Development 122, 121-129 (1996) 121 Printed in Great Britain © The Company of Biologists Limited 1996 DEV3255 Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4 Anne Vaahtokari*, Thomas Åberg and Irma Thesleff Institute of Biotechnology and Institute of Dentistry, PO Box 56, SF-00014 University of Helsinki, Finland *Author for correspondence SUMMARY Apoptosis was localized in developing mouse teeth from interface indicating that epithelial-mesenchymal tissue initiation of morphogenesis to completion of cusp interactions prevent apoptosis. Epidermal growth factor formation by using modified TUNEL method for serial (EGF) and fibroblast growth factor-4 (FGF-4) inhibited sections and Nile Blue staining for whole mounts. Apoptosis apoptosis in the dental mesenchyme when applied locally was first detected at bud stage (E12-E13) in the central cells using agarose or heparin-coated acrylic beads, suggesting of the invaginating dental epithelium suggesting involve- involvement of these or related growth factors in the pre- ment of cell death in epithelial budding morphogenesis. vention of apoptosis in dental tissues in vivo. During cusp development, apoptotic cells were located in The spatially and temporally restricted distribution the enamel knots, which are transient clusters of dental patterns of apoptotic cells suggest multiple roles for pro- epithelial cells proposed to act as signaling centers grammed cell death in dental development. Of particular directing the morphogenesis of tooth cusps. Apoptosis was interest is the removal of the enamel knots by apoptosis also detected in other restricted epithelial cell populations which may terminate their tasks as regulators of the pat- including the dental lamina, ameloblasts, as well as stratum terning of the tooth cusps. The apical ectodermal ridge intermedium and stellate reticulum cells suggesting that (AER) of the limb bud has similar signaling characteristics the removal of these epithelial cells occurs by apoptosis. as the enamel knot, and it also undergoes apoptosis. Hence, Apoptotic cells, presumably osteoclasts, were also located apoptosis may be a general mechanism for the silencing of on the surfaces of the developing alveolar bone. embryonic signaling centers. When dissected E13 dental epithelium or mesenchyme were cultured in isolation, apoptotic cells were abundant Key words: Tunel, Nile Blue, enamel knot, tooth cusp, ameloblasts, throughout the tissues, whereas when cultured together, epithelial-mesenchymal interactions, osteoclasts, organ development, apoptosis was inhibited in both tissues close to their programmed cell death, mouse INTRODUCTION derived ameloblasts undergo terminal differentiation and start the secretion of dentine and enamel, respectively. The epi- The developing molar tooth is a good model for studying the thelial cells of the enamel organ, including ameloblasts, stratum regulation of organ morphogenesis (Fig. 1). The main stages intermedium and stellate reticulum cells disappear by the time of tooth development are presented in Fig. 1. At the site of the tooth erupts to the oral cavity (about 2-3 weeks after birth). future tooth anlage, the oral epithelium thickens indicating the Programmed cell death (PCD) probably constitutes an initiation of tooth development (E10-E11 for mouse lower first important mechanism of embryonic development. In PCD, molars). At bud stage (E12-E13), the epithelium invades the death of a cell is triggered by the appearance or loss of an underlying neural crest-derived mesenchyme and forms an external signal, leading to the activation of an internal cell epithelial bud, around which the mesenchymal cells condense. death program (Schwartz and Osborne, 1993). One form of As a result of epithelial folding, cap stage is reached (E14-E15) PCD is apoptosis (Kerr et al., 1972; Wyllie, 1987), and its char- and a transient structure called the primary enamel knot acteristic morphological features include fragmentation of the appears in the middle of the enamel epithelium. At bell stage cell into membrane-bound apoptotic bodies, nuclear and cyto- (E16-P7), secondary enamel knots appear at the tips of the plasmic condensation, and endonucleolytic cleavage of DNA developing cusps. The enamel knots have been suggested to (Steller, 1995). regulate the shape of the tooth germ and, in particular, cusp Although it is often stated in the literature that apoptosis morphogenesis (Jernvall et al., 1994). The epithelial dental controls cell number and tissue shape, during organ morpho- lamina, which connects the tooth to the oral epithelium, genesis these processes have been rather poorly studied. For becomes disrupted during advancing morphogenesis. Around example, apoptosis in the developing kidney was reported birth, the mesenchymal-derived odontoblasts and epithelial- relatively recently despite intensive research on kidney mor- 122 A. Vaahtokari, T. Åberg and I. Thesleff EARLY BELL (E16-NB) CAP (E14-E15) BUD (E12-E13) oe INITIATION sek (E10-E11) dl sr oe oee de dc si de pab jm iee dm A. B. C. D. DPdm dm pob pek LATE BELL (P1-P7) c sr