Fig 1.
pzg genetically interacts with pro-apoptotic genes.
(A-C) Control eyes after overexpression of lacZ and pzg, as well as pzg-RNAi induction. (D-F) Overexpression of hid, rpr and grim during eye development causes small and rough eyes in the adults. (G-I) Overexpression of pzg ameliorates the small eye size resulting from ectopically expressed pro-apoptotic genes, whereas depletion of pzg activity by RNAi enhances the phenotypes (J-L). Genotypes analyzed: Gmr-Gal4/+; UAS-lacZ/+. Gmr-Gal4/+; EP-pzg/+. Gmr-Gal4/+; UAS-pzg-RNAi/+. Gmr-hid/Gmr-Gal4; UAS-lacZ/+. Gmr-Gal4/+; Gmr-rpr or Gmr-grim/ UAS-lacZ. Gmr-hid/Gmr-Gal4; EP-pzg/+. Gmr-Gal4/+; Gmr-rpr or Gmr-grim/EP-pzg. Gmr-hid/Gmr-Gal4; UAS-pzg-RNAi/+. Gmr-Gal4/+; Gmr-rpr or Gmr-grim/ UAS-pzg-RNAi.
Fig 2.
pzg depletion autonomously triggers the apoptotic signaling cascade in wing imaginal discs.
RNAi mediated depletion of pzg was induced in the posterior part of the wing disc using en-Gal4. Rpr-lacZ, DIAP1 and caspase activity (in red) was monitored as indicated. (A-A''') A strong activation of the pro-apoptotic gene rpr (arrows) as well as the Drosophila activated caspases Driceact (C-C''', arrow), Caspase 3act (D-D''', arrow) and Dcp-1act (E-E''', arrows) is detected in the posterior half of the disc, whereas DIAP1 protein level is reduced (B-B''', repressive arrow). (A-A''') en-Gal4 UAS-GFP UAS-pzg-RNAi/+; rpr-lacZ/+. (B-E''') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. Pzg protein is shown in blue (anti-Pzg, A-E and A'''- E'''); GFP in green (en-Gal4 GFP) marks the posterior compartment (A-E and A''-E''). Posterior is to the right and dorsal up. The antero-posterior compartment boundary is marked with a dashed line. Scale bars: 100 μm.
Fig 3.
pzg depletion results in an inappropriate JNK-signaling activation.
pzg was downregulated in the posterior part of the wing disc (Pzg protein is shown in blue). (A-B'') puc-lacZ activity is induced in pzg-RNAi mutant cells reflecting JNK-signaling activity (red in A-A', B-B', arrows) (en-Gal4 UAS-GFP UAS-pzg-RNAi/+; puc-lacZ/+). In addition, especially in late third instar larval discs, a non-autonomous activity can be detected in the anterior compartment (open arrows in A', B'). (C-F'') Further consequences of JNK-mediated developmental apoptosis induction can be observed: (C-D'') Dilp8 protein is secreted in the pzg depleted cells, however not before late third larval instar (red in D, D', arrow). (E-E'') In pzg-RNAi mutant cells, Arm protein accumulation is disturbed (red, repressive arrow). (F-F'') pzg mutant cells penetrate into the anterior compartment while retaining their posterior identity (green, arrows in F, F'). (C-F'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
Fig 4.
pzg mutant cells show characteristics of genuine dying cells and induce AiP.
pzg was downregulated in the posterior part of the wing disc. Pzg protein is shown in blue (C, C''), the posterior compartment is marked in green with GFP (A-D; A''-D''; A'''-B'''). (A-A''') DNA-synthesis visualized with EdU-labeling (red) is amplified anteriorly along the A/P compartment boundary (arrows) upon pzg depletion (A-A') compared to en-Gal4 UAS-GFP control (A'''), whereas a loss can be observed within the posterior compartment (open arrow, A-A'). (B-B''') Cell division was visualized with anti-PH3 (red). Compared to the control (B'''), many cells in the posterior domain lost this marker upon pzg depletion (arrowhead, B-B'), whereas a strip of cells anterior to the A/P boundary shows stronger PH3 labeling (arrows, B-B'). (C-C'') Expression of Wg protein was monitored (red): it is interrupted at the dorso-ventral boundary in response to pzg-RNAi depletion (arrowhead in C'), whereas ectopic induction of Wg is observed in the posterior compartment outside the normal Wg expression domain (arrows, C'). (D-D'') dpp-lacZ expression (red) is unchanged by pzg depletion in the posterior compartment. Genotypes: (A'''-B''') en-Gal4 UAS-GFP/+. (A-C'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. (D-D'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+; dpp-lacZ/+. Wing discs are oriented posterior rightwards and dorsally upwards. The A/P compartment boundary is marked with a dotted line. Scale bars represent: (A, B, D) 100 μm, (C) 50 μm.
Fig 5.
pzg-RNAi induces AiA in late larval stages.
