Fig. 2. Immunohistochemical localization of cytoskeletal and junctional complex proteins in Smo mutant molars and incisors at 1 dpp. Frontal sections of control (A,C,E,G,I,K,M,O) and Smo mutant (B,D,F,H,J,L,N,P) teeth. Parasagittal sections of control (Q) and mutant (R) incisors. ZO-1 (A-H), E-cadherin (I-P) and ß-tubulin (Q, R). Sections at the middle segment of incisors (E,F,M,N) and at their anterior-most segment (G,H,O,P). The boxed areas in A and B are shown in C and D, respectively. In the control molars (A,C) and incisors (E), ZO-1 accumulates between polarizing ameloblasts (a) and the stratum intermedium (si) as well as at the apicolateral membrane domain of polarizing ameloblasts (arrowhead in C) facing predentin matrix. The outer dental epithelium (ode) shows gaps (arrow in C) with no ZO-1 staining. At this time, ZO-1 is absent from the apical and basal aspect of Smo mutant ameloblasts (a) in molars (arrowhead in D) and incisors (F), whereas it accumulates in the ode (arrow in D), which remains as a continuous layer (B,D). At the anterior-most aspect of the control incisors, ZO-1 accumulation is polarized in the apicolateral and basolateral poles of secretory ameloblasts (G) but is absent in mutant ameloblasts (H). Note that by this stage mutant ameloblasts are severely shrunken, and the strong staining visible is associated with cells of the stratum intermedium and ode (H). E-cadherin staining highlights the gaps in the ode (arrow in K) of control molars (I,K) which are absent (arrow in L) in the mutant molars (J,L). Boxed areas in I and J are shown in K and L, respectively. In some cusps of mutant molars, ameloblasts show some E-cadherin staining at their apicolateral pole (red arrow in L). However, these cells do not show as strong a basolateral E-cadherin accumulation (arrowhead in L) as in controls (arrowhead in K). In the middle segment of control incisors, accumulation of E-cadherin is polarized at the apicolateral and basolateral membrane domains of polarizing ameloblasts (M) but is barely detectable in the mutant ameloblats (N). E-cadherin staining decorates the papillary layer (arrow in O) and accumulates at the apicolateral pole of secretory ameloblasts (O). In mutant incisors, E-cadherin is barely detectable in ameloblasts, and the papillary layer (arrow in P) is unrecognizable. ß-tubulin is virtually absent in mutant ameloblasts (a), whereas it is present in odontoblasts (od) (R). Immuno-positive sites are purple and immuno-negative sites are stained in blue. Scale bars: 200 µm (A,B,I,J); 50 µm (C-H,K-R).

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