Biomedical Research (Tokyo) 36 (3) 205-217, 2015 A systematic analysis for localization of predominant growth factors and their receptors involved in murine tooth germ differentiation using in situ hybridization technique 1 1 2 3 3 Meri HISAMOTO , Marie GOTO , Mami MUTO , Junko NIO-KOBAYASHI , Toshihiko IWANAGA , and Atsuro 1 YOKOYAMA 1 Department of Oral Functional Prosthodontics, Division of Oral Functional Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan; 2 Department of Orthodontics, Division of Oral Functional Science, Graduate School of Dental Medicine, Hokkaido University, Sapporo, Japan; and 3 Department of Anatomy, Graduate School of Medicine, Hokkaido University, Sapporo, Japan (Received 22 April 2015; and accepted 2 May 2015) ABSTRACT Tooth development is regulated by various growth factors and their receptors. However, the over- all 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 periodonti- um 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 inter- action of tooth germ differentiation, and their expressions are precisely controlled. Tooth germ differentiation commonly progresses in genesis, partially due to insufficient in vivo system- the following order: the initiation, bud, cap, bell, and atic analyses of the expression of such factors. root formation stages. According to the stages, pro- The transforming growth factor-β (TGF-β) super- genitor constituents of the tooth germ proliferate and family may take a leading role in the regulation of are differentiated to form specialized parts of teeth cell proliferation, differentiation, and apoptosis, also and associated tissues. Tooth development is pre- being one of the major signals for tooth and peri- cisely regulated by cross-talk between adjacent tis- odontium morphogenesis. Immunohistochemical sues and various growth factors secreted in autocrine studies of the mouse tooth germ detected TGF-β1 in and paracrine modes. Recent studies in this research the inner enamel epithelium, ameloblasts, and odon- field have identified many molecules which play im- toblasts; the expression of TGF-β receptor-1 (TGF- portant roles in signaling associated with tooth germ βR1) was weak in the enamel epithelium but differentiation, but it is largely unknown which increased in intensity in differentiated ameloblasts growth factors are the main contributors to odonto- (6). An in situ hybridization study using mouse em- bryos identified an intense mRNA expression of TGF-β2/3 in the odontoblast layer (20). Among Address correspondence to: Dr. Meri Hisamoto, Depart- bone morphogenetic proteins (BMPs) which belong ment of Oral Functional Prosthodontics, Division of Oral Functional Science, Graduate School of Dental to the TGF-β family, it has been reported that the Medicine, Hokkaido University, North 13, West 7, Kita- expression sites of BMP2 and BMP4 mRNA in the ku, Sapporo 060-8586, Japan mouse tooth germ shift from the enamel knot to Tel: +81-11-706-4270, Fax: +81-11-706-4270 dental papilla and odontoblasts, and their signalings E-mail: [email protected] are required for the epithelium-mesenchyme interac- 206 M. Hisamoto et al. tion during tooth development (1). Another immu- factor) show characteristic expressions in develop- nohistochemical study using the rat tooth germ ing tooth germs. CTGF is a member of the CCN showed that the immunoreactivity of BMP receptor- family involved in cell proliferation, differentiation, 1b (BMPR1b) increased in intensity with the differ- and angiogenesis (2, 8, 12). Shimo et al. demon- entiation of ameloblasts and odontoblasts (15). strated by an in situ hybridization analysis of mice Therefore, BMPs may also act as key regulators in that CTGF and Fisp12 (fibroblast-inducible secreted the differentiation of tooth germs (1, 15). However, protein) are expressed in the dental follicle as well the precise direction and comparative intensity of as inner/outer enamel epithelium (22). Furthermore, signaling by TGF-βs and BMPs have not been fully FGF4 displays a unique expression pattern in the elucidated. enamel knot which regulates the patterning of the Platelet-derived growth factors (PDGFs) mainly tooth crown, including cusp formation, and releases regulate the migration and multiplication of mesen- many signaling molecules such as BMP2/4, Shh, chymal cell lineages. PDGF signaling consists of and Wnt as well as FGF4 (9, 10, 14, 24). The pri- four