Nonautonomous apoptosis is triggered by local cell cycle progression during epithelial replacement in Drosophila

Abstract

Tissue remodeling involves collective cell movement, and cell proliferation and apoptosis are observed in both development and disease. Apoptosis and proliferation are considered to be closely correlated, but little is known about their coordinated regulation in physiological tissue remodeling in vivo. The replacement of larval abdominal epidermis with adult epithelium in Drosophila pupae is a simple model of tissue remodeling. During this process, larval epidermal cells (LECs) undergo apoptosis and are replaced by histoblasts, which are adult precursor cells. By analyzing caspase activation at the single-cell level in living pupae, we found that caspase activation in LECs is induced at the LEC/histoblast boundary, which expands as the LECs die. Manipulating histoblast proliferation at the LEC/histoblast boundary, either genetically or by UV illumination, indicated that local interactions with proliferating histoblasts triggered caspase activation in the boundary LECs. Finally, by monitoring the spatiotemporal dynamics of the S/G₂/M phase in histoblasts in vivo, we found that the transition from S/G₂ phases is necessary to induce nonautonomous LEC apoptosis at the LEC/histoblast boundary. The replacement boundary, formed as caspase activation is regulated locally by cell-cell communication, may drive the dynamic orchestration of cell replacement during tissue remodeling.

Fig. 1.

Single-cell level caspase activation dynamics in LECs during epithelial replacement. (A) Schematic illustration of the dorsal abdominal epidermis of a pupa. (B) Expression pattern of an epithelial driver, tsh-Gal4, in the pupal abdomen (A, anterior; P, posterior; L, lateral; M, medial). In each dorsal segment, there are anterior dorsal histoblast (ADH) nests and posterior dorsal histoblast (PDH) nests. The boxed area (segments A2 and A3) was monitored. The expression of fluorescent proteins (SCAT3) in histoblasts is not clear because the expression was low. (C to G) Measurement of FRET ratio changes at the single-cell level. Nuclei are marked by ubiquitously expressed His2Av-mRFP (magenta), and the abdominal epidermis is labeled by SCAT3 (Venus image) (green). SCAT3 ratio images are shown in the lower panels. Arrows indicate caspase-activated LECs. Time-lapse imaging was begun at 18 h APF. The elapsed time is shown. The dashed lines indicate the boundary between the histoblasts and LECs. (F) Typical kinetics of the FRET ratio of an LEC. (G) Ratio images and corresponding morphological changes. Two representative nuclei are outlined by a dashed line. The kinetics of the FRET ratio of a single LEC (*) is drawn in panel F, and the arrows labeled a to d in panel F correspond to panels a to d in panel G. The genotype of the SCAT3 flies was tsh-Gal4 UAS-SCAT3/His2Av-mRFP. (H and I) Confirmation of FRET reduction for SCAT3 imaging. (H) FRET ratio images of the caspase noncleavable SCAT3, SCAT3(DEVG). The fly genotype was tsh-Gal4 UAS-SCAT3(DEVG)/CyO. The time-lapse imaging was begun at 18 h APF. The elapsed time is shown in each panel. (I) FRET ratio images from a fly expressing p35 in the posterior compartment. The genotype was en-Gal4 UAS-SCAT3/His2Av-mRFP; UAS-p35/+. The dashed line shows the boundary of histoblast nests. L, lateral; M, medial. Scale bars, 50 μm (B, C, D, E, H, and I) and 20 μm (G).

Fig. 2.

