a new era by discovery of the regulatory role of growth factors in dental morphogenesis. Thesleff and colleagues5,33,44,45 have reviewed recent advances in this area of developmental biology. The earliest growth factor signal emanating from the presumptive dental lamina epithelium is bone morphogenetic protein 4 (BMP-4)5,46 (Fig 1-11). Epithelial cells make BMP-4 until the cap stage, when the production of BMP-4 shifts to the condensed ectomesenchymal cells. Soon thereafter, a new bone morphogenetic protein (BMP-2) appears in the epithelial cells. These shifts in BMP expression may account for the transfer of instructional activity from the epithelium to the dental papilla ectomesenchyme at the cap stage.
It has been proposed that BMP-4 activates Msx genes in the adjacent ectomesenchymal cells46 (see Fig 1-11). The Msx genes are “muscle segment” members of the homeobox genes (regulators of segmentation) that have been implicated as regulators of the mesiodistal axis of tooth bud placement. Msx gene products are believed to be transcription activators that regulate the expression of BMPs, syndecan, and peptide growth factors in the condensing ectomesenchyme (see Fig 1-11). At the bell stage, Msx2 is active in secondary enamel knots (EKs) and in the dental papilla.
Fig 1-11 Proposed model of molecular mechanisms in early tooth bud development, illustrating the role of bone morphogenetic protein 4 (BMP-4) in activating Msx gene expression and a cascade of differentiation within the underlying ectomesenchyme. With the activation of Msx genes, the inductive potential is transferred to the dental ectomesenchyme. Reciprocal interactions involving signaling growth factors, matrix molecules, and cell surface receptors regulate cell differentiation. Enamel knot signaling centers appear in the enamel organ prior to cusp formation. (FGF-8) Fibroblast growth factor 8. (Based on the findings of Vainio et al.38,46)
Transcription products of Msx1 function during later stages of tooth development, possibly regulating the differentiation of ameloblasts and odontoblasts.20 Animals that lack the Msx1 gene fail to develop teeth.22
An especially important discovery was the identification of the enamel knot as a signaling center within the enamel organ.24,25,47 The enamel knot, a component of the enamel organ previously believed to be unimportant, has achieved prominence as a potential regulatory center of cell proliferation involved in cusp formation. The EK is a small group of closely packed, nondividing cells located adjacent to the IEE, and, in a single-cusped tooth, close to the center of the enamel organ (Figs 1-6 and 1-12).
Fig 1-12 Possible role of the enamel knot (EK) in cusp formation. (arrows) Direction of growth. During the cap stage, the epithelium grows laterally around the dental mesenchyme. A single EK coordinates the development of the early cap stage. In multicusped teeth, secondary EKs are formed over future cusps to coordinate development during the late cap stage to the bell stage. (Adapted from Jernvall et al24 with permission.)
The earliest sign of EK formation appears to be the localized expression of BMP-2 and BMP-7 in epithelial cells of the dental lamina and enamel organ. In situ hybridization techniques demonstrate that EK cells produce fibroblast growth factor 4 (FGF-4), several bone morphogenetic proteins (BMP-2, BMP-4, and BMP-7), and sonic hedgehog (Shh) protein.26,27,48 Fibroblast growth factor 4 is a potent stimulator of epithelial and mesenchymal cell proliferation.25 Epithelial and ectomesenchymal cells adjacent to the EK continue to divide in response to FGF-4, while the EK cells, which produce FGF-4, remain nondividing. The cells of the EK are retained in the G1 phase of the cell cycle by a high level of expression of the cyclindependent kinase inhibitor, p21. Bone morphogenetic protein 4 may regulate EK activity via its ability to sustain high levels of p21 expression.47
By secreting growth factors, the EK promotes cell proliferation along a proximodistal axis, leading to the formation of a cusp. In this sense, the EK is akin to the apical ectodermal ridge that controls limb bud development. In establishing coronal form, embryonic dental tissues follow a pattern of polarized growth. Cells in the cervical loop proliferate and move away from older differentiating cells located nearer to the cusp tip.
The best example of polarized growth is found in the developing limb. The specific genes that participate in determining the anteroposterior axis of developing limbs are also expressed in cap to bell stage tooth buds. The Shh gene responsible for polarizing activity in developing limbs is active in the enamel knot (see Fig 1-12) and in differentiating odontoblasts and ameloblasts.48 Proof that genes that regulate polarized growth, such as Shh, are active in the tooth bud was obtained when tooth buds were grafted to developing limbs. The grafted tooth buds induced the formation of additional digits, revealing a capacity for polarizing growth in an anteroposterior axis.48
In multicusped teeth, secondary EKs are formed over the tips of the future cusps (see Fig 1-12). In mouse molar teeth, the EKs remain active for about 24 hours before undergoing apoptosis.49 Programmed cell death is also responsible for the removal of the dental lamina after tooth bud formation.
Growth and Differentiation Factors That Regulate Tooth Formation
Bone morphogenetic factors, Shh, and FGFs are also important during the later stages of tooth development.47 Both BMP-2 and BMP-7 are expressed in the IEE across from the differentiating odontoblasts, suggesting that they may have an inductive role. Secretory odontoblasts express BMP-4 and BMP-7, while BMP-5 appears to be restricted to fully differentiated ameloblasts. Bone morphogenetic protein 3 is localized in the cells of the dental follicle.
Activin A, a protein structurally related to BMPs and a member of the TGF-β superfamily of cytokines, has been implicated in signaling during tooth development.50 Mice deficient in activin A have craniofacial abnormalities and failure of incisor tooth development.
Vitamin A and its metabolic derivatives, retinol and retinoic acid (RA), are essential regulators of epithelial cell proliferation and differentiation and have special impact on tooth development.51–54 The importance of vitamin A in the initiation of tooth development was underscored by the observation that when endogenous vitamin A is blocked in vitro, the dental lamina fails to develop in organ cultures of mouse embryonic mandibles.55 Early studies of the effect of vitamin A on tooth development showed that a deficiency of the metabolite leads to defective enamel and dentin.56 In contrast, excessive vitamin A increases the chance for tooth bud fusion and/or the formation of supernumerary teeth.57,58
In organ cultures of embryonic mandibular explants, retinol and retinoic acid increase epithelial proliferation and stimulate the formation of extra tooth buds. Retinoic acid exerts its effect by binding to nuclear transcription factors (RA receptors [RARs]) located near retinoid response elements on various target genes, one being the gene that produces epidermal growth factor (EGF) (Figs 1-13a and 1-13b).59 Retinoic acid also increases the expression of midkine (MK) protein, a regulator of cell proliferation.
Cellular retinol-binding proteins