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. 2021 Jan 28;17(1):e1009300. doi: 10.1371/journal.pgen.1009300

Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants

Nanami Akai 1,2,#, Shizue Ohsawa 1,2,*,#, Yukari Sando 1, Tatsushi Igaki 1,*
Editor: Norbert Perrimon3
PMCID: PMC7842893  PMID: 33507966

Abstract

Highly reproducible tissue development is achieved by robust, time-dependent coordination of cell proliferation and cell death. To study the mechanisms underlying robust tissue growth, we analyzed the developmental process of wing imaginal discs in Drosophila Minute mutants, a series of heterozygous mutants for a ribosomal protein gene. Minute animals show significant developmental delay during the larval period but develop into essentially normal flies, suggesting there exists a mechanism ensuring robust tissue growth during abnormally prolonged developmental time. Surprisingly, we found that both cell death and compensatory cell proliferation were dramatically increased in developing wing pouches of Minute animals. Blocking the cell-turnover by inhibiting cell death resulted in morphological defects, indicating the essential role of cell-turnover in Minute wing morphogenesis. Our analyses showed that Minute wing discs elevate Wg expression and JNK-mediated Dilp8 expression that causes developmental delay, both of which are necessary for the induction of cell-turnover. Furthermore, forced increase in Wg expression together with developmental delay caused by ecdysone depletion induced cell-turnover in the wing pouches of non-Minute animals. Our findings suggest a novel paradigm for robust coordination of tissue growth by cell-turnover, which is induced when developmental time axis is distorted.

Author summary

Animal development can be disturbed by various stimuli such as genetic mutations, environmental fluctuations, and physical injuries. However, animals often accomplish normal tissue growth and morphogenesis even in the presence of developmental perturbations. Drosophila Minute mutants, a series of fly mutants for a ribosomal protein gene, show significantly prolonged larval period but develop into essentially normal flies. We found an unexpected massive cell death and subsequent compensatory cell proliferation in developing wing discs of Minute animals. This ‘cell-turnover’ was essential for normal wing morphogenesis in Minute flies. We found that the cell-turnover was induced by elevated Wg expression in the wing pouch and JNK-mediated Dilp8 expression that causes developmental delay. Indeed, cell-turnover was reproduced in non-Minute animals’ wing discs by overexpressing Wg using the wg promoter together with developmental delay caused by ecdysone depletion. Our findings propose a novel paradigm for morphogenetic robustness by cell-turnover, which ensures normal wing growth during the abnormally prolonged larval period, possibly by creating a flexible cell death and proliferation platform to adjust cell numbers in the prospective wing blade.

Introduction

Multicellular organisms accomplish normal tissue growth in a highly reproducible manner even in the presence of various perturbations such as genetic mutations, environmental fluctuations, and physical injuries [1]. To achieve such robust development, cells in the developing tissue must behave in a plastic and coordinated manner to correct perturbations and ensure time-dependent morphogenesis. For instance, when abnormally massive cell death occurs in developing Drosophila imaginal epithelia, dying cells trigger compensatory proliferation of surrounding viable cells by secreting growth factors to ensure normal morphogenesis [2–4]. In addition, in response to a physical injury induced in Drosophila imaginal epithelia, the larval period is extended to allow injured tissue time to regenerate before entering metamorphosis [5]. During the extended larval period, other intact tissues stop growing until the injured or delayed tissue catches up in development [6]. Thus, multicellular organisms possess buffering mechanisms against perturbations in the developmental time axis through systemic and/or short-range cell-cell communications that govern cell proliferation and cell death.

Reproducible animal morphogenesis is ensured by the precise progression of developmental timing, and thus a distortion in the developmental time axis potentially causes developmental defects. It has been well documented that Drosophila mutants heterozygous for ribosomal proteins, called Minute/+ (M/+) mutants, show significant delay in the progression of their larval period (~1.5 times longer than wild-type larvae) [7]. Intriguingly, despite the significant delay in their development, M/+ animals are essentially normal flies without any significant morphological defects except for relatively thinner bristles than wild-type [8]. This suggests that M/+ animals must enforce morphogenetic robustness during the abnormally prolonged larval period. However, the mechanism by which M/+ animals exert morphogenetic robustness against the developmental time distortion remained unknown. Here, we find evidence that both cell death and compensatory cell proliferation are dramatically increased in the M/+ wing imaginal epithelium, which are essential for robust wing development in M/+ animals. Our data suggest that massive cell-turnover creates a flexible cell death/proliferation platform to adjust cell numbers in the developing tissue.

Results

Massive cell-turnover occurs during robust morphogenesis of M/+ tissue

To explore how animals ensure robust coordination of tissue growth, we analyzed cellular behaviors in the developing M/+ wing imaginal disc. It has been well documented by clonal analysis that the growth rate of M/+ cell clones is lower than that of wild-type clones in developing wing discs [9]. The lower growth rate of M/+ animals has been largely assumed to stem from a lower cell division rate of M/+ cells, without any defects in cell proliferation and cell death. Surprisingly, however, we found that cell mitoses were significantly increased in the wing pouch of RpS3/+, one of the M/+ mutants, compared to wild-type, as visualized by M phase marker phospho-Histone H3 (Fig 1A and 1B, quantified in Fig 1C; the wing pouch is pale green-marked oval domain that becomes the adult wing blade) and EdU labeling for S phase cells (Fig 1D and 1E, quantified in Fig 1F) (hereafter, developmental stages of M/+ and wild-type animals examined were adjusted to the wandering larval stage by using the sgsΔ3-GFP reporter [10]). Similar increased cell proliferation was also observed in the wing pouches of other M/+ strains such as RpS17/+ and RpL19/+ (S1A and S1B Fig, quantified in S1C Fig). We further examined this using the cell cycle monitoring probe S/G2/M-Green that labels cells in S/G2/M phases [11,12], which showed that the number of cells in either S/G2 or M phase significantly increased in the RpS3/+ pouch compared to wild-type (Fig 1G and 1H). Furthermore, time-lapse imaging of cultured wing discs confirmed that the number of dividing cells was significantly increased in the RpS3/+ pouch (S2 Movie, quantified in Fig 1I), while the interval between S/G2 and M phases in the dividing cells in the RpS3/+ pouch was comparable to wild-type (S1 Movie, quantified in Fig 1J). These data reveal that cells in slow-growing M/+ wing pouch proliferate more rapidly than cells in wild-type wing pouch.

Fig 1. Massive cell-turnover occurs during wing development in M/+ animals.

Fig 1

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(A and B) Wing disc of wild-type (A) or RpS3/+ (B) flies were stained with anti-phospho-histone H3 (pH3) (Ser10) antibody (white). Wing pouches were marked in pale green. Scale bar, 100 μm. (C) Boxplot with dots representing pH3 positive cells in the pouch in genotypes shown in (A) (n = 34, number of wing pouches) and (B) (n = 31). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (D and E) S-phase cells in the wing discs of wild-type (D) or RpS3/+ (E) flies were visualized by EdU staining (nuclei of proliferating cells) (white). Wing pouches were marked in pale green. Scale bar, 100 μm. (F) Boxplot with dots representing the EdU-positive area per wing pouch in genotypes shown in (D) (n = 13, number of wing pouches) and (E) (n = 11). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (G and H) Dividing cells were visualized by the S/G2/M-Green probe. Wing disc of nub-Gal4, UAS-S/G2/M-Green (G) or RpS3/+, nub-Gal4, UAS-S/G2/M-Green (H) is shown. Scale bar, 100 μm. (I) Quantification of the manually counted number of dividing cells in the wing pouch of genotypes shown in (G) (n = 7, number of wing pouches) and (H) (n = 10). Error bars, SEM; **, p<0.01; non-parametric Mann-Whiteney U-test. (J) Duration time (min) of S/G2 to M phase in cells in the wing pouch of wild-type (n = 19, number of wing pouches) or RpS3/+ (n = 42) larvae were manually measured. Time-laps imaging was performed for 7 hours. Error bars, SEM; n.s, not significant; non-parametric Mann-Whiteney U-test. (K and L) Dying cells in the wing disc were visualized by anti-Dcp1 antibody staining. Wing disc of wild-type (K) or RpS3/+ (L) is shown. Arrowheads indicate massive cell death occurred along the D/V axis in the wing pouch. Scale bar, 100 μm. (M) Boxplot with dots representing cleaved-Dcp-1-positive dying cells per pouch in genotypes shown in (K) (n = 12, number of wing pouches), and (L) (n = 19). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. Scale bar, 100 μm. (N and O) The activated-caspase-3 indicator CD8-PARP-Venus was expressed in wild-type (N) or RpS3/+ (O) flies, and dying cells in the wing disc were visualized by anti-cleaved PARP staining (white). Arrowheads indicate massive cell death. The nuclei were visualized by DAPI staining (blue). Scale bar, 100 μm. (P) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (A) (n = 22, number of wing pouches) and (B) (n = 12). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. Genotypes are as follows: nub-Gal4/+; UAS-CD8-PARP-Venus/+ (A, D), nub-Gal4, UAS-CD8-PARP-Venus, RpS3Plac92/+ (B, E), nub-Gal4/+; UAS-S/G2/M-Green/+ (G), nub-Gal4/+; UAS-S/G2/M-Green/RpS3Plac92 (H), w1118 (K), FRT82B, Ubi-GFP, RpS3Plac92/+ (L), Actin-Gal4, UAS-CD8-PARP-Venus/+ (N), and Actin-Gal4, UAS-CD8-PARP-Venus/FRT82B, Ubi-GFP, RpS3Plac92 (O).

