Ecdysone regulates cell–cell dissociation during FBR through the loss of CIVICs. (A–C) Confocal images of fat bodies from 3 h APF pupae expressing Lpp-Gal4+UAS-Myr-td-Tomato+Viking-GFP +control (A) or +UAS-EcRDN⁶⁸⁶⁹ (B; side a and side b shown in planar and lateral view; merge of single Z plane [left] and Viking-GFP channel of single Z plane or of projection of 10 Z planes 2.5–5 μm from cell surface [middle and right panels, respectively]). Quantification of mean Viking-GFP–positive CIVIC numbers (A′, B′, from thresholded Z projection images of 10 Z planes of Viking-GFP channel [2.5–5 μm from cell surface], n: 10 tissues, 3 Z projection images per tissue and side, data paired by tissue). Paired two-sided t test, ****P < 0.0001 and ***P < 0.001. Quantification of CIVIC numbers for control and UAS-EcRDN⁶⁸⁶⁹ on side b (C). Unpaired two-sided t test, ****P < 0.0001. (D) Proposed model of FBR. The fat body in the third instar larva displays an apical-basal cell polarity, which regulates collagen IV–mediated cell–cell adhesion. Ecdysone induces FBR in the prepupa resulting in the loss of apical-basal polarity and CIVICs by 3 h APF. Cells then dissociate and initiate amoeboid swimming migration in pupae around 14 h APF. Scale bars: 20 µm (A and B).