Expression of Bmp4 in the Developing Human Tooth Germ
Among the several Bmp genes that are expressed in the developing mouse tooth, Bmp4 has been extensively studied and is thought to play a central role in tooth development (Thesleff and Mikkola, 2002). In mice, Bmp4 expression is initially restricted to the dental epithelium at E11, but then it shifts to the dental mesenchyme during the subsequent bud stage (Vainio et al., 1993; Zhang et al., 2000). During the cap and bell stages, Bmp4 expression remains in the dental papilla mesenchyme, the dental pulp cells and odontoblast. At these stages, expression is also found in the enamel epithelium, first in the enamel knot and then in the ameloblasts (Feng et al., 2002). In the developing human tooth germ, similar to that in the mouse, BMP4 expression was detected in the dental papilla mesenchyme as well as in the dental epithelium at the cap stage in both incisors and premolars (Fig. 1A,B). BMP4 expression in the inner enamel epithelium became significant in both incisors and premolars at the bell stage, while the expression in the dental papilla was maintained at a relatively lower level (Fig. 1C–F). It was previously reported that BMP4 expression was detected in the dental papilla and dental pulp cells of human embryonic tooth germs at the cap and bell stage at a lower level by in situ hybridization (Heikinheimo, 1994). The different results are very likely due to the different sensitivities of the probes and the methods used.
Figure 1. Expression of BMP4 in the developing human tooth. A:BMP4 expression was detected in the dental papilla cells and also in the dental epithelium of the second incisor (at the cap stage) of a 12-week-old human embryo. B:BMP4 expression was seen in both the dental mesenchyme and the dental epithelium of the second premolar (at the cap stage) of a 12-week gestation embryo. C,D: Expression of BMP4 was seen in dental papilla cells and inner enamel epithelium of the second incisor (C) and the second premolar (D) of a 14-week gestation embryo. Both teeth are at the bell stage. E: Higher magnification of the designated area in C showing BMP4 expression in the inner enamel epithelium and dental papilla cells. F: Higher magnification of the designated area in D showing BMP4 expression in the inner enamel epithelium and dental papilla cells. G: A section through a 14-week premolar germ graft that had been cultured under the mouse kidney capsule for 2 months showing BMP4 expression in odontoblasts and ameloblasts. Note the presence of dentin. H: A section through a graft of a 14-week premolar germ 2 months after renal culture showing DSPP expression in odontoblasts. Dentin formed in the graft. D, dentin; DP, dental papilla; IEE, inner enamel epithelium. Scales bars = in 100 μm in A,B,G,H, 200 μm in C,D.
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To examine gene expression in the human tooth germ at a late differentiation stage, the premolar tooth germs from embryos of 14-week gestation were grafted underneath the kidney capsule of nude mice. Grafts were harvested 2 months after the renal culture. Histological examination demonstrated that the grafted teeth exhibit well-differentiated dental structures with the formation of dentin (Fig. 1G,H, and data not shown). This conclusion is supported by the specific expression of DSPP in the odontoblasts of the grafted teeth (Fig. 1H). BMP4 expression was also detected in the odontoblasts and ameloblasts of the grafted teeth (Fig. 1G), suggesting a persistent role for BMP4 in the development and differentiation of both odontoblasts and ameloblasts.
MSX1 Expression in the Human Tooth Germ
In mice, Msx1 is expressed in the dental mesenchyme, including the dental papilla of developing tooth throughout the lamina, bud, cap, and bell stages of odontogenesis (MacKenzie et al., 1991). Identical to that found in the mouse tooth germ, MSX1 expression was restricted to the dental papilla mesenchyme of the primary tooth germ at the cap stage in humans (Fig. 2A,B). Both the incisor and the premolar exhibited a similar expression pattern. Of interest, at the bell stage, while remaining in the dental papilla cells, MSX1 expression was also seen in the inner enamel epithelium at a very high level in incisor and premolar teeth (Fig. 2C–E). During the late differentiation stage, similar to the expression pattern of BMP4, MSX1 transcripts were also detected in the odontoblasts and ameloblasts of a premolar that had been grafted and cultured in the kidney capsule for 2 months.
