Fig. 8. Schematic drawing showing Shh expression (stippled green) and responsiveness (yellow) as well as the cytological changes occuring in the enamel organ and odontoblast layer during the gradual differentiation process in a normal (upper panel) and in a Smo mutant (lower panel) tooth. Proliferating cells are represented by smiling nuclei and post-mitotic cells are represented by non-smiling or plain nuclei. In a normal tooth, at stage 4, preameloblasts facing the first layer of predentin matrix (PD, pink) withdraw from the cell cycle and become postmitotic (PmA). This occurs about 24 hours after the adjacent odontoblasts become postmitotic (stage 2), polarize (stage 3) and secrete PD (stage 4). Polarizing ameloblasts (PoA) continue to grow, they attain their full size at stage 6 and become secretory ameloblasts (SA) displaying Tomes' processes and secrete enamel matrix (E, dark red). Cells of the stratum intermedium overlying PoA and SA increase in size and become cuboidal (cSI). Shortly before enamel secretion, gaps appear in the outer dental epithelium (ODE) and blood vessels and fibroblasts from the dental sac (DS) start to invade the stellate reticulum (SR). Shh is produced in large amounts in proliferating preameloblasts (PA), PmA, PoA, squamous stratum intermedium (sSI) and cSI. Shh expression and Shh synthesis decline thereafter in presecretory ameloblasts (not represented) and SA (stage 6). In the normal tooth, both the enamel organ and dental mesenchyme are responsive to Shh signaling. In the Smo mutant tooth, Shh responsiveness, cell proliferation, growth and differentiation are preserved in the dental mesenchyme, including the dental papilla (not represented), dental sac and the odontoblast layer. However, Shh responsiveness is absent in the enamel organ at all stages (stages 1-6). Mutant preameloblasts become post-mitotic prematurely (stage 3) before predentin secretion by the adjacent odontoblasts. Mutant ameloblasts fail to grow in size, are unpolarized and unable to produce enamel matrix. The mutant stratum intermedium cells remain small and squamous. The outer dental epithelium layer remains continuous, and the stellate reticulum remains avascular. In addition, a premature gradual decrease of Shh production occurs in the mutant ameloblasts, stratum intermedium and stellate reticulum. A, ameloblast; BM, basement membrane (light green); D, dentin (dark pink); O, odontoblast; PmO, post-mitotic odontoblast; PO, proliferating preodontoblast; PoO, polarizing odontoblast.