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).