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First published online 16 December 2004
doi: 10.1242/dev.01541

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Coordination of trigeminal axon navigation and patterning with tooth organ formation: epithelial-mesenchymal interactions, and epithelial Wnt4 and Tgfß1 regulate semaphorin 3a expression in the dental mesenchyme

¹ Division of Anatomy and Cell Biology, Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, Norway
² Department of Experimental Medicine and Cancer Research, The Hebrew University, Jerusalem, 91120, Israel
³ Laboratories of Integrated Biology, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan
⁴ Group of Developmental Neurobiology, Division of Biological Science, Nagoya University Graduate School of Science, Chikusa-ku, Nagoya, 464-8602, Japan
⁵ Biocenter Oulu and Department of Biochemistry, Faculties of Science and Medicine, University of Oulu, 90014, Finland
⁶ Department of Biochemistry and Molecular Biology, Graduate School of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

View larger version (145K): [in a new window]   Fig. 6. Wnt4 and Tgfß1 induce Sema3a in the mandibular and dental mesenchymes. Bright- and dark-field images. (A1-B2) Fgf8 induces Pax9 in the presumptive molar mesenchyme at E10, while no effects on Sema3a expression are observed. (C1-E2) A prominent Sema3a expression is seen around the Wnt4-producing cells in the E10.5 and E11 mandibular mesenchyme and E12 dental mesenchyme explants. (F1,F2) Endogenous expression of Sema3a in E12.5 dental mesenchyme explants. (G1,G2) Tgfß1-releasing bead (arrow indicates the site where the bead was located) stimulates Sema3a expression in the dental mesenchyme area of E12.5 lower jaw mesenchyme explants. (J1-K2) Wnt4 cells stimulate Msx1 in the proximal presumptive molar mesenchyme at E10.5, while no expression is observed around the NIH3T3 cells. (L1-P2) No Sema3a stimulation is seen in the mesenchyme surrounding the Wnt6-producing or control NIH3T3 cells or around the Fgf9- or BSA-soaked beads. Wnt6 cells show some endogenous Sema3a hybridization signal. (H1-I2,Q1-Q2) Bmp4, Fgf4 and Fgf9 induce the expression of target genes in control explants. Scale bars: 100 µm. c, cell cluster.

View larger version (79K): [in a new window]   Fig. 7. Tgfß1 stimulates proliferation of the dental mesenchymal cells as detected by BrdU incorporation from tissue sections (A,B) and whole mounts (C). The E12 mandibular mesenchyme or dental recombinant explants were cultured for 24 hours with protein-soaked agarose beads. Increased cell proliferation is seen in the mesenchymal cells next to the Tgfß1 (A-C) and control Fgf2 (D)-releasing beads, as well as cells next to the dental epithelium (E12), which also shows BrdU incorporation. Near the control BSA (1 mg/ml) bead (E), hardly any BrdU-positive cells are seen. B shows proliferating cells in A at a higher magnification. Arrows indicate the first molar mesenchyme region where the beads were located. (G,H) Induction of tubulogenesis in isolated E11 metanephric mesenchyme by Wnt4- and Wnt6-expressing cells after 5 days in culture. NIH3T3 control cells do not support survival and differentiation of the mesenchyme. (I) Stimulation of Gli1 expression in E11 mandibular mesenchyme by Shh-containing beads. b, bead; c, cell cluster; e, dental epithelium; m, dental mesenchyme. Scale bars: 200 µm in A,E,I; in C, 100 µm for C,D; 50 µm in B; in H, 500 µm for C,F,H.

View larger version (108K): [in a new window]   Fig. 1. Sema3a regulates the timing of tooth innervation and dental axon guidance and patterning. Expression of Sema3a in the embryonic mandibular molar tooth germ (A1-A5) at the epithelial thickening (E11.5) (A1), early bud (E12.5) (A2), bud (E13.5) (A3), cap (E14.5) (A4) and bell (E18.5) (A5) stages compared with the localization of nerve fibers in corresponding stages of wild-type (Sema3a+/+) (B1-B5) and Sema3a null mouse embryos (Sema3a-/-) (C1-C5) using peripherin antibodies. (A1-A5) The `molar nerve' is indicated by arrows in B2. b, developing alveolar bone; cdm, condensing dental mesenchyme; cm, condensed dental mesenchyme; de, dental epithelium; dp, dental papilla mesenchyme; df, dental follicle; tn, trigeminal inferior alveolar nerve; pm, presumptive dental mesenchyme. Arrowheads mark nerve fibers. Scale bars: 100 µm.

View larger version (172K): [in a new window]   Fig. 2. Npn1 mediates Sema3a signaling in dental axons. Frontal sections of E13.5 trigeminal ganglia (A,B) and tooth germs (C,D). Bright- (A1,B1) and dark-field (A2,B2) images. (A1-B2) A prominent Npn1 expression is seen in the trigeminal ganglion, while no specific expression of Npn2 is observed. (C) Npn1 immunoreactivity is seen in the dental nerve fibers (arrows) next to the condensed dental mesenchyme (dm) and in the trigeminal superior alveolar nerve trunk (sn). (D) E12.5 Npn1-/- heads analyzed by peripherin antibodies show that ectopic nerve fibers are prematurely present next to the dental epithelium and in the area of the condensing dental mesenchyme (arrows). Trigeminal inferior (in) and superior alveolar nerve trunks are abnormally defasciculated. Scale bars: 100 µm.