ligands, PDGF-A to D, and two different recep- mary enamel knot induced at the late bud stage un- tors, PDGF receptor-α (PDGFRα) and PDGFRβ. dergoes apoptosis during the cap stage, and then the PDGFRα binds to PDGF-A/B/C, and PDGFRβ has secondary enamel knot occurs at the future cusps to affinity for PDGF-B/C/D (5, 27). Concerning tooth trigger the folding of the inner enamel epithelium, development, a recent immunohistochemical study leading to cusp formation (14). In relation to cusp using a tooth germ organ culture system reported formation, rodent molars exhibit specific regions at that expressions of PDGFA and PDGFB were in- the tips of cusps, called the enamel-free area (23). tense and extensive in the oral and dental epithelia Although various morphological studies have dealt and underlying mesenchyme, while their receptors with the enamel-free area, it is unknown what mole- (PDGFRα and PDGFRβ) were expressed mainly in cules are specifically expressed there and related to the mesenchyme (27). Moreover, this organ culture signaling for tooth development. Among FGFs, study suggested that PDGFAA, a disulfide-linked FGF2 is detected immunohistochemically in the homodimer of PDGFA, accelerates cusp formation, dental mesenchyme and stellate reticulum of the while PDGFBB induces mesenchyme proliferation mouse enamel organ (3, 21). Thus, we examined the (27). PDGFA and PDGFRα may regulate the size expression profiles of FGF2/4 and CTGF in associa- and stage of tooth development via an autocrine tion with other growth factors in the present study. mechanism (5). However, it is still unclear where Information from systematic analyses of predomi- the PDGF signals arise and are directed. nant growth factors is insufficient to fully understand Insulin-like growth factors (IGFs) are involved in tooth germ differentiation. To comprehend its differ- the differentiation, proliferation, and morphogenesis entiation, we need to elucidate the stage-dependent of various tissues, including odontogenesis (4, 30). expressions of growth factors with respect to interac- The IGF-1 receptor (IGF-1R) and high-affinity IGF- tion with associated factors and signaling directions. binding proteins (IGFBPs) mediate most of the ac- The present study, using an in situ hybridization tions of IGFs. IGFBPs, primarily IGFBP3, inhibit technique, investigated cellular expressions of vari- the bioactivity of IGFs by preventing interaction ous growth factors and their receptors in the fetal with IGF-1R. On the other hand, IGFBPs function and neonatal tooth germs of mice. Our method over- as carrier proteins to direct IGFs from the circula- comes the shortage of specific antibodies and may tion to target tissues and extend their half-lives by provide useful information on growth factors in protecting them from enzymatic degradation (7, 13, tooth development. 16). Morphologically, an in situ hybridization and im- munohistochemical study using rat incisors showed METHODS that IGF-1/2 and IGF-1R/2R are expressed in the enamel organ and ameloblasts, suggesting an impor- Animals and tissue sampling. Pregnant ddY mice tant role of the IGF family in the differentiation and were supplied by Japan SLC (Shizuoka, Japan). physiological activity of ameloblasts (30). Another Mice were sacrificed by the intraperitoneal injection immunohistochemical study using human third mo- of an overdose of pentobarbital sodium (Schering- lars reported that IGFBP3 was localized in the den- Plough Animal Health, the Netherlands). Heads of tal follicle (13). E16.5 embryos and day-1 neonates were used in the Among other growth factors, CTGF (connective present study. The tissues were directly embedded tissue growth factor) and FGF (fibroblast growth into a freezing medium (OCT compound; Sakura Fi- Growth factors in tooth germ 207 Table 1 Targeted nucleotide residues of probes used in this study Gene code Accession no. Residues Gene code Accession no. Residues Tgfb1 NM_011577 1182–1226 Smad1 NM_008539 601–645 1839–1883 1561–1605 Tgfb2 NM_009367 1566–1610 Smad2 NM_010754 841–885 2877–2921 1401–1445 Tgfb3 NM_009368 1546–1590 Smad3 AF016189 481–525 2311–2355 1261–1305 Tgfbr1 MM_009370 373–417 Smad5 NM_008541 431–475 1218–1262 1451–1495 Tgfbr2 NM_00937 685–729 Igf1 NM_001111276 341–375 1585–1629 574–618 Tgfbr3 NM_011578 691–735 Igf2 NM_010514 621–665 1921–1965 869–913 Bmp2 NM_007553 1221–1265 Igf1r NM_010513 1521–1565 2261–2305