<b>A systematic analysis for localization of predominant growth factors and their receptors involved in murine tooth germ differentiation using </b><i><b>in situ</b></i><b> hybridization </b><b>technique </b>

Abstract

Tooth development is regulated by various growth factors and their receptors. However, the overall mechanism of growth factor-mediated odontogenesis remains to be elucidated. The present study examined expression sites and intensities of major growth factors and receptors in the tooth germ of murine fetuses and neonates. Signals of TGF-β and CTGF in fetuses were released from the enamel epithelium, while their neonatal signals arose in odontoblasts. Moreover, BMP/Smad signaling may affect the differentiation of ameloblasts, in contrast to PDGFα whose signals may cause odontoblast differentiation. Growth factors associated with the formation of the periodontium were IGF1, IGF2, IGFBP3, CTGF, and PDGFα. Concerning cusp formation, the enamel knot selectively expressed FGF4, BMP2, and BMP4 with an expression of PDGFα in the enamel-free area. It is concluded that many molecules play critical roles in the epithelium-mesenchyme interaction of tooth germ differentiation, and their expressions are precisely controlled.Tooth germ differentiation commonly progresses in the following order: the initiation, bud, cap, bell, and root formation stages. According to the stages, progenitor constituents of the tooth germ proliferate and are differentiated to form specialized parts of teeth and associated tissues. Tooth development is precisely regulated by cross-talk between adjacent tissues and various growth factors secreted in autocrine and paracrine modes. Recent studies in this research field have identified many molecules which play important roles in signaling associated with tooth germ differentiation, but it is largely unknown which growth factors are the main contributors to odontogenesis, partially due to insufficient in vivo systematic analyses of the expression of such factors. The transforming growth factor-β (TGF-β) superfamily may take a leading role in the regulation of cell proliferation, differentiation, and apoptosis, also being one of the major signals for tooth and periodontium morphogenesis. Immunohistochemical studies of the mouse tooth germ detected TGF-β1 in the inner enamel epithelium, ameloblasts, and odontoblasts; the expression of TGF-β receptor-1 (TGF-βR1) was weak in the enamel epithelium but increased in intensity in differentiated ameloblasts (6). An in situ hybridization study using mouse embryos identified an intense mRNA expression of TGF-β2/3 in the odontoblast layer (20). Among bone morphogenetic proteins (BMPs) which belong to the TGF-β family, it has been reported that the expression sites of BMP2 and BMP4 mRNA in the mouse tooth germ shift from the enamel knot to dental papilla and odontoblasts, and their signalings are required for the epithelium-mesenchyme interac-