The fact that M/+ cells have a higher cell division rate suggests that there must be a compensatory mechanism that adjusts cell number in developing M/+ wing pouch. Strikingly, we found that massive cell death occurred in the developing RpS3/+ wing pouch, while only a few dying cells were detected in the wild-type pouch (Fig 1K and 1L, arrowheads, quantified in Fig 1M; Fig 1N and 1O, quantified in Fig 1P), using the antibody against cleaved-Dcp1 or CD8-PARP-Venus probe that visualizes caspase activity in the imaginal disc [13–20]. Similar massive cell death was also observed in the wing pouches of other M/+ strains, such as RpS17/+ and RpL19/+ animals (S1D and S1E Fig). The incidence of cell death in the M/+ wing pouch increased as development proceeded and peaked during the middle to late 3rd instar (S1F and S1G Fig). Consistent with these observations, blocking effector caspases by overexpression of baculoviral p35 [21] in the wing pouch resulted in wing disc overgrowth (S2C Fig, compared to S2A and S2B Fig) and severe morphological defects with blisters in the adult RpS3/+ wing (Fig 2C, compared to Fig 2A and 2B, quantified in Fig 2F), while p35-overexpression alone had no effect on wild-type or RpS3-rescued RpS3/+ wing development (Fig 2D and 2E, quantified in Fig 2F). This morphological defect was likely due to the generation of “undead cells”, which produce secreted growth factors such as Wingless (Wg; a Wnt homolog) and Decapentaplegic (Dpp, a Dpp homolog) via elevated initiator caspase activity [21–23]. p35-overexpression in the wing pouch similarly resulted in severe morphological defects with blisters in other M/+ such as RpS17/+ and RpL19/+ (S2D and S2E Fig, quantified in S2F Fig). This further demonstrates massive cell death occurring in M/+ wing pouch. Time-lapse imaging of ex vivo wing morphogenesis indicated that the RpS3/+ wing pouch expressing p35 overgrew and therefore failed to appose the ventral and dorsal surfaces of the wing blade correctly (S2G and S2H Fig, quantified in S2I Fig; S3–S5 Movies), which is consistent with the adult wing blister phenotype [24].

Fig 2. Epithelial cell-turnover is essential for robust wing development in M/+ animals.

Fig 2

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(A-E) Adult wings of wild-type (A), RpS3/+ (B), RpS3/+, nub-Gal4, UAS-p35 (C), nub-Gal4, UAS-p35 (D), or RpS3/+, nub-Gal4, UAS-p35, UAS-RpS3 (E) fly. Scale bar, 500 μm. (F) The rate of defective wings in the genotypes shown in (A) (n = 150), (B) (n = 271), (C) (n = 128), (D) (n = 125), and (E) (n = 44). (G and H) Dying cells were visualized by anti-cleaved PARP staining (white) in the wing discs of RpS3/+, H99/+ (G) or RpS3/+, nub-Gal4, UAS-DroncDN (H). Undead cells were not generated in these situations. Scale bar, 100 μm. (I) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (Fig 1B) (n = 12, number of wing pouches), (G) (n = 16), and (H) (n = 33). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (J and K) Wing disc of RpS3/+, H99/+ (J) or RpS3/+, nub-Gal4, UAS-DroncDN (K) were stained with anti-phospho-histone H3 (pH3) (Ser10) antibody (white). Wing pouches are marked in pale green. Scale bar, 100 μm. (L) Boxplot with dots representing pH3 positive cells per pouch in genotypes shown in (Fig 1A) (n = 34, number of wing pouches), (Fig 1B) (n = 31), (J) (n = 19), and (K) (n = 27). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (M) The rate of defective wings in the genotypes of wild-type (n = 70), RpS3/+ (n = 86), RpS3/+, H99/+ (n = 83), H99/+ (n = 100), RpS3/+, nub-Gal4, UAS-DroncDN (n = 63), and nub-Gal4, UAS-DroncDN (n = 109). (N) Adult wing phenotypes were classified as following five types: (a) normal, (b) vein (weak) (weakly having a brunched vein in the point of “landmark 14” (arrow) [69]), (c) vein (strong) (having other additional vein phenotypes (arrowheads) in addition to “landmark 14”), and (d) severe. Genotypes are as follows: w1118 (A), FRT82B, Ubi-GFP, RpS3Plac92/+ (B), nub-Gal4/+; FRT82B, Ubi-GFP, RpS3Plac92/UAS-p35 (C), nub-Gal4/+; UAS-p35/+ (D), nub-Gal4/UAS-RpS3; UAS-p35/FRT82B, Ubi-GFP, RpS3Plac92 (E), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/Df(3L)H99 (G, J), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/UAS-DroncDN (H, K), nub-Gal4/+; UAS-CD8-PARP-Venus/+ (wild-type), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/+ (RpS3-/+), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/Df(3L)H99 (RpS3-/+, H99/+), nub-Gal4/+; UAS-CD8-PARP-Venus/Df(3L)H99 (H99/+), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/UAS-DroncDN (RpS3-/+, nub>DroncDN), and nub-Gal4/+; UAS-CD8-PARP-Venus/UAS-DroncDN (nub>DroncDN) in (M).

The increased cell death and proliferation suggests that M/+ cells in the wing pouch trigger apoptosis-induced compensatory cell proliferation [3,4]. Indeed, we found that the increased cell proliferation depended on the induction of cell death, as cell proliferation was significantly reduced by blocking cell death in the RpS3/+ wing pouch using heterozygous chromosomal deletion H99 (which removes cell death genes reaper, hid, and grim) [25–27] (Fig 2G, quantified in Fig 2I) or dominant-negative form of the Drosophila initiator caspase Dronc (DroncDN) [28] (Fig 2H, quantified in Fig 2I), as assessed by phospho-Histone H3 staining (Fig 2J and 2K, quantified in Fig 2L). These data indicate that the M/+ wing pouch exhibits greatly enhanced cell-turnover that is induced by massive apoptosis and subsequent compensatory cell proliferation.

Cell-turnover is required for normal wing morphogenesis in M/+ animals

We next investigated whether this cell-turnover is essential for normal wing morphogenesis in M/+ animals. Significantly, reducing cell death and compensatory cell proliferation, namely cell-turnover, in the RpS3/+ wing pouch by introducing H99/+ or by overexpressing DroncDN resulted in morphological defects in the adult wing, while H99/+ or DroncDN alone had no effect on wing development (Fig 2M and 2N). These data suggest that M/+ wing disc induces cell-turnover to ensure robust wing morphogenesis.

