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Figure 1. Development of the apical end of mouse incisor in organ culture. (a) Schematic appearance of the incisor tooth in a sagittal section through the mouse mandible. Enamel (red) is covered by odontogenic epithelium (blue) that forms the cervical loop in the apical end of the tooth. Dentin is shown in green. (b) The appearance of the dissected incisor tooth of a 2-d-old mouse in stereomicroscope. (c and d) Histology of the apical end of the incisor showing the different epithelial layers in the cervical loop and the gradual differentiation from the apical to incisal direction of epithelial ameloblasts and mesenchymal odontoblasts depositing enamel (red) and dentin (green), respectively. (e–i) Development of the dissected apical end of a 2-d-old mouse incisor in organ culture for 7 d as seen in stereomicroscope. (j) H-E–stained section of the explant in i, and (m) higher magnification of ameloblasts and enamel. (k) Whole mount alizarin red staining of an explant (l) cultured for 5 d shows mineralization of the extracellular matrices formed in vitro. Abbreviations: a, ameloblast; be, basal epithelium; d, dentin; e, enamel; iee, inner enamel epithelium; o, odontoblast; p, pulp cell; si, stratum intermedium; sr, stellate reticulum. Bars: (b) 1 mm; (d and i) 500 μm; (j) 200 μm; (l) 700 μm; and (m) 100 μm.
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Figure 2. Analysis of the cell fate of cervical loop epithelial cells. DiI was injected in the center of the cervical loop and the explants were cultured for 5 d. (a) Appearance of the explant in phase-contrast and (b) fluorescent microscope. DiI-labeled cells have extended from the point of injection incisally, and the highest intensity of fluorescence is in the inner enamel epithelium corresponding to the location of rapidly dividing cells. Arrowheads show the point of injection of DiI at the onset of culture. Bar, 250 μm.
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Figure 3. Observations of cultured apical explants after removal of the cervical loop epithelium or inner enamel epithelium and ameloblasts. (a–f) After the removal of the cervical loop epithelium, no apical extension of the epithelium was seen during 9 d of culture. Epithelial cells differentiated and produced enamel matrix, which calcified (f). The arrows point to the cut end of the epithelium. (g–l) After removal of all dental epithelium except the cervical loop, new epithelium was produced by the cervical loop, which grew rapidly to incisal direction during 7 d of culture. Large arrowheads indicate the cervical loop. Small arrowheads point to the edge of the regenerating epithelium. Bars: (a–d and f–l) 300 μm; (e) 100 μm.
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Figure 4. Analysis of cell division in the dental epithelium by BrdU labeling. (a–c) After 3 h labeling, BrdU incorporating cells are located in the cervical loop (c) and inner enamel epithelium extending to the zone of terminal ameloblast differentiation (b). (d–i) After 24 (d–f) and 72 h of labeling (g–i), BrdU incorporation is seen also among the newly differentiated ameloblasts (arrows). Particularly high incorporation was evident in the zone of inner enamel epithelium (arrowheads). Bars: (a, d, and g) 200 μm; (b, c, e, f, h, and i) 100 μm.
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Figure 5. Localization of stem cells by BrdU pulse–chase analysis and by anti-adenovirus receptor antibody (anti-CVADR). 3 h BrdU labeling was followed by 7 d chase period. (a and b) Labeled cells were detected in the cervical loop at the border between basal epithelium and stellate reticulum. (a and c) Labeled cells represent ameloblasts derived from the last cell division of inner enamel epithelial cells. Stellate reticulum cells in cervical loop expressed CVADR strongly (d and e). The expression in basal epithelium was at lower level. The staining at the border of the frozen section is due to nonspecific binding of antibodies. m, mesenchyme; be, basal epithelium; sr, stellate reticulum; black dots, basement membrane. Bars: (a and d) 200 μm; (b) 100 μm; (c and e) 50 μm.
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Figure 6. In situ hybridization analysis of Notch1, 2, 3, Serrate1, and lunatic fringe expression in the mandibular incisor of 2-d-old mice. (a–a′′) Notch1 is expressed in stellate reticulum in cervical loop (a) and in stratum intermedium and stellate reticulum in the zone of cell differentiation (a′′). (b–b′′) Notch2 is expressed in outer enamel epithelium and stellate reticulum in cervical loop (b) and in stratum intermedium and stellate reticulum in the zone of cell differentiation (b′′). (c–c′′) Notch3 is absent from the cervical loop (c), but is expressed in stellate reticulum and in the zone of cell differentiation (c′′). (d–d′′) Serrate1 is absent from the cervical loop (d), but is expressed in ameloblasts (d′′). (e-e′′) Lunatic fringe is expressed in basal epithelium in cervical loop continuing in the inner enamel epithelium (e′), and is downregulated in differentiated ameloblasts (e′′). Bar, 1 mm (χ). Bar, 200 μm (χ′ and χ′′).
