Fig. 3. Pio, but not other wing-blister gene products, is required for the transalar arrays of microtubule bundles in pupal wings. The distribution of tubulin in late pupal wings (60-70 hours after puparium formation) is shown in white or green in each panel except B, which shows an adult wing. All cells express tubulin-GFP in A,C-E. In a confocal section of a wild-type folded pupal wing (A), the prominent microtubule bundles spanning the wing cells can be seen. Pairs of attached cells are marked with yellow arrows, with the arrowheads basal. (B-E) Clones of mutant cells are marked with a mutation in shavenoid (sha), which disrupts the formation of wing hairs but does not cause blistering, as shown in a bright-field micrograph of the adult wing (B). In the absence of Pio, the microtubule bundles are not seen: (C) a section across the apical surface, combined with differential interference contrast, showing the absence of microtubules and wing hairs in the sha pio mutant clone; (D) the absence of the microtubule bundles in sha pio mutant cells (indicated with a white horizontal bracket); opposing wild-type cells also appear overcontracted. The microtubule-binding protein Shot, which localizes to both ends of the microtubule bundles as detected by antibody staining (F), is not required to maintain the bundles (E); this image combines tubulin-GFP fluorescence and differential interference contrast, and the white horizontal bracket shows the cells lacking Shot (sha shot). (G-I) The MARCM technique was used to express tubulin-GFP in just the mutant clones of cells. A control wild-type clone shows microtubule bundles in one layer of the wing (G). Clones of cells lacking Pot (H) or the integrin ßPS subunit (I, mys) contain microtubule bundles. These bundles are disordered in the absence of integrins, presumably owing to the separation between the two cell layers (the other layer is not visible because it does not contain a clone). Bars, 10 µm.