Aberrant Wg signaling gradient is essential for massive cell-turnover

We next sought to address how the M/+ wing pouch induces massive cell-turnover. We noticed that most cell death occurred in the RpS3/+ wing pouch along the DV axis at a constant distance from the DV boundary, where the morphogen Wingless (Wg; a Wnt homolog) is expressed (Fig 3A and 3B). Wg signaling is required in the wing pouch for cell specification, tissue growth, patterning, and morphogenesis during wing development (Reviewed in [29,30]). The expression pattern of Wg was indistinguishable between RpS3/+ and wild-type wing pouches (Fig 3A”and 3B”). Notably, we found that the Wg expression level was elevated in the M/+ wing pouch, as genetic rescue of RpS3 in the posterior compartment of the RpS3/+ wing disc resulted in decreased Wg transcription and protein expression compared to the anterior RpS3/+ control (S3A and S3D Fig, quantified in S3C and S3F Fig). In addition, Wg signaling activity was elevated much more broadly in the RpS3/+ pouch compared to the same-stage wild-type (wondering larval stage), as assessed by the nmo-lacZ reporter [31] (Fig 3C and 3D). The elevation of Wg signaling activity in the RpS3/+ wing pouch was further confirmed by the genetic rescue experiment whereby RpS3 was overexpressed in the posterior compartment of the RpS3/+ disc, which showed decreased expression of Wg activity reporters nmo-lacZ [32] and nkd-lacZ [33] compared to the anterior control (Fig 3F and 3H), while overexpression of RpS3 alone did not affect Wg signaling activity in the wild-type wing disc (Fig 3E and 3G). Interestingly, the areas of massive cell death in the RpS3/+ wing pouch always corresponded to the areas of relatively lower Wg signaling activity (Fig 3D). This raises the possibility that massive cell death could be induced by Wg-dependent cell competition, a phenomenon by which cells with higher Wg signaling activity eliminate neighboring cells with lower Wg signaling activity in the wing disc [34]. We assumed that in the RpS3/+ wing pouch, ectopically upregulated Wg signaling at a distant site from the DV boundary might induce cell death in neighboring distal cells with relatively lower Wg signaling activity. Indeed, cell death was markedly suppressed when the aberrant Wg signaling gradient was reduced either by deleting one copy of the wg gene or by overexpressing Wg in the entire wing pouch (Fig 3I and 3J, quantified in Fig 3L). Furthermore, reduction of the Wg signaling gradient by downregulating dishevelled (dsh) or dtcf, downstream effectors of Wg signaling [35–39], in the RpS3/+ wing pouch also strongly suppressed cell death (Fig 3K, quantified in Fig 3L; S3G and S3H Fig, quantified in Fig S3K). Similarly, gentle upregulation of Wg signaling activity in the entire wing pouch by introducing one copy of wgGla-1, a gain of function allele of wg, or by mildly overexpressing an active form of ArmadilloS10 (ArmS10) [40] significantly suppressed cell death (S3I and S3J Fig, quantified in S3K Fig). These data suggest that an aberrant Wg signaling gradient generated in the M/+ wing pouch is required for the induction of massive cell death.

Fig 3. Aberrant Wg signaling gradient is essential for the induction of massive cell-turnover.

Fig 3

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(A-B”) The relative position between dying cells (cleaved-PARP, magenta) and Wg expression (anti-Wg, green) in the wild-type (A) or RpS3/+ (B) wing pouch. Scale bar, 100 μm. (C-D”) The relative position between dying cells (cleaved-PARP, magenta) and Wg signaling activity (anti-β-galactosidase, green) in the nmo-LacZ/+ (C) or RpS3/+, nmo-LacZ/+ (D) wing pouch. These images are Z-stacked images from 8 (C) and 8 (D) confocal images (3.02 μm and 2.39 μm, respectively) for whole wing pouch that is acquired every 1.01 μm or 0.8 μm, respectively. Scale bar, 50 μm. (E-F’) RpS3 was overexpressed in the posterior compartment of wing discs of nmo-lacZ/+ (E) or RpS3/+, nmo-lacZ/+ (F) flies using the en-Gal4 driver. nmo expression was visualized by anti-β-galactosidase staining (white). Scale bar, 100 μm. (G-H’) RpS3 was overexpressed in the posterior compartment of wing discs of nkd-lacZ/+ (G) or RpS3/+, nkd-lacZ/+ (H) flies using the en-Gal4 driver. nkd expression was visualized by anti-β-galactosidase staining (white). Scale bar, 100 μm. (I-K’) Dying cells were visualized by anti-cleaved PARP staining (white) in the wing discs of RpS3/+, wg/+ (I), RpS3/+, nub-Gal4, UAS-Wg (J), or RpS3/+, nub-Gal4, UAS-dsh-RNAi (K) flies expressing CD8-PARP-Venus. Wg expression was visualized by anti-Wg staining (white). Scale bar, 100 μm. (L) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (B) (n = 12, number of wing pouches), (I) (n = 9), (J) (n = 10), and (K) (n = 11). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. Genotypes are as follows: nub-Gal4/+; UAS-CD8-PARP-Venus/+ (A), nub-Gal4, UAS-CD8-PARP-Venus, RpS3Plac92/+ (B), nub-Gal4/+; UAS-CD8-PARP-Venus/nmoP1 (C), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/nmoP1 (D), en-Gal4, UAS-GFP/+; UAS-RpS3/nmoP1 (E), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/nmoP1 (F), en-Gal4, UAS-GFP/+; UAS-RpS3/nkd04869a (G), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/nkd04869a (H), nub-Gal4/wgl-8; UAS-CD8-PARP-Venus, RpS3Plac92/+ (I), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/UAS-Wg (J), and nub-Gal4/UAS-dsh-RNAi; UAS-CD8-PARP-Venus, RpS3Plac92/+ (K).

JNK-Dilp8-mediated developmental delay is required for massive cell-turnover

From the results presented so far, we postulated that aberrant Wg gradient through upregulation of endogenous Wg expression could be sufficient to cause massive cell death in the wing pouch. However, Wg-overexpression using the endogenous wg promoter did not induce cell death in the wing pouch (S4A and S4B Fig, quantified in S4C Fig). This indicates that the M/+ wing pouch must induce an additional event(s) essential for causing massive cell death. A strong candidate for the missing event is developmental delay, as the prolonged larval period of M/+ animals (about 40 hours longer than that of wild-type [7–9]) may enhance the formation of an aberrant Wg signaling gradient. It has been reported that M/+ imaginal discs upregulate Drosophila insulin-like peptide 8 (dilp8), which causes a developmental delay [41,42]. Indeed, in RpS3/+ animals, dilp8 was upregulated in various imaginal discs, especially in the wing pouch (Fig 4A and 4B). In addition, consistent with previous reports [42–45], blocking JNK signaling by overexpression of the JNK phosphatase Puckered (Puc) abolished dilp8 upregulation in the RpS3/+ wing pouch (S4D Fig). Consistent with these results, developmental delay in the larval period of RpS3/+ animals was partially suppressed by reducing dilp8 gene dosage or downregulating dilp8 expression in the wing pouch, as well as by blocking JNK signaling in the wing pouch (Fig 4C). Moreover, the RpS3/+ wing pouch indeed elevated JNK activity, as genetic rescue of RpS3 expression in the posterior compartment of the RpS3/+ disc resulted in decreased expression of JNK sensor TRE-DsRed [46] and JNK target genes misshapen (msn) [47] compared to the anterior control (S4F and S4H Fig); overexpression of RpS3 on its own did not affect JNK activity in wild-type wing disc (S4E and S4G Fig). These data indicate that elevated JNK activity in the M/+ wing pouch causes Dilp8-mediated developmental delay in the M/+ larvae. The developmental delay in M/+ animals, however, could be initially triggered by additional unknown causes, since developmental delay was not fully rescued by blocking the JNK-Dilp8 axis in M/+ wing discs.

Fig 4. JNK-Dilp8-mediated developmental delay is required for massive cell-turnover.

Fig 4

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(A and B) The expression of dilp8 in wing discs of dilp8::eGFP/+ (A) or RpS3/+, Dilp8::eGFP/+ (B) flies were visualized with GFP (green). Scale bar, 100 μm. (C) Pupariation curves for wild-type (n = 390), RpS3/+ (n = 666), RpS3/+, dilp8EX/+ (n = 46), RpS3/+, nub>dilp8RNAi (n = 215), or RpS3/+, nub>Puc (n = 302) flies. p>0.001, comparing RpS3/+ to the other flies (log-rank analysis). Error bars, SEM. (D) 20-Hydroxyecdysteroid titer was measured in wild-type or RpS3/+ larvae. (E) Pupariation curves of wild-type or RpS3/+ larvae that were fed food containing 0.75 mg/ml 20E or control. Error bars, SEM. (F) Dying cells were visualized by anti-cleaved PARP staining (white) in the wing discs of RpS3/+ larvae that were fed food containing 0.75 mg/ml 20E. Scale bar, 100 μm. (G) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (Fig 3A) (n = 22), (Fig 3B) (n = 12), and (F) (n = 14). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (H-I) Dying cells were detected by anti-cleaved PARP staining in the wing discs of RpS3/+, dilp8EX/+ (H), RpS3/+, nub-Gal4, UAS- dilp8-RNAi (I), or RpS3/+, nub-Gal4, UAS-Puc (J) flies expressing CD8-PARP-Venus. Scale bar, 100 μm. (K) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (Fig 3B) (n = 12), (H) (n = 12), (I) (n = 9), and (J) (n = 9). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (L-N) Dying cells were visualized by anti-cleaved Dcp-1 staining in the wing discs (white). ecd1 is a temperature-sensitive ecd mutant allele that blocks biosynthesis of the active-form of the hormone 20-Hydroxyecdysone at 29°C. Non-heat-shock treated flies was grown at 18°C (L). For heat-shock treatment, ecd1/ecd1 (M), or ecd1/ecd1, wg-Gal4, UAS-Wg (N) fly culture was transferred to 29°C for 48 hours during the 3rd instar larval stage. (O) Boxplot with dots representing cleaved-Dcp-1-positive dying cells per pouch in genotypes shown in (L) (n = 14), (M) (n = 15) and (N) (n = 15). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. Scale bar, 100 μm. Genotypes are as follows: dilp8::eGFP/+ (A), RpS3Plac92/dilp8::eGFP (B), nub-Gal4/+; UAS-CD8-PARP-Venus/+ (black), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/+ (red), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/dilp8EX (green), nub-Gal4/UAS-dilp8-RNAi; UAS-CD8-PARP-Venus, RpS3Plac92/+ (orange), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/UAS-Puc (blue) (C), w1118 (D: wild-type), FRT82B, Ubi-GFP, RpS3Plac92/+ (D; RpS3/+), nub-Gal4/+; UAS-CD8-PARP-Venus/+ (E: wild-type), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/+ (E: RpS3/+, F), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/dilp8EX (H), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/UAS-dilp8-RNAi (I), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/UAS-Puc (J), ecd1/ecd1 (L, M), and wg-Gal4/UAS-Wg; ecd1/ecd1 (N).

