Figure 5.
Figure 5. DIAP1 functions as a caspase inhibitor in the early stage of shaft cell differentiation. (A) Wild-type bristle. (B and C) Down-regulation of the diap1 level induces a loss-of-bristle phenotype. The sockets (arrowheads) were present and only the bristles were missing. Bars, 10 µm. (D) In the Reaper-expressing SOP lineage, PRAP did not accumulate at detectable levels in the socket or shaft cells, and the shaft cell eventually migrated away. Arrowheads indicate the shaft cell. Dashed lines outline the cell nuclei. (E) The sequential variation of Venus intensity in the shaft cell of the Reaper-expressing SOP lineage (dark lines) and the wild-type SOP lineage (faint lines). The time of the shaft cell's birth was defined as 0:00. The shaft cell disappeared 4:35 ± 0:58 h (mean ± SD) after its birth, accompanied by rapid PRAP degradation (n = 18). In the wild-type lineage, PRAP accumulation starts 3:53 ± 1:18 h (n = 13) after the cell's birth. Five representative lines from each genotype are shown. (F) Caspase activation is observed in the shaft cell before its nuclear fragmentation (arrows). Caspase activity was examined by the imaging of a FRET-based probe, nls-SCAT3, and shown in pseudocolor. Arrowheads indicate the shaft cell, and fragmented nuclei are indicated by arrows. Bar, 5 µm. (G) Genetic interaction of DIAP1dsRNA-expressing flies with cell death signaling components. The loss-of-bristle phenotype was completely rescued by the dominant-negative form of Dronc. 109.68-GAL4 UAS-DIAP1-IR/CyO flies were crossed to flies with the indicated genotypes. The number of F1 progeny adult flies exhibiting at least one loss-of-bristle lineage on the notum was counted, and the percentage (%) was calculated. The sample number (n) is indicated at the right side of each bar. The genotypes were as follows: w1118 (A), 109.68-GAL4 UAS-DIAP1-IR/CyO (B), 109.68-GAL4/UAS-Reaper (C), 109.68-GAL4/UAS-Reaper; UAS-PRAP/UAS-Histone2B-ECFP (D, and +Reaper in E), 109.68-GAL4/+; UAS-PRAP/UAS-Histone2B-ECFP (wild-type in E), 109.68-GAL4/UAS-Reaper; UAS-nls-SCAT3/+ (F). The genotypes of flies used in G were as follows: 109.68-GAL4/UAS-Histone2B-ECFP (Histone2B-ECFP), 109.68-GAL4 UAS-DIAP1-IR/CyO (+), UAS-mycDIAP1/+; 109.68-GAL4 UAS-DIAP1-IR/+ (DIAP1), 109.68-GAL4 UAS-DIAP1-IR/+; UAS-Dronc DN/+ (Dronc DN).

DIAP1 functions as a caspase inhibitor in the early stage of shaft cell differentiation. (A) Wild-type bristle. (B and C) Down-regulation of the diap1 level induces a loss-of-bristle phenotype. The sockets (arrowheads) were present and only the bristles were missing. Bars, 10 µm. (D) In the Reaper-expressing SOP lineage, PRAP did not accumulate at detectable levels in the socket or shaft cells, and the shaft cell eventually migrated away. Arrowheads indicate the shaft cell. Dashed lines outline the cell nuclei. (E) The sequential variation of Venus intensity in the shaft cell of the Reaper-expressing SOP lineage (dark lines) and the wild-type SOP lineage (faint lines). The time of the shaft cell's birth was defined as 0:00. The shaft cell disappeared 4:35 ± 0:58 h (mean ± SD) after its birth, accompanied by rapid PRAP degradation (n = 18). In the wild-type lineage, PRAP accumulation starts 3:53 ± 1:18 h (n = 13) after the cell's birth. Five representative lines from each genotype are shown. (F) Caspase activation is observed in the shaft cell before its nuclear fragmentation (arrows). Caspase activity was examined by the imaging of a FRET-based probe, nls-SCAT3, and shown in pseudocolor. Arrowheads indicate the shaft cell, and fragmented nuclei are indicated by arrows. Bar, 5 µm. (G) Genetic interaction of DIAP1dsRNA-expressing flies with cell death signaling components. The loss-of-bristle phenotype was completely rescued by the dominant-negative form of Dronc. 109.68-GAL4 UAS-DIAP1-IR/CyO flies were crossed to flies with the indicated genotypes. The number of F1 progeny adult flies exhibiting at least one loss-of-bristle lineage on the notum was counted, and the percentage (%) was calculated. The sample number (n) is indicated at the right side of each bar. The genotypes were as follows: w1118 (A), 109.68-GAL4 UAS-DIAP1-IR/CyO (B), 109.68-GAL4/UAS-Reaper (C), 109.68-GAL4/UAS-Reaper; UAS-PRAP/UAS-Histone2B-ECFP (D, and +Reaper in E), 109.68-GAL4/+; UAS-PRAP/UAS-Histone2B-ECFP (wild-type in E), 109.68-GAL4/UAS-Reaper; UAS-nls-SCAT3/+ (F). The genotypes of flies used in G were as follows: 109.68-GAL4/UAS-Histone2B-ECFP (Histone2B-ECFP), 109.68-GAL4 UAS-DIAP1-IR/CyO (+), UAS-mycDIAP1/+; 109.68-GAL4 UAS-DIAP1-IR/+ (DIAP1), 109.68-GAL4 UAS-DIAP1-IR/+; UAS-Dronc DN/+ (Dronc DN).

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