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First published online 30 March 2005
doi: 10.1242/dev.01790

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The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila

Dongbin Xu^1,*, Ying Li^1,*, Michael Arcaro¹, Melinda Lackey¹ and Andreas Bergmann^1,*,

¹ The University of Texas M.D. Anderson Cancer Center, Department of Biochemistry and Molecular Biology, 1515 Holcombe Boulevard – Unit 117, Houston, TX 77030, USA

View larger version (68K): [in a new window]   Fig. 2. dronc mutants suppress the GMR-hid- and GMR-reaper-induced small-eye phenotype in recessive clones. (A) The unsuppressed GMR-hid ey-FLP (GheF) eye ablation phenotype. (B) Suppression of the GheF phenotype in ey-FLP/FRT-induced clones of droncI24. The exact genotype of this fly is GheF/w; droncI24 FRT80/w+ FRT80. This dronc allele gives rise to strong suppression of GMR-hid and is molecularly a null allele. Similar results were obtained for droncI29 and dronc2. Despite the fact that mutant clones are phenotypically w– (see Fig. 1), these flies still produce red eye pigment because the GMR-hid transgene is marked with w+ (not indicated in Fig. 1). (C) Suppression of the GheF phenotype in ey-FLP/FRT-induced clones of droncL32. The exact genotype of this fly is GheF/w; droncL32 FRT80/w+ FRT80. This dronc allele gives rise to medium-strong suppression of GMR-hid, and is thus a hypomorphic allele. (D) dronc mutants fail to dominantly modify the GMR-hid eye ablation phenotype. The genotype of this fly is GheF/w; droncI24 FRT80/+. (E) The unsuppressed GMR-reaper ey-FLP eye ablation phenotype. Genotype of this fly: ey-FLP/w; CyO, 2xGMR-reaper/+. (F,G) Suppression of GMR-reaper in ey-FLP/FRT-induced clones of droncI24 (F) and droncL32 (G). Genotypes: ey-FLP/w; CyO, 2xGMR-reaper/+; droncI24 FRT80/w+ FRT80 (F) and ey-FLP/w; CyO, 2xGMR-reaper/+; droncL32 FRT80/w+ FRT80. (H) Partial restoration of the GMR-hid eye ablation phenotype in dronc mutant clones by a dronc+ transgene. The genotype of this fly is GheF/w; GMR-Gal4/+; droncI29 FRT80 UAS-proDronc/w+ FRT80.

View larger version (12K): [in a new window]   Fig. 1. The GheF screening method. Males of the indicated genotype were treated with the chemical mutagen EMS as described in Materials and methods. The mutagenized males were mated to the GMR-hid ey-FLP (GheF), FRT80 tester females. F1 offspring of this cross were screened for a modification, usually a suppression, of the GMR-hid eye ablation phenotype. Suppressor mutants were recovered, retested and established as balanced stocks. Only recessive suppressors were maintained; dominant suppressors were discarded.

View larger version (82K): [in a new window]   Fig. 4. dronc is essential for cell death in wing and eye development. (A,B) Abnormal wing phenotype of homozygous adult dronc mutants. The wings are less transparent and curved. The held-out wing in A is occasionally observed and not typical. Genotype: (A) droncI24/droncI24; (B) droncI29/droncI29. (C) en::GFP expression in a wing of a freshly eclosed wild-type male (less than 1 hour old). (D) en::GFP expression in a wing of a 24-hour-old wild-type male. No GFP expression is detectable. (E) en::GFP expression in a wing of a 24-hour-old droncI24/droncI29 male. GFP expression is still detectable. (F) Anti-Dlg labeling in a wild-type pupal retina disc to visualize cell outline. (G) Anti-Dlg expression in pupal retina of a droncI24 mutant clone. Additional inter-ommatidial cells are present, suggestive of lack of apoptosis.

View larger version (13K): [in a new window]   Fig. 3. Location of point mutations in the dronc gene. The domain structure of Dronc with CARD, large and small subunits is depicted. Essential residues for maturation of Dronc are indicated. Asp113, Asp135, Asp324, and Glu352 are putative caspase cleavage sites. The molecular lesions of the isolated dronc mutations are shown.

View larger version (100K): [in a new window]   Fig. 5. Cell death analysis in wild-type and dronc mutant embryos. (A,D) Wild-type (wt) embryos stained with TUNEL (A) and CM1 antibody (D). CM1 labels activated DrICE (Yu et al., 2002). (B-F) Maternally and zygotically mutant droncI24 (B,E) and droncI29 (C,F) embryos labeled with TUNEL (B,C) and CM1 antibody (E,F). Despite the strong reduction in labeling signal, there were still a few TUNEL- and CM1-positive cells in dronc mutant embryos.

View larger version (80K): [in a new window]   Fig. 6. dronc mutant embryos contain additional cells. (A,C,E) Wild-type embryos stained for the midline glia (A), Krüppel (C), and Elav (E). (B,D,F) Maternally and zygotically droncI29 mutant embryos stained for midline glia (B), Krüppel (D) and Elav (F). Similar results were obtained for droncI24 embryos (data not shown). (C,D) Ventral views of the CNS. Note the enlarged band in D compared with C (white bar). The numbers in E and F indicate the number of chordotonal cells in each cluster.

View larger version (46K): [in a new window]   Fig. 7. Ovarian atrophy caused by weak diap1 mutations is rescued by dronc. (A) The diap16B/diap18 mutant ovariole is poorly developed and atrophied. (B) By comparison, the droncI24 diap18/droncL32 diap16B double-mutant ovariole shows improved differentiation of nurse cells and advanced maturation of the oocyte. (C) This improvement is visible in a global view of single mutant (left) and double mutant (right) ovaries.