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2009 Annual Meeting Abstracts


Discovery of a New Molecular Pathway in Palatogenesis: IRF6 is an Important Downstream Effector of TGF-beta3 Signaling in Palatal Fusion
Gregory E. Lakin, M.D.1, Philip W. Zoltick, M.D.2, Allison Zajac, B.S.3, Richard E. Kirschner, M.D.1, Hyun-Duck Nah, D.M.D., Ph.D.1.
1Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia and The University of Pennsylvania School of Medicine, Philadelphia, PA, USA, 2The Children's Center for Fetal Research, The Children's Hospital of Philadelphia, Philadelphia, PA, USA, 3Division of Plastic and Reconstructive Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.

PURPOSE:
Transforming growth factor beta 3 (TGF-beta3) is known to be a critical regulator of palatogenesis, however the downstream mechanisms of TGF-beta3 signaling in palatal development are not fully understood. Evidence suggests that TGF-beta3 induces Interferon Regulatory Factor 6 (IRF6) gene expression in the medial edge epithelium (MEE) of fusing palatal shelves. IRF6 gene mutations are known to cause Van der Woude syndrome, the most common syndromic form of cleft palate. We hypothesized that IRF6 is an important downstream effector of TGF-beta3 signaling in palatogenesis. To test this hypothesis we determined whether adenoviral delivery of IRF6 to the MEE can rescue the fusion defect of the TGF-beta3-/- mouse palate in organ culture.
METHODS:
TGF-beta3+/- mice were time-mated to produce a Mendelian distribution of offspring. 198 embryos were harvested from 32 litters between 13.5-14 days of gestation. Their palatal shelves were micro-dissected and tails were saved for genotyping. Palatal shelves were placed together in organ culture and used as uninfected controls or were infected with one of the following human adenovirus serotype 5 vectors: a negative control vector encoding GFP (rAd-GFP) to trace viral infection, a positive control vector encoding murine TGF-beta3 (rAd-TGF-beta3), or an experimental vector encoding murine IRF6 (rAd-IRF6). Both TGF-beta3 and IRF6 vectors were engineered to also express GFP. Only palates with WT or KO genotypes were further analyzed. GFP expression was examined by fluorescent stereomicroscopy after 24, 48, and 72 hours in organ culture. Specimens were prepared for histologic examination of palatal shelf fusion by H/E staining. MEE disappearance was confirmed by immunofluorescent staining for an epithelial cell surface marker, E-cadherin. GFP expression detected by immunofluorescent staining determined the extent of transduction.
RESULTS:
By fluorescent stereomicroscopy, high levels of GFP expression were detected along the palatal midline 24 hours after vector administration. Three days after the addition of rAd-TGF-beta3, GFP expression decreased and the palates fused. GFP expression persisted in negative controls in which there was no fusion. There was patchy disappearance of GFP expression in palates infected with the experimental rAd-IRF6. Examination of H/E stained and E-cadherin immunostained sections demonstrated that in all uninfected WT specimens (n=6) palatal fusion was complete. In all KO specimens infected with rAd-GFP (n=4) or sham infected (n=1), palates were unfused. In all KO specimens transduced with rAd-TGF-beta3 (n=3), palatal fusion was complete. In the experimental rAd-IRF6 infected KO organ cultures, palatal fusion varied from unfused (n=1) to MEE adhesion (n=4) to fusion with focal MEE disappearance (n=3). GFP immunostaining confirmed palatal MEE cells were tranduced with adenoviral vectors.
CONCLUSION:
Adenoviral mediated transduction of IRF6 to the MEE induces palatal fusion in the TGF-beta3-/- mouse organ cultures, indicating that IRF6 is an important downstream effector of TGF-beta3 signaling in palatogenesis. The varying degree of fusion induced by IRF6 suggests that there may be additional downstream effectors for TGF-beta3 or may be related to the transduction rate of the MEE. Our results contribute to the current understanding of the molecular regulation of palatogenesis and potential therapeutic targets in palatal development.


 

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