Fig. 6. Premature cell-cycle exit and differentiation of Smo mutant preameloblasts. Molars (A-H) and incisors (I-L) from control (A,C,E,G,I,K) and mutant (B,D,F,H,J,L) pups. Parasagittal sections (C-F). Frontal sections (A,B,G-L). Immunohistochemistry showing phosphorylated histone H3 (PH-H3; A,B) and calbindin D28K (G,H) distribution. The insets in A, and B show high magnifications of the boxed areas. Arrows in A and B point to preameloblasts adjacent to post-mitotic odontoblasts prior to predentin secretion. The arrowhead in A indicates differentiated ameloblasts in a more mature cusp. In situ hybridization for cyclin D1 (C,D) and amelin (E,F,I-L). In control molars, cyclin D1 expression is lost in post-mitotic ameloblasts facing the first layer of predentin matrix in the most advanced cusp (red arrow in C), whereas preameloblasts and the overlying stratum intermedium (black arrowhead in C) facing post-mitotic odontoblasts (black arrow in C) continue to express cyclin D1. cyclin D1 expression is prematurely lost in Smo mutant preameloblasts and stratum intermedium (black arrowhead in D) adjacent to post-mitotic odontoblasts (arrow in D) prior to predentin secretion. Asterisks in C and D show proliferating cells at the cervical loops and red arrowheads show preodontoblasts. In D the section is through the less-developed cusps of the first molar region. In control molars (E), at the level of the less mature cusps, amelin is expressed in polarizing odontoblasts and is barely detectable in preameloblasts adjacent to them. In the Smo mutant molars (F), amelin is expressed prematurely at high amounts in preameloblasts facing polarizing odontoblasts in several cusps. Note that the cusps in the molar in F are at the same developmental stage as the left cusp of the molar in E. Arrow in H indicates preameloblasts adjacent to preodontoblasts. The insets in G and H show high magnifications of the boxed areas. Sections through the middle segment of the incisors showing absence of amelin expression in control preameloblasts (I) and premature expression by mutant preameloblasts (J). Sections through the anterior segment of incisors (K,L) showing amelin expression by ameloblasts. Scale bars: 200 µm (A-L).