The localization of twist protein in EMT during palatal fusion

Objectives: Epithelial-mesenchymal transformation (EMT) plays an important role in the disappearance of medial edge epithelial cells (MEE) during fusion of secondary palate. This process is controlled by a series of interactions between growth factors and transcription factors. Twist protein, a basic helix-loop-helix (bHLH) transcription factor has been reported to have major roles during mesoderm differentiation, myogenesis and EMT. In some recent studies Twist mRNA was detected in the palatal mesenchyme of normal mice by in situ hybridization. In this study we analyzed the distribution of Twist protein of paraffin embedded normal mouse palates from E13.5-15 by immunofluorescence staining. Methods: The tissues were stained with primary antibodies specific for Twist (Santa Cruz Biotechnology) and Anti-Laminin (Sigma) and detected with appropriate fluorescence conjugated secondary antibodies. Negative controls received only PBS and secondary antibodies. Positive Twist tissue controls were mouse cranial sutures. Sections were viewed and scanned with Leica TCS SP-2 confocal microscope using 40X (n.a. = 1.3) water dipping objective. Results: Twist protein was found in the epithelial layer of MEE and the future nasal and oral epithelium at different stages studied. At E 13.5, only a few Twist labeled cells were found in the epithelium of the open horizontal palatal shelves. Signals were more intense when two palatal shelves approximated and made contact in the mid line. As the fusion proceeded and the epithelial seam broke down, scattered islands of labeled cells were found along the midline, as well as in the oral and nasal epithelium. The MEE and oral epithelium had relatively more labeled cells than the nasal counterpart. After the mesenchyme converged completely, labeled cells were decreased. Positive twist cells were also found in the adjacent mesenchyme. Conclusion: Our observations demonstrate that Twist protein was up regulated in MEE prior to EMT and palatal fusion.