The development of diastema teeth could potentially be due to events either at the placode or at the bud stage. An expansion of the dental lamina at an early stage could lead to the development of more teeth. Later events, such as survival of the diastema bud, can also lead to diastema teeth [Klein et al., 2006]. Because recent work has focused on signaling events at the bud-cap transition leading to survival of the diastema bud, we have focused on this below.
Whether the diastema bud normally has a functional enamel knot is still a matter of controversy. It appears, though, that in mutants with diastema teeth, the presence of a functional enamel knot is essential for maintenance of the diastema bud. The pathways that are important in this process are diagrammed in figure 3, and they include members of the FGF, EDA, BMP and Wnt signaling families [reviewed in Tucker and Sharpe, 2004]. As an example of how changes in the function of one of these pathways can lead to development of diastema teeth, we focus below on the role of the FGF signaling pathway in this process.
The FGF signaling pathway appears to mediate epithe-lial-mesenchymal interactions at several stages of tooth morphogenesis in mammals and other vertebrates [Thesleff and Sharpe, 1997; Jernvall and Thesleff, 2000; Mandler and Neubuser, 2001; Jackman et al., 2004]. In the developing mouse molar, at least five different FGF ligands (FGF3, FGF4, FGF8, FGF9, and FGF10) and two receptors (FGFR1 and FGFR2) are expressed in complex, overlapping patterns in the epithelium and/or mesenchyme [Niswander and Martin, 1992; Neubuser et al., 1997; Kettunen et al., 1998; Kettunen and Thesleff, 1998; Kettunen et al., 2000]. Molar development is thought to be initiated by signaling via FGF8 [Trumpp et al., 1999], but Fgf8 does not appear to have a function later in tooth development, because Fgf8 expression is not detected at later stages [Kettunen and Thesleff, 1998].
Subsequent development of the tooth is thought to depend on signaling via other FGFs (fig. 3). This hypothesis has been based primarily on gain of function studies in organ culture and gene expression analyses. Epithelial cell proliferation and morphogenesis throughout the cap and bell stages is stimulated by FGF3 and FGF10 produced in the dental mesenchyme from the bud stage onwards [Jernvall et al., 1994; Kettunen and Thesleff, 1998; Kettunen et al., 2000]. These FGFs signal to the epithelium by activating the 'b' isoforms of FGFR1 and FGFR2, which are expressed exclusively in the epithelium [Kettunen et al., 1998]. Conversely, FGF4 and FGF9 produced in the epithelium are presumed to be key mediators of enamel knot activity in coordinating tooth morphogenesis. These signals bind to and activate the 'c' isoforms of FGFR1 and FGFR2 expressed in the mesenchyme [Kettunen et al., 1998]. Their major function is to maintain Fgf3 and Fgf10 expression in the dental mesenchyme, which - as discussed above - is thought to be critical for sustaining tooth morphogenesis. Some of the downstream targets of this FGF signaling cascade have been identified (fig. 3). Of particular interest, two genes known to be involved in dental anomalies in humans - Msxl and Runx2 - are thought to be transcriptional targets of FGF signaling [Bei and Maas, 1998; Aberg et al., 2004]. In addition to being a mesenchymal target of FGF signaling, Runx2, the gene responsible for CCD, has been proposed to directly induce mesenchymal Fgf3 expression [Aberg et al., 2004]. Thus, Runx2 appears to be a critical link in the epithelial-mes-enchymal FGF signaling loop. In patients with CCD, which is caused by haploinsufficiency for RUNX2 [Mundlos et al., 1997], supernumerary teeth arise from the permanent teeth, representing a third dentition [Jensen and Kreiborg, 1990]. The underlying molecular mechanism is proposed to result from an incomplete resorption of the dental lamina of the secondary dentition [Lukinmaa et al., 1995]. In Runx2 homozygous null mutant mice, molar development ceases at the bud stage, a time during which there is normally strong expression of Runx2 in the dental mesenchyme [D'Souza et al., 1999]. However, in Runx2 heterozygote mice there appears to be the beginning of successional tooth development: lingual epithelial buds
with active Shh signaling are present [Wang et al., 2005]. Thus, in both mice and humans, an important role of the Runx2 protein appears to be prevention of excess budding of successional laminae.
