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First published online 3 March 2004
doi: 10.1242/dev.01049

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Activation of the cAMP/PKA signaling pathway is required for post-ecdysial cell death in wing epidermal cells of Drosophila melanogaster

Ken-ichi Kimura, Akitoshi Kodama, Yosihiro Hayasaka and Takumi Ohta^*

Laboratory of Biology, Iwamizawa Campus, Hokkaido University of Education, Iwamizawa, Hokkaido 068-8642, Japan

View larger version (121K): [in a new window]   Fig. 1. (A-M) Programmed cell death of wing epidermal cells in en-Gal4 UAS-GFPN flies. Wing epidermal cells were visualized by nuclear-localized GFP. GFP was apparent in wing epidermal cells of the posterior compartment at 0 hours (A) and 0.5 hours (B) after eclosion, but disappeared by 2 hours after eclosion (C). Higher magnification images of cell death at 0 minutes (D), 10 minutes (E), 20 minutes (F), 30 minutes (G), 40 minutes (H) and 60 minutes (I) after wing spreading. At wing spreading, wing epidermal nuclei are labeled by GFP (D). The GFP signal was then dispersed into the cytoplasm from the nucleus following breakdown of the nuclear membrane (E,F; arrows). GFP was undetectable in the nuclei of all cells after 30 minutes (G), and the cells were detached from the wing cuticle and floated in the cavity formed by the dorsal and ventral wing surfaces (H). After 60 minutes, the wing epidermal cells had disappeared and GFP was detectable only in the nuclei of vein cells (I, arrow). At wing spreading, GFP in the nuclei of wing epidermal cells (J) corresponded to nuclei stained with DAPI (K). At 20 minutes after wing spreading, GFP was dispersed in the cytoplasm (L, arrows). DAPI staining showed that the chromatin of these cells was fragmented (M, arrows). (N-Q) Transverse sections of adult wings in wild-type flies. At eclosion, two layers of wing epidermal cells were stained with DAPI (N) but were not labeled with TUNEL (O). At 20 minutes after wing spreading, the wing epidermal cells were stained with DAPI (P) and were labeled with TUNEL (Q), except for vein cells (arrows; P,Q). Transmission electron microscopy in a wing epidermal cell at 0 hours after eclosion (R) and at 20 minutes after wing spreading (S). The arrow (S) indicates representative condensed chromatin structures. Arrowheads (S) denote cytoplasmic vacuoles that contain cellular structure including mitochondria. Scale bars: 1 µm in R,S. Ectopic expression of p35 inhibited the death of wing epidermal cells in en-Gal4 UAS-GFPN /UAS-p35 flies at 2 hours (T) or even at 48 hours after eclosion (U).

View larger version (69K): [in a new window]   Fig. 2. Effects of neck ligation or wing isolation on the death of wing epidermal cells in en-Gal4 UAS-GFPN flies. Induction of cell death was examined at 2 hours after neck ligation (A-C) or wing isolation (D-F). Neck ligation or wing isolation in flies just after eclosion suppressed cell death (A,D, respectively), but treatment at 20 minutes after eclosion did not prevent the death (B,E). The percentage of flies showing wing epidermal cell death increased with time of treatment (C,F). Black and gray bars indicate the percentage of flies showing cell death extensively or in restricted domains of the wing, respectively (see Materials and methods). The number of flies examined is shown in parentheses.

View larger version (71K): [in a new window]   Fig. 3. Effects of hemolymph injection on the death of wing epidermal cells in en-Gal4 UAS-GFPN flies. Hemolymph collected from wild-type flies at various times after eclosion was injected into flies neck-ligated at eclosion and wings were examined for induction of the cell death 2 hours later (A-C). Cell death was not induced by injection of hemolymph from flies at eclosion (A), but was induced by it at 30 minutes after eclosion (B). Hemolymph from flies at 30 minutes after eclosion was injected into pharate adults neck-ligated at various stages and induction of the cell death was examined 2 hours after injection (D-F). Injection of hemolymph into the flies at S stage did not induce cell death (D), but it did at W stage (E). (C,F) Black and gray bars indicate the percentage of flies showing cell death extensively or in restricted domains of the wing, respectively. The number of flies examined is shown in parentheses.