Initiation of caspase activation in LECs starts mainly at the boundary between LECs and histoblasts. (A) Categories for showing the location of caspase-activated LECs. Magenta circles show caspase-activated LECs at the “boundary,” and yellow circles show the “nonboundary” caspase-activated LECs. Scale bar, 50 μm. (B) Panel a, distribution of caspase-activated LECs at 2-hour intervals, categorized as in panel A (n = 214 LECs; 3 segments). Panel b, distribution of caspase-activated cells in the larval epidermis during abdominal replacement in the pupa (left) and in the AS during DC in the embryo (right) (n = 42 cells; 3 flies). (C) Kinetics of the FRET ratio of individual caspase-activated boundary LECs. Data for 65 individual LECs are shown (gray lines). Three representative LECs exhibiting caspase activation at early, intermediate, or late replacement times are highlighted in green, red, and blue, respectively. The arrows indicate the time of histoblast apposition for each highlighted LEC. The genotype was tsh-Gal4 UAS-SCAT3/His2Av-mRFP. (D and E) SCAT3 is expressed in the epidermis of an embryo. CFP (D) and FRET ratio (E) images of a SCAT3-expressing embryo are shown. Snapshots show the spatiotemporal pattern of caspase activation in AS cells during DC. The dashed line shows the boundary between AS cells and the LE cells of the lateral epidermis. The elapsed time (min) is shown. The arrows show AS cells with activated caspase. The genotype was UAS-SCAT3/+; 69B-Gal4/+. Scale bars, 50 μm.

Fig. 3.

Histoblast nest expansion regulates the frequency of caspase activation in the “boundary” LECs. (A to C) Defective LEC death in histoblast proliferation mutants. Time-lapse imaging was performed from 18 h APF. The elapsed time is shown for each panel. Histoblasts and LECs are shown in magenta and green pseudocolor, respectively. The genotypes were as follows: (A) Wild type (WT) (hh-Gal4 UAS-nls-SCAT3/+); (B) cdc2 mutant (Dmcdc2^E1-24/Dmcdc2^B47; hh-Gal4 UAS-nls-SCAT3/+); (C) esg mutant (esg^VS8/esg^G66B; hh-Gal4 UAS-nls-SCAT3/+). L, lateral; M, medial. (D to F) Monitoring system for the effects of genetically modified histoblasts on caspase activation in LECs. SCAT3 is expressed by the LexA/lexAop system, and the GAL4/UAS system is used for histoblast-specific expression. The elapsed time (h:min) is shown for each panel. (E and F) UAS-dacapo (dap) or EcR (Ecdysone Receptor)-RNAi constructs were overexpressed by the esg-Gal4 driver. Arrows indicate caspase-activated LECs at the boundary between the LECs and histoblasts. (G to J) Anterior regions and ADH nests are the focus of the following results. (G) Expansion of the histoblast nest over the 8-h interval between 16 h and 24 h APF. Histoblast nest expansion was severely impaired when histoblast proliferation was inhibited. (H) LECs located adjacent to the ADH in the A3 segment at the initial stage (16 h APF, ellipses) were analyzed. (I) Frequency of caspase activation in the boundary LECs. The ratio of the caspase-activated LECs at the boundary to the total boundary LECs is shown (WT, n = 5 flies; Dap, n = 4 flies; EcR-RNAi, n = 4 flies). (J) Frequency of caspase activation in nonboundary LECs. The ratio of nonboundary caspase-activated LECs to the total LECs in the vicinity of wild-type or proliferation-defective histoblasts is shown (WT, n = 4 flies; Dap, n = 4 flies; EcR-RNAi, n = 4 flies). Fifty to 75 LECs in the anterior region were counted for each individual. (G, I, and J) One-way analysis of variance (ANOVA) with Tukey-Kramer test: ***, P < 0.0001; **, P < 0.01; n.s., not significant). Genotypes: WT, tubP-LexA::GAD; esg-Gal4 lexAop-SCAT3/CyO; Dap, tubP-LexA::GAD/+; esg-Gal4 lexAop-SCAT3/UAS-dap; EcR-RNAi, tubP-LexA::GAD/+; esg-Gal4 lexAop-SCAT3/+; UAS-EcR-RNAi/+. Scale bars in panels A to F, 50 μm.

Fig. 4.