We thus examined whether Dilp8-dependent developmental delay contributes to the induction of massive cell death in the M/+ wing pouch. It has been reported that Dilp8 causes a developmental delay by inhibiting ecdysone biosynthesis in the prothoracic gland [41,42]. Indeed, the peak of the active form of ecdysone 20-Hydroxyecdysone (20E) level was delayed in the RpS3/+ 3rd instar larvae, compared to wild-type (Fig 4D). In addition, massive cell death as well as the developmental delay of RpS3/+ larvae [48] were rescued by feeding larvae with 20E (Fig 4E and 4F, quantified in Fig 4G). In addition, reduction of dilp8 expression by heterozygosity for the dilp8 gene or by dilp8-RNAi strongly suppressed cell death (Fig 4H and 4I, quantified in Fig 4K). Similar results were obtained when dilp8 expression was blocked by overexpression of Puc (Fig 4J, quantified in Fig 4K). These data suggest that the developmental delay caused by the JNK-Dilp8 module contributes to the induction of cell-turnover in the M/+ wing pouch.

Developmental delay in conjunction with Wg upregulation causes massive cell-turnover

Finally, we asked whether massive cell-turnover could be reproduced in a non-M/+ wing pouch by inducing both developmental delay and Wg upregulation. The temperature-sensitive ecdysoneless (ecd) mutant flies fail to produce ecdysone and therefore have a prolonged larval period upon heat-shock treatment [49]. We found that the number of dying cells was moderately increased in ecd mutant wing discs (Fig 4M, compare to Fig 4L, quantified in Fig 4O). Strikingly, a forced increase in endogenous Wg expression in conjunction with developmental delay caused massive cell death in the wing pouch along the DV axis (Fig 4N, quantified in Fig 4O), which was accompanied by increased cell mitoses (S4I and S4J Fig, quantified in S4K Fig). In addition, depletion of ecdysone in wild-type 3rd instar larvae using the fly food with erg-2 mutant yeast (erg-2Δ) [50,51] resulted in prolonged larval period and significantly increased number of dying cells in the wing disc (S4L Fig, compared to S4A Fig, quantified in S4N Fig). A forced increase in endogenous Wg expression in conjunction with erg-2Δ-induced developmental delay resulted in more increased cell death in the wing pouch along the DV axis (S4M Fig, quantified in S4N Fig). Furthermore, induction of homozygous mutations in an apicobasal polarity gene scribble (scrib-/-) in the entire eye discs, which resulted in the formation of tumors and extended larval period [52], significantly increased the number of dying cells in the wing discs (S4O Fig, compared to S4A Fig, quantified in S4Q Fig). A forced increase in endogenous Wg expression in conjunction with the tumor-induced developmental delay resulted in more increased cell death in the wing pouch (S4P Fig, quantified in S4Q Fig). Together, these data suggest that M/+ wing imaginal epithelium undergoes massive cell-turnover through the combination of upregulated Wg expression and developmental delay, thereby ensuring normal wing morphogenesis (Fig 5).

Fig 5. A model for ensuring robust coordination of tissue growth in M/+ animals.

Fig 5

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During prolonged larval period of M/+ animals, JNK signaling and Wg expression are upregulated in wing discs, which triggers a developmental delay and an aberrant Wg signaling gradient, respectively. These cooperate to induce massive cell turnover in the wing pouch, which permits normal wing morphogenesis.

Discussion

Our genetic study of Drosophila M/+ mutants proposes a novel paradigm for morphogenetic robustness of the epithelial tissue by cell-turnover. The massive cell-turnover induced in the M/+ wing pouch ensures normal wing growth during the abnormally prolonged larval period, possibly by creating a flexible cell death and proliferation platform to adjust cell numbers in the prospective wing blade, just like the process of tissue regeneration [53]. The fact that impairment of cell-turnover in M/+ animals induces phenotypic variations (Fig 2M and 2N) suggests that cell-turnover may provide the flexibility to change developmental programs. Interestingly, it has been shown that a molecular chaperone heat-shock protein 90 (Hsp90) acts as a ‘capacitor’ for morphological evolution by buffering genetic variations during animal and plant development [54–56]. Thus, our findings in M/+ flies suggest that massive cell-turnover may act as a buffering system for developmental time distortion, which could allow morphological variations. Such buffering system through cell-turnover could be also important for restore organ allometry, defined as the scaling of organ growth with body size [57], in response to developmental time distortion.

Our data suggest that massive cell-turnover in the M/+ wing pouch is caused by an aberrant Wg signaling gradient generated by Wg upregulation. Wg upregulation gives rise to ectopic cell population with high Wg signaling activity in the wing pouch, likely triggering non-autonomous cell death of nearby cells with lower Wg signaling activity, which could be similar mechanism with cell competition. The prolonged larval period of M/+ animals would further promote differences in Wg signaling activity between cells, as Wg signaling is continuously activated during larval development. Indeed, forced Wg upregulation using the endogenous wg promoter caused massive cell death when combined with developmental delay. While cell competition is thought to be involved in tissue homeostasis, tumor suppression, and the selection of fitter stem cells [58–60], its physiological role still remains elusive. Our data may suggest a role of cell competition in ensuring robust coordination of tissue growth during development.

The mechanism by which JNK activation in the M/+ wing disc induces dilp8 expression is an important open question for future studies. It has been reported that JAK/STAT signaling acts downstream of JNK signaling to coordinate regenerative cell proliferation and developmental delay through upregulating dilp8 expression during regeneration of the Drosophila fragmentated leg imaginal disc [61]. Interestingly, the activation pattern of JAK/STAT signaling in the RpS3/+ wing disc is indistinguishable from that in the wild-type wing discs (S5A and S5B Fig), raising the possibility that another unknown signaling pathway or basal level of JAK/STAT signaling could regulate dilp8 expression, downstream of JNK in the M/+ wing disc.

There are 88 Drosophila genes encoding cytoplasmic ribosomal proteins, and 65 of those heterozygous mutants (M/+) manifest phenotypes of a 2–3 day delay in larval development as well as short thin bristles. We found that different M/+ strains showed similar massive cell death in their developing wing pouches, suggesting a general role for cell-turnover in morphogenetic robustness in ribosomal protein mutant animals. Intriguingly, heterozygous mutations in human genes encoding ribosomal proteins or ribosomal biogenesis factors are associated with genetic disorders with developmental defects, which are collectively called ribosomopathies [62]. For instance, heterozygous mutations in human RpS19, RpS24, RpS17, RpL5, RpL11 or RpL35A are linked to diamond-Blackfan anaemia (DBA), and some DBA patients exhibit tissue-specific developmental defects, including limb defects, cleft palate, and abnormal heart development. Heterozygous mutations in the human RpSA gene have been identified in patients with isolated congenital asplenia (ICA), a disorder characterized by the absence of a spleen at birth [63]. Future studies on the molecular mechanism by which massive cell-turnover ensures morphogenetic robustness may help in understanding the etiology of ribosomopathies.

