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Runx2 (Cbfa1) Inhibits Shh Signaling in the Lower but not Upper Molars of Mouse Embryos and Prevents the Budding of Putative Successional Teeth

¹ Developmental Biology Programme, Institute of Biotechnology, Viikki Biocenter, PO Box 56, FIN-00014, University of Helsinki, Finland; and
² Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel;

View larger version (108K): [in a new window]   Figure 1. p21 (A–D) and Fgf4 (E–H) are expressed similarly in the molars of Runx2/Runx3 double-knockout and Runx2 knockout mice. (A,E,I) E13 wild-type, (B,F,J) E14 wild-type, (C,G,K) E14 Runx2 knockout mice, and (D,H) E14 Runx2/Runx3 double-knockout mice. (I–K) Runx3 expression is very weak in the wild-type tooth (I,J), but is intensely up-regulated in the dental mesenchyme of E14 Runx2 mutant upper molars (K). Arrows point to the down-regulation of p21 (C,D) and Fgf4 (G,H) in Runx2 knockout and Runx2/Runx3 double-knockout lower molars. Scale bar: 200 µm.

View larger version (98K): [in a new window]   Figure 2. Shh signal pathway genes are down-regulated in the lower molars, but not in the upper molars of Runx2 mutants and Runx2/Runx3 double-mutants. (A,B,F,J,N) E13 wild-type, (C,G,K,O) E14 wild-type, (D,H,L,P) E14 Runx2 knockout, and (E,I,M,Q) Runx2/Runx3 double-knockout. (A–C) Shh is expressed intensely at the tip of the dental epithelium corresponding to the enamel knot region. (D–E) Shh transcripts are absent in Runx2 knockout and Runx2/Runx3 double-knockout mouse lower molars, while weak expression is apparent in the upper molars. Ptc1 (F,G) and Gli1 (N,O) are co-expressed in the dental epithelium and mesenchyme. Ptc2 (J–M) transcripts are mainly in the dental epithelium. In Runx2 knockout (H,L,P) and Runx2/Runx3 double-knockout (I,M,Q) mouse upper molars, Ptc1 (H,I), Ptc2 (L–M), and Gli1 (P–Q) are expressed as in the wild-types, but are dramatically down-regulated in the lower molars. (R) Quantitative real-time PCR confirmed that Shh, Ptc1, Ptc2, and Gli1 transcripts are reduced in Runx2 mutant lower molars (n = 3). Bar graph represents the difference in the number of PCR cycles for the Runx2 knockouts to reach a threshold (ct) compared with wild-types. Brackets represent highest and lowest ct. Arrows in A and B point to the tip of the tooth bud. Arrows in C point to the lingual cervical loops of the cap-stage tooth germs. The dental epithelium is outlined with yellow. Scale bar: 200 µm (A–Q).

View larger version (94K): [in a new window]   Figure 3. Cell proliferation is reduced in Runx2 mutant teeth, and their mesenchyme responnds to Shh signal. (A) Runx2 mutant dental mesenchyme is able to respond to Shh signal. (a–d) Upper molar dental mesenchyme. (e–l) Lower molar dental mesenchyme. Shh-soaked beads induced in Runx2 mutant dental mesenchyme intense expression of Ptc1 (b,f) and Gli1 (d,h), which were comparable with the wild-types (a,e,c,g). There was no signal around the BSA control beads (i–l). (B) Cell proliferation is reduced in Runx2 mutant molars. Proliferating cells incorporating BrdU are stained in brown. (a) E14 wild-type molars (percent proliferating cells: [upper molar] epithelium 58%, mesenchyme 86%; [lower molar] epithelium 61%, mesenchyme 85%), (b) E14 Runx2 mutant molars (upper molar, epithelium 33%, mesenchyme 53%; lower molar, epithelium 19%, mesenchyme 36%). Scale bar: 200 µm.

View larger version (98K): [in a new window]   Figure 4. Runx2 knockout (C,F,I,L,O) and Runx2/Runx3 double-knockout mice (G,J,M,P) exhibit prominent lingual epithelial buds in the upper but not the lower molars. (A,D) E13 wild-type, (B,E,H,K,N) E14 wild-type, (C,F,I,L,O) E14 Runx2 knockout, and (G,J,M,P) Runx2/Runx3 double-knockout. (A–C) Freshly dissected upper molars observed in the dissection microscope outlined with yellow dashed lines. Arrows indicate the location of sections stained with hematoxylin and eosin in D–F. Shh (H,J), Gli1 (I), Ptc1 (K–M), and Ptc2 (N–P) transcripts are expressed intensely in the lingual epithelial buds in Runx2 knockout (I,L,O) and Runx2/Runx3 double-knockout (J,M,P) upper molars. (Q–R) E14 Runx2 heterozygous mice also exhibit a prominent lingual bud at the anterior region of the upper molars. (S) There is intense Shh expression in the lingual epithelial bud (arrow) of Runx2 heterozygotes. Shh is also expressed intensely at the tip of the tooth bud, indicating the initiation of the enamel knot. (T) The cap-stage first molar tooth germ intensely expresses Shh in the enamel knot. Scale bar: 200 µm (D–G, Q); 100 µm (H–P, R–T).