Cell cycle dynamics of histoblasts monitored by S/G2/M-Green. (A) S/G2/M-Green, a fluorescent probe that labels cells in the S/G₂/M phases. In the construct, mAG (monomeric Azami Green) is fused to the deletion mutant of human Geminin (57). Accumulation of S/G2/M-Green in the nucleus indicates that the cell is in the S/G₂ phase. Distribution of S/G2/M-Green into the cytoplasm corresponds to the initiation of M phase. (B and C) S/G2/M-Green accumulated in histoblasts in the L3 larval stage and before the first mitosis upon pupation (0 h APF). (D) Histoblasts undergo synchronous division at around 2 h APF. (E) Time-lapse observation of cell cycle changes of histoblasts during the replacement stage (from 15 h APF). Cellular outlines are marked by Nrg-GFP. White lines indicate the example. (F) Average cell doubling time (C.D.T) and duration of the S/G₂ phases of the histoblasts (C.D.T., n = 47; S/G₂ phase, n = 127). S/G₂ phase was monitored by examining S/G2/M-Green flies, and C.D.T. was also counted by time-lapse analysis using His2Av-RFP lines (from 16 h APF). (G) Left, at the initial stage of replacement, most histoblasts were in the S/G₂ phase. Right, cell cycle progression occurs dynamically in histoblasts throughout the expanding nest. The white dashed lines indicate histoblast nests. (H and I) S/G2/M-Green indicates the S/G₂ and M phases but not the G₁ phase. (H) Cyclin E accumulated in histoblasts that did not show an accumulation of S/G2/M-Green in the nucleus. (I) M phase cells were recognized by their round or dividing shape using a cell shape marker (Nrg-GFP). These cells showed a cytoplasmic distribution of S/G2/M-Green and coincided with PH3-positive cells. (J) Frequency of G₂/M transition in histoblasts surrounding LECs. The number of histoblasts passing from G₂ to M phase surrounding LECs was counted in 2-h intervals. Three individual flies were tested. The frequency of the G₂/M transition of histoblasts was not different significantly between time courses (one-way ANOVA with Tukey-Kramer test). Cells with large nuclei are LECs in panels E and G. The genotype of S/G2/M-Green flies was Nrg-GFP; act-Gal4 UAS-S/G2/M-Green/CyO. Scale bars, 10 μm (B to E, H, and I) and 50 μm (G).

Fig. 5.

Normal cell cycle progression of histoblasts is necessary to induce nonautonomous apoptosis in neighboring LECs. (A and B) After UV exposure, S/G2/M-Green accumulated in the nuclei of histoblasts for a much longer time than in the nontreated control. (A) In vivo monitoring of S/G2/M-Green accumulation after UV laser illumination. (B) Duration of S/G₂ phases of histoblasts. Each bar shows the time required for histoblasts to pass through S/G₂ phases under several conditions according to the laser (405-nm) power (control, n = 127; 30%, n = 64; 50%, n = 27; 80%, n = 49). (C) Stills from a time-lapse movie showing that the cell cycle progression of histoblasts occurs during neighboring LEC apoptosis. LEC apoptosis is defined as delamination from the epithelium (coincident with nuclear fragmentation). The arrow indicates a dividing histoblast. The white line indicates the shape of a dying LEC. (D) Stills from a time-lapse movie showing defects in the apoptosis of LECs neighboring S/G₂ phase-arrested histoblasts. LECs in contact with S/G₂ phase-arrested histoblasts (red dots) did not show delamination during the observation. The white dashed lines indicate histoblast nests. Magenta dashed lines indicate the UV-illuminated region. (E) The number of histoblasts surrounding apoptotic LECs was similar to the number of S/G₂ phase-arrested histoblasts surrounding LECs after UV laser exposure. The graph shows the distribution of the number of histoblasts surrounding each LEC for the following conditions. The number of normal, untreated histoblasts surrounding each LEC during apoptosis was 4.71 ± 1.76 (histoblasts per LEC; n = 77 LECs). The number of UV-illuminated histoblasts surrounding individual boundary LECs was 4.42 ± 1.42 (histoblasts per LEC; n = 38 LECs). The genotype of S/G2/M-Green flies was Nrg-GFP; act-Gal4 UAS-S/G2/M-Green/CyO. Scale bars, 50 μm (A and D) and 10 μm (C).

