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Expression of Bone Morphogenetic Proteins and Msx Genes during Root Formation
1 Developmental Biology Program, Institute of Biotechnology, Viikki Biocenter, PO Box 56, FIN-00014 University of Helsinki, Finland; Correspondence: *corresponding author, yamat{at}md.okayama-u.ac.jp
Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.
Key Words: epithelial-mesenchymal interactions • root development • dentinogenesis • cementogenesis
Tooth development is characterized by sequential and reciprocal interactions between the dental epithelium and mesenchyme. Many signal molecules and growth factors have been implicated in the mediation of these interactions (Thesleff and Mikkola, 2002). Bone morphogenetic proteins (BMPs) are secretory signal molecules and members of the TGF-beta superfamily of growth and differentiation factors. During early tooth morphogenesis, the expression patterns of Bmp2, Bmp4, and Bmp7 are associated with the epithelial-mesenchymal interactions and shift between the tissues (Vainio et al., 1993; Åberg et al., 1997; Thesleff and Åberg, 1999). In organ culture, beads releasing BMP2 and BMP4 proteins mimic the inductive epithelial and mesenchymal signals. During tooth initiation, BMP4 induces the expression of Msx1 and Msx2 in dental mesenchyme (Vainio et al., 1993), and during bud- to cap-stage transition, it induces, in the epithelium, the expression of Msx2 as well as the formation of the enamel knot signaling center (Jernvall et al., 1998). Msx1 and Msx2 are homeobox-containing transcription factors, and their expression patterns are also associated with epithelial-mesenchymal interactions in early tooth development (MacKenzie et al., 1992; Jowett et al., 1993; Thesleff, 1995). Mice deficient for Msx1 or Msx2 show altered tooth phenotypes (Satokata and Maas, 1994; Satokata et al., 2000). Bmp4 is a major downstream target of Msx1 in the dental mesenchyme, and BMP can rescue the dental phenotype of Msx1 null mice (Bei et al., 2000). Hence. the BMP-Msx pathway mediates reciprocal interactions between the epithelium and mesenchyme during tooth initiation and crown morphogenesis. After the tooth crown has formed, the outer and inner enamel epithelia form a double-layered Hertwig’s epithelial root sheath which proliferates apically and directs root morphogenesis. Histological observations indicate that there is a close association between the epithelial root sheath and the initiation of root dentin formation, and the peripheral pulpal mesenchymal cells differentiate into pre-odontoblasts along the inner surface of the epithelial root sheath. However, the molecular mechanisms regulating this interaction have not been identified in the process of root development (Thomas, 1995). As root formation proceeds, the epithelial root sheath disrupts, allowing mesenchymal cells from the dental follicle to come into contact with the root surface and differentiate into cementoblasts which deposit cementum (Thomas, 1995; Ten Cate, 1996). The function of this fragmented epithelium, known as epithelial cell rests of Malassez, is not known, but it has been speculated that it may induce cementoblast differentiation and perhaps later regulate their function (Thomas, 1995; Bosshardt and Schroeder, 1996; Kagayama et al., 1998). The aim of this study was to investigate the possible roles of BMP signaling and Msx family transcription factors during root morphogenesis and the formation of root dentin and cementum. We performed an in situ hybridization analysis of temporospatial expression of Bmp2, Bmp3, Bmp4, Bmp7, Msx1, and Msx2. As a phenotypic marker for terminally differentiated mineralizing cells, we also analyzed the distribution of bone sialoprotein (Bsp) mRNA.
Processing of Tissues The Animal Welfare Committee of the University of Helsinki approved the experimental procedures. Heads of 14-day-old NMRI mice were dissected in Dulbecco’s phosphate-buffered saline. The tissues were fixed in 4% paraformaldehyde at 4°C overnight, and decalcified in 12.5% EDTA containing 2.5% paraformaldehyde for 2 wks. They were dehydrated, embedded in paraffin, and serially sectioned at 7 µm. Keratin was used as a marker for epithelial cells, and it was detected by immunohistochemistry with polyclonal pan-keratin antibodies (DAKO, A575, Glostrup, Denmark) (Kaneko et al., 1999).
