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 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 and , proven antagonists of Wnt signaling. The functional interactions between Pax9 and Dkk1 are shown by the genetic rescue of secondary palate clefts in embryos. The controlled intravenous delivery of small-molecule Wnt agonists (Dkk inhibitors) into pregnant mice restored Wnt signaling and led to the growth and fusion of palatal shelves, as marked by an increase in cell proliferation and osteogenesis, 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 development of dentition is a fascinating process that encompasses a complex series of epithelial-mesenchymal interactions involving growth factors, transcription factors, signal receptors and other soluble morphogens. It is not surprising that such a complex process is prone to disturbances and may result in tooth agenesis. Initial discoveries indicating that the homeo-domain protein MSX1 and the paired-domain transcription factor PAX9 are causative genes in tooth morphogenesis were made in mice. Both genes are co-expressed in dental mesenchyme and either one, when homozygously deleted, results in an arrest at an early developmental stage. Heterozygous Pax9 or Msx1 mice have normal teeth, however, double heterozygous Pax9/Msx1 mice show a phenotype of arrested tooth development which can be rescued by transgenic expression of Bmp4, a very influential signaling factor in many developmental processes. We have obtained mounting evidence for a partnership between PAX9 and MSX1 within the tooth-specific Bmp4 signaling pathway. In humans, unlike in mice, a heterozygous mutation in either PAX9 or MSX1 suffices to cause tooth agenesis of a predominantly molar or more premolar pattern, respectively. Our laboratory and others have identified several PAX9 and MSX1 mutations in families with non-syndromic forms of autosomal dominant posterior tooth agenesis. We have also identified families with tooth agenesis in whom PAX9 and MSX1 mutations have been excluded opening up the possibilities for the discovery of other genes that contribute to human tooth agenesis.

Mutations of the Ectodysplasin-A (EDA) gene are generally associated with the syndrome Hypohidrotic Ectodermal Dysplasia (HED, MIM 305100) but they can also manifest as selective, non-syndromic tooth agenesis (MIM300606). We have performed an in vitro functional analysis of six selective tooth agenesis causing EDA mutations (one novel and five known) which are located in the C-terminal tumor necrosis factor (TNF) homology domain of the protein. Our study reveals that expression, receptor binding or signaling capability of the mutant EDA1 proteins is only impaired in contrast to syndrome-causing mutations, which we have previously shown to abolish EDA1 expression, receptor binding or signaling. Our results support a model in which the development of the human dentition, especially of anterior teeth requires the highest level of EDA-receptor signaling while other ectodermal appendages, including posterior teeth have less stringent requirements and form normally in response to EDA mutations with reduced activity.

The development of dentition is a fascinating process that encompasses a complex series of epithelial-mesenchymal interactions involving growth factors, transcription factors, signal receptors and other soluble morphogens. It is not surprising that such a complex process is prone to disturbances and may result in tooth agenesis. Initial discoveries indicating that the homeo-domain protein MSX1 and the paired-domain transcription factor PAX9 are causative genes in tooth morphogenesis were made in mice. Both genes are co-expressed in dental mesenchyme and either one, when homozygously deleted, results in an arrest at an early developmental stage. Previous studies have shown a down regulation of Bmp4 gene expression in Pax9 and Msx1 single mutant mice. Therefore, we chose to explore the molecular relationship between Pax9, Msx1 and Bmp4. In humans, unlike in mice, a heterozygous mutation in either PAX9 or MSX1 suffices to cause tooth agenesis of a predominantly molar or more premolar pattern, respectively. Our laboratory and others have identified several PAX9 and MSX1 mutations in families with non-syndromic forms of autosomal dominant posterior tooth agenesis. We have also identified families with tooth agenesis in whom PAX9 and MSX1 mutations have been excluded opening up the possibilities for the discovery of other genes that contribute to human tooth agenesis.

Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the 18 mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants, the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wild-type PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.