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First published online 3 August 2006
doi: 10.1242/dev.02495

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The Drosophila caspase Ice is important for many apoptotic cell deaths and for spermatid individualization, a nonapoptotic process

¹ Division of Biology, MC 156-29, California Institute of Technology, Pasadena, CA 91125, USA.
² Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742, USA.
³ Department of Biology, Kyung Hee Institute of Age-related and Brain Diseases, Kyung Hee University, Seoul, 130-701, Korea.
⁴ Department of Neurology, Brain Research Institute, The David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA.

View larger version (19K): [in a new window]   Fig. 1. Ice1 is a null allele of Ice. (A) Genomic organization of the Ice region. The P transposon element EPGE28489 is inserted at base 1711 in the Ice transcription unit. The Ice1 excision line removes the entire Ice-coding region, fusing small remaining fragments of the Ice 5'- and 3'-UTR sequences. A 1.5 kb fragment of EPGE28489 also remains. The gray region indicates the Ice-coding region; the black boxes indicate the UTR sequences of Ice and of two neighboring genes. Locations of the EPGE28489 element, the Ice1 deletion and the Ice genomic rescue construct (Ice+2.4) are indicated to scale. (B) Western blot of adults of various genotypes probed with anti-Ice antibodies. The genotypes are wild type, Ice1, Ice1; Ice+2.4 and dcp-1Prev1 (Dcp-1), a null allele of dcp-1.

View larger version (94K): [in a new window]   Fig. 2. Ice is required for normal pupal development. (A) The percent viability to adulthood for wandering third instar larvae of several different genotypes is shown: wild type, Ice1, Ice+2.4; Ice1, dcp-1Prev1 and dcp-1Prev1; Ice1. (B-G) Paraffin sections of pupae aged 24 hours after puparium formation (apf). (B) In control pupae, salivary glands and larval muscle have both disappeared and the larval mid-gut has degenerated, as previously described (Lee and Baehrecke, 2001). (C) Seventy-three percent of Ice1 pupae develop normally. By 24 hours apf, only a few remaining small degraded fragments of salivary glands and larval muscles are observed and the mid-gut is normal. (D) Twenty-seven percent of Ice1 pupae were arrested in development. Of those that arrested, one-third did not undergo head eversion and arrested prior to the commencement of salivary gland cell death. The remaining pupae appear to have arrested 1 or 2 hours after head eversion, and thus the salivary glands and larval muscle are not degraded, and the larval gut has failed to condense properly. Ice1 pupae have abnormal masses in the head (C,E), wing and leg discs (C,F), and abdomen (G). Arrows indicate abnormal masses; arrowheads indicate muscle; red circles are placed around salivary glands, salivary gland fragments and regions where degraded salivary glands would be if they had failed to die. Animals homozygous for Ice1 lack zygotic but not maternal Ice. Scale bars: 200 µm in B-D; 40 µm in E-G.

View larger version (81K): [in a new window]   Fig. 3. Adult Ice1 show multiple defects suggestive of defects in developmental cell death. (A-E) SEM images of arista of different genotypes. (A) Wild type, (B) Ice1, (C) th1, (D) th1, Ice1 and (E) dcp-1Prev1/+; Ice1. (F) Ventral view of the abdomen of a wild-type male, The genitalia (G) and analia (A) are marked. (G) Similar view of the abdomen of a Ice1 adult. Genitalia are markedly rotated. (H) Dorsal view of the abdomen of a wild-type adult male. (I) Similar view of the abdomen of a Ice1 adult male, showing a pale line that runs along the dorsal abdominal midline. Animals homozygous for Ice1 lack zygotic but not maternal Ice.

