Extended Data Fig. 4: Inhibiting cell proliferation in the embryonic murine incisor interferes with incisor growth and anisotropic stress development.
(a) Measured proliferation profiles from the centre in the epithelium at E11.5, 12.5, 14.5 and 15.5. (Mean values plotted from n = 3 per stage). (b) EdU and eGFP or BrdU and E-Cad immunostaining after a 1 h EdU or BrdU pulse chase in E12 murine incisors cultured with DMSO or Aphidicolin for 7 h (Top panel) and 40 h (Bottom panel). (n = 6 per condition) (c) Morphology of E12 incisors at 0 h and 40 h after culture with DMSO or aphidicolin. Dashed line outlines the incisor in (b, c). (Ctrl: n = 4, 6 and Aphi: n = 4, 6 for 0 and 40 hours respectively) (d) Quantification of the epithelial area (outlined in yellow dotted line) from all the buds represented in c. (n values in c). (e, f) Anisotropy analysis of the live E12 murine incisors cultured for (e) 7 h or (f) 30 h with DMSO or aphidicolin. (7 h – n = 5; 30 h - n = 3) (g) Representation of droplet orientation and cell anisotropy (orientation) maps (reproduced from Fig. 3c, c’) and quantification of droplet orientation with reference to the regional cell orientation in live 7 h DMSO or aphidicolin treated incisors. Compilation of average data from 3–6 h after injection. Orientation angle values for DMSO treated incisors reproduced from Fig. 2f E12 data. (Ctrl: n = 15 in LC and n = 14 in HC; Aphi: n = 13 in LC and n = 15 in HC) LC, low coherence region; HC, high coherence region. (h) Quantification of the tissue stress anisotropy measured by the oil droplets in the LC region of E12 incisors cultured for 7 hours with DMSO or aphidicolin (Ctrl: n = 15 from 2 h; Aphi: n = 13). Dashed line outlines the incisor. Scale bars, 25 µm. epi, epithelium; mes, mesenchyme. Representative 2D central plane shown in all images. Data are represented as mean ± s.e.m. Statistical analysis was done using 2 way ANOVA for d, unpaired two-tailed Student’s T test (assuming unequal variance) with Welch’s correction for g and 2 tailed Mann-Whitney test for h. Source data are available for all plots. n represents number of embryos; one incisor measured per embryo.