Fig. 6.

Local interactions with proliferating histoblasts regulate the frequency of caspase activation in neighboring LECs. (A to C) The frequency of caspase activation in the boundary LECs is regulated by their local interactions with proliferating histoblasts. (A) Medial boundary LECs located adjacent to ADH at the initial stage (ellipses) were analyzed under four UV-illuminated conditions: a, nontreated control; b, entire ADH; c, medial one-third of the ADH; and d, lateral one-third of the ADH. L, lateral; M, medial. (B) Expansion of the histoblast nest (bar a, n = 4 flies; bar b, n = 4 flies; bar c, n = 5 flies; bar d, n = 6 flies) (one-way ANOVA with Tukey-Kramer test: ***, P < 0.0001; **, P < 0.01; *, P < 0.05; n.s., not significant). (C) Ratio of the caspase-activated LECs to the total boundary LECs from 18 to 24 h APF (bar a, n = 5 flies; bar b, n = 7 flies; bar c, n = 6 flies; bar d, n = 8 flies) (one-way ANOVA with Tukey-Kramer test: ***, P < 0.0001; **, P < 0.01; n.s., not significant). (D and E) Local interactions with proliferation-defective histoblasts delay caspase activation in neighboring LECs. (D) FLP-Out/FRT UAS-PTEN clones were generated in histoblasts marked with SCAT3. Venus (panel a) and FRET ratio (panel b) images of SCAT3 are shown. The yellow region and white dashed lines indicate PTEN-overexpressing clones in histoblasts; the magenta dashed line indicates neighboring wild-type histoblasts. Arrows indicate caspase-activated LECs in contact with wild-type neighbors. Scale bar, 20 μm. (E) Frequency of caspase activation in boundary LECs interacting with PTEN-overexpressing clones (PTEN) or wild-type neighbors (WT); the ratio of caspase-activated LECs to boundary LECs (WT, n = 5 flies; PTEN, n = 6 flies) is shown. Observations were made between 16 and 24 h APF. (Unpaired t test, P = 0.0187 [<0.05].) (F and G) Local interactions between LECs and histoblasts are restricted by the developmental A/P compartment boundary during epithelial replacement. (F) Snapshots showing an individual with a PDH nest that was entirely UV illuminated. In the upper panels, posterior cell nuclei are marked by an enhancer trap line with nls:DsRed (magenta), and the abdominal epidermis is labeled by SCAT3 (Venus image) (green). Ratio images of SCAT3 are shown in the lower panels. Time-lapse imaging began around 18 h APF. White dashed lines indicate the ADH boundary; yellow arrows indicate posterior LECs in contact with only ADH cells. Elapsed time (h:min) is shown. Scale bars, 50 μm. (G) Duration of anterior or posterior LEC interactions with ADH cells before LEC caspase activation (P LECs, n = 16; A LECs, n = 33) (unpaired t test: ***, P < 0.0001.) (H) Model summarizing the abdominal epithelial replacement. Apoptosis at the “replacement boundary,” which moves as the LECs are eliminated, couples histoblast proliferation with LEC apoptosis. Boundary apoptosis creates the spatiotemporal pattern of tissue removal and maintains the epithelial integrity as the histoblasts replace the LECs. Stochastic, nonboundary apoptosis also occurs at a low frequency. The A/P compartment (yellow dashed lines) is maintained between histoblasts and histoblasts/LECs during tissue remodeling. The genotypes used for the analysis were tsh-Gal4 UAS-SCAT3/+; His2Av-mRFP/+ (A to C), hs-flp¹²²/+; AyGal4 UAS-SCAT3/UAS-PTEN (D and E), and tsh-Gal4 UAS-SCAT3/+; hh^PyR215/+ (F and G).