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Sonic Hedgehog Signaling is Important in Tooth Root Development

¹ Section of Molecular Craniofacial Embryology,
² Section of Cellular Physiological Chemistry, Department of Maxillofacial Biology, Graduate School, Tokyo Medical and Dental University, and
³ 21st Century Center of Excellence (COE) Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8549, Japan

View larger version (90K): [in a new window]   Figure 1. Expression of Shh and its target genes, Ptc1 and Gli1, in the developing tooth root. Whole-mount in situ hybridization revealed that Shh (A), Ptc1 (B), and Gli1 (C) were expressed at P8 in the outline of the epithelial edge of the growing medial and distal roots in the lower first molar. Tooth buds are oriented with the bottom side up and the medial side on the left. Original color images were digitally converted to gray scale. Scale bar: 200 µm.

View larger version (159K): [in a new window]   Figure 2. Localization of Shh signaling genes in the developing lower molar. The buccal side is to the left. P1 (A-G*), P5 (H-N*), and P9 (O-U*). In situ hybridization of Shh (A, A*, H, H*, O, O*), Ptc2 (B, B*, I, I*, P, P*), Ptc1 (C, C*, J, J*, Q, Q*), Smo (D, D*, K, K*, R, R*), and Gli1 (E, E*, L, L*, S, S*). Immunohistochemistry for PCNA (F, F*, M, M*, T, T*) and BrdU (G, G*, N, N*, U, U*). (A*-U*) Higher magnification of boxed areas in (A-U). The expression of Shh signaling genes gradually became localized to the proliferating apical end of the tooth root. Ptc1 and Gli1 transcripts were found in areas overlapping extensively with the cell proliferation markers PCNA and BrdU. AB, ameloblast; B, bone; D, dentin; DM, dental mesenchyme; E, enamel; HERS, Hertwig’s epithelial root sheath; IEE, inner enamel epithelium; IR, incisor root; OB, odontoblast; SR, stellate reticulum; T, tongue; WF, whisker follicle. Scale bars: 200 µm in (A) for (A-U) and 50 µm in (A*) for (A*-U*).

View larger version (126K): [in a new window]   Figure 3. Repressed cell proliferation around the HERS of homozygous Ptcmes mutants. The buccal side is to the left. (A,B) BrdU incorporation (four-hour labeling) in the apical end of the developing lower first molar of a control littermate (A) and a mutant (B) at P7. Several BrdU-positive cells were identified along the HERS in the control (arrows in A), but not in the mutant. (C,D) Hematoxylin and eosin staining of a control littermate (C) and a mutant (D) at P9. Double-headed arrows indicate the distance between the cemento-enamel junction and the apical end of root dentin. AB, ameloblast; B, bone; Ctrl, control; D, dentin; DF, dental follicle; E, enamel; HERS, Hertwig’s epithelial root sheath; MN, mandibular nerve; OB, odontoblast. Scale bars: 50 µm in (A) and 100 µm in (C).

View larger version (43K): [in a new window]   Figure 4. Disturbance of tooth eruption and root formation in homozygous Ptcmes mutants at P28. The medial side is to the left in (A-E). (A,B) Lingual view of the lower molars of a control littermate (A) and a mutant (B). Soft tissues were carefully removed. Eruption of all molars of the mutant was delayed, especially the third molar (black arrow in B), which had not reached the occlusal plane (white arrowhead in B). (C-E) Lingual view of the extracted first (C), second (D), and third (E) molars. In each panel, the tooth on the left is from the control, and the one on the right is from the mutant. All roots were shorter in the mutant than in the control, especially the third molar (white arrow in E). Lines indicate the measured position. (F,G) Mean root length (F) and width (G) ± SD of medial (M) and distal (D) roots of the lower first (M1) and second (M2) molars and single root of the third (M3) molar of controls (n = 11) and mutants (n = 7). Asterisks (*) indicate values significantly different from controls according to Student’s t test (P < 0.001). (H,I) BrdU incorporation (24-hour labeling) was detected inside the apical region of the lower third molar of the mutant (arrowhead in I), whereas it was not observed in the control (H). The basal layer of the oral mucosa in the same section was observed as a positive control (inset in H). Almost no cementum was deposited in the mutant in contrast to the control (double-headed arrow in H). AF, apical foramen; BL, basal layer; C, cementum; Ctrl, control; D, dentin; DL, dermal layer; P, dental pulp; PL, periodontal ligament. Scale bars: 600 µm in (A) for (A-E) and 50 µm in (H).