Figure 2. MSX1 expression in human tooth germ. A:MSX1 expression was detected in the dental mesenchyme of an incisor at the cap stage of a 12-week gestation embryo. B: A section through a premolar at the cap stage of a 12-week-old human embryo showing localization of MSX1 transcripts to the dental mesenchyme. C,D: The second incisor (C) and the second premolar (D) at the bell stage from a 14-week-old embryo exhibited MSX1 expression in the dental papilla cells and the inner enamel epithelium. E: A higher magnification of the designated region in C. F: A section through a graft of a 14-week-old premolar 2 months after renal culture showed MSX1 expression in both the odontoblasts and ameloblasts. D, dentin; AB, ameloblasts; DE, dental epithelium; DP, dental papilla; IEE, inner enamel epithelium. Scale bars = 100 μm in A,B,F, 200 μm in C.D.
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Figure 3. Expression of PITX2 in the mouse and human tooth germ. A:Pitx2 expression was restricted to the dental epithelium of an E14.5 mouse molar. B–E: In the human developing tooth germ, PITX2 expression was also restricted to the dental epithelium, as seen in an 8-week-old second incisor (B), a 12-week-old first premolar (C), a 14-week-old first incisor (D), and a 14-week-old second premolar (E). F:PITX2 expression was detected in the ameloblasts of a graft of 14-week-old premolar 2-month after renal culture. Scale bars: in B, 50 μm; in C and F, 100 μm; in D and E, 200 μm.
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Mouse and Human Exhibit Similar Expression Pattern of PITX2 in the Developing Tooth
Mutations in PITX2, a member of the PITX/RIEG family of bicoid-related homeobox genes, are responsible for the autosomal-dominant disorder Rieger syndrome, which exhibits defects in the tooth and eye (Semina et al., 1996). In mice, Pitx2 is expressed in the presumptive dental epithelium before tooth formation and is continued in the dental epithelium throughout the entire tooth developmental process (St. Amand et al., 2000; Fig. 3A). Our in situ hybridization results show an expression pattern of PITX2 in the developing human tooth germ identical to that in mice. In both the incisor and the premolar, PITX2 expression was detected only in the dental epithelium at the late bud stage (Fig. 3B), the cap stage (Fig. 3C), and the bell stage (Fig. 3D,E). In the well-differentiated tooth (14-week-old premolar graft after 2 months in renal culture), the expression of PITX2 was restricted to the ameloblasts (Fig. 3F). These results support a role for PITX2 in the development of dental epithelium and differentiation of the enamel organ in the human tooth.
Expression of FGF8, PAX9, and SHOX2 in Developing Human Tooth Germ
In the developing mouse tooth, strong Fgf8 expression is detected in the presumptive dental epithelium before and during tooth initiation. The expression becomes slightly down-regulated, but it persists there throughout the bud stage, basically restricted to the distal part of the tooth bud (Kettunen and Thesleff, 1998; Fig. 4A). However, Fgf8 expression is not detectable in the cap stage tooth germ (Kettunen and Thesleff, 1998). In the human tooth germ at the bud stage, FGF8 expression is slightly different from that in mice. FGF8 transcripts were strongly detected in the dental epithelium but were also observed at a much lower level in the dental mesenchyme (Fig. 4B). In addition, FGF8 expression in the dental epithelium appears to be restricted to the central portion where the enamel knot will form. At the cap stage, FGF8 expression appeared in the dental papilla mesenchyme and the dental epithelium (Fig. 4C), an expression pattern that is not seen in the mouse (Kettunen and Thesleff, 1998).
Figure 4. Comparison of the expression of FGF8, PAX9, and SHOX2 in the mouse and human tooth germ. A:Fgf8 expression was mainly detected in the distal part of the dental epithelium in an embryonic day (E) 13.5 mouse molar. B,C:FGF8 expression was detected mainly in the dental epithelium and also weakly in the dental mesenchyme of the second incisor (B) of an 8-week-old human embryo and the first premolar (C) of a 12-week-old human embryo. D:Pax9 expression was restricted to the dental mesenchyme of an E14.5 mouse molar. E,F: In the human tooth germ, PAX9 expression was detected in both the dental mesenchyme and the dental epithelium, as seen in the incisor (E) and premolar (F) of a 12-week-old embryo. G:Shox2 expression was detected in the dental epithelium of an E14.5 mouse molar. H,I: in the human embryonic tooth germ, SHOX2 expression was detected in both the dental epithelium and dental mesenchyme, as seen in an 8-week-old second incisor (H) and a 12-week-old premolar (I). Scale bars = 50 μm in B,H, 100 μm in C,E,F,I.