Probes and in situ Hybridization
Root Development Fig. 1  shows the histological appearance of a sagittal section of the lower jaw of a 14-day-old mouse. In the section, 1st, 2nd, and 3rd molars show different stages of root development (Fig. 1A). The crowns of the 1st and 2nd molars are almost fully developed, and approximately one-half and one-third of the root has been formed in the 1st and 2nd molars, respectively. At the apical end of the growing root, epithelial root sheath is present. Odontoblasts are columnar cells lining the pulpal surface of dentin (Fig. 1B). In this study, we defined the apically located odontoblasts as "early odontoblasts" and the coronally located odontoblasts as "late odontoblasts". Pre-odontoblasts are the odontoblast precursors in the apical end of the root (Fig. 1B). We confirmed the identity of epithelium by immunohistochemistry using pan-keratin antibodies. Immunoreactivity was observed in the epithelial root sheath and the epithelial cell rests of Malassez. In the 3rd molar, root development has not been initiated, and the epithelial root sheath is continuous with the dental epithelium of the crown (Fig. 1C).
Expression of Bmps in Developing Root and Surrounding Bone Bmp2, Bmp3, and Bmp7 were expressed in early odontoblasts and overlapped each other in the apical region (Figs. 2A, 2B, 2C, 2D, 2G, 2H ; Table). Their expression in odontoblasts was down-regulated with the advancement of differentiation, and transcripts were not present in late odontoblasts (Figs. 2A, 2C, 2G). Bmp2 was also intensely expressed in the central mesenchyme of the pulp (Fig. 2A).
Strong Bmp3 expression was detected in cementoblasts in the 1st and 2nd molars, as well as the dental follicle around all three molars (Figs. 2C, 2D  ). On the active bone-forming surface, intense expression of Bmp3 was observed in the osteoblasts (Figs. 2C, 2D).
Bmp4 expression appeared early in the pre-odontoblastic cells adjacent to the epithelial root sheath (Figs. 2E, 2F
Bmp7 and Bmp4 were also detected in ameloblasts (Figs. 2E, 2G
Expression of Msx1, Msx2, and Bsp
Bsp was strongly expressed in the cells lining the cementum and alveolar bone (Figs. 3E, 3F ). Bsp expression was also intense in the early odontoblasts in the apical regions, whereas no expression was detected in the terminally differentiated odontoblasts lining dentin in either the root or the crown areas (Figs. 3G, 3H).
All four BMPs examined showed dynamic expression patterns and were associated with root morphogenesis and/or differentiation of the hard-tissue-forming cells. All BMPs were expressed in the odontoblast lineage cells at some stage. Bmp4 and Bmp7 were expressed in ameloblasts, and only Bmp3 was found in cementoblasts. Of the Msx genes, Msx2 was associated with both morphogenesis and ameloblast differentiation, whereas Msx1 showed very weak expression in pulp mesenchyme with no obvious association with root development. The findings indicate that some but not all functions of these genes are similar in crown and root development. During the initiation of tooth morphogenesis, BMP2, BMP4, and BMP7 act as important epithelial signals regulating the differentiation of the neural-crest-derived mesenchyme into odontogenic lineage (Thesleff and Åberg, 1999). BMPs induce in the mesenchyme the expression of Msx1 and Msx2 (Vainio et al., 1993). The functions of these two genes are redundant in early stages in tooth development, since, in single-gene knockouts, tooth development is normal until the bud stage, whereas in double knockouts, tooth morphogenesis is arrested at the dental lamina stage (Bei and Maas, 1998). Interestingly, Msx1 and Msx2 transcripts were completely absent from the apical part of the root mesenchyme, and transcripts of Bmps were not detected in the epithelium of the root sheath. These findings indicate that BMPs and Msx1 and Msx2 do not have similar functions in root and crown initiation. Apparently, the induction of mesenchyme by epithelial BMPs during tooth initiation—‘odontogenic competence’—is maintained during root development (Vainio et al., 1993; Tucker et al., 1998). Msx1 mutant tooth development arrests at the bud stage (Satokata and Maas, 1994). At this time, Msx2 has been down-regulated in the mesenchyme (Jowett et al., 1993), and therefore it does not compensate for the function of Msx1. An important function of Msx1 in the mesenchyme is to regulate the expression of Bmp4, which acts as a reciprocal signal on epithelium and regulates the development of the enamel knot. The genes regulated by BMP4 in the enamel knot include Msx2 and p21. The enamel knot acts as a signaling center which regulates subsequent epithelial morphogenesis during the cap stage (Jernwall et al., 1998; Thesleff and Åberg, 1999; Bei et al., 2009). This primary enamel knot as well as the later-forming secondary enamel knots regulate the complex epithelial folding morphogenesis which determines the cusp pattern