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.

Hand2 is required in the epithelium for palatogenesis in mice.

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 (Hand2(LoxP/-)) 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 Hand2(LoxP/-) 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.

Mice with an anterior cleft of the palate survive neonatal lethality.

Many genes are known to function in a region-specific manner in the developing secondary palate. We have previously shown that Shox2-deficient embryos die at mid-gestation stage and develop an anterior clefting phenotype. Here, we show that mice carrying a conditional inactivation of Shox2 in the palatal mesenchyme survive the embryonic and neonatal lethality, but develop a wasting syndrome. Phenotypic analyses indicate a delayed closure of the secondary palate at the anterior end, leading to a failed fusion of the primary and secondary palates. Consistent with a role proposed for Shox2 in skeletogenesis, Shox2 inactivation causes a significantly reduced bone formation in the hard palate, probably due to a down-regulation of Runx2 and Osterix. We conclude that the secondary palatal shelves are capable of fusion with each other, but fail to fuse with the primary palate in a developmentally delayed manner. Mice carrying an anterior cleft can survive neonatal lethality.

Wnt5a regulates directional cell migration and cell proliferation via Ror2-mediated noncanonical pathway in mammalian palate development.

Tissue and molecular heterogeneities are present in the developing secondary palate along the anteroposterior (AP) axis in mice. Here, we show that Wnt5a and its receptor Ror2 are expressed in a graded manner along the AP axis of the palate. Wnt5a deficiency leads to a complete cleft of the secondary palate, which exhibits distinct phenotypic alterations at histological, cellular and molecular levels in the anterior and posterior regions of the palate. We demonstrate that there is directional cell migration within the developing palate. In the absence of Wnt5a, this directional cell migration does not occur. Genetic studies and in vitro organ culture assays further demonstrate a role for Ror2 in mediating Wnt5a signaling in the regulation of cell proliferation and migration during palate development. Our results reveal distinct regulatory roles for Wnt5a in gene expression and cell proliferation along the AP axis of the developing palate, and an essential role for Wnt5a in the regulation of directional cell migration.

Application of lentivirus-mediated RNAi in studying gene function in mammalian 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.