Notum live imaging was performed as described previously [3 (link)]. Briefly, the pupae were collected at the early stage (0–6 h after pupal formation), aged at 29∘C, glued on double sided tape on a slide and surrounded by two metal spacers of approx. 0.650 mm. The pupal case was opened up to the abdomen using forceps and mounted with a 20 × 40 mm #1.5 coverslip where we buttered halocarbon oil 10S. The coverslip was then tapped on the spacers using regular tape. Pupae were collected 48 or 72 h after clone induction and dissected 16-18h after pupae formation (APF). Pupae were imaged at 29∘C for 22 h on a LSM 880 scanning laser confocal microscope (Carl Zeiss A.G.) equipped with a fast Airyscan module using an oil 40X objective (NA 1.3), Z stacks (1 μm/slice), every 5 min using autofocus. The autofocus was performed using the autofluorescence of the cuticle in far red (using a Zen Macro developed by Jan Ellenberg laboratory, MyPic). Movies were performed in the nota close to the scutellum region containing the midline and the aDC and pDC macrochaetae. The experiment presents a pupae with endoCad::GFP signal and groups of cell over-expressing UAS-yorkie S11A S168A S250A V5 clones and nuclei in red over the control of the GAL80TS thermosensitive. The cross and the progeny were kept at 18∘C, and the pupae were switched to 29∘C 8 h prior to the movie for conditional activation.
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