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First published online October 22, 2003
doi: 10.1242/10.1242/jcs.00783

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DIAP1 suppresses ROS-induced apoptosis caused by impairment of the selD/sps1 homolog in Drosophila

Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain

View larger version (112K): [in a new window]   Fig. 1. Genetic mosaics of selDptuf in the eye. (A) selDptuf clones were recovered to adulthood and resulted in aberrant eyes. The mutant area (light red sectors; arrowheads) was scarred and almost no ommatidia differentiated. (B) Tangential sections of selDptuf clones revealed no ommatidial cells present in the mutant area. Right half: mutant sector; left half: normal tissue. (C) selDptuf homozygous clones in the eye imaginal disc stained with the anti-Elav neuronal cell marker (red; left) indicated that differentiation of photoreceptors is altered. In the middle panels mutant clones (black) lack ß-galactosidase staining (green). Right panels: merged images. A confocal section at the normal level of Elav pattern (upper panels) showed no labeling in the selDptuf clone; however, deeper in the mutant tissue (lower panels) Elav staining occurs, showing abnormal ommatidia (arrows). (D) TUNEL staining (red) in a selDptuf clone of an eye imaginal disc. It shows that cells in the selDptuf clone (dark area lacking ß-galactosidase labeling) entered apoptosis. In addition to wild-type apoptosis in the vicinity of the morphogenetic furrow (arrow), ectopic apoptotic figures were observed in cells adjacent to the clone (arrowhead). (E) In vivo detection of ROS with DHE (red) in selDptuf clones (dark area lacking GFP labeling) DHE labeled cells were present in the clone and also in cells adjacent to it (arrowhead).

View larger version (92K): [in a new window]   Fig. 2. Phenotypes resulting from genetic interactions of the Hid and the Ras pathway. (A) Wild type eye. (B) GMRhid. (C) GMRhid/selDptuf. (D) GMRhid/selDptuf; Catn1. (E) GMRhid; sev-rasV12. (F) GMRhid/selDptuf; sev-rasV12. (G) GMRyanAct. (H) GMRyanAct/ selDptuf. (I) sev-rasV12. (J) selDptuf; sev-rasV12/ Catn1. Anterior part of the eye is to the left.

View larger version (93K): [in a new window]   Fig. 3. Transcription pattern of reaper in selDptuf cells. (A) Widespread rpr transcription (blue) in a selDptuf homozygous mutant imaginal disc where the cell surface was outlined using anti-Fasciclin III antibody (green). (B,C) rpr transcription (blue) in selDptuf clones (area lacking the green -Myc labeling) in the wing (B) and the eye (C) disc, respectively. (D) Detail of some cells of a gl-Dmp53 eye disc showing Dmp53 stabilization (red) and rpr transcription (blue). Panels on the right correspond are merged images.

View larger version (129K): [in a new window]   Fig. 4. Dmp53 stabilization in selDptuf cells. (A) Detection of Dmp53 (red) in an eye disc overexpressing Dmp53 under glass enhancer sequences. (B) High magnification of nuclear confocal section counterstained with YOYO nuclear marker (green) showed co-localization of Dmp53 (red) into the nuclei. (C) Dmp53 accumulation (red) in a selDptuf homozygous mutant imaginal disc stained with YOYO. (D) Detail of a high magnification image focusing on two nuclei stained with YOYO nuclear marker (green, middle panel) showed co-localization of Dmp53 (red upper panel) into disrupted selDptuf nuclei. Lower panel shows merged images.

View larger version (118K): [in a new window]   Fig. 5. DRONC and DRICE caspases are involved in selDptuf apoptosis. (A) Overexpression of DRONC caspase under the control of GMR-GAL4 driver gives rise to slightly rough and mottled eyes as a result of ectopic cell death. white patches result from the ablation of pigment cells. (B,C) Reduction of one dose of selD or catalase, respectively, enhanced the mottled eye phenotype. Wider areas of white tissue were observed indicating that an increase in ROS levels enhances DRONC apoptotic activity. (D) Combined reduction of selD and catalase strongly enhanced DRONC overexpression phenotype and few pigmented cells were observed. (E) Active DRICE (red) in a selDptuf homozygous mutant imaginal disc. The cell surface was outlined using anti-Fasciclin III antibody (green). (F) Active DRICE in selDptuf clones (dark area lacking ß-galactosidase labeling, green) in the eye disc. Consistent with the finding of non autonomous apoptosis and ROS, active DRICE was found as well outside the clone. (G) Overexpression of Dmp53 triggers apoptosis through DRICE caspase as shown by the presence of active DRICE (red) in gl-Dmp53 discs. In E, F and G lower panels are merged images.

View larger version (17K): [in a new window]   Fig. 6. Rescue of selDptuf clone size by DIAP1 overexpression. selDptuf clones were induced in the posterior DIAP1 expressing compartment and in the anterior compartment of the wing pouch. Bars represent mean values of the area of selDptuf clones as a percentage of the area of their wild-type selD+ twin clone±s.d., n=number of mutant and wild-type twin clone pairs analyzed in the anterior and posterior compartment. Overexpression of DIAP1 in the posterior compartment almost doubled selDptuf clonal size. Mean area of anterior and posterior selDptuf clones, 34% and 58% of their wild-type twin clone respectively, were significantly different (t-test: P<0.001).

View larger version (10K): [in a new window]   Fig. 7. A model for ROS-induced apoptosis in cells devoid of selD/sps1. We propose that the increase in ROS caused by the selDptuf mutation leads to stabilization of Dmp53 and activation of Rpr caspase-dependent apoptosis. We cannot discount the possibility that other pathways such Ras/MAPK downregulation or a ROS-induced caspase-independent pathway may have a minor contribution to selDptuf apoptosis.