Mitsuko Moriguchi et al.: Axin2 and p-Smad3 in Molar Germ Journal of Hard Tissue Biology 21[2] (2012) p113 - 120 © 2012 The Hard Tissue Biology Network Association Printed in Japan, All rights reserved. CODEN-JHTBFF, ISSN 1341-7649 Original Immunolocalization of Axin2 and p-Smad3 in Developing Rat Molar Germ Mitsuko Moriguchi1), Marie Yamada2), Yasuo Miake1) and Mayu Koshika1) 1) Department of Ultrastructural Science, Tokyo Dental College, Chiba, Japan 2) Department of Physical Therapy, School of Health Science, Niigata University of Health and Welfare, Niigata, Japan (Accepted for publication, February15, 2012) Abstract: Wnt is involved in odontoblast and ameloblast differentiation and matrix protein expression in dentin and enamel, but there have been only a few reports on Axin2 in the Wnt signaling pathway. Axin2 promotes Smad3 activation in the transforming growth factor beta (TGF-ß) signaling pathway, and the presence of cross- talk between the Wnt and TGF-ß signaling pathways has been reported, but there have been no reports on that in odontogenic cells. Thus, we examined immunohistochemically the factors in the 2 signaling pathways using serial sections of rat first molar germ at embryonic day 19 and 10 days after birth to investigate whether there is cross-talk between the 2 signaling pathways through Axin2 and Smad3. Factors in the Wnt signaling pathway: Wnt10, Dishevelled (Dvl), and Axin2, and those in the TGF-ß signaling pathway: TGF-ß receptor 1 (TGF-ß- R1), Smad3, and its active type, phosphorylated Smad3 (p-Smad3), showed similar immunoreactions; in detail, odontoblasts possessing secretory function, rather than dental papilla cells and preodontoblasts, and secretory- stage ameloblasts, rather than inner enamel epithelium, showed stronger reactions, whereas the reaction of maturation-stage ameloblasts was very weak. In addition, the reaction of Axin2 was completely consistent with that of pSmad3, and odontoblasts and secretory ameloblasts showed the strongest positive reactions. Accordingly, it was suggested that the TGF-ß signaling pathway as well as Dvl- and Axin2-mediated Wnt10 signaling pathway may are involved in odontoblast and ameloblast differentiation and the secretory function of these cells. In addition, the reaction of Axin2 was completely consistent with that of pSmad3, and odontoblasts and secretory ameloblasts showed the strongest positive reactions, suggesting that a cross-talk between the 2 signaling pathways through Axin2 and Smad3 may be involved in the odontoblast and ameloblast differentiation and secretory function of these cells. Keywords: Immunolocalization, Developing rat molar germ, Axin2, p-Smad3 Introduction Axin2 is considered to control Wnt signaling by forming a negative In the Wnt/β-catenin pathway, when Wnt binds to its receptors, feedback loop5). Regarding the involvement of Wnt in tooth Frizzled and low-density lipoprotein receptor-related protein 5/6 development, many studies have reported its involvement in the (LRP5/6), glycogen synthase kinase-3 beta (GSK-3β) activity is early stage of tooth germ formation, and it is considered to initiate inhibited through Dishevelled (Dvl), and scaffold protein of β- tooth germ formation and control proliferation and differentiation catenin destruction complex, Axin, becomes unstable due to the of tooth germ epithelial cells and mensenchymal cells6), 7), 8), 9). On inactive-type GSK-3β. The complex is then destroyed and releases the other hand, in the late bell stage in which dentin and enamel β-catenin, resulting in its intracellular accumulation. Accumulated are formed, Wnt-10a mRNA is expressed in preodontoblasts and β-catenin is considered to transfer to the nucleus, bind to transcript odontoblasts and dentin sialophosphoprotein (Dspp) mRNA is factor; lymphoid enhancer-binding factor / T cell factor (LEF/ expressed by odontoblasts, on the basis of which Wnt-10a has TCF), and induce and regulate gene expression1). Axin2 is a been reported to be involved in odontoblast differentiation and negative regulator of the Wnt signaling pathway2). Under Wnt dentin formation by regulating Dspp10). It has been reported that signals, it is released from the complex and binds to LRP5/6, Wnt-10a and Axin2 appeared in the incisor tooth germ leading to Axin degradation3). In contrast, the Wnt/β-catenin ameloblasts11), Wnt10, Dvl, inactive-type GSK-3β and β-catenin pathway is known to induce Axin2 mRNA transcription4), and distributed in secretory-stage ameloblast and progenitors of the more tooth germ12), and β-catenin/LEF1 activates the appearance Correspondence to: Mitsuko Moriguchi, PhD. Department of Ultrastruc- tural Science, Tokyo Dental College, 1-2-2 Masago Mihama-ku, Chiba of enamelin in ameloblast-like cells13), indicating involvement