View larger version (145K): [in a new window]   Fig. 4. Ice is required for normally occurring cell death in the adult wing and pupal retina. (A) en::GFP expression in the wing of a wild-type adult (eclosed for less than 1 hour). (B) en::GFP expression in the wing of a wild-type adult (eclosed for 2 hours). (C) en::GFP expression in the wing of a Ice1 adult (eclosed for 24 hours). (D,E) Forty-hour pupal eyes stained with anti-Dlg. (D) Wild type; (E) Ice1. The area in which the number of interommatidial cells was counted for each genotype is indicated in D by the broken line. Several regions with extra cells are indicated with arrows in the mutant (E). Animals homozygous for Ice1 lack zygotic but not maternal Ice.

View larger version (56K): [in a new window]   Fig. 5. Ice is required for some normally occurring cell death during embryogenesis. (A-C) TUNEL stains of stage 14 embryos of different genotypes. (A) Wild type; (B) Ice1; (C) Ice+2.4; Ice1. (D-F) Stage 17 embryos of different genotypes, each of which carries the P[sli-1.0-lacZ] enhancer trap line, which is expressed in midline glia. (D) Wild type, (E) Ice1 and (F) Ice+2.4; Ice1. Embryos homozygous for Ice1 lack maternal and zygotic Ice.

View larger version (61K): [in a new window]   Fig. 6. Ice is required for cell death induced by X-irradiation and inhibition of protein synthesis. (A-F) TUNEL-stained third instar wing discs of various genotypes. (A) Untreated wild type; (B) X-irradiated wild type; (C) untreated Ice1; (D) X-irradiated Ice1; (E) X-irradiated Ice+2.4; Ice1; (F) X-irradiated dcp-1Prev1. (G) Wild-type hemocytes exposed to cyclohexamide died within 4 hours. Removal of zygotic Ice (Ice1)prevented almost all this death, which was restored in the presence of a wild-type Ice rescue transgene (Ice1,Ice+2.4). Animals homozygous for Ice1 lack zygotic but not maternal Ice. Animals homozygous for dcp-1Prev1 lack both maternal and zygotic dcp-1.

View larger version (82K): [in a new window]   Fig. 7. Ice is important for cell death induced by rpr, hid, grim and loss of Th, but not Nc overexpression. (A-J) Adult eyes of various genotypes: (A) GMR-rpr; (B) GMR-hid; (C) GMR-grim; (D) GMR-Th-RNAi; (E) GMR-Nc; (F) GMR-rpr; Ice1; (G) GMR-hid;Ice1; (H) GMR-grim; Ice1; (I) GMR-Th-RNAi; Ice1; (J) GMR-Nc; Ice1. (K-M) Tangential sections through adult eyes of various genotypes: (K) wild type; (L) GMR-Nc; (M) GMR-Nc, Ice1. Pigment cells (red) and photoreceptors (blue) form a regular array in the wild-type adult eye (K). These cells are largely absent in the eyes of flies expressing Nc, in the presence (L) or absence (M) of Ice. Animals homozygous for Ice1 lack zygotic but not maternal Ice.

View larger version (171K): [in a new window]   Fig. 8. Ice is important for spermatid individualization. (A-C) EM sections of elongated cysts of different genotypes: (A) wild type; (B) Ice1; (C) Ice1. Scale bars: 1 µm in A,B; 2.4 µm in C. Arrows in C indicate some regions within cysts in which individualization has failed. Animals homozygous for Ice1 lack maternal and zygotic Ice.

View larger version (100K): [in a new window]   Fig. 9. Antibodies that recognize cleaved versions of Ice are accurate reporters of Ice activation in irradiated wing discs, but not in embryos and spermatids. (A-R) X-irradiated wing discs of various genotypes stained for TUNEL (A,C,D,F,G,I,J,L,M,O,P,R), anti-active Ice (B,C,H,I,N,O) or CM1 (E,F,K,L,Q,R). Genotypes are as indicated in the figure. Wild-type (S,U) or Ice1 (T,V) embryos stained with anti-active Ice (S,T) or CM1 (U,V). (W-Z) Wild-type (W,Y) or Ice1 testis stained with anti-active Ice (W,X) or CM1 (Y,Z).