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

Abstract

Objective: Reported expression patterns for TGF-β receptors (TβR-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 TβRs during palatal fusion.

Methods: Using organ culture of mouse palatal shelves, expression levels of TβR-I, -II, and -III were suppressed by transfecting the siRNAs siTβR-I, -II, and -III, respectively. Phosphorylation of SMAD2 was examined as an indicator of downstream signalling via each TβR. Linkage between TGF-β 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-β receptors.

Results: The siRNA treatment decreased the expression level of each receptor by more than 85%. When treated with either siTβR-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 siTβR-I or -II had fusion from one-half or one-third of the palatal region. Treatment with siTβR-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 siTβRs altered the pattern of SMAD2 phosphorylation. Palatal shelf cultures treated with siTβR-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 TβR during palatogenesis.

Keywords: MMP-13 expression; Palatal development; SMAD2 phosphorylation; TGF-β receptor; TGF-β signalling; siRNA.