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Figure 7. Localization of Fgf-3 and Fgf-10 and the receptors Fgfr1b and Fgfr2b (Kgfr) mRNA in the mandibular incisor of 2-d-old mice. (a and c) Fgf-10 is intensely expressed in the dental mesenchyme adjacent to cervical loop and inner enamel epithelium. (b and d) Fgfr1b is expressed throughout cervical loop epithelium with high intensity at the border between basal epithelial cells and stellate reticulum. (e) Fgf-3 is expressed in dental mesenchyme under the inner enamel epithelium in a more restricted area than Fgf-10. (f) Fgfr2b (Kgfr) is expressed in the cervical loop in similar locations as Fgfr1b. c and d show higher magnification of the cervical loop in a and b, respectively. Bars: (a, b, e, and f) 1 mm; (c and d) 200 μm.
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Figure 8. Effect of FGF10 on the growth of cervical loop epithelium. Beads releasing FGF-10 (a, c, and e) and BSA (b, d, and f) were placed in contact with cervical loop and inner enamel epithelium, and the explants were cultured for 48 h. A stimulation of growth of the cervical loop epithelium by FGF-10 beads is evident. (g and h) FGF-10 beads (large arrowheads) stimulated BrdU incorporation when placed on epithelium isolated from the apex of the incisor. The control BSA beads (no arrows) did not affect cell division. The stimulatory effect of FGF-10 was more pronounced when beads were placed on cervical loop epithelium (g, small arrowheads) than on inner enamel epithelium (h). Bars: (a–f) 250 μm; (g and h) 200 μm.
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Figure 9. Lunatic fringe mRNA expression in dental epithelium is stimulated by dental mesenchyme and by FGF-10. (a) The epithelium and mesenchyme from the apical end of the 2-d-old mouse incisor were separated and cultured as recombinants for 24 h. Stimulation of lunatic fringe expression was seen in the cervical loop epithelium and inner enamel epithelium in close contact with mesenchyme. (b) FGF-10 beads stimulated lunatic fringe expression when placed adjacent to cervical loop epithelium. (c) FGF-10 beads did not induce lunatic fringe expression when placed on the zone of differentiated ameloblasts. No specific expression was observed around BSA beads (large arrowhead). Red points in a represent the border of epithelial tissue. Green dots in b and c represent beads (the beads were detached during processing). Small arrowheads point to cervical loop epithelium. Bar, 200 μm.
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Figure 10. A model for the molecular mechanisms involved in the generation of the ameloblast cell lineage from stem cells. (a) Stem cell kinetics. The stem cell divides slowly and gives rise to one daughter cell remaining in the stem cell pool in the cervical loop, whereas the other daughter cell enters the zone of rapidly dividing inner enamel epithelial cells (transit-amplifying cell population). During rounds of divisions, these cells move toward the incisal direction and differentiate into ameloblasts forming enamel matrix. They are in close contact with dental mesenchyme expressing FGF-10, which stimulates the division of both stem cells and transit-amplifying cells (inner enamel epithelial cells), and FGF-3 stimulating division of transit-amplifying cells only. (b) Signaling pathways for cell fate specification in ameloblast cell lineage. Mesenchymal FGF-10 (green dots) stimulates lunatic fringe (blue dots) expression in the basal epithelium. The stem cells are located in the stellate reticulum expressing Notch1 (red stripes). Lunatic fringe modulates Notch signaling in the stem cells. When the daughter of a stem cell enters the zone of lunatic fringe expression in basal epithelium it will be incorporated to the ameloblast cell lineage interacting with adjacent mesenchymal cells. In the zone of differentiated cells (left), the Notch signaling pathway regulates interactions between ameloblasts expressing Serrate1 and stratum intermedium cells expressing Notch1. Abbreviations: a, ameloblast; be, basal epithelial cell; bm, basement membrane; d, dentin; e, enamel; iee, inner enamel epithelial cell; m, mesenchymal cell; o, odontoblast; si, stratum intermedium; and sr, stellate reticulum.
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