Fig. 5. Shh expression and Shh responsiveness during late tooth development in Smo mutants. Section through control (A,C,E,G,I,K,M,O,Q,S,U,W,Y) and Smo mutant (B,D,F,H,J,L,N,P,R,T,V,X,Z) teeth at 1 dpp. In situ hybridizations for Shh (A,B,M,N,Q,R); Ptc1 (C,D,S,T); Gli1 (E,F,O,P,W,X); Hip1 (G,H); Ptc2 (I,J,U,V); Gli2 (Y,Z). Immunohistochemistry with Ab80 showing SHH protein distribution (K,L). Parasagittal (A-H,K,L) and frontal (I,J) sections through the molar region. Frontal sections at the middle (M-P) and anterior (Q-Z) segments of incisors. Shh expression is dramatically decreased in mutant ameloblasts facing predentin (arrow in B). The stratum intermedium continues to express Shh for only a short period before expression is severely decreased (asterisk in N). The signal in the stellate reticulum (SR, arrowhead in G) is artefactual and is due to refractile properties of displaced red blood cells and not to silver grains in SR cells. In I, the arrow points to the inner dental epithelium at the cervical loop and the arrowhead shows preameloblasts. In L, the arrowhead indicates preameloblasts adjacent to polarized odontoblasts and the arrow, preameloblasts adjacent to predentin matrix. In the mutant molar (L), Shh protein has accumulated in large amounts in the predentin facing preameloblasts, which show low amounts of Shh staining. This is probably due to minute amounts of Shh protein, emanating from preameloblasts and possibly also from the SI, which become trapped within predentin matrix, as these mutant cells express extremely low levels of Ptc2 and Ptc1 and thus may be unable to sequester Shh protein. The insets in K and L show high magnifications of the boxed areas. In Q, the arrowhead points to polarizing ameloblasts and the arrow to presecretory ameloblasts. a, ameloblasts; ode, outer dental epithelium; si, stratum intermedium; sr, stellate reticulum. Scale bars: 200 µm.

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