Akira Nakajima1, Yoshihiro Ito2, Eiji Tanaka3, Remi Sano4, Yoko Karasawa4, Masao Maeno5, Koichi Iwata5, Noriyoshi Shimizu6, Charles F Shuler7. 1. Department of Orthodontics, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 1018310, Japan; Dental Research Center, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 1018310, Japan. Electronic address: nakajima.akira@nihon-u.ac.jp. 2. Department of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, BCC-239, La Jolla, CA 92037, USA. 3. Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, Tokushima University Graduate School, 3-18-5 Kuramoto-cho, Tokushima 7708504, Japan. 4. Nihon University Graduate School of Dentistry, Nihon University, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 1018310, Japan. 5. Dental Research Center, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 1018310, Japan. 6. Department of Orthodontics, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 1018310, Japan; Dental Research Center, Nihon University School of Dentistry, 1-8-13 Kanda Surugadai, Chiyoda-ku, Tokyo 1018310, Japan. 7. Department of Oral Biological and Medical Sciences, Faculty of Dentistry, University of British Columbia, 2194 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.
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.
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.
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