An Examination of TGF-b3 and Apoptotic Mechanisms in Palate Fusion

Objectives: Various theories have been proposed to explain the disappearance of medial edge epithelium during palate fusion. Although epithelial-mesenchymal transformation has been documented, programmed cell death may also play a role in palate fusion. The goal of this study was to examine the expression of different apoptotic factors during fusion of the palatal shelves in vitro. Methods: Eight day old chicken embryos (developmental stages 32-34) were removed, decapitated, and the palatal shelves dissected and placed into organ culture along with TGF-b3 for 48, 72, or 96 hours to achieve palatal fusion. Individual palatal shelves were also cultured separately to serve as unfused controls or in the presence of inhibitors to either the PI-3 kinase or p38 MAP kinase pathways (n=12/group). Tissues were then collected, RNA extracted, and RT-PCR performed to examine changes in the expression of the anti-apoptotic factors bcl-2 and bcl-xl and the pro-apoptotic factors BAX and caspase-3. The RT-PCR band densities were quantified from three separate experiments of four samples each. Results: Densitometric analysis of the RT-PCR results indicated that a significant increase occurred in the mRNA levels of BAX and caspase-3 (p£0.01) in palates cultured with TGF-b3 in comparison to control unfused palatal tissues at all time periods investigated. However, Bax and caspase-3 levels were significantly decreased (p£0.05) after treatment with either inhibitor in culture after 72 and 96 hours. However, there were no significant differences in the expression of bcl-2 and bcl-xl between fused and unfused palatal tissues at any time period or between any of the treatment groups. Conclusions: This study demonstrates that the expression of some pro-apoptotic factors is significantly increased during fusion of the palate in vitro, which can be affected by inhibitors to the TGF-b3 pathway. This study was supported by NIH/NIDCR Traineeship T35 DE07188 and the Baylor Oral Health Foundation.