Functional role of TGF-β receptors during palatal fusion in vitro

Objective: Reported expression patterns for TGF-beta receptors (T beta R-I, -II, and -III) during palatogenesis suggest that they play essential roles in the mechanisms leading to palatal fusion. The purpose of this study was to compare the functions of the three T beta Rs during palatal fusion. Methods: Using organ culture of mouse palatal shelves, expression levels of T beta R-I, -II, and -III were suppressed by transfecting the siRNAs siT beta R-I, -II, and -III, respectively. Phosphorylation of SMAD2 was examined as an indicator of downstream signalling via each T beta R. Linkage between TGF-beta signalling and critical events in palatal fusion led to the use of MMP-13 expression as an outcome measure for the function of the TGF-beta receptors. Results: The siRNA treatment decreased the expression level of each receptor by more than 85%. When treated with either siT beta R-I or -II, palatal shelves at E13 + 72 h were not fused, with complete clefting in the anterior and posterior regions. The middle palatal region following treatment with either siT beta R-I or -II had fusion from one-half or one-third of the palatal region. Treatment with siT beta R-III resulted in a persistent midline seam of medial edge epithelium (MEE) in the anterior region with islands of persistent MEE in the middle and posterior regions of the midline. Treatment with all three siT beta Rs altered the pattern of SMAD2 phosphorylation. Palatal shelf cultures treated with siT beta R-I or -II, but not -III, showed altered MMP-13 expression levels. Conclusion: The ability to identify and recover MEE and palatal mesenchymal cells during palatal fusion will aid in the evaluation of the different mechanistic events regulated by each T beta R during palatogenesis. (C) 2014 Elsevier Ltd. All rights reserved.