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Keywords:

  • human tooth germ;
  • gene expression;
  • transcriptional factors;
  • growth factors

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

In the developing murine tooth, the expression patterns of numerous regulatory genes have been examined and their roles have begun to be revealed. To unveil the molecular mechanisms that regulate human tooth morphogenesis, we examined the expression patterns of several regulatory genes, including BMP4, FGF8, MSX1, PAX9, PITX2, and SHOX2, and compared them with that found in mice. All of these genes are known to play critical roles in murine tooth development. Our results show that these genes exhibit basically similar expression patterns in the human tooth germ compared with that in the mouse. However, slightly different expression patterns were also observed for some of the genes at certain stages. For example, MSX1 expression was detected in the inner enamel epithelium in addition to the dental mesenchyme at the bell stage of the human tooth. Moreover, FGF8 expression remained in the dental epithelium at the cap stage, while PAX9 and SHOX2 expression was detected in both dental epithelium and mesenchyme of the human tooth germ. Our results indicate that, although slight differences exist in the gene expression patterns, the human and mouse teeth not only share considerable homology in odontogenesis but also use similar underlying molecular networks. Developmental Dynamics 236:1307–1312, 2007. © 2007 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Mammalian tooth development is regulated by means of sequential and reciprocal interactions between the cranial neural crest-derived mesenchymal cells and the stomadial epithelium. The developing tooth has thus served as an excellent model system for studying fundamental processes and mechanisms of vertebrate embryogenesis, including induction, differentiation, and pattern formation (Thesleff and Pispa, 1998). In mice, the molar tooth germ is a model system from which most of our current knowledge on tooth development is derived. The determination of the tooth forming site and tooth type occurs around embryonic day (E) 10 (Neubuser et al., 1997; Tucker et al., 1998; Peters and Balling, 1999). At E11.5, the dental placode forms as the dental epithelium becomes thickened. Subsequently, the dental epithelium proliferates and invaginates into the subjacent mesenchyme, forming the epithelial bud at E12.5 and E13.5. Meanwhile dental mesenchymal cells condense around the epithelial bud. At the E14.5 cap stage, the epithelial bud undergoes specific folding and forms a transient epithelial cluster called the enamel knot in the central region of the bud. The enamel knot expresses several signaling molecules and is, therefore, considered to be a signaling center in the developing tooth controlling the patterning of the tooth cusps (Thesleff and Mikkola, 2002). Following the cap stage is the bell stage, starting around E16.5, when ameloblasts and odontoblasts differentiate.

Human odontogenesis shares considerable homology with that of the mouse. Around the sixth week of human embryonic development, the oral epithelium at the sites of presumptive dentition forms a thickened U-shaped epithelial band, called the dental lamina, on the upper and lower jaws. Around the seventh week, 10 centers of epidermal cell proliferation form at intervals within the U-shaped epithelial band. Cells in these centers proliferate and invaginate into the subjacent dental mesenchyme to form the epithelial buds of the primary teeth (two incisors, one canine, and two premolars in each quadrant; Larsen, 2001). From the eighth to the ninth week of gestation, the dental epithelium continues to invaginate into the subjacent dental mesenchyme, forming a tooth germ at the late bud stage. The cap stage tooth germ appears in the tenth week of embryonic development, and the bell stage begins in the fourteenth week of gestation.

The recent development of new molecular technologies has resulted in a rapid escalation of studies on molecular mechanisms underlying tooth morphogenesis in mice, and the spatial and temporal expression pattern of a large number of genes has been revealed. The roles of many of these genes in tooth development have also been studied using transgenic, tissue recombination, and gene expression assay approaches (Zhang et al., 2005). Among them are genes encoding growth factors and transcription factors. It is generally believed that growth factors play a critical role in odontogenesis by mediating inductive interactions between the odontogenic tissue layers, while transcription factors function to regulate the expression of growth factors and other genes. The tooth phenotype seen in mouse mutants is sometimes found in humans carrying mutations in the counterpart genes. For example, mutations in either Msx1 or Pax9 in mice lead to the arrest of tooth development at the bud stage (Satokata and Maas, 1994; Chen et al., 1996; Peters et al., 1998). In humans, mutations in the MSX1 gene or PAX9 gene also cause tooth agenesis or oligodontia, respectively (Vastardis et al., 1996; Van den Boogaard et al., 2000; Lidral and Reising, 2002; Stockton et al., 2002). These results indicate that tooth development in mice and humans not only shares many similarities in the morphological processes but that they may also use similar molecular mechanisms.