sr dm E. F. G. si ab enamel dentin ob dm Fig. 1. Schematic representation of different stages of mouse tooth development until completion of cuspal morphogenesis. Dental follicle, which surrounds the tooth germ from cap stage until the crown morphogenesis is completed, is omitted for the sake of clarity. Drawings of (A,E) sagittal sections, (B-D) frontal sections, (F) a transversal section, (G) a higher magnification of a cusp. ab, ameloblasts; c, cusp; dc, developing cusp; de, dental epithelium; dm, dental mesenchyme; dl, dental lamina; iee, inner enamel epithelium; jm, jaw mesenchyme; ob, odontoblasts; oe, oral epithelium; oee, outer enamel epithelium; pek, primary enamel knot; sek, secondary enamel knot; si, stratum intermedium; sr, stellate reticulum. phogenesis (Koseki et al., 1992; Coles et al., 1993). In the day 10 (E10) to postnatal day 7 (P7) mice (CBA × NMRI) were kidney, apoptosis is speculated to be associated with cell dissected (vaginal plug = day 0) and fixed overnight in 4% differentiation and to match the numbers of epithelial and mes- paraformaldehyde (PFA) in phosphate-buffered saline, pH 7.3 (PBS). enchymal cells (Coles et al., 1993). Tissues from postnatal mice were decalcified in 0.3 mM ethylenedi- Apoptosis has been proposed to have a role in the produc- aminetetraacetic acid (EDTA), pH 7.0 for about 3 weeks at 4°C. After the tissues were embedded in paraffin and sectioned, they were either tion of tooth anomalies associated with cleft lip and palate used for terminal deoxynucleotidyl transferase (TdT)-mediated nick (Sharma and Kharbanda, 1991). However, as there is little end labeling (TUNEL) or for localization of tartrate resistant acid information concerning apoptosis in tooth development, and phosphatase (TRAP) as described by Reponen et al. (1994). the existing studies have focused on apoptosis in postnatal ameloblasts (Joseph et al., 1994 and references therein), we TUNEL staining have now analyzed apoptosis in detail during mouse tooth To detect apoptotic cells from tissue sections, we used digoxigenin- development. based modification of the original TUNEL method introduced by There is an increasing amount of data suggesting that growth Gavrieli et al. (1992). TUNEL is based on the enzymatic incorpora- ′ factors acting through tyrosine kinase receptors are important tion of labeled nucleotides into the free 3 ends of DNA. The apoptotic survival factors suppressing apoptosis (Collins et al., 1994). cells due to their DNA fragmentation get heavily labeled and can be specifically detected with antibody staining. Several steps in our There is even some evidence indicating that as a default, all procedure were applied from the in situ hybridization method cells undergo apoptosis unless they are rescued by survival described by Wilkinson (1992). factors (Raff, 1992; Steller, 1995). As epidermal growth factor The paraffin sections were heated at 60°C for 30 minutes where- (EGF) and fibroblast growth factor-4 (FGF-4) are expressed in after they were deparaffinized in xylene twice for 10 minutes. They tooth germs, we investigated their possible involvement in the were dehydrated in 100% ethanol for 5 minutes, then transferred to regulation of apoptosis in cultured dental tissues (Snead et al., ethanol/acetic acid (2:3) for 20 minutes, incubated in 0.5% H2O2 in 1989; Niswander and Martin, 1992; Heikinheimo et al., 1993). MeOH for 30 minutes, and rehydrated with 94%, 70%, 50% and 30% MeOH in PBS for 5 minutes each. After washing the sections in PBS containing 0.1% Triton X-100 (PBT) twice for 5 minutes each, they were incubated in 50 mM Tris-HCl, 5 mM EDTA, pH 8.0, contain- MATERIALS AND METHODS ing 7 µg/ml proteinase K (Sigma P-4914) for 10 minutes and washed twice in PBT for 5 minutes each. Preparation of tissues for TUNEL and localization of TRAP The sections were fixed in fresh 4% PFA in PBS for 20 minutes Heads, jaws, lower first molar regions and limb buds from embryonic and rinsed twice with PBT for 5 minutes each. The sections were Apoptosis in tooth development 123 labeled for 1 hour at 37°C in a labeling mix (TdT buffer (100 mM and murine FGF-4 are 82% homologous in amino acid sequence. The cacodylate, pH 6.8, 1 mM CoCl2, 0.1 mM dithiothreitol, 100 µg/ml beads were rinsed in PBS and placed in intimate contact with the BSA) containing 1 nM dig-11-dUTP (Boehringer Mannheim, 1093 separated dental mesenchyme or epithelium. Sometimes more than 088), 1 nM dATP, 0.5% 3-[(3-cholamidopropyl)-dimethylammonio]- one mesenchyme or epithelium were cultured together because then 1-propanesulfonate (CHAPS) (Sigma C-3023), and 27 U/100 µl the tissues grow better. We also cultured separated mesenchymes and terminal transferase (TdT) (Promega)). The labeling mix used for the epithelia either together or in isolation. negative controls did not contain any TdT. The reaction was stopped The explants were cultured on 0.1 µm pore-size Nuclepore filters by incubating the slides in 300 mM NaCl, 30 mM sodium citrate, (General Electron) placed on metal grids in Eagle’s Minimum 0.1% CHAPS for 15 minutes with gentle rocking.