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When wing feathers were asynchronously molted on the right wing, we used the scores of the left wing.
Because several morphological variables were measured per wing, we used the conservative Bonferroni correction for multiple tests.
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In case of wing discs, we used sample areas of 75 × 75 pixels from the wing pouch exclusively.
To quantify individual variation and FA of wing size, we used the within-sample variance of the centroid size [25] of the wings and the variance of the (right−left) difference of centroid size.
For ROS analysis of wing discs we used CellRox Deep Red (Invitrogen) as described.
For Vha44 expression in the wing disc, we used ptc-Gal4, dpp-Gal4, nub-GAL4 (all Bloomington) or ap-Gal4 (a gift of Marek Mlodzik) drivers.
To activate EY03046 expression in the wing pouch, we used the following Gal4 drivers: actin-Gal4, dad4-Gal4, salE-Gal4, dpp-Gal4, and ptc-Gal4.
To analyze the localization of apical tubulin in early third-instar wing pouch we used the cross correlation method as previously described (Matis et al., 2012).
To quantify wing size, we used centroid size, which is a measure of the spread of landmarks around their centre of gravity [ 35].
To demonstrate the potential for the FINGR method to refine existing GAL4 expression patterns and make it possible to study localized sections of wing tissues, we used ET-FLPx2 36A to target clusters of cells in which to reduce expression of the PCP gene pk during wing development.
For the proximal-distal wing section analysis we used the forewing data to perform an ANOVA by wing section, with Stage, Wing Section, and Stage*Wing Section as fixed effects and Morph as a random effect.
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