In Utero Transduction of Tgfβ3-/- Mouse Palatal Epithelium with IRF6 Induces Disappearance of Peridermal Cells and Rescues Palatal Fusion Defect
Chadwick Wu, MD1, Byung Ho Yang, PhD2, Imad Salhab, MS2, Richard E. Kirschner, MD2, Hyun D. Nah, PhD2.
1Hospital of the University of Pennsylvania, Philadelphia, PA, USA, 2The Children's Hospital of Philadelphia, Philadelphia, PA, USA.
PURPOSE: One of the critical steps in palatogenesis is the fusion between two developing palatal shelves. Prior to palatal fusion at embryonic day 14.5 (E14.5) in mice, peridermal cells along the surface of medial edge epithelium (MEE) undergo apoptosis. Exposed MEE cells form filopodia on their surface, which have been suggested to promote adhesion between the opposing palatal shelves and palatal fusion occurs thereafter. Transforming growth factor beta 3 (TGFβ3) is essential in palatal fusion; however, the mechanism by which TGFβ3 promotes palatal fusion is not fully understood. Interferon regulatory factor 6 (IRF6), mutations of which are linked to Van der Woude syndrome, is transiently expressed in the MEE of the palatal shelves at a critical time of palatal fusion (E14.5 in mice), immediately following the increased expression of TGFβ3 (E13.5 in mice). The purpose of the experiment was to determine whether IRF6 is a downstream effector to TGFβ3 and its potential role in palatogenesis.
METHODS: Adenovirus co-expressing IRF6 and green fluorescent protein (GFP) (rAd-IRF6-GFP) was microinjected at E13.5, 14, or 14.5 into the amniotic cavity of fetuses from pregnant time-mated Tgfβ3+/- female mice. Adenovirus expressing GFP (rAd-GFP) was used as a control vector. Embryos were harvested at E16.5, genotyped and assessed for the degree of palatal fusion by fluorescent stereomicroscopy, histology, and scanning electron microscopy (SEM).
RESULTS: Ad-IRF6 vector was introduced to 207 mouse embryos with a 62% survival and 73% infectivity rate. Intra-amniotic injection of Ad-IRF6 at E14 induced partial fusion of the palatal shelves in 38% of Tgfβ3-/- embryos. Palatal shelf fusion was not observed, however, when the vector was delivered to Tgfβ3-/- embryos at E13.5 or E14.5. Furthermore, over-expression of IRF6 in E13.5 wild type and heterozygous mice led to palatal fusion defect in about 20% of embryos. The Ad-GFP control vector did not have any effect on palatal fusion in mouse embryos. Based on SEM, Tgfβ3-/- palates transduced with IRF6 had disappearance of peridermal cells along the palatal edge with exposure of underlying basal MEE cells with filopodia. In contrast, Tgfβ3-/- palates transduced with GFP control vector had intact peridermal cells.
CONCLUSION: These in utero gene transfer data support the hypothesis that IRF6 may be an important downstream effector of TGFβ3 signaling in palatal fusion. It appears that transient upregulation of IRF6 in the MEE must be strictly restricted to the precise time of palatal fusion and that premature expression of IRF6 in the MEE may in fact have an adverse effect on palatal fusion. Based on SEM and histology, IRF6 induces the disappearance of peridermal cells, allowing exposure of the underlying basal MEE cells for palatal adhesion.