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We also examined the expression patterns of PAX9 and SHOX2 in the human embryonic tooth germ. PAX9, a member of the paired domain family genes, was found to be expressed in both the dental mesenchyme and the dental epithelium of incisors and premolars at the cap stage (Fig. 4E,F). This expression pattern is different from that in mice in which Pax9 expression is confined to the dental mesenchyme (Fig. 4D). The similar situation was also observed for the expression of SHOX2, a member of the short stature homeobox gene family (Blaschke et al., 1998; Semina et al., 1998). In the developing mouse tooth, Shox2 expression is restricted to the dental epithelium from the early bud stage to the cap stage (Fig. 4G and data not shown). In the human tooth germ at the bud stage, SHOX2 expression was indeed mainly localized in the dental epithelium (Fig. 4H). However, in the tooth germ at the cap stage, SHOX2 expression not only remained in the dental epithelium, but also expanded to the subjacent dental papilla mesenchyme (Fig. 4I).
The bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) signaling pathways have been shown to play critical roles in almost every aspect of mouse tooth development, from the determination of tooth forming sites and tooth types to tooth morphogenesis, differentiation, and pattern formation (Zhang et al., 2005). The evidence that BMP4 and FGF8 exhibit expression patterns in the human tooth germ that are similar to those observed in mice suggests that these two factors may play similar functions in tooth development in both humans and mice. Bmp4 was shown to be involved in cusp formation and differentiation of the ameloblasts in mice (Tabata et al., 2002). In our studies, we found that BMP4 is strongly expressed in the inner enamel epithelium of the human tooth at the bell stage, when differentiation begins, and also in the ameloblasts of tooth (tooth graft) at the late differentiation stage, thus implicating an active involvement of BMP4 in the differentiation of ameloblasts. Several other members of the BMP family, including BMP2 and BMP6, have also been shown to be expressed in the human tooth germ in a stage-specific manner, suggesting distinct roles for different BMP members in human tooth development (Heikinheimo, 1994). The expression of BMP4 and FGF8 in both the dental epithelium and dental mesenchyme of the developing tooth suggests multiple roles in tooth morphogenesis, particularly in the epithelial–mesenchymal interactions during odontogenesis.
Functional studies have demonstrated that many transcription factors are essential for tooth development in mice. Knockout of Msx1, Pax9, or Pitx2 in mice causes failed tooth development (Satakata and Maas, 1994; Peters et al., 1998; Lin et al., 1999; Lu et al., 1999). In humans, mutations in each of these genes are also associated with tooth phenotypes, including tooth agenesis and oligodontia (Semina et al., 1996; Vastardis et al., 1996; Van den Boogaard et al., 2000; Lidral and Reising, 2002; Stockton et al., 2002).
In the developing mouse tooth, these transcription factors and growth factors are closely linked together and regulate each other, forming a molecular network that controls tooth formation. For example, Bmp4 induces Msx1 but represses Pax9 expression, whereas Fgf8 induces Pax9 expression during the determination of tooth forming sites (Vainio et al., 1993; Chen et al., 1996; Neubuser et al., 1997) On the other hand, Msx1 and Pax9 regulate the expression of Bmp4 (Chen et al., 1996; Peters et al., 1998). Fgf8 positively but Bmp4 negatively regulates Pitx2 expression (St. Amand et al., 2000), while Pitx2 in turn positively regulates Fgf8 expression (Lin et al., 1999; Lu et al., 1999). Furthermore, BMP activity was shown to be necessary for Shox2 expression in developing mouse palatal shelves (Yu et al., 2005). While the genes that were examined in this study exhibit, in general, similar expression patterns in the developing teeth in both humans and mice, several genes, including MSX1, FGF8, PAX9, and SHOX2, show some slightly different expression patterns. It is likely that similar, if not identical, regulatory mechanisms and networks involving these factors are used in human and mouse tooth development based on the gene expression patterns and tooth phenotypes associated with mutations in these genes in both mice and humans. Unveiling the expression patterns of genes and their role in the development of human tooth will provide an important insight for understanding the genetically related dental abnormalities and the realization of tooth regeneration in humans.