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. 2014 Mar 3;204(5):839–856. doi: 10.1083/jcb.201306088

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© 2014 Shamir et al.

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Figure 5. — Twist1 induced robust dissemination of normal epithelial cells. (A) Organoids were isolated from CMV::rtTA;TRE-Twist1 mice, and Twist1 was induced in half of the organoids with doxycycline. (B and C) In basal medium, control organoids maintained epithelial organization (B), whereas Twist1 expression induced robust dissemination (C; Video 4). Disseminating cells (red arrowheads) migrated away from the epithelium with extensive protrusions (C’). (D and E) In FGF2-containing medium, control organoids completed branching morphogenesis (D), whereas Twist1 expression blocked branching and induced robust dissemination (E; Video 5). Red arrowheads in E indicate disseminated cells. With FGF2, disseminated cells proliferated to form secondary epithelial sites (blue arrows; E’; 6/9 biological replicates). (F) Less than 1% of Twist1⁺ organoids branched (***, P = 0.0006, two-tailed Student’s t test with equal variance), whereas 97% disseminated (****, P = 4 × 10⁻⁷, two-tailed Student’s t test with equal variance). n, total number of organoids; r, number of biological replicates. Error bars indicate SD. (G) Both luminal (K8⁺; red arrows) and myoepithelial (K14⁺; green arrows) cells disseminated. (H) Both disseminated single cells and cells within the main epithelial group were Twist1⁺. (I) Myoepithelial cells (SMA⁺) and basement membrane (laminin 332; arrows) were inappropriately localized to the organoid interior. Gamma adjustments were performed in H, H′, and I to improve image clarity. Bars: (B–E) 20 µm; (G–I) 10 µm.