Wnd/DLK and Nmnat protect axons through parallel mechanisms downstream of Hiw.

<p>(A) Representative muscle 4 NMJs at 24 h after injury immunostained for Futsch (green) and HRP (neuronal membrane, red) for the following genotypes: WT <i>(Canton S)</i>, neuronally overexpressed <i>nmnat (BG380-Gal4; UAS-HA-nmnat/+)</i>, or overexpressed <i>nmnat</i> in a <i>wnd</i> mutant background <i>(BG380-Gal4; UAS-HA-nmnat/+; wnd¹/wnd²)</i>. Overexpression of <i>nmnat</i> protected NMJs from degeneration and this protection was not compromised by mutations in <i>wnd</i>. (B) Quantification of NMJ degeneration in the above genotypes. <i>(C) UAS-mCD8::GFP/+; m12-Gal4/+</i> labeled singles axons (green) 24 h after injury in WT <i>(UAS-Dcr2; UAS-mCD8::GFP/+; m12-Gal4/+)</i>, when overexpressing <i>wnd (UAS-Dcr2; UAS-mCD8-GFP/+; m12-Gal4/UAS-wnd)</i>, or when overexpressing <i>wnd</i> in conjunction with <i>nmnat RNAi (UAS-Dcr2; UAS-mCD8-GFP/UAS-nmnat-RNAi; m12-Gal4/UAS-wnd)</i>. Reducing Nmnat levels by this method had no effect upon the protection caused by overexpression of <i>wnd</i>. (D) Degeneration index of the <i>m12-Gal4, UAS-mCD8::GFP</i> labeled single axons at different time points after injury in the above genotypes. (E) Representative images of NMJs at muscle 4 18 h after injury stained for Futsch (green) and HRP (red) in the following genotypes: <i>(hiw^ND8,BG380-Gal4; UAS-Dcr2/+), (hiw^ND8, BG380-Gal4;UAS-Dcr2/+;UAS-wnd-RNAi/+), (hiw^ND8, BG380-Gal4; UAS-Dcr2/UAS-nmnat-RNAi), (hiw^ND8, BG380-Gal4; UAS-Dcr2/UAS-nmnat-RNAi; UAS-wnd-RNAi/+)</i>. (F) Quantification of the percentage of NMJs that are completely degenerated, partially degenerated, or intact at 12 h or 18 h after injury, for the genotypes described above. (G) Model: Wnd and Nmnat inhibit axonal degeneration through independent pathways downstream of Hiw. Scale bars = 12. 5 µm, error bars represent standard error; *<i>p</i><0.05; ns, not significant, <i>p</i>>0.05 in <i>t</i>-test.