(A) Tubulin staining in third-instar wing pouch. Note the asymmetric accumulation of tubulin on the proximal side of the cell in early third-instar wing pouch. Scale bar, 2.5 Δm. White arrows show the proximal distal axis in the wing disc, with distal in the center of the wing pouch, and proximal tissue forming a ring around the future wing blade. (A′) Correlation analysis of image (A) for tubulin and Dsh. Note the increasing overlap of signal when the image of tubulin is shifted leftward (proximal) relative to the Dsh image, resulting in a peak at distance A. A rightward shift (distal) initially shows a decrease in correlation of tubulin and Dsh signals, with a peak at larger distance B, indicating that, on average, the tubulin signals are closer to the proximal than to the distal sides of cells. Similar dorsal or ventral translocations show that tubulin signals are, on average, equidistant between dorsal and ventral sides of the cell. (B–D) Orientations of apical MTs correlate with core PCP protein polarization and Fj/Ds gradients throughout wing development. Spatiotemporal correlation of core PCP protein polarity (Dsh::GFP) with MT alignment (TyrTUB) at time points from 19 to 30 hAPF. Rose plots show distributions of MT orientation (P-D axis, corresponding to the Fj and Ds gradients, is plotted as horizontal (90°); Plots, derived from OrientationJ, are composed of 36 bins of 5° each). Note that orientation remains polarized until 30 hAPF, at which time orientations become randomized. Scale bars: 5 μm.