of the crown (Jernvall and Thesleff, 2000). Interestingly, Bmp4 was expressed in the mesenchyme lining the root sheath epithelium, which in turn expressed Msx2. This may be indicative of a similar mesenchymal-epithelial interaction regulating epithelial morphogenesis in both the crown and the root. Like the crown shape, the shape of the root is complicated, particularly in the multirooted molar teeth. Interestingly, during the preparation of this manuscript, Ohshima et al. (2002) reported that Msx2 null mutant mice have irregularly shaped molar roots. This provides direct evidence that Msx2 is involved in root morphogenesis. Classic heterotypic tissue recombination experiments between molars and incisors have shown that, besides crown morphogenesis, the mesenchyme also directs root development (Kollar and Baird, 1970). Therefore, it is conceivable that mesenchymal factors regulate the growth and morphogenesis of the root sheath epithelium which determines the root shape. We suggest that BMP4 and Msx2 are involved in the mediation of this mesenchymal-epithelial interaction. BMPs were expressed sequentially at different stages of odontoblast differentiation. Bmp4 was expressed in the early pre-odontoblasts lining the root sheath epithelium. Subsequently, Bmp2 and Bmp7 were expressed in pre-odontoblasts and odontoblasts during a relatively short period of differentiation and were absent from mature odontoblasts on both crown and root dentin surfaces. Bmp3 was expressed in the pre-odontoblasts and odontoblasts in the root area, but it was absent from differentiating and secretory odontoblasts in the crown, as previously reported (Åberg et al., 1997). As suggested for BMP4, BMP2, and BMP7 may affect epithelial differentiation (Tabata et al., 2002). All BMPs may also have autocrine or paracrine effects on differentiating odontoblasts. Bmp3 was expressed intensely in the dental follicle cells in the root area, as earlier reported in the crown follicle (Åberg et al., 1997). Particularly intense Bmp3 expression was found in cementoblasts. In addition, it was intensely expressed in osteoblasts at sites of active deposition. BMP3 may regulate the function of the osteoblasts and cementoblasts. It is a negative regulator of trabecular bone formation, and in vitro findings indicate an inhibitory role for BMP3 in BMP2-mediated differentiation of osteoprogenitor cells into osteoblasts (Daluiski et al., 2001). It is possible that BMP3 has a similar inhibitory function in cementum formation. A possible target of BMP regulation is bone sialoprotein, which was co-expressed with Bmp2, Bmp3, and Bmp7 in differentiating odontoblasts and with Bmp3 in cementoblasts and osteoblasts. Expression of Bsp by osteoblasts on the active bone-forming surface as well as by cementoblasts has been demonstrated previously (D’Errico et al., 1997). Our careful analysis showed that Bsp expression was restricted to the apical differentiating pre-odontoblasts and that it was down-regulated with advancement of the differentiation, while other matrix proteins such as osteocalcin and type I collagen show intense expression in all odontoblasts during similar developmental stages (D’Errico et al., 1997). The differentiation of cementoblasts from dental follicle cells, and their deposition of cementum at the dentin surface, is believed to be regulated by the epithelial cells derived from the root sheath, i.e., the cell rests of Malassez (Thomas, 1995). None of the BMPs analyzed was expressed by the Malassez epithelial cells, and therefore the putative signal remains to be identified. However, the close association between the Bmp3-expressing cementoblasts and Msx2-expressing Malassez epithelial cells raises the possibility that perhaps cementoblasts signal to the Malassez epithelial cells and regulate their functions. Msx2 has previously been associated with the maintenance of the proliferative potential and inhibition of differentiation (Hu et al., 2001). It is an intriguing possibility that Msx2 has a similar function in Malassez epithelial cells, which do not proliferate and stay apparently undifferentiated. The earlier observation that the epithelial cell-rest cells bind EGF intensely is in line with this proposal (Thesleff, 1987). Very little is currently known of the roles of the Malassez epithelial cells and the patterns of their gene expression (Yamashiro et al., 2000).
We thank Merja Mäkinen and Riikka Santalahti for excellent technical assistance. This study was supported by the Academy of Finland and the Sigrid Juselius Foundation. T. Yamashiro was supported by the Graduate School of Medicine and Dentistry, Okayama University, Japan. Received for publication May 31, 2002. Revision received December 16, 2002. Accepted for publication December 17, 2002.
Journal of Dental Research, Vol. 82, No. 3,
172-176 (2003) This article has been cited by other articles:
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