(A-D'') Strong caspase activity, either visualized with anti-Caspase 3act (red in A-A', B-B') or anti-Dcp-1act (red in C-C', D-D'), can be detected non-autonomously in late larval wing disc in the anterior compartment (arrowheads in A-D'). Preventing cell death execution with p35 amplifies the overgrowth effect (asterisks in B'') but still induces AiA in the anterior half of the disc (B-B'', D-D'', arrowheads). Genotypes: (A-A'') and (C-C'') en-Gal4 UAS-GFP UAS-pzg-RNAi/+. (B-B'') and (D-D'') UAS-p35; en-Gal4 UAS-GFP UAS-pzg-RNAi/+. The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
Fig 6.
Apoptotic consequences of Notch depletion in wing imaginal discs.
N-RNAi was induced in the posterior compartment of the wing disc, N protein is shown in blue (A, B, D and A'', B'', D''), the posterior compartment is marked in green with GFP (A-F and C'', E'', F''). (A-B'') Caspase activity, visualized with anti-Dcp-1act (red in A, A' and B, B') is detectable in early (96 h AEL) and late third instar wing discs (120 h AEL) autonomously (arrows in A, A' and B, B') and non-autonomously (open arrows in A, A' and B, B'). The effect of AiA is enhanced in later phases of development (B, B'). (Genotype: UAS-N-RNAi; en-Gal4 UAS-GFP/+). (C-C'') JNK-signaling readout visualized with puc-lacZ expression is seen in both compartments (red in C, C' arrows; UAS-N-RNAi; en-Gal4 UAS-GFP/+; puc-lacZ/+). The wild type expression of puc-lacZ at the stalk region is marked with an asterisk (C, C'). (D-D'') Cells within S-phase are labeled with EdU (red); a minor autonomous reduction and weak increase in most central cells abutting the A/P compartment boundary is observed (arrow in D, D'; genotype as in A). (E-E'') The expression of Wg is lost at the dorso-ventral boundary in the posterior compartment (red in E, E', repressive arrow; genotype as in A). (F-F'') dpp expression is not affected by downregulation of N in the posterior compartment (red in F, F'; UAS-N-RNAi; en-Gal4 UAS-GFP/+; dpp-lacZ/+). The A/P compartment boundary is marked with a dotted line. Scale bars: 100 μm.
Fig 7.
Loss of Dref activity entails cell cycle arrest, cell death and AiP.
Dref was downregulated in the posterior compartment of the wing disc, which is marked in green with GFP (A-F; A''-F''). (A-B'') Dref-RNAi induction in the posterior compartment of the wing disc is correlated with an autonomous (arrow) and non-autonomous (open arrow) cell death induction, monitored with anti-Dcp1act (red in A, A' and B, B'). Although AiA can be detected already in early third instar wing discs (96 h AEL, A' open arrow), the amount of non-autonomous cell death is strongly increased in later stages (120 h AEL, B' open arrows). (C-C'') puc-lacZ is ectopically induced in both compartments (red in C, C'; closed and open arrows). Asterisk highlights the wild type expression in the stalk region of the disc. (D-D'') Replication is disturbed autonomously after Dref-RNAi depletion, visualized with EdU staining (red in D, D', repressive arrow), whereas enhanced proliferation is induced anteriorly (open arrow). (E-F'') Ectopic induction of Wg (red in E, E') can be detected in the Dref-depleted compartment (arrows in E, E'), whereas dpp-lacZ expression appears unchanged (red in F-F'). Genotypes: (A-B'') and (D-E'') en-Gal4 UAS-GFP/+; UAS-Dref-RNAi/+. (C-C'') en-Gal4 UAS-GFP/+; UAS-Dref-RNAi/puc-lacZ. (F-F'') en-Gal4 UAS-GFP/+; UAS-Dref-RNAi/dpp-lacZ. Posterior is right, dorsal upwards. The dashed line marks the A/P compartment boundary. Scale bars: 100 μm.
Fig 8.
Dref overexpression ameliorates pzg-RNAi apoptosis defects.
Comparison of the apoptotic consequences of pzg depletion in the presence (A-E’) or absence (B-F’) of ectopic Dref expression. (A-D') Compared to the control (B,B',D,D'), overexpression of Dref in pzg-RNAi depleted cells ameliorates the cell cycle arrest defect (A,A',C,C'), visualized either with EdU (S-phase, red in A-B') or anti-PH3 (M-phase, red in C-D'). Repression of cell division by pzg depletion (repressive arrow in B', D'), is absent (C') or less pronounced in the Dref overexpression background (open repressive arrow in A'). Activation of Dcp-1 (anti-Dcp-1act, red in E-F') upon pzg downregulation (arrow in F'), is much weaker when Dref is overexpressed (open arrow in E'). Genotypes: (A, A'; C, C'; E, E') en-Gal4 UAS-GFP UAS-pzg-RNAi/+; UAS-Dref/+. (B, B'; D, D'; F, F') en-Gal4 UAS-GFP UAS-pzg-RNAi / UAS-lacZ. Posterior is right and dorsal up. The A/P compartment boundary is marked with a dashed line. Scale bars: 100 μm.