Continued research of the signaling events initiated by growth factors and matrix molecules will soon lead to a more complete understanding of tooth development. According to Slavkin,73 “Recent advances towards identifying epigenetic signals such as growth factors, regulatory or homeotic genes, and the significant advances towards understanding how cis- and trans-regulating elements control differential gene expression during development provide enormous optimism for future research in craniofacial genetics and developmental biology.”
Establishing Coronal Form (Cusp Formation)
As noted earlier, the three-dimensional plane of the IEE basal lamina sets the position of the dentinoenamel junction and thus the anatomic shape of the crown. From the cap stage, the enamel organ continues to increase in size until it assumes a bell-shaped structure, almost completely enclosing the dental papilla (see Fig 1-1). The three-dimensional shape of the enamel organ, at various stages of its development, has been precisely reconstructed from serial sections of human embryos. In extensive studies of human embryos, Ooe74 has demonstrated that secretion and mineralization of dentin and enamel matrices begin only after the shape of the crown has been determined in soft tissues.
Numerous factors under genetic control, including rates of cell division, assembly of cytoplasmic contractile filaments in differentiating preameloblasts, and the osmotic pressure of the surrounding tissues, act to shape the three-dimensional topography of the basement membrane between the IEE and the dental papilla. Cusp outline is set by the three-dimensional folding of the IEE basement membrane, setting the position of the future dentinoenamel junction. Cells in both the preameloblast and preodontoblast compartments must stop dividing to differentiate into matrix-producing ameloblasts (enamel) and odontoblasts (dentin) (Fig 1-15). Proliferation is controlled from primary and secondary enamel knots established over the tips of the future cusps. The FGF-4 and EGF produced by the nondividing cells of the EK may diffuse laterally to regulate cell proliferation in the IEE and the underlying preodontoblasts (see Fig 1-12).
Fig 1-15 Proliferation of preodontoblasts and preameloblasts from undifferentiated precursors in the dental papilla and inner enamel epithelium located in the cervical loop area. Cell cohorts leave the proliferation compartment and differentiate into mature secretory cells. Odontoblast differentiation and dentin deposition occur slightly in advance of ameloblast differentiation and enamel matrix secretion.
Apoptosis of epithelial cells in the EK terminates cusp growth.49 As the enamel knot begins its apoptotic decline, its function is transferred to the stratum intermedium. Progressing away from the tip of the cusp, in the proximodistal direction, a wave of signaling activity occurs in the cells of the stratum inter-medium that promotes the cell proliferation necessary to complete the morphodifferentiation of the bell-shaped crown.
Cell division at the cervical loop extends the size of the enamel organ until it reaches its mature state as a bell-shaped organ almost encompassing the dental papilla. Harada et al75 have demonstrated the presence of stem cells in the stellate reticulum of the cervical loop. Each division of a stem cell creates two daughter cells; one remains within the stem cell pool while the other cell enters the transit-amplifying pool (preameloblasts) within the IEE. A signaling pathway involving Notch and its ligand (Lunatic fringe) plays a central role in determining daughter cell entry into the differentiation pathway.75,76 Odontoblasts differentiate slightly in advance of ameloblasts, forming a thin layer of predentin prior to the start of enamel secretion.
Cell migration
Embryonic development involves orderly and precisely timed cell migrations. In many cases, cells must move over long distances. Some migrations contain large cohorts of cells moving over relatively long distances, as in the migration of neural crest cells from specific sites in the neural tube of the head region to their final destination in the developing face and jaws. Another example is the migration of pigment cells from the neural crest to sites throughout the epidermis. Tooth development requires the migration of neural crest ectomesenchyme to appropriate locations in the developing jaw. During root development, cells of the dental sac migrate toward the newly deposited dentin surface prior to cementogenesis.
For decades, developmental biologists sought answers to the following questions: What is the basis of cell motility? What guides a migrating cell to its ultimate destination? Although the answers to these questions are still incomplete, rapid progress is being made in understanding the molecular basis of cell migration. Directed cell locomotion is a complex process. It requires plasma membrane cycling or flow, the interaction of cell surface integrins with components of the extracellular matrix as well as the cytoskeleton, and the contraction of actin and myosin filaments.77,78 It also requires receptor-ligand signaling systems to detect and respond to gradients of chemotactic molecules.
Some cells types are relatively stationary, while other types engage in locomotion (neutrophils and lymphocytes).79 Transmigration through the extracellular matrix is a result of the cell’s capacity to explore its immediate environment. It does this through the extension of probing cytoplasmic processes (lamellae and filopodia).80 Lamellae are flat folds of cytoplasm sent out across a broad area, while filopodia are narrow fingerlike protrusions (Fig 1-16).
The extension and retraction of lamellae and filopodia are, in part, responses to two fundamental properties of the cell: the continuous turnover of the plasma membrane, and the contractility of cytoplasmic microfilaments. When cell processes from a region of the cell boundary make adhesive contact with a substrate, cytoplasmic polarity is established toward the substrate, and new membrane is transported toward that surface. This region of the cell surface has the potential of becoming the leading edge if there is no impediment to prevent the cell from moving forward in that direction. New membrane is added to the leading edge of the cell and retrieved toward the center of the cell.
Fig 1-16 Changes in shape and cell-to-substrate contacts made by chick heart fibroblasts explanted onto plastic culture dishes. (A) In the early phase of migration, the cells exhibit a clear leading lamella devoid of dense focal contacts. Only close contacts are made at this stage. (B) With time, the cells establish filopodia and focal contacts at the leading edge. A tail of trailing cytoplasm is characteristically found on migrating fibroblasts. (C) After 3 days in culture, most cells no longer have the migratory phenotype, no leading lamella is observed, and many well-developed focal adhesions are present in many regions of the cells. (Adapted from Couchman and Rees81 with permission from The Company of Biologists.)
It has been calculated that the lipid phase of the plasma membrane of a fibroblast turns over in about 50 minutes. Some intramembrane proteins are caught up in this flow, while others remain in place because of their association with the internal cytoskeleton or with extracellular substrates.
Protrusion of lamellae and filopodia at the leading edge is driven by rapid polymerization of actin filaments (see chapter 11 for a discussion of actin filament formation). Assembly of linear actin bundles may push the membrane outward or cause an increase in local hydrostatic pressure to deform the membrane outward at the leading edge. Because calcium triggers actin polymerization, it has been proposed that