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. 2015 Oct 6;11(10):e1005576. doi: 10.1371/journal.pgen.1005576

Fig 6. Matrix metalloproteinase and ECM involvement in hh-Gal4 UAS-gyc76C-RNAi phenotypes.

Fig 6

(A-D’) Increased posterior MMP2::GFP levels (anti-GFP staining) in gyc76C knockdown wings (A-B’), compared with wild type (C-D’), at 24–25 hours AP (A,A’,C,C’) and 27–28 hours AP (B,B’,D,D’). The posterior increase was most obvious in apical focal planes (apical) and in projections of cross-sections summed along the proximo-distal axis of the image (X project.). (E) Ratios of posterior to anterior Mmp2::GFP intensity in apical portions of epithelia, corresponding to boxes in (A), in 23–25 hour AP Mmp2::GFP (Control) and hh-Gal4 UAS-gyc76C-RNAi (RNAi) wings. Error bars show standard deviation. The ratio was significantly higher in RNAi wings at p<0.001, using single-tailed Student’s T and Mann-Whitney tests. (F-I’) Increased anti-MMP1 levels in posterior veins and interveins of gyc76C knockdown wings at 23–28 hours AP, even as Trol levels decrease in posterior intervein pockets at 27–28 hours AP (F-H’). At 32–24 hours AP, when Trol is completely lost from intervein pockets, so is Mmp1 (I,I’). See S9S–S9X Fig for lower magnification images and anti-Mmp1 in control wings. (J,K) Rescue of PCV in gyc76C knockdown wings by expression of UAS-TIMP, as detected with anti-pMad (H) or anti-Trol (I). Ectopic pMad in proximal wing correlates roughly with the proximal region where abnormal levels of Trol accumulate. (L-P’) Rescue of PCV and ECM abnormalities in gyc76C knockdown wings by expression of UAS-Mmp2-RNAi. (L-N) Nearly normal PCVs, detected using anti-pMad (L,M) and anti-DSRF (N) at 24, 26 and 28 hours AP, respectively. (O-P’) Nearly normal anti-Trol (O,O’) and anti-LanB2 (P) staining in posterior veins and intervein pockets at 30 hours AP, although posterior has abnormal aggregates of 6G7 anti-CgIV staining (P’). (Q-T’) Effects of overexpression of UAS-trol (EP insertion into trol locus). (Q) Broadening of adult PCV caused by hh-Gal4- UAS-trol. (R) Rescue of adult PCV in gyc76C knockdown wing by UAS-trol, despite increased wing blistering. (S) Rescue of PCV in 34 hour AP gyc76C knockdown wing by UAS-trol, as assayed by downregulation of DSRF. (T,T’) Abnormal accumulation of diffuse 6G7 anti-CgIV staining proximal to PCV between L4 and L5 (T), and failure to rescue posterior intervein pockets (2.4x magnification detail, T’) after UAS-trol expression in 34 hour AP gyc76C knockdown wing.