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First published online 19 May 2004
doi: 10.1242/dev.01163

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Restriction of sonic hedgehog signalling during early tooth development

¹ Department of Craniofacial Development and Orthodontics, GKT Dental Institute, King's College London, Floor 28, Guy's Hospital, London SE1 9RT, UK
² Department of Craniofacial Development, GKT Dental Institute, King's College London, Floor 28, Guy's Hospital, London SE1 9RT, UK

View larger version (62K): [in a new window]   Fig. 1. Expression of Shh pathway genes in the murine mandibular process. E11.5 mandibular explants cultured for 24 hours in the presence (A,C,E,G) or absence (B,D,F,H) of epithelium. (A) Shh in incisor and molar epithelium (arrowed); large arrow indicates diastema. (B) Loss of Shh in mandibular arch mesenchyme. (C) Ptc1 in the incisor and molar regions (arrowed). (D) Upregulation of Ptc1 in isolated mesenchyme (arrowed). (E) Gli1 in the incisor and molar regions (arrowed). (F) Upregulation of Gli1 in isolated mesenchyme (arrowed). (G) Hhip1 in the incisor and molar regions (arrowed). (H) Loss of Hhip1 in isolated mesenchyme. (I-L) Timed culture in the absence of epithelium. (I) Downregulation of Ptc1, principally in the molar regions (arrowed), after 12 hours. (J) Loss of Ptc1 in both the incisor and molar regions after 16 hours. (K) Upregulation of Ptc1 after 20 hours. (L) Ectopic Ptc1 expression established after 22 hours. All figures show whole-mount in situ hybridisation. Scale bar in A: 600 µm for A-L.

View larger version (75K): [in a new window]   Fig. 2. Ectopic Ptc1 expression is localised to diastema mesenchyme. (A-D) E11.5 mandibular mesenchyme cultured for 24 hours. (A) Barx1 marks the molar regions. (B) Double-labelled (digoxigenin-fluorescein) in situ hybridisation marks Ptc1 (blue staining) diastema regions as distinct from Barx1-expressing (red staining) molar regions. (C) Adjacent sections through Ptc1-expressing domain (upper) demonstrate an absence of Barx1 (lower). (D) Adjacent sections through Barx1-expressing molar region (upper) demonstrate an absence of Ptc1 (lower). (E-H) Ectopic expression of Ptc1 and Gli1 in the diastema is due to Shh signalling. E11.5 mandibular mesenchyme cultured for 24 hours in the presence of 2H3 control antibody (E,G; normal Ptc1, Gli1 expression, respectively) or 5E1 Shh-blocking antibody (F,H; loss of Ptc1, Gli1 expression, respectively). (I,J) E11.5 mandibular explants cultured from Pro1(II)-lacZ transgenic mice demonstrating lacZ expression in Meckel's cartilage. (I) In the presence of epithelium Meckel's cartilage extends as two symmetrical rods. (J) In the absence of epithelium Meckel's cartilage extends further distally (arrowed), but no ectopic cartilages are visible after 24 hours. (K,L) Ihh expression is absent at E11.5 in the mandibular arch in both the presence (K) and absence (L) of epithelium after 24 hours. Scale bars: in A, 600 µm for A,B,E-L; in C, 600 µm for C,D.

View larger version (138K): [in a new window]   Fig. 3. Shh protein is detectable in epithelium and mesenchyme of the developing tooth germ in a wider distribution than epithelial-restricted Shh. (A,C,E,G) Immunohistochemistry; (B,D,F,H) section in situ hybridisation. (A,B) E11.5 epithelial thickening. (C,D) E12.5 early bud. (E,F) E13.5 late bud. (G,H) E14.5 cap. Shh protein is undetectable in the diastema in the presence of epithelium. (I,J) E11.5 mandibles cultured for 24 hours. (I) Section through the early molar epithelial thickenings (arrows indicate Shh protein in odontogenic epithelium). (J) Section through the diastema demonstrating a lack of detectable Shh protein. (K,L) Immunohistochemistry. Timed-culture of E11.5-isolated mandibular mesenchyme demonstrating the dynamics of Shh detection in the diastema. (K) Shh is first detectable after approximately 20 hours. (L) Shh is strongly detected in the diastema after 24 hours. Shh immunohistochemistry does not cross-react with Ihh. (M,N) E16.5 mandibular condyle. (M) Ihh; (N) adjacent section stained for Shh demonstrating no cross-reaction with Ihh. mc, Meckel's cartilage; t, tongue. Scale bars: in A, 50 µm for A-F; in G, 150 µm for G,H; in I, 600 µm for I,J; in K, 600 µm for K,L; in M, 500 µm for M,N.