View larger version (61K): [in a new window]   Fig. 4. Effects of 8-Br-cAMP injection on the death of wing epidermal cells in en-Gal4 UAS-GFPN flies neck-ligated at eclosion. Cell death was induced by injection of 8-Br-cAMP at a concentration of 10–1 mol/l (A) but not by injection of PBS (B). Injection of 8-Br-cAMP produced a dose-dependent effect on the induction of cell death (C). 8-Br-cAMP (10–1 mol/l) was injected into pharate adult flies neck-ligated at various stages and wings were examined for cell death 2 hours after injection (D). (C,D) Black and gray bars indicate the percentage of flies showing cell death extensively or in restricted domains of the wing, respectively. The number of flies examined is shown in parentheses. 8-Br-cAMP (10–1 mol/l) was injected into intact flies at EP stage (just before eclosion). A control fly injected with PBS spread its wings normally (E). A fly injected with 8-Br-cAMP had blistered wings (F, arrow), in which the epidermal cells had died prior to spreading (G). Arrows (G) indicate the persistence of GFP in vein cells.

View larger version (91K): [in a new window]   Fig. 5. Effects of ectopic expression of the wild-type Gs subunit in en-Gal4 UAS-GFPN / +; UAS-Gs/+ flies (A), or a constitutively active form of Gs (Gs*) in en-Gal4 UAS-GFPN / +; UAS-Gs*/+ flies (B,C). Ectopic expression of wild-type Gs produced no visible phenotype (A), whereas Gs* caused wing blisters (B, arrow). Precocious cell death had occurred in blistered wings at the time of wing spreading (C). The arrow (C) indicates GFP remaining in the vein cells. The onset of precocious cell death induced by ectopic expression of Gs* was examined at various stages in pharate adults (D). Black and gray bars indicate the percentage of flies showing cell death extensively or in restricted domains of the wing, respectively. The number of flies examined is shown in parentheses. Effects of elimination of Gs activity on the cell death of wing epidermal cells (E,F). Mutant clones of dgs were marked by the sha– phenotype of missing or smaller hairs (E, enclosed by white lines). The cells of the clones remained at 2 hours after wing spreading, although neighboring cells had died (F). The arrow (F) indicates the persistence of GFP in anterior wing margin cells.

View larger version (86K): [in a new window]   Fig. 6. Effects of reduction or elimination of PKA activity on the death of wing epidermal cells. Ectopic expression of a dominant-negative form of the regulatory subunit of PKA (R*) inhibited cell death at 2 hours (A) or even at 8 hours (B) after wing spreading in en-Gal4 UAS-GFPN /+; UAS-R*/+ flies. Mutant clones of DC0 were marked by the stc– phenotype of smaller hairs or of tufts of hairs (C, enclosed by white lines). Cells of the clones remained 2 hours after wing spreading, although neighboring cells had died (D). Effects of ectopic expression of a constitutively active form of the PKA catalytic subunit (mC*) in en-Gal4 UAS-GFPN /UAS-mC* flies (E-G). Ectopic expression of mC* caused wing blisters (E, arrows). Precocious cell death had occurred in blistered wings by the time of wing spreading (F). The arrow (F) indicates GFP in vein cells. Precocious cell death induced by ectopic expression of mC* was examined at various stages of pharate adult (G). Black and gray bars indicate the percentage of flies showing cell death extensively or in restricted domains of the wing, respectively. The number of flies examined is shown in parentheses.

View larger version (136K): [in a new window]   Fig. 7. Effects of a rickets mutation on the death of wing epidermal cells in rk1 cn1 bw1; His-GFP/+ flies. In rk1 mutants, cell death was inhibited at 2 hours (A) or even at 8 hours (B) after eclosion. In rk1 mutants neck-ligated at eclosion, cell death was induced by injection of 8-Br-cAMP (10–1 mol/l) (C), but not by injection of hemolymph from wild-type flies at 30 minutes after eclosion (D).