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Inhibition of Twist expression blocks palatal fusion
Author(s) -
Yu Wenli,
Svoboda Kathy
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a233-e
Subject(s) - lipofectamine , transfection , fusion protein , microbiology and biotechnology , chemistry , downregulation and upregulation , biology , biochemistry , recombinant dna , gene , vector (molecular biology)
Epithelial‐mesenchymal transformation (EMT) plays an important role in the disappearance of medial edge epithelial cells (MEE) during fusion of secondary palate. TGF beta3 regulates the disappearance of MEE during this process. Twist protein has a major role during EMT and our previous studies demonstrated that twist protein was upregulated just prior to palatal fusion. In this study, we analyzed Twist function in palatal fusion by inhibiting its expression with Twist siRNA in vitro. The palatal shelves from E13.5 CD1 mice were dissected and placed on filter paper with the medial edges in contact. They were cultured in BGJb medium with 100, 200nM siRNA in 0.004% of transfection reagent, Lipofectamine. TGF beta3 was added (10ng/ml) in some groups. Transfection time was 24hr and medium was changed to control conditions. Tissues were harvested at 48hr and 72hr. Morphology was examined by H&E staining and immuno‐histology. Down‐regulation of Twist protein was demonstrated using western blots. Palate fusion was inhibited when the tissue was transfected with Twist specific siRNA. In the presence of 100nM Twist siRNA, there were some MEE islands in the midline. In the 200nM Twist siRNA group, the MEE remained intact. When TGF beta3 was added with the Twist siRNA, the MEE remained in the midline after 72hrs. In conclusion, Twist was necessary for basement membrane breakdown and disappearance of MEE cells during palatal fusion. Additional TGF beta3 did not rescue palatal fusion when Twist was down regulated.