Methods

Fly strains

The following strains were used: dilp8ex (Pierre Leopold), 10xSTAT-GFP (Erika A. Bach), UAS-CD8-PARP-Venus (Yash Hiromi), UAS-S/G2/M-Green (Kaoru Sugimura), hh-gal4 (Tetsuya Tabata), UAS-DroncDN (Sharad Kumar), wg-gal4 (Jean-Paul Vincent), scrib1 [64], UAS-Puc [65], UAS-RpS3 [66], RpS3Plac92, RpL19K03704, RpS174, dsh3, nmoP1 (nmo-lacZ), nkd04869a (nkd-lacZ), wg17en40cP1 (wg-lacZ), msn06946 (msn-lacZ), dilp8MI00727 (eGFP trap), UAS-p35, UAS-Wg, UAS-dsh-RNAi, Df(3L)H99, nub-gal4, en-gal4, wgGla-1, TRE-DsRed, act-gal4 (Bloomington Stock Center), UAS-ArmS10, ecd1, wgl-8, FRT82B, GMR-hid, CL3R (the Drosophila Genomics and Genetic Resources, DGGR), UAS-tcf-RNAi, UAS-dsh-RNAi (National Institute of Genetics Stock Center), and UAS-dilp8-RNAi (Vienna Drosophila RNAi Center, VDRC).

Histology

Larval tissues were stained with standard immunohistochemical procedures using rabbit anti-cleaved PARP antibody (Cell Signaling Technology, CST, 1:200), mouse anti-Wg antibody (Developmental Studies Hybridoma Bank (DSHB), 1:500), rabbit anti-phospho-histone H3 (Ser10) antibody (CST, 1:100), rabbit anti-β-galactosidase antibody (MP Biomedicals, 1:2000), mouse anti-β-galactosidase antibody (Sigma, 1:500), and rabbit anti-Cleaved Drosophila Dcp-1 (Asp216) antibody (CST, 1:100) for 3 days. For EdU labeling, larvae were dissected in Schneider’s Drosophila medium (GIBCO) containing 5% FBS and incubated in the same media with 12.5 μM EdU for 30 min. Detection of EdU was performed with Click-iT Plus EdU Alexa Fluor 647 Imaging Kit (Thermo Fisher Scientific; C10640), according to the manufacture’s instruction. Samples were mounted with DAPI-containing SlowFade Gold Antifade Reagent (Molecular Probes). Images were taken with a Leica SP5, SP8, or SPE confocal microscope.

Quantification of EdU-positive area

For the analysis of EdU-positive cells, total area of EdU-positive cells and the size of the wing pouch area were automatically measured using Z-stacked images with Fiji and calculated with Microsoft Excel for Mac, as previously described [67].

Detection of dying cells

To detect dying cells in the wing pouch, the activated-caspase-3 indicator CD8-PARP-Venus were expressed in the pouch under the control of nub-gal4 driver. The anti-Dcp1 antibody was used in the experiments using the wg-gal4 driver for overexpression of Wg to genetically enhance Wg gradient.

Quantification of the relative number of phospho-Histone H3 positive cells or dying cells

Cleaved Dcp1 signals or phospho-histone H3 signals were taken as 3D projection images from the whole wing pouch. Cleaved PARP signals were acquired at the confocal plane with the highest signal. The number of signals in the wing pouch was automatically or manually measured with Fiji and calculated with Microsoft Excel for Mac.

Time-lapse imaging and quantification

To monitor cell cycle, late 3rd instar larvae were dissected and cultured in Schneider’s Drosophila medium (GIBCO) with 5% FBS and 2% low-melting agarose/PBS using the Glass Bottom Dish (MATSUNAMI). Imaging was conducted at 10-min intervals for 7 hours with a Leica SP8 confocal microscope [19,68]. For quantitative analysis, the number of dividing cells and duration time (min) between S/G2 and M phase were manually counted and measured in the apical surface of wing pouches (3–5 stacked images in 50 μm x 50 μm area). To observe wing eversion, white-pupae were dissected and cultured in M3 Insect Medium (Sigma) with 2% FBS and 0.5% methyl-cellulose over 12 hours [68].

Measurement of adult wings

Left and right wings of female flies were rinsed with xylene and mounted in a Canada Balsam (Nacalai Tesque). Images of the wings were captured using Leica M205C stereo microscope.

Quantification of Wg and wg-lacZ signals

Wg or wg-lacZ signals were acquired with Leica SPE confocal microscope. Intensity was measured with Fiji and the intensity plot lines were generated using the data of average fluorescence intensities (n>6) in the area marked in pale green. Statistical analysis was done using Microsoft Excel.

Developmental time analysis

For collecting embryos, flies were allowed to lay eggs for 2 hours at 25°C on grape juice agar plates. 1st instar larvae were collected 24 hours after egg laying and were transferred to fresh yeast-based food. Developmental time from egg to pupariation was calculated in 4 hours intervals. Each experiment was performed more than three times. For adding 20E (20-Hydroxyecdysone; SIGMA), around twenty larvae were transferred to 20E-containing food (0.75 mg/ml) 92hr after egg laying.

Ecdysteroid measurements

Ecdysteroid levels were measured using 20-Hydroxyecdysone Enzyme Immunoassay (EIA) kit (Bertin Bioreagent) according to the manufacturer's protocol. Briefly, ten larvae were frozen and homogenized with a Pellet Mixer (Treff Lab) in 250 ml methanol. Ecdysone was re-extracted twice with 125 ml methanol. 50 ml of the methanol extract was evaporated and dissolved in EIA Buffer. Further procedures were curried out according to the EIA protocol. The absorbance was read at 405 nm using ARVO X3 (PerkinElmer).

Heat-shock treatment

ecdysoneless mutant flies were grown at 18°C for 5 days and heat-shocked at 29°C for 48 hours. Flies were dissected right after heat-shock treatment at wandering stage.

Fly cultures with ecdysteroid synthesis defect

To inhibit the ecdysteroid synthesis, larvae were reared in the food with erg-2 sterol mutant strain (a gift from Tomonori Katsuyama). Given that the erg-2 gene encodes the Δ8-Δ7-sterol isomerase, flies feeding food with erg-2 yeast impair production of a sufficient titer of ecdysteroid hormone necessary for pupation, leading to the developmental arrest in the larval stage. To collect embryos, flies were allowed to lay eggs for 8 hours at 25°C on acetic acid agar plates. Embryos were rinsed, dechorinated with 3% bleach, and rewashed with MilliQ. Embryos were then put on a cover glass and transferred into erg-2 Δ yeast food.

Supporting information

S1 Fig. Related to Fig 1. Massive cell-turnover occurs during wing development in M/+ animals.

(A and B) Wing disc of RpS17/+ (A) or RpL19/+ (B) flies were stained with anti-phospho-histone H3 (pH3) (Ser10) antibody (white). Wing pouches were marked by pale green. Scale bar, 100 μm. (C) Boxplot with dots representing pH3 positive cells in the pouch in genotypes shown in (Fig 1A) (n = 34, number of wing pouches), (A) (n = 46) and (B) (n = 27). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (D and E) The activated-caspase-3 indicator CD8-PARP-Venus was expressed in RpS17/+ (D) or RpL19/+ (E) flies and dying cells in the wing disc were visualized by anti-cleaved PARP antibody. The nuclei were visualized by DAPI staining. Arrowheads indicate massive cell death occurred along the D/V axis in the wing pouch. Scale bar, 100 μm. (F and G) Developing wing disc of wild-type or RpS3/+ flies are shown. Dying cells visualized by anti-cleaved Dcp-1 staining (white). Wg expression was visualized by anti-Wg staining (green). Flies were allowed to lay eggs for 2 hours at 25°C on yeast-based food. Larvae were dissected at the indicated time points after egg laying (AEL). All flies were third instar larvae. Scale bar, 100 μm. Genotypes are as follows: nub-Gal4/+; UAS-CD8-PARP-Venus/RpS174 (A, D), nub-Gal4/RpL19K03704; UAS-CD8-PARP-Venus/+ (B, E). SgsΔ3–GFP/+ (F), SgsΔ3–GFP/FRT82B, Ubi-GFP, RpS3Plac92 (G).

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S2 Fig. Related to Fig 2. Epithelial cell-turnover is essential for robust wing development in M/+ animals.

(A-C) Wing discs of wild-type (A), RpS3/+ (B), or RpS3/+, nub-Gal4, UAS-p35 (C) flies. Nuclei were stained with DAPI. Scale bar, 100 μm. (D and E) Adult wings of RpS17/+, nub-Gal4, UAS-p35 (D), or RpL19/+, nub-Gal4, UAS-p35 (E) fly. Scale bar, 500 μm. (F) The rate of defective wings in the genotypes shown in (D) (n = 32) and (E) (n = 31). (G and H) Time-lapse of wing eversion in RpS3/+ (G) or RpS3/+, nub-Gal4, UAS-p35 (H). Arrows indicate the direction of extension. Arrowheads indicate abnormal wrinkled structures. (I) The percentage of abnormal pupal wings in RpS3/+ (n = 53) or RpS3/+, nub-Gal4, UAS-p35 (n = 36), which were classified according to the level of severity. Genotypes are as follows: nub-Gal4/+; UAS-CD8-PARP-Venus/+ (A), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/+ (B), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/ UAS-p35 (C), nub-Gal4/+; UAS-p35/RpS174 (D), nub-Gal4/RpL19K03704; UAS-p35/+ (E), nub-Gal4/+; FRT82B, Ubi-GFP, RpS3Plac92/+ (G), and nub-Gal4/+; FRT82B, Ubi-GFP, RpS3Plac92/UAS-p35 (H).