View larger version (17K): [in a new window]   Fig. 4. Schematic of epithelial transplantation experiments. (A) Diastema epithelium harvested from the mandible of E11.5 ROSA-26 mice and transplanted unilaterally onto E12.5 wild-type early bud stage incisors. (B) Tongue epithelium harvested from ROSA-26 mice and transplanted unilaterally onto E12.5 wild-type incisors in control experiments. x-y demonstrates the plane of section through incisor regions.

View larger version (86K): [in a new window]   Fig. 5. Diastema epithelium can inhibit transcription of Ptc1 and Shh in the mandibular arch. (A-F) E12.5 mandibular explants following 24 hours of culture. (A,B,D,E) ß-Galactosidase staining marks transplanted E11.5 tongue and diastema epithelium, over the left incisor tooth germs. (C) Ptc1 expression is normal in the presence of tongue epithelium. (F) Ptc1 is downregulated in the presence of diastema epithelium. (G,H) Isolated E11.5 incisor explants following 24 hours of culture. (G) In the presence of E11.5 tongue epithelium, Shh expression is normal (right incisor, arrowhead), as compared with the control (left incisor, arrow). (H) In the presence of E11.5 diastema epithelium, Shh expression is lost (right incisor, arrowhead), as compared with the control (left incisor, arrow). The Shh-blocking antibody 5E1 can downregulate Shh transcription. (I) E11.5 mandibular explant cultured for 24 hours in the presence of a 2H3 bead (left) and a 5E1 bead (right) placed adjacent to sites of incisor development; in the right incisor adjacent to the 5E1 bead, Shh is downregulated. (J-M) Isolated diastema mesenchyme is able to transcribe Ptc1, but not Shh. E13.5 tongue explants following 24 hours of culture. (J,L) GFP-label marks the transplant. (K) Transplanted diastema mesenchyme transcribes Ptc1. (M) Transplanted diastema mesenchyme fails to transcribe Shh. (N) Ectopic Ptc1 expression in isolated diastema mesenchyme following separation of the incisor and molar regions prior to epithelial removal and culture for 24 hours. Scale bars: in A, 600 µm for A,D; in B, 100 µm for B,C,E,F; in G, 600 µm for G,H,J-M; in I, 600 µm for I,N.

View larger version (61K): [in a new window]   Fig. 6. Expression and regulation of Gas1 in the murine first arch. (A,B) Coronal sections through the branchial regions at E11.5 (downregulation of Gas1 in odontogenic mesenchyme arrowed). (C-L) Timed culture of E11.5 mandibular mesenchyme. (C-F) Gas1 progressively downregulates in the diastema (arrowed), whereas expression is maintained in the non-odontogenic mesenchyme underlying the incisor and molar regions. (G-L). Adjacent section in situ hybridisation demonstrates progressive Gas1 downregulation (G,I,K) and Ptc1 upregulation (H,J,L) in isolated diastema mesenchyme between 18 and 24 hours of culture. (M-P) E11.5 mandibular mesenchyme cultured for 24 hours following co-electroporation of Gas1 and GFP constructs (M,N), or electroporation of a GFP construct alone (O,P). Electroporation fluorescence (M,O); Ptc1 expression (N,P). Note downregulation of Ptc1 in the presence of ectopic Gas1 expression (N, arrowed), as opposed to controls (P). Scale bars: in A, 200 µm for A,B; in C, 600 µm for C-F,M-P; in G, 600 µm for G-L.

View larger version (26K): [in a new window]   Fig. 7. Shh signalling along the dental axis during initiation of odontogenesis. (A) In the presence of epithelium, Shh transcription is limited to the incisor and molar-forming epithelium, and is established by autoregulation (dashed line). Shh protein diffuses from epithelium into mesenchyme, where it induces both Ptc1 and Hhip1. Ptc1 and Hhip1 protein restrict Shh signalling to the sites of incisor and molar odontogenesis, but some Shh protein is able to diffuse into the diastema mesenchyme. In the presence of diastema epithelium, Gas1 sequesters and masks the activity of Shh protein in the diastema mesenchyme, thus maintaining restriction of Shh signalling to odontogenic regions. Ptc1 and Hhip1 transcription has not been shown in the tooth germs for simplicity. (B) In the absence of epithelium, Gas1 is lost in diastema mesenchyme, leaving localised Shh signalling in this region, which activates transcription of the target gene Ptc1.