View larger version (122K): [in a new window]   Fig. 3. Expression of Sema3a in 1- (PN1) and 4-day postnatal (PN4) first (I), second (II) and third (III) lower molar tooth germs. Bright-(A1,C1) and dark-field (A2,C2) images of sagittal sections as well as (B) three-dimensional reconstruction of Sema3a (in red) expression in PN1 first molar tooth germ shown from below. Sema3a is absent from the sites of the secondary anterior (mesial) and posterior (distal) secondary apical foramina. The inner dental epithelium (gray) is 30% transparent to better visualize Sema3a expression in the dental papilla around the secondary apical foramina. Three-dimensional reconstruction was rendered using a perspective camera view. Scale bars: 200 µm. White arrows indicate Sema3a expression in dark-field images. a, developing alveolar bone; cl, cervical loop; d, dentin; e, enamel; dp, dental papilla; ode, outer dental epithelium; p, developing pulp floor; sr, stellate reticulum.

View larger version (155K): [in a new window]   Fig. 4. Ngf, Lanr, Gdnf, Ncam and Net3 expression is not altered in the Sema3a-/- tooth. Bright- and dark-field images of frontal sections of the E14-late bud/early cap stage wild-type (A1-A2,C1-C2,E1-E2,G1-G2,I1-I2) and Sema3a mutant (B1-B2,D1-D2,F1-F2,H1-H2,J1-J2) upper and lower first molar tooth germs. Abbreviations: de, dental epithelium; dp, dental papilla. Scale bar: 100 µm.

View larger version (146K): [in a new window]   Fig. 5. Epithelial-mesenchymal interactions regulate Sema3a expression in the mandibular and dental mesenchyme. Expression of Sema3a in the E10.5 head (A1,A2) and E11.5 (E1,E2) and E12.5 (I1-I2) mandibular molar tooth germs, as well as in cultured mandibular and dental mesenchymes and tissue recombinants analyzed by in situ hybridization from frontal sections after 24 hours (B1-D2,F1,F2,H1,H2,J1,J2,K1,K2), 2 days (G1-G2) and 3 days (L1-L2) of culture. Bright- and dark-field images. (B1-B2) No specific Sema3a expression is seen in the E10.5 mandibular mesenchyme cultured alone without epithelium (compare also images in Fig. 5J1-L2 cultured with BSA-soaked beads and Wnt6 and NIH3T3 cells), while in the cultured intact mandible a prominent mesenchymal Sema3a expression is present (C1-C2). (D1-D2) E10.5 oral epithelium has induced Sema3a in the underlying E10.5 molar area of mandibular mesenchyme, which is devoid of Sema3a (arrows in A1). (F1,F2) In cultured E11 mandibular mesenchyme, Sema3a transcripts are largely absent from the presumptive molar area (arrow), whereas the remaining Sema3a is seen in the deep aboral mesenchyme. (G1-G2,L1-L2) Sema3a induction is observed next to the E11 and E12 dental epithelia, which were placed onto the proximal, molar region of E10.5 mandibular mesenchyme. (H1-K2) A prominent Sema3a expression is seen in the E11 jaw and E12 dental mesenchyme under the E11 and E12 dental epithelia, respectively, while some endogenous Sema3a is observed in the dental mesenchyme explant. The expression patterns of Sema3a in these homochronic recombinant explants correlate with the Sema3a expression in in vivo developed teeth (compare E1,E2 with I1,I2). de, dental epithelium; dm, dental mesenchyme; m, mandibular mesenchyme; Md, mandibular process, Mx, maxillary process; oe, oral epithelium. Scale bars: 100 µm (200 µm for D2).

View larger version (30K): [in a new window]   Fig. 8. Schematic model for coordination of early tooth organogenesis and establishment of nerve supply by epithelial-mesenchymal interactions. The Sema3a exclusion areas (in red) regulate timing of tooth innervation and the innervation pattern. Prior to the histological onset of tooth formation (E10.5), the odontogenic oral epithelium, which instructs tooth formation and also possesses information to control tooth-specific nerve supply, induces (mediated by Wnt4) Sema3a in the presumptive dental mesenchyme. During subsequent morphogenesis epithelial signaling and Wnt4 and Tgfß1 continue to control Sema3a expression domains in the dental mesenchyme target area. Wnt4 and Tgfß1 contribute to the regulation of tooth morphogenesis by maintaining Msx1 (the effect of Wnt4 on Msx1 expression at E11.5 is hypothetical) and stimulating dental mesenchymal cell proliferation, respectively. The trigeminal molar nerve located in the mesenchymal axon pathway and tooth target fields are indicated in black.