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. Author manuscript; available in PMC: 2016 Apr 20.
Published in final edited form as: Dev Cell. 2015 Apr 9;33(2):125–135. doi: 10.1016/j.devcel.2015.02.021

Figure 6. The BMP-Smad4 signaling cascade inhibits SHH-Gli1 signaling activity to control Sox2+ epithelial stem cell maintenance during molar development.

Figure 6

(A, B) Immunofluorescence of Gli1 (green) and Sox2 (blue) in tetO-Cre;Smad4fl/fl;Gli1-LacZ (T;S;Gli1-LacZ; control) and KRT14-rtTA;tetO-Cre;Smad4fl/fl;Gli1-LacZ (K;T;S;Gli1-LacZ) lower first molars at PN0.5. Detection of Gli1 expression was performed using anti-beta galactosidase antibody. Boxed areas in (A) and (B) are shown magnified in (A’-A’’) and (B’-B’’), respectively. Arrows indicate co-expression of Gli1 and Sox2 (light blue). Broken lines indicate the cervical loop. (C-F) In situ hybridization of Shh in control (C, E) and KRT14-rtTA;tetO-Cre;Smad4fl/fl (K;T;S) (D, F) lower first molars at PN7.5 and PN12.5. Arrows indicate expression of Shh. Broken lines indicate the HERS (C, E) or the cervical loop (D, F). (G, H) LacZ expression assayed by X-gal staining (blue) in tetO-Cre;Smad4fl/fl;Gli1-LacZ (control) and KRT14-rtTA;tetO-Cre;Smad4fl/fl;Gli1-LacZ lower first molars at PN21.5. Arrows indicate activated Gli1 expression in the dental epithelium. (I) Timing of doxycycline induction and sample harvest in control, KRT14-rtTA;tetO-Cre;Smad4fl/fl (K;T;S) and KRT14-rtTA;tetO-Cre;Smad4fl/fl;Shhfl/fl (K;T;S;Shhfl/fl) mice. (J, K) MicroCT 3D reconstructions (J) and sagittal images (K) of the lower first molars in PN21.5 control, KRT14-rtTA;tetO-Cre;Smad4fl/fl and KRT14-rtTA;tetO-Cre;Smad4fl/fl;Shhfl/fl mice. (L-N) H&E staining of control, KRT14-rtTA;tetO-Cre;Smad4fl/fl and KRT14-rtTA;tetO-Cre;Smad4fl/fl;Shhfl/fl lower first molars at PN21.5. (O-Q) Immunofluorescence of Sox2 (red) in control (O), KRT14-rtTA;tetO-Cre;Smad4fl/fl (P) and KRT14-rtTA;tetO-Cre;Smad4fl/fl;Shhfl/fl (Q) lower first molars at PN21.5. Boxed insert in (Q) shows positive Sox2 expression in the oral epithelium of the same section of KRT14-rtTA;tetO-Cre;Smad4fl/fl;Shhfl/fl lower first molar. White arrows indicate expression, whereas white arrowheads indicate absence of expression. DE: Dental epithelium; M1: Lower first molar; P: Pulp; PDL: Periodontal ligament; R: Root. Scale bars (A-F, O-Q): 50μm. Scale bars (A’-A’’, B’-B’’): 25μm. Scale bars (G, H, L-N): 100μm.