The paired-related homeobox genes, Prx1 and Prx2, encode transcription factors critical for orofacial development. Prx1 -/-/Prx2 -/-neonates have mandibular hypoplasia and malformed mandibular incisors. Although the mandibular incisor phenotype has been briefly described (ten Berge et al., 1998(ten Berge et al., , 2001Lu et al., 1999), very little is known about the role of Prx proteins during tooth morphogenesis. Since the posterior mandibular region was relatively normal, we examined molar tooth development in Prx1 -/-/Prx2 -/-embryos to determine whether the tooth malformation is primary to the loss of Prx protein or secondary to defects in surrounding tissues. Three-dimensional (3D) morphological reconstructions demonstrated that Prx1 -/-/Prx2 -/-embryos had molar malformations, including cuspal changes and ectopic epithelial projections. Although we demonstrate that Prx1 protein is expressed only mesenchymally, 3D reconstructions showed important morphological defects in epithelial tissues at the cap and bell stages. Analysis of these data suggests that the Prx homeoproteins are critical for mesenchymal-epithelial signaling during tooth morphogenesis.
The paired-related homeobox genes, Prx1 and Prx2, are important for normal skeletal and cardiovascular development as well as adult vascular remodeling. The identification and characterization of Prx downstream targets is crucial to understanding their function in normal developmental processes and congenital malformations. To identify Prx2 regulated genes, stably transfected NIH3T3 clones expressing Prx2 sense or antisense transcripts were generated. Expression profiles initially were established for two of the clones using Affymetrix GeneChip arrays. Over 6,400 genes were screened by the microarray approach, and approximately 500 genes differed in expression by a factor of two or more. Fifteen genes were chosen for further analysis. RT-PCR of the two transfectants used in the GeneChip analysis demonstrated that five out of the 15 genes were differentially expressed. However, after screening additional stable transfectant clones only one of the 15 genes, Protease Nexin-1 (PN-1), was differentially expressed. Subsequent Northern blot, RT-PCR, and further GeneChip analysis of additional stable transfectants confirmed that PN-1 expression is increased at least fivefold when Prx2 is overexpressed. It was demonstrated that Prx2 directly regulates PN-1 because (1) Prx2 binds to a cis element in the PN-1 promoter in vitro, and (2) Prx2 regulates the PN-1 promoter in transient transfection assays. The GeneChip analysis generated a prioritized list of other potential targets. The utility and limitations of cell culture models combined with microarray analysis for elucidating complex regulatory cascades are discussed.
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