Interestingly, both FGF receptor levels and Runx2 activity are modulated by Twist-1 [Bialek et al., 2004; Gue-nou et al., 2005], a transcription factor involved in Sae-thre-Chotzen syndrome, and patients with this condition have been reported to have dental anomalies [Goho, 1998]. The relief of a functional antagonism between Runx2 and Twist-1 proteins leads to the onset of osteoblast differentiation [Bialek et al., 2004], suggesting a potential mechanism for the formation of supernumerary
teeth in human CCD. Presently, experiments are underway to evaluate whether the formation of accessory tooth buds in Runx2 homozygous null mice are due to a relative overabundance of Twist-1. The latter may result in a prolonged survival of the dental lamina. Tooth development progresses normally in Runx2/Twist-1 double heterozygote mutant mice, suggesting that the in vivo genetic interaction between the two molecules is critical for tooth morphogenesis (fig. 4). Interactions between Runx2 and Twist-1 proteins may thus modulate a variety of events in both development and homeostasis.
For each of the major intercellular signaling pathways in development, antagonists have been identified. Signal ing via FGFRs is inhibited by a molecule called Sprouty (spry), which was first identified in a screen for mutations that affect tracheal branching in Drosophila melanogas-ter [Hacohen et al., 1998]. Because Sprouty is a negative feedback regulator of FGF signaling, the FGF pathway affects the expression of its own antagonist and thereby limits the range over which FGF signaling is active. When subsequent experiments in Drosophila showed that spry also regulates epidermal growth factor receptor signaling and other receptor tyrosine kinase pathways, the notion arose that spry is a general inhibitor of receptor tyrosine kinase signaling pathways [Casci et al., 1999; Kramer et al., 1999; Reich et al., 1999].
Four mouse genes have sequence similarity to Drosophila spry, and all have human orthologs [de Maximy et al., 1999; Minowada et al., 1999]. Three of the four mouse Sprouty genes, Spry1, Spry2, and Spry4, are expressed at various stages of embryonic development, whereas Spry3 expression has been detected only in the adult [Minowada et al., 1999]. As Sprouty gene expression is induced by FGF signaling, it is observed in association with FGF signaling centers throughout the embryo in numerous developing organs, including the brain, lungs, digestive tract, kidneys and limb buds [Minowada et al., 1999; Zhang et al., 2001]. Sprouty family members act in-tracellularly to negatively regulate FGF and other receptor tyrosine kinase signaling through diverse biochemical mechanisms, primarily via effects on the mitogen-ac-tivated protein kinase pathway [Dikic and Giordano, 2003; Guy et al., 2003; Kim and Bar-Sagi, 2004].
In the tooth buds that form in the wild-type embryonic diastema, the genetic program that normally controls progression from the bud to the cap stage is not active. One mechanism by which diastema bud development is normally suppressed is via inhibition of FGF gene expression, including Fgf4 in the enamel knot and Fgf3 in the dental mesenchyme. Sprouty genes are required to prevent diastema tooth development even though there is little or no FGF gene expression in wildtype diastema buds. It is likely that the normal function of Spry2 is to prevent the relatively low level of signaling via FGF10 produced in diastema bud mesenchyme from inducing/maintaining Shh expression. Likewise, the normal function of Spry4 in the mesenchyme is to prevent any epithelial FGF signals, including FGF4 and FGF9 produced in the adjacent M1 tooth germ, from inducing/maintaining Fgf3 expression. As a result of the combined activities of Spry2 and Spry4, the diastema bud regresses and there are no teeth in the adult dia-stema.
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