APOPTOSIS IN THE DEVELOPING TOOTH - ASSOCIATION WITH AN EMBRYONIC SIGNALING CENTER AND SUPPRESSION BY EGF AND FGF-4

Apoptosis was localized in developing mouse teeth from initiation of m orphogenesis to completion of cusp formation by using modified TUNEL m ethod for serial sections and Nile Blue staining for whole mounts. Apo ptosis was first detected at bud stage (E12-E13) in the central cells of the invaginating dental epithelium suggesting involvement of cell d eath in epithelial budding morphogenesis. During cusp development, apo ptotic cells were located in the enamel knots, which are transient clu sters of dental epithelial cells proposed to act as signaling centers directing the morphogenesis of tooth cusps. Apoptosis was also detecte d in other restricted epithelial cell populations including the dental lamina, ameloblasts, as well as stratum intermedium and stellate reti culum cells suggesting that the removal of these epithelial cells occu rs by apoptosis. Apoptotic cells, presumably osteoclasts, were also lo cated on the surfaces of the developing alveolar bone. When dissected E13 dental epithelium or mesenchyme were cultured in isolation, apopto tic cells were abundant throughout the tissues, whereas when cultured together, apoptosis was inhibited in both tissues close to their inter face indicating that epithelial-mesenchymal tissue interactions preven t apoptosis. Epidermal growth factor (EGF) and fibroblast growth facto r-4 (FGF-4) inhibited apoptosis in the dental mesenchyme when applied locally using agarose or heparin-coated acrylic beads, suggesting invo lvement of these or related growth factors in the prevention of apopto sis in dental tissues in vivo. The spatially and temporally restricted distribution patterns of apoptotic cells suggest multiple roles for p rogrammed cell death in dental development. Of particular interest is the removal of the enamel knots by apoptosis which may terminate their tasks as regulators of the patterning of the tooth cusps. The apical ectodermal ridge (AER) of the limb bud has similar signaling character istics as the enamel knot, and it also undergoes apoptosis. Hence, apo ptosis may be a general mechanism for the silencing of embryonic signa ling centers.