Prrx1 Regulates ECM Expression During Secondary Palate Formation

Methods: Prrx deficient mice and WT littermates were evaluated for secondary palate formation during fetal development. Three-dimensional (3-D) reconstructions were used to examine the extent of palate dysmorphogenesis in Prrx1 mutant mice. Immunohistochemistry (IHC) was used to determine Prrx1 and versican expression in palates of WT and Prrx deficient embryos. Transient co-transfections, EMSA and ChIP assays were performed to assess whether Prrx transcription factors could bind to and regulate the versican promoter in vitro and in vivo.

Results: The Prrx1 homeobox transcription factor was expressed in the secondary palatal shelves during fetal development and 100% of Prrx1-/- embryos displayed a cleft palate phenotype at birth. 3-D reconstructions revealed that in Prrx1 deficient embryos the secondary palatal shelves had formed but did not elevate by E14.5 as seen in the WT littermates. IHC of serial sections through the secondary palate at both E13.5 and E14.5 in Prrx1 deficient mice revealed a dramatic increase in the levels of versican compared to WT littermates. In vitro (EMSA) and in vivo (ChIP) analysis demonstrated that Prrx1 proteins bind to a phylogenetically conserved cis element of the mouse versican promoter. Transient transfection analysis of WT and mutated versions of the versican promoter combined with Prrx expression constructs, also demonstrated that versican was directly regulated by Prrx homeodomain proteins.

Conclusions: Palate dysmorphogenesis in the Prrx deficient mice may be due to altered hydration of the ECM of palatal shelves due to misregulation of versican. Prrx mutant mice may reveal the genetic circuitry involved in normal secondary palate formation as well as cleft dysmorphogenesis.

This work was supported by the SC COBRE for Oral Health Research NIH/NCRR-P20RR017696 and the MUSC Dental Research Training Grant NIH/NIDCR-T32DE017551