Genome wide analysis of Msx1- and Pax9-dependent gene expression profiles during murine tooth development

Objectives: Mutations in PAX9 and MSX1 have been associated with human tooth agenesis. Pax9 and Msx1 mutant mice also have tooth developmental arrest at the bud stage, indicating that they are critical for tooth development, and provide good models for evaluating therapeutic studies. Previous work in our lab showed that Pax9 and Msx1 interact synergistically to regulate the expression of Bmp4, but the mechanisms underlying the tooth developmental arrest in the mutant mice are incompletely understood. The purpose of this study is to further explore target genes and molecular networks regulated by Pax9 and Msx1 in order to identify potential candidates for novel therapies.
Methods: Developing tooth germs at E13.5, E14.5 were micro-dissected from Pax9^-/- and Msx1^-/- mutant embryos as well as their control littermates. After genotyping, total RNAs were extracted and microarray analyses were performed to identify differentially expressed genes by comparing the global gene expression profiles between Pax9 or Msx1 mutant vs. their control littermates.
Results: Edar, Shh, Fgf4 and Lef1 were down-regulated in both Pax9^-/- and Msx1^-/- tooth germs while Wnt signaling inhibitors, such as Dkk1, Dkk2, Sfrp1 and Sfrp2, were up-regulated. Real-time PCR was performed to verify the regulated expression of selected target genes, including Dkk, Sfrp, Lef1, Foxf1a, Tcfap2b, Msx1, Inhba, and Spock1. In-situ hybridization experiments confirmed the expression patterns of some candidate molecules. These studies reveal that the Wnt signaling inhibitors were upregulated in Pax9 and Msx1 mutant tooth germs while Msx1 and Inhba were significantly down-regulated in Pax9 mutant tooth organs.
Conclusions: The high throughput microarray analyses provide many candidate target genes regulated by Pax9 and Msx1. Gene functional annotation analysis revealed that genes involved in Bmp, Wnt, and Eda signaling pathways showed differential patterns of expression in control and mutant tooth organs. Hence, Pax9 and Msx1-dependent signaling pathways are potential candidates for therapeutic targets to treat craniofacial developmental defects caused by Pax9 and Msx1 mutations.