of 261-8502, Japan. Tel: +81-43-270-3755, Fax: +81-43-270-3756, Email: the Wnt signaling pathway in the ameloblast differentiation and [email protected] 113 J.Hard Tissue Biology Vol. 21(2):113-120, 2012 Figure 1. Immunohistochemical observation of the rat first molar germ on embryonic day 19. a Hematoxylin and eosin (H-E) staining. Inner enamel epithelium (arrows) and mesenchymal cells of the dental papilla distributed densely along this epithelium are observed. b, c Staining with anti-Wnt10 (b) or anti-Dvl (c) was positive in the inner enamel epithelium and in the cervical loop. In the dental papilla, positive reactions were observed in mesenchymal cells in contact with the enamel epithelium. d Negative control for figures (b) and (c). No clear reaction is noted. e, f Staining with anti-Axin2 (e) or anti-TGF-ß-R1 (f) was strongly positive in the inner enamel epithelium and in the cervical loop. Moderate reactions were observed in the dental papilla cells in contact with the enamel epithelium. g, h Staining with anti-p-Smad3 (g) or anti-Smad3 (h) was also strongly positive in the inner enamel epithelium and in the cervical loop. In the dental papilla, moderate reactions were also observed in mesenchymal cells in contact with the enamel epithelium. These stainings were strongest in the nucleus (white arrowhead). The boxed areas are enlarged to the right base of each panel. i Negative control for figures (e) - (h). No signal was detected. Arrows Inner enamel epithelium. Black arrowheads Dental papilla cells in contact with the enamel epithelium. DP Dental papilla, CL Cervical loop. Bars, 100 ìm or 20ìm. matrix protein secretion in enamel. Regarding the TGF-β signaling degrade Smad3 by phosphorylation and modulate TGF-ß signaling pathway, the Smad pathway is well known, in which Smad3 is by controlling Smad3 stability28). However, there have been no activated through TGF-β-R1, and active-type Smad3 and Smad4 reports on investigations of cross-talk between the 2 signaling form a complex, move into the nucleus, and regulate gene pathways in tooth development. In this study, to investigate expression, through which the action is exhibited. Regarding tooth whether cross-talk between the Wnt and TGF-β signaling development, it has been reported that TGF-β and its genes and pathways is involved in ameloblast and odontoblast differentiation receptor are localized in the inner enamel epithelium, cervical and the function of these cells, we immunohistochemically loop, ameloblasts, and odontoblasts14), 15), 16), 17), and TGF-β1 induces examined the distributions of factors involved in the Wnt signaling ameloblast and odontoblast differentiation18),19), which is involved pathway (Wnt10, Dvl, and Axin2) and in the TGF-β signaling in the production of matrix proteins, such as amelogenin, pathway (TGF-β-R1, Smad3, and p-Smad3) in the early bell stage enamelysin, collagen, and Dspp20), 21), 22), 23), 24). It has also been immediately before differentiation of these cells, as well as in the reported that Smad3 and Smad4 are distributed in inner enamel late bell stage in which hard tissue formation is underway, using epithelium and dental mesenchyme and regulate tooth rat molar tooth germ. development 24), 25), and that Smad3 is necessary for mineralization of enamel26). Materials and Methods Reportedly, there is cross-talk between the Wnt and TGF-β Tissue preparation signaling pathways. It has been reported that interaction between The experiment was performed using Sprague–Dawley (SD) Axin and Smad3 promotes Smad3 activation in the TGF-β rats at embryonic day 19 and 10 days after birth, in accordance signaling pathway27), and that Axin and active-type GSK3-β with the Guidelines for the Handling of Experimental Animals of 114 Mitsuko Moriguchi et al.: Axin2 and p-Smad3 in Molar Germ Figure 2. Immunohistochemical observation of the rat maxillary first molar germ 10 days after birth. a H-E staining. Enamel, dentin, and Hertwig’s epithelial sheath had formed. Secretory ameloblasts and odontoblasts were distributed, and differentiation from secretory ameloblasts into maturation ameloblasts and from preodontoblasts into odontoblasts was observed near the cusp or apical end. b, c Staining with anti-Wnt10 (b) or anti-Dvl (c) was strongly positive in secretory ameloblasts and odontoblasts, and also moderate in preodontoblasts, but weak in maturation ameloblasts, showing stronger reactions with differentiation into odontoblasts, and weaker reactions from secretory ameloblasts to maturation ameloblasts. d Negative control for figures (b) and (c). No clear reaction is noted. e Staining with anti-Axin2 was most strongly positive in the secretory ameloblasts and odontoblasts, and also moderate in preodontoblasts, showing stronger reactions