Leg disc development was imaged using an inverted spinning disk confocal microscope (CSU-X1, Yokogawa, coupled to a Leica or Zeiss microscope) mounted with 20×/0.8 multi-immersion 40×/1.2 oil or 20×/0.8 air objectives and equipped with 488 nm and 561 nm LEDs and a piezo stage. Images were acquired over time with an EMCCD camera (Hamamatsu) controlled by the Metamorph or Zen software, at a rate of one z-stack every 5 to 15 min. Images were processed with the ImageJ software for registration (StackReg plug-in from Thévenaz and co-workers, EPFL, Switzerland), bleaching correction by histogram matching (Bleach Correction plug-in from Miura, EMBL, Germany), stitching (Pairwise Stitching plug-in Preibisch et al., 2009 (link), background correction and smoothing. The length of the PE and that of the leg (Fig. 1C and Fig. S1) were measured on image z-stacks, from the dorsal tip to the distal pole (PE) and from the femur to the distal pole (leg), respectively, as indicated in Fig. 1B. The occurrence time of a particular event (Fig. 1D) was defined as the first timepoint when the event was visible.
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