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S3 Fig. Related to Fig 3. Aberrant Wg signal gradient is essential for the induction of massive cell-turnover.

(A-B’) RpS3 was overexpressed in the posterior compartment of wing discs of RpS3/+, wg-lacZ/+ (A) flies using the en-Gal4 driver. wg expression was visualized by anti-β-galactosidase staining (white). Arrowheads indicate relatively lower expression of wg. Scale bar, 100 μm. (C) Quantification of wg intensity in the pale green-marked area shown in (A) (n = 6) or (B) (n = 7). The intensity was automatically or manually measured using ImageJ (Fiji) software as described in the previous manuscript [67]. (D-E’) RpS3 was overexpressed in the posterior compartment of wing discs of RpS3/+ (D) flies using the en-Gal4 driver. Wg expression was visualized by anti-Wg staining (white). Arrowheads indicate relatively lower expression of Wg. Scale bar, 100 μm. (F) Quantification of Wg intensity in the pale green-marked area shown in (D) (n = 8) or (E) (n = 7) are shown. The intensity was automatically or manually measured using ImageJ (Fiji) software as described in the previous manuscript [67]. (G-J) Dying cells were visualized by anti-cleaved PARP staining in RpS3/+, dsh3/+ (G), RpS3/+, nub-Gal4, UAS-tcf-RNAi (H), RpS3/+, wgGla-1/+ (I), or RpS3/+, nub-Gal4, UAS-ArmS10, TG80 ts7(J) wing disc. Massive cell death was significantly suppressed by reducing Wg signal gradient. Scale bar, 100 μm. (K) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (Fig 3B) (n = 12, number of wing pouches), (G) (n = 8), (H) (n = 15), (I) (n = 31), and (J) (n = 24). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. Genotypes are as follows: en-Gal4, UAS-GFP/wg17en40cP1; RpS3Plac92, UAS-RpS3/+ (A), en-Gal4, UAS-GFP/wg17en40cP1; RpS3Plac92/+ (B), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/+ (D), en-Gal4, UAS-GFP/+; RpS3Plac92/+ (E), dsh3/+; nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/+ (G), nub-Gal4/UAS-tcf-RNAi; UAS-CD8-PARP-Venus, RpS3Plac92/+ (H), nub-Gal4/wg gla-1; UAS-CD8-PARP-Venus, RpS3Plac92/+ (I), and UAS-ArmS10/+; nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/ TG80 ts7 (J).

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S4 Fig. Related to Fig 4. JNK-Dilp8-mediated developmental delay is required for massive cell-turnover.

(A and B) Wg was overexpressed using the endogenous wg promoter (wg-Gal4). Dying cells in the wing discs of wild-type (A) or wg-Gal4, UAS-Wg (B) flies were visualized by anti-cleaved Dcp-1 staining. Scale bar, 100 μm. (C) Boxplot with dots representing cleaved-Dcp-1-positive dying cells per wing pouch of genotypes shown in (A) (n = 17, number of wing pouches) and (B) (n = 16). Error bars, SEM; n.s, not significant; non-parametric Mann-Whiteney U-test. (D) The expression of dilp8 in wing discs of RpS3/+, Dilp8::eGFP/+, nub-Gal4, UAS-Puc flies were visualized with GFP (green). Scale bar, 100 μm. (E-F’) RpS3 was overexpressed in the posterior compartment of the wing disc of TRE-DsRed/+ (E) or RpS3/+, TRE-DsRed/+ (F) flies using the en-Gal4 driver. JNK-activated cells were visualized by TRE-DsRed reporter (white). (G-H’) RpS3 was overexpressed in the posterior compartment of the wing discs of msn-lacZ/+ flies (G) or RpS3/+, msn-lacZ/+ (H) flies using the en-Gal4 driver. JNK activity was visualized by anti-β-galactosidase staining (white). Scale bar, 100 μm. (I and J) Wing disc of ecd1/ecd1 (I) or ecd1/ecd1, wg-Gal4, UAS-Wg (J) were stained with anti-phospho-histone H3 (pH3) (Ser10) antibody (white). Wing pouches were marked by pale green. ecd1 is a temperature-sensitive ecd mutant allele that blocks biosynthesis of the active-form of the hormone 20-Hydroxyecdysone at 29°C. For heat-shock treatment, fly culture was transferred to 29°C for 48 hours during the 3rd instar 1arval stage. Scale bar, 100 μm. (K) Boxplot with dots representing pH3 positive cells per pouch in genotypes shown in (I) (n = 13, number of wing pouches) and (J) (n = 13). Error bars, SEM; *, p<0.05; non-parametric Mann-Whitney U-test. (L and M) Wg was overexpressed using the endogenous wg promoter (wg-Gal4) in 3rd instar larvae fed with food containing erg-2 mutant yeast. Dying cells in the wing discs of wild-type (L) or wg-Gal4, UAS-Wg (M) flies were visualized by anti-cleaved Dcp-1 staining. Scale bar, 100 μm. (N) Boxplot with dots representing dying cells of cleaved-Dcp-1-positive cells per pouch in genotypes shown in (A) (n = 17, number of wing pouches), (B) (n = 16), (L) (n = 17), and (M) (n = 14). Error bars, SEM; ***, p<0.001; **, p<0.01; non-parametric Mann-Whitney U-test. Scale bar, 100 μm. (O and P) Dying cells in the wing disc were visualized by anti-Dcp1 antibody staining (white). Wing disc of wild-type (O) or wg-Gal4, UAS-Wg (P) shown in flies bearing the eye disc in which scrib mutant clones were generated and surrounding wild-type cells were simultaneously removed by a combination of GMR-hid and a recessive cell-lethal mutation, CL3R. Scale bar, 100 μm. (Q) Boxplot with dots representing cleaved-Dcp-1-positive dying cells per pouch in genotypes shown in (O) (n = 14, number of wing pouches) and (P) (n = 7). Error bars, SEM; *, p<0.05; non-parametric Mann-Whitney U-test. Scale bar, 100 μm. Genotypes are as follows: wg-Gal4/+ (A, L), wg-Gal4/+;UAS-Wg/+ (B, M), nub-Gal4/+; RpS3Plac92, dilp8::eGFP/UAS-Puc (D), en-Gal4, UAS-GFP/+; TRE-DsRed/UAS-RpS3 (E), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/ TRE-DsRed (F), en-Gal4, UAS-GFP/+; UAS-RpS3/msn06946 (G), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/msn06946 (H), ecd1/ecd1 (I), wg-Gal4/UAS-Wg; ecd1/ ecd1 (J), eyFLP1/+;; FRT82B,scrib1/FRT82B, GMR-hid.CL3R (O), and eyFLP1/+; wg-Gal4/UAS-Wg; FRT82B,scrib1/FRT82B, GMR-hid.CL3R (P).

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S5 Fig. The activation pattern of JAK/STAT signaling in the M/+ wing disc is indistinguishable from that in the wild-type wing disc.

(A-B’) The wild-type (A) or RpS3/+ (B) wing disc bearing the 10xSTAT-GFP reporter (green). The nuclei were visualized by DAPI staining (blue) Scale bar, 100 μm. Genotypes are as follows: 10xSTAT-GFP/+ (A) and 10xSTAT-GFP/ RpS3Plac92 (B).

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S1 Movie. Live imaging of wing discs with the cell-cycle probe S/G2/M-Green.

The S/G2/M-Green probe was expressed in the wing pouches of wild-type larvae. Wing discs were dissected and a part of the wing pouch was subjected to time-lapse imaging at 10-min intervals for 7 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: nub-Gal4/+; UAS-S/G2/M-Green/+. Each experiment was replicated at least 3 times.

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S2 Movie. Live imaging of wing discs with the cell-cycle probe S/G2/M-Green.

The S/G2/M-Green probe was expressed in the wing pouches of or RpS3/+ larvae. Wing discs were dissected and a part of the wing pouch was subjected to time-lapse imaging at 10-min intervals for 7 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: nub-Gal4/+; UAS-S/G2/M-Green/RpS3Plac92. Each experiment was replicated at least 3 times.

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S3 Movie. Live imaging of wing disc eversion.

GFP-expressing wing discs of wild-type larvae were dissected and subjected to time-lapse imaging at 10-min intervals for over 12 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: FRT82B, Ubi-GFP/+. Each experiment was replicated at least 3 times.

