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

Abstract

The initiator caspase Dronc is the only Drosophila caspase that contains a caspase activation and recruitment domain (CARD). Although Dronc has been implicated as an important effector of apoptosis, the genetic function of dronc in normal development is unclear because dronc mutants have not been available. In an EMS mutagenesis screen, we isolated four point mutations in dronc that recessively suppress the eye ablation phenotype caused by eye-specific overexpression of hid. Homozygous mutant dronc animals die during pupal stages; however, at a low frequency we obtained homozygous adult escapers. These escapers have additional cells in the eye and wings that are less transparent and slightly curved down. We determined that this is due to lack of apoptosis. Our analyses of dronc mutant embryos suggest that dronc is essential for most apoptotic cell death during Drosophila development, but they also imply the existence of a dronc-independent cell death pathway. We also constructed double mutant flies for dronc and the apoptosis inhibitor diap1. dronc mutants can rescue the ovarian degeneration phenotype caused by diap1 mutations, confirming that dronc acts genetically downstream of diap1.

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.

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 dronc^I24. The exact genotype of this fly is GheF/w; dronc^I24 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 dronc^I29 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 dronc^L32. The exact genotype of this fly is GheF/w; dronc^L32 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; dronc^I24 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 dronc^I24 (F) and dronc^L32 (G). Genotypes: ey-FLP/w; CyO, 2xGMR-reaper/+; dronc^I24 FRT80/w⁺ FRT80 (F) and ey-FLP/w; CyO, 2xGMR-reaper/+; dronc^L32 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/+; dronc^I29 FRT80 UAS-proDronc/w⁺ FRT80.

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.

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) dronc^I24/dronc^I24; (B) dronc^I29/dronc^I29. (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 dronc^I24/dronc^I29 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 dronc^I24 mutant clone. Additional inter-ommatidial cells are present, suggestive of lack of apoptosis.

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 dronc^I24 (B,E) and dronc^I29 (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.

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 dronc^I29 mutant embryos stained for midline glia (B), Krüppel (D) and Elav (F). Similar results were obtained for dronc^I24 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.

Fig. 7

Ovarian atrophy caused by weak diap1 mutations is rescued by dronc. (A) The diap1^6B/diap1⁸ mutant ovariole is poorly developed and atrophied. (B) By comparison, the dronc^I24 diap1⁸/dronc^L32 diap1^6B 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.