoptosis in the medial edge epithelium (MEE) but unaltered cell proliferation in mutant palatal shelves. At the molecular level, we show that Fgf10 is epistatic to Jagged2 and Tgfbeta3 in the developing palate. Notably, the expression of Jagged2 is downregulated throughout the palate epithelium in Fgf10 mutants while Tgfbeta3 is misexpressed in the palatal epithelium at the oral side. Our results demonstrate that mesenchymally expressed Fgf10 is necessary for the survival of MEE cells and for the normal expression of Jagged2 and Tgfbeta3 in the palatal epithelium during mammalian palatogenesis.

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 Ϫ/Ϫ humans and Wnt9b Ϫ/Ϫ mutant mice, indicate that Wnt3 and Wnt9b signal through the canonical Wnt signaling pathway to regulate midfacial development and lip fusion.

Summary The basic helix-loop-helix (bHLH) transcription factor Hand2 has been implicated in the development of multiple organs, including craniofacial organs. Mice carrying Hand2 hypomorphic alleles (Hand2LoxP/−) display a cleft palate phenotype. A specific deletion of the Hand2 branchial arch-specific enhancer also leads to a hypoplastic mandible and cleft palate formation in mice. However, the underlying mechanism of Hand2 regulation of palate development remains unknown. Here we show that Hand2 is expressed in both the epithelium and mesenchyme of the developing palate. While mesenchymal specific inactivation of Hand2 has no impact on palate development, epithelial specific deletion of Hand2 creates a cleft palate phenotype. Hand2 appears to exert distinct roles in the anterior and posterior palate. In the anterior palate of Hand2LoxP/− mice, premature death of periderm cells and a down-regulation of Shh are observed in the medial edge epithelium (MEE), accompanied by a decreased level of cell proliferation in the palatal mesenchyme. In the posterior palate, a lower dose of Hand2 causes aberrant periderm cell death on the surface of the epithelium, triggering abnormal fusion between the palatal shelf and mandible and preventing palatal shelf elevation. We further demonstrate that BMP activities are essential for the expression of Hand2 in the palate. We conclude that Hand2 is an intrinsic regulator in the epithelium and is required for palate development.

Bmp4 is a downstream gene of Msx1 in early mouse tooth development. In this study, we introduced the Msx1-Bmp4 transgenic allele to the Msx1 mutants in which tooth development is arrested at the bud stage in an effort of rescuing Msx1 mutant tooth phenotype in vivo. Ectopic expression of a Bmp4 transgene driven by the mouse Msx1promoter in the dental mesenchyme restored the expression of Lef-1 and Dlx2 but neither Fgf3 nor syndecan-1 in the Msx1 mutant molar tooth germ. The mutant phenotype of molar but not incisor could be partially rescued to progress to the cap stage. The Msx1-Bmp4 transgene was also able to rescue the alveolar processes and the neonatal lethality of the Msx1 mutants. In contrast, overexpression of Bmp4 in the wild type molar mesenchyme down-regulated Shh and Bmp2 expression in the enamel knot, the putative signaling center for tooth patterning, but did not produce a tooth phenotype. These results indicate that Bmp4 can bypass Msx1 function to partially rescue molar tooth development in vivo, and to support alveolar process formation. Expression of Shh and Bmp2 in the enamel knot may not represent critical signals for tooth patterning.

Mammalian tooth development depends on activation of odontogenic potential in the presumptive dental mesenchyme by the Msx1 and Pax9 transcription factors. We recently reported that the zinc finger transcription factor Osr2 was expressed in a lingual-to-buccal gradient pattern surrounding the developing mouse molar tooth germs and mice lacking Osr2 developed supernumerary teeth lingual to their molars. We report here generation of a gene-targeted mouse strain that allows conditional inactivation of Pax9 and subsequent activation of expression of Osr2 in the developing tooth mesenchyme from the Pax9 locus. Expression of Osr2 from one copy of the Pax9 locus did not disrupt normal tooth development but was sufficient to suppress supernumerary tooth formation in the Osr2−/− mutant mice. We found that endogenous Osr2 mRNA expression was significantly downregulated in the developing tooth mesenchyme in Pax9del/del mice. Mice lacking both Osr2 and Pax9 exhibited early tooth developmental arrest with significantly reduced Bmp4 and Msx1 mRNA expression in the developing tooth mesenchyme, similar to that in Pax9del/del mutants but in contrast to the rescue of tooth morphogenesis in Msx1−/−Osr2−/− double mutant mice. Furthermore, we found that Osr2 formed stable protein complexes with the Msx1 protein and interacted weakly with the Pax9 protein in co-transfected cells. These data indicate that Osr2 acts downstream of Pax9 and patterns the mesenchymal odontogenic field through protein-protein interactions with Msx1 and Pax9 during early tooth development.

RNA interference (RNAi) has recently become a powerful tool to silence gene expression in mammalian cells, but its application in assessing gene function in mammalian developing organs remains highly limited. Here we describe several unique developmental properties of the mouse molar germ. Embryonic molar mesenchyme, but not the incisor mesenchyme, once dissociated into single cell suspension and re-aggregated, retains its odontogenic potential, the capability of a tissue to instruct an adjacent tissue to initiate tooth formation. Dissociated molar mesenchymal cells, even after being plated in cell culture, retain odontogenic competence, the capability of a tissue to respond to odontogenic signals and to support tooth formation. Most interestingly, while dissociated epithelial and mesenchymal cells of molar tooth germ are mixed and re-aggregated, the epithelial cells are able to sort out from the mesenchymal cells and organize into a well-defined dental epithelial structure, leading to the formation of a well-differentiated tooth organ after sub-renal culture. These unique molar developmental properties allow us to develop a strategy using a lentivirus-mediated RNAi approach to silence gene expression in dental mesenchymal cells and assess gene function in tooth development. We show that knockdown of Msx1 or Dlx2 expression in the dental mesenchyme faithfully recapitulates the tooth phenotype of their targeted mutant mice. Silencing of Barx1 expression in the dental mesenchyme causes an arrest of tooth development at the bud stage, demonstrating a crucial role for Barx1 in tooth formation. Our studies have established a reliable and rapid assay that would permit large-scale analysis of gene function in mammalian tooth development.