Background: Palatal formation is a complex process that requires intricate temporal and spatial coordination of a myriad of developmental, cellular and molecular events in early embryonic stage. One of the critical steps in this process is the fusion between two developing palatal shelves, which is characterized by a series of cellular events; adhesion of the medial edge epithelium (MEE), epithelial seam formation, disappearance of the seam and mesenchymal confluence. It has been shown that transforming growth factor beta 3 (TGFβ3) signaling is essential in palatogenesis, particularly during palatal fusion; however, the mechanism by which TGFβ3 promotes palatal fusion is not fully understood. Interferon regulatory factor 6 (IRF6), whose mutations are linked to Van der Woude syndrome (most common syndromic form of cleft lip and palate), is transiently expressed in the MEE of palatal shelves at a critical time of palatal fusion (E14.5 in mice) immediately following the increased expression of TGFβ3 (E13.5 in mice). Furthermore, IRF6 expression level is significantly reduced in the Tgfβ3^-/- MEE. We hypothesize that IRF6 is a downstream effector of TGFβ3-mediated palatal fusion signaling. To test this hypothesis we determined whether transduction of the MEE with an Ad-IRF6 vector could rescue the palatal fusion defect in Tgfβ3^-/- mouse embryos.
Methods: Our laboratory has recently shown that intra-amniotic delivery of adenoviral vector encoding a transgene allows efficient transduction of the embryonic palatal MEE. We therefore prepared high titer adenoviral stocks co-expressing IRF6 and green fluorescent protein (GFP) (rAd-IRF6-GFP). Viruses were microinjected between E13.5, E14, and E14.5 into the amniotic cavity of pregnant Tgfβ3^+/- female mice time-mated with Tgfβ3^+/- male mice. Adenovirus expressing GFP (rAd-GFP) was used as a control vector. Embryos were harvested at E16, genotyped and assessed for the degree of palatal fusion by fluorescent stereomicroscopy and histology.
Results: Ad-IRF6 vector was introduced to 144 mouse embryos with a 62% survival and 70% infectivity rate. Intra-amniotic injection of Ad-IRF6 at E14 allowed transduction of the MEE and was able to induce partial fusion of the palatal shelves in 42% of Tgfβ3^-/- embryos. The fusion however was not observed when the vector was delivered to Tgfβ3^-/- embryos at E13.5 or E14.5. Furthermore, premature expression of IRF6 in E13.5 wild type and heterozygous MEE led to fusion defect in about 30% of infected embryos. The Ad-GFP control vector did not have any effect on palatal fusion in mouse embryos.
Conclusion: Our in utero gene transfer data support our hypothesis that IRF6 may be a critical downstream effector of TGFβ3 signaling in palatal fusion. It however appears that the IRF6 expression in the MEE must be strictly restricted to a transient period at the time of palatal fusion and premature expression of IRF6 in the MEE may in fact have an adverse effect on palatal fusion.