Understanding the molecular mechanisms underlying human odontogenesis is a prerequisite for the realization of human tooth regeneration in the future. As a first step toward this goal, we surveyed in the human developing tooth germ the expression of genes known to play a critical role in the mouse odontogenesis. Here we report the expression of several regulatory genes in the human embryonic tooth germ. We also compare the expression patterns in mice and human.

RESULTS AND DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

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.

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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.

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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).

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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.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

Samples

The surgically and medically terminated human embryos of 8th to 14th week gestation were provided by the Hospital for Women and Children of Fujian Province. The use of human embryos including mouse kidney capsule culture of human embryonic tissues in this study was ethically permitted by the Ethics Committee of Fujian Normal University. The embryos were washed in PBS before being fixed in 4% paraformaldehyde (PFA) at 4°C overnight and then processed for paraffin sectioning at 10 μm. To obtain human tooth germs at a late differentiation stage, premolar tooth germs were dissected out from 14-week-old human embryos and grafted under the kidney capsule of adult nude mice according to the method described previously (Zhang et al., 2003). Grafts were harvested 2 months after the subrenal culture, fixed in 4% PFA, decalcified in 0.1 M ethylenediaminetetraacetic acid at room temperature for 2 days before dehydration, paraffin embedding, and sectioning. Mouse embryos were harvested from timed pregnant females and processed for in situ hybridization as described previously (Zhang et al., 1999).

Probes and In Situ Hybridization

All the probes that were used for examining human gene expression in this study with the exception of BMP4 were amplified from the exons of each gene using human genomic DNA, as detailed below. The probe for the human PITX2 gene was amplified from exon 3 using the primers (upper, 5′-cgtcgggccaaatggagaaa-3′; lower, 5′-tccctttctttagtgcccacgac-3′) that gave rise to a 654-bp product. The probe for the human FGF8 gene was amplified from exon 5 using the primers (upper, 5′-agcaacggcaaaggcaagga-3′; lower, 5′-agcaacggcaaaggcaagga-3′) that produced a 454-bp product. The 429-bp probe for human MSX1 was amplified using the primers (upper, 5′-ctgtggcgcccgtgggactct-3′; lower, 5′-tgcgcttttcttgcctggtgt-3′) that targeted to exon 2. The 500-bp probe for human PAX9 was amplified from exon 4 using the primers (upper, 5′-accctaccccagcccaagtg-3′; lower, 5′-ctttgaggggtgtaggtttctttgt-3′). The 500-bp probe for human SHOX2 was amplified from exon 4 using the primers (upper, 5′-cgcacgcgcaccaccacctg-3′; lower, 5′-actcccccaaacccgctcctacaa-3′). The 479-bp probe for human DSPP was amplified from exon 4 using the primers with the following sequences: upper, 5′-ggggacacaggaaaagcagaaaca-3′; and lower, 5′-ccattccctgcttcttcatcttca-3′). The 1,259-bp probe for human BMP4 was a gift from Dr. Guangquan Zhao of UT Southwestern Medical Center. Section in situ hybridization was performed as described previously (Zhang et al., 1999).

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES

We thank the Hospital for Women and Children of Fujian Province for providing aborted human embryos for this study. We also thank Mr. Catalin Anghelina of the Chen laboratory for proofreading of the manuscript. Y.D.Z. was funded by the Ministry of Science and Technology of China, the National Natural Science Foundation of China, and Fujian Provincial Department of Science and Technology, China. Y.P.C. was funded by the NIH.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS AND DISCUSSION
  5. EXPERIMENTAL PROCEDURES
  6. Acknowledgements
  7. REFERENCES