(A-C) Immunofluorescence of Ki67 (red) in control and KRT14-rtTA;tetO-Cre;Smad4fl/fl (K;T;S) lower first molars at PN7.5. Quantitation of the percentage of Ki67-labeled nuclei in the dental epithelium (indicated by broken lines) of control and KRT14-rtTA;tetO-Cre;Smad4fl/fl lower first molars. N=3. *, p < 0.05. Error bars, SD. (D, E)
In situ hybridization of Amelogenin in control and KRT14-rtTA;tetO-Cre;Smad4fl/fl lower first molars at PN7.5. Black arrow indicates Amelogenin expression in control ameloblasts. Black arrowhead indicates lack of expression of Amelogenin in KRT14-rtTA;tetO-Cre;Smad4fl/fl mice. (F-I)
In situ hybridization of dental epithelial stem cell niche markers Notch1 or Lfng in control (F, H) and KRT14-rtTA;tetO-Cre;Smad4fl/fl (G, I) lower first molars at PN7.5. Black arrows indicate expression, whereas black arrowheads indicate lack of detectable expression. (J-M) Immunofluorescence of Sox2 (green) in control and KRT14-rtTA;tetO-Cre;Smad4fl/fl lower first molars at PN7.5 (J, K) and PN21.5 (L, M). White arrows indicate expression, whereas white arrowheads indicate absence of expression. (N) Timing of doxycycline induction and sample harvest for cell culture in vitro in control and KRT14-rtTA;tetO-Cre;Smad4fl/fl (K;T;S) mice. (O) Schematic diagram depicting dissection of PN3.5 lower first molar, separation of the epithelium and mesenchyme, dissociation of the epithelium into single cells, and culture of dental epithelial cells. Four days after plating, single stem cells in KRT14-rtTA;tetO-Cre;Smad4fl/fl molars proliferated to produce colonies. Colonies were not detectable in controls. (P, Q) Colony-forming assay after 7 days of culture in control versus KRT14-rtTA;tetO-Cre;Smad4fl/fl molars. Total number of colonies was quantified. N=3. *, p < 0.05. Error bars, SD. (R) Immunofluorescence of Sox2 (red) and Keratin 14 (K14; green) in epithelial cell colony from KRT14-rtTA;tetO-Cre;Smad4fl/fl molars after 7 days culture. (S) Immunofluorescence of Bmi1 (red) in epithelial cell colony from KRT14-rtTA;tetO-Cre;Smad4fl/fl molars after 7 days culture. Scale bars (A, B, D-M): 50μm. Scale bars (O, R, S): 100μm. See also Figure S2.