Quantification of planar polarity

Stacks from stage 7 to early stage 8 embryos were acquired with LSM 710 or LSM880, 40 ×/NA 1.3 Plan-Apochromat oil objective, Carl Zeiss. Mean fluorescence intensities of all borders were measured with ImageJ’s line tool (linewidth 3) at 300% zoom. To ensure that the entire apical border was measured, stacks of five planes 0.3 µm apart were used. The ImageJ Stacks>Z project function was used to construct a Z-axis sum intensity projection. Background mean intensity was subtracted to obtain the final value for each cell. Borders were sorted by angles (relative to embryo DV axis). AP borders, 0°–29°; DV borders, 60°–90°. Ratios from three or more experiments were averaged.