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S4 Movie. Live imaging of wing disc eversion.

GFP-expressing wing discs of RpS3/+ larvae were dissected and subjected to time-lapse imaging at 10-min intervals for over 12 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: FRT82B, Ubi-GFP, RpS3Plac92/+. Each experiment was replicated at least 3 times.

(MOV)

Click here for additional data file. (3.4MB, mov)
S5 Movie. Live imaging of wing disc eversion.

GFP-expressing wing discs of RpS3/+, nub-Gal4, UAS-p35 larvae were dissected and subjected to time-lapse imaging at 10-min intervals for over 12 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: nub-Gal4/+; FRT82B, Ubi-GFP, RpS3Plac92/UAS-p35. Each experiment was replicated at least 3 times.

(MOV)

Click here for additional data file. (2.6MB, mov)

Acknowledgments

We thank Pierre Leopold, Laura Boulan, and John Vaughen for comments on the manuscript; Yayoi Wada, Aya Betsumiya, Kanako Nakagawa, Megumi Nakayama, Sayaka Kikuhara, Toshiharu Sawada, Kanako Baba, Miho Tanaka, Minoru Koijima for technical support; Carl Thummel, Vincent Henrich, Erika Bach, Yash Hiromi, Kaoru Sugimura, Tetsuya Tabata, Sharad Kumar, Jean-Paul Vincent, Masayuki Miura, Tian Xu, the Bloomington Drosophila Stock Center (Indiana), the Vienna Drosophila Resource Center (Vienna), the National Institute of Genetics Stock Center (Mishima), and the Drosophila Genomics and Genetic Resources (Kyoto) for fly stocks.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by Grant-in-Aid for Scientific Research (A) (Grant No. 16H02505) to T.I, Grant-in-Aid for Scientific Research on Innovative Areas (Grant Nos. 15H05862, 26114002, 20H04866) to S.O and T.I, Grant-in-Aid for challenging Exploratory Research (Grant No. 19K22423) to S.O, the Naito Foundation to S.O and T.I, the Takeda Science Foundation to S.O and T.I, Japan Agency for Medical Research and Development (Project for Elucidating and Controlling Mechanisms of Aging and Longevity, Grant Number 17938731) to T.I., Inamori Foundation to S.O., Toray Science Foundation to S.O., Senri Life Science Foundation to S.O., Yamada Science Foundation to S.O. and the Mitsubishi Foundation to S.O. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Norbert Perrimon, Gregory P Copenhaver

12 Jul 2020

Dear Tatsushi,

Thank you very much for submitting your Research Article entitled 'Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants' to PLOS Genetics. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some  concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review again a revised version. We cannot, of course, promise publication at that time.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: This manuscript by Akai et al. reports a study of wing disc development in Minute Drosophila mutant larvae. The authors found abnormally high levels of both cell proliferation and cell death in the wings of these animals. They call this “massive cell turnover”. Dissection of this phenomenon showed that it is an instance of regeneration through apoptosis-induced compensatory proliferation, ensuring normal wing development through the known JNK-Wg axis. Furthermore, developmental delay through the also known JNK-Dilp8 axis contributes to the phenomenon. Overall, this is a well-written manuscript reporting a study that is both interesting and expertly conducted, with exquisite quantification of phenotypes. My major concerns have to do with the interpretation of the results, or sometimes lack of it, as I detail below.

1. It is not clear to me what is the ultimate reason that Minute animals show this increased cell death. The model the authors propose is that in Minute/+ animals cell death induces both compensatory proliferation through Wg and developmental delay through Dilp8, and that developmental delay somehow contributes to amplify the response. This is my take from the text. However, the authors are carefully and deliberately ambiguous about the relations among these elements in the model in Fig. 5, grouping proliferation and death together as “cell turnover”. “Minute” appears upstream in the diagram, but it is not clear whether this refers to Minute disc or Minute larva. Then, prolonged larval period is predicated of the larva, but it is not clear whether this is the same as the “developmental delay” downstream of Dilp8 or a different, more upstream event. I think a clear possibility is that the whole thing is a consequence of developmental delay and that developmental delay through JNK-Dilp8 is an amplifying event, whereas developmental delay of unknown origin (Dilp8 cannot account for all of it) is the ultimate cause of the whole thing. 4F shows a level of rescue of cell death by ecdysone in the Minute discs that is compatible with this. Furthermore, ecdysoneless mutants in 4M show an amount of cell death that is again compatible with this possibility. This should be explicitly acknowledged and discussed.

2. An alternative interpretation would be that Minute mutant discs have autonomously elevated cell death, intrinsically and independent of developmental delay. In that case, developmental delay induced by cell death would amplify cell death and cell proliferation, but would not be the upstream triggering event. The timing of cell death in Minute discs in Fig S1 suggests that autonomous causes are responsible for elevated cell death and developmental delay later contributes to amplifying it. My understanding is that the field assumes Minute mutant disc cells develop slower but fine. This I thought was the reason the authors repeatedly state that their results are “surprising”, but do not explain what is surprising about this. Do the authors agree? Is there anything in their data or in the literature suggesting that Minute cells die at a higher rate in a context-independent way? If that is the interpretation of the authors, there are important consequences for the field and the concept of cell competition itself that the authors should again discuss.

Other comments:

A. Line 260: “Our genetic study of M/+ mutants proposes a novel paradigm…” What happens in these discs seems to me very similar to the rn>egr model of regeneration that Iswar Hariharan’s lab established. This should be acknowledged and his papers cited.

B. Line 267: “Interestingly, it has been shown that a molecular chaperone heat-shock protein 90 (Hsp90) acts as a ‘capacitor’ for morphological evolution by buffering genetic variations during animal and plant development (52-54). Thus, our findings in M/+ flies suggest that massive cell-turnover may act as a buffering system for developmental time distortion, which could allow morphological variations.”

The results here can be understood in the conceptual framework of regeneration through apoptosis-induced proliferation plus injury-induced developmental delay. I really don’t understand the Hsp90 reference here and the relation with morphological evolution.

C. The authors in the abstract write “Minute/+ mutants”. They should write instead “Minute mutants”, later clarifying in the introduction that these are dominant haploinsufficient mutations and that the analysis corresponds to Minute heterozygous (or hemizygous) animals (M/+).

D. Expressions like “surprisingly”, “strikingly”, “interestingly” or “intriguingly” should be used more sparingly by the authors if they want them to achieve any effect.

E. Semi-quantitative words should be avoided:

Massive cell turnover, massive cell death

Line 89: much lower

Line 111: only occasionally

Line 244: slightly increased

F. Others:

Line 180: a phenomenon BY WHICH

Line 214: PARTIALLY suppressed

Line 250: number of DYING CELLS

Reviewer #2: The manuscript by Akai et al., provides a detailed molecular genetic analysis of the developmental delay phenotype caused by heterozygosity for ribosomal mutants in Drosophila. These mutations, collectively referred to as Minutes, are known to cause pronounced developmental delay, but with the exception of a thin bristle phenotype, produce largely normal adults. Akai et al., were curious how normally proportioned adults where produced by Minute heterozygous mutants even though they experience substantial developmental delay. In recent years it has been discovered that Minute mutants induce expression of the insulin/relaxin like factor dilp8. Dilp8 is a stress signal that attenuates the production of the steroid hormone ecdysone, the metamorphosis inducing factor, thus prolonging developmental time. Therefore, a simple explanation for the relatively normal adult body size and patterning of Minute mutants might have been that in Minute heterozygous larvae all mitotic cells grow and divide slowly as a result of a reduced ribosome pool. This leads induction of the dilp8 stress signal resulting in delayed metamorphosis. Thus, the slow growth rate is compensated by developmental delay leading to the formation of essential normal adults. What the authors found, however, was something much more intriguing. When they examined the proliferation rates of Minute heterozygous imaginal tissue, they discovered that it was actually significantly increase rather than decreased as the simple model might predict. Therefore, in order to achieve proper size and pattern, the enhanced proliferation must be compensated by some additional mechanism. The authors found that the compensatory mechanism was dramatic induction of apoptosis. They then go on to investigate the molecular mechanism responsible for the Ying and Yang of proliferation/apoptosis and found that found that Minute heterozygosity leads to up-regulation of Wingless at the D/V compartment which triggers cell-competition as a result of an altered Wg gradient. Intriguingly, they further found that simply altering the Wg gradient in wildtype background is not sufficient to induce the massive apoptosis but that it also requires the dip8 mediated developmental delay. When they artificially altered the Wg gradient and slowed developmental time in a wt background, then apoptosis was induced. The authors conclude that robust cell turnover helps maintaining tissue homeostasis by interfacing with developmental timing mechanisms.

Overall, I find this to be a remarkable set of observations which leads to many additional questions. Obviously, these could be the subject of future studies, but perhaps a few additional experiments could lend a bit of additional clarity to the results.

The authors investigate JNK signaling as a means of inducing dilp8 but recognize that there must be some additional cues. Several other inputs that regulate dilp8 expression have been identified including Hippo and Jak/Stat signaling, the transcription factor Xrp1 and the Ecdysone Receptor. Although the authors mention that Jak/Stat signaling does not seem to be involved, have the examined the requirement of these other factors and pathways?

Another key regulator of of imaginal disc growth is Dpp. Did the authors examine Dpp expression to see if it was altered in addition to Wg?

Another question concerns the degree to which the wing disc response is directly related to developmental time. The authors say it is perturbation of developmental time that is an important aspect of the process and to illustrate this they perturb timing by using the ecd ts mutant while over-expressing Wg in its endogenous pattern which produces strong apoptosis in the wing pouch. But, is it really timing or simply the basal level of ecdysone which is sensed by the disc? Can the authors see the same effect if they block ecdysone reception in the disc itself by either expressing dominant negative EcR or perhaps overexpress RNAi of ECI, the ecdysone importer. This would require using two different conditional expression systems but would add some clarity to the actual mechanism. Likewise, the ecd ts mutant, while convenient, is not the best way to specifically alter E production since it is a splicing factor that may affect many other processes in addition to ecdysone production. Again, the use of two different conditional systems, one to knockdown E production in the PG and the other to overexpress Wg would be cleaner than using the ecd ts mutation.

Another issue is whether heterozygosity for a Minute is only affecting cell turnover in the imaginal discs. Have the authors examined if other mitotic tissues such as the brain, or mitotic portions of the gut and/or the histoblasts also affected?

For the rescue experiments shown in Fig 4C, the authors find that heterozygosity for dilp8 partially overcomes the developmental delay produced by heterozygosity for RpS3 as does nub>dilp8 RNAi. However, the rescue observed when they feed ecdysone (Fig. 4E) is much better (less delay). Is this difference simply because dilp8 expression was not fully attenuated, or are there other signals involved? What happens to the delay if dilp8 RNAi is ubiquitously expressed?

Minor point: line 244 “…found that the number of cell death…” please reword

Reviewer #3: In this manuscript, the authors examine the Minute (M/+) phenotype in more detail using wing imaginal discs in Drosophila. M mutations affect ribosomal genes and cause a significant developmental delay. It was assumed that this delay is caused by reduced proliferation rates due to decreased ribosomal activity. However, contrary to this assumption, the authors found that at least one M mutation (RpS3-/+) is characterized by increased proliferation rates. Likewise, the authors observed significantly increased apoptosis in this and two other M/+ mutants. The authors then demonstrate that M/+ wing pouches exhibit greatly enhanced cell turnover that is induced by massive apoptosis and subsequent compensatory proliferation. In search for the apoptosis-inducing signal, the authors found that increased expression of Wg in an aberrant (steeper) gradient in the wing disc triggers apoptosis in M/+ discs. However, the steeper Wg gradient alone is not sufficient for apoptosis. The authors further found that the developmental delay of M/+ mutants contributes to the aberrant Wg gradient and apoptosis. Consistently, down-regulation of dilp8 which promotes the developmental delay, reduces the amount of apoptosis in M/+ discs. In addition, feeding ecdysone (E20) to M/+ larvae rescues the developmental delay and apoptosis. Finally, overexpression of wg under wg control which in wild-type larvae has very little effect on apoptosis, triggers strong apoptosis in ecdysoneless mutant larvae which are developmentally delayed independently of M/+.

This is a very interesting manuscript. It reports the very surprising discovery that M/+ wing discs are characterized by massive cell turnover due to increased apoptosis and compensatory proliferation. This cell turnover is dependent on the developmental delay and ensures developmental robustness in M/+ mutants. The data are very convincing and often different methods were applied to ensure consistency. The manuscript is very well written. I do have a few experimental suggestions and comments/questions to further improve the manuscript.

1. It would be good if the authors can show the increased proliferation in more than one M/+ mutant. They showed the increased cell death for 3 M/+ strains, but the proliferation phenotype was only shown for one.

2. If I understand this correctly, the aberrant steeper Wg gradient is the result of the continuous wg expression during the delay period in M/+ wing discs. If correct, it would follow that the massive apoptosis is only triggered during the period of the developmental delay (so quite late in larval development). However, the authors write that “cell death increased as development preceded and peaked during middle to late 3rd instar” (lines 117-118). Is that consistent with the dependence of apoptosis on the aberrant Wg gradient and the developmental delay? When do the authors detect the aberrant Wg gradient first in development?

3. Wg expression along the DV border is dependent on Notch signaling. Did the authors observe any abnormalities of Notch signaling in M/+ discs?

4. Is the enhanced cell turnover also observed in other discs such as eye discs? If so, would there be a similar dependence on Wg in this tissue?

5. In the ecd;wg>wg experiment where the authors detect increased apoptosis (Fig. 4N), is there also compensatory proliferation?

6. Explain in the manuscript, what the erg-2 mutant yeast in the fly food accomplishes.

**********

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Decision Letter 1

Norbert Perrimon, Gregory P Copenhaver

7 Dec 2020

Dear Tatsushi,

We are pleased to inform you that your manuscript entitled "Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants" has been editorially accepted for publication in PLOS Genetics. Congratulations! Please note a few minor suggestions from one of the reviewer (see below).

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: My concerns have been addressed, but, in the interest of clarity, I suggest rewording changed sentences as follows:

Line 108: It has been well documented by clonal analysis that the growth rate of M/+ cell clones is lower than of wild-type clones in developing wing discs (9). The lower growth rate of M/+ implied that M/+ cells simply have a lower cell division rate without any defects in proliferation and cell death.

SUGGESTION: It has been well documented by clonal analysis that the growth rate of M/+ cell clones is lower than THAT of wild-type clones in developing wing discs (9). The lower growth rate of M/+ ANIMALS HAS BEEN LARGELY ASSUMED TO STEM FROM a lower cell division rate OF M/+ cells, without any defects in proliferation and cell death.

Line 242: These data indicate that elevated JNK activity in the M/+ wing pouch causes Dilp8-mediated developmental delay in the M/+ larvae. The developmental delay in M/+ animals could be triggered by unknown origin in M/+ larvae and is amplified by the JNK-Dilp8 axis, since developmental delay was not fully rescued by blocking the JNK-Dilp8 axis in M/+ wing discs.

SUGGESTION: These data indicate that elevated JNK activity in the M/+ wing pouch causes Dilp8-mediated developmental delay in the M/+ larvae. The developmental delay in M/+ animals, HOWEVER, could be INITIALLY triggered by ADDITIONAL unknown CAUSES, since developmental delay was not fully rescued by blocking the JNK-Dilp8 axis in M/+ wing discs.

Reviewer #2: The authors addressed all my concerns

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes: Michael B O'Connor

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Acceptance letter

Norbert Perrimon, Gregory P Copenhaver

13 Jan 2021

PGENETICS-D-20-00918R1

Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants

Dear Dr Igaki,

We are pleased to inform you that your manuscript entitled "Epithelial cell-turnover ensures robust coordination of tissue growth in Drosophila ribosomal protein mutants" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Related to Fig 1. Massive cell-turnover occurs during wing development in M/+ animals.

    (A and B) Wing disc of RpS17/+ (A) or RpL19/+ (B) flies were stained with anti-phospho-histone H3 (pH3) (Ser10) antibody (white). Wing pouches were marked by pale green. Scale bar, 100 μm. (C) Boxplot with dots representing pH3 positive cells in the pouch in genotypes shown in (Fig 1A) (n = 34, number of wing pouches), (A) (n = 46) and (B) (n = 27). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. (D and E) The activated-caspase-3 indicator CD8-PARP-Venus was expressed in RpS17/+ (D) or RpL19/+ (E) flies and dying cells in the wing disc were visualized by anti-cleaved PARP antibody. The nuclei were visualized by DAPI staining. Arrowheads indicate massive cell death occurred along the D/V axis in the wing pouch. Scale bar, 100 μm. (F and G) Developing wing disc of wild-type or RpS3/+ flies are shown. Dying cells visualized by anti-cleaved Dcp-1 staining (white). Wg expression was visualized by anti-Wg staining (green). Flies were allowed to lay eggs for 2 hours at 25°C on yeast-based food. Larvae were dissected at the indicated time points after egg laying (AEL). All flies were third instar larvae. Scale bar, 100 μm. Genotypes are as follows: nub-Gal4/+; UAS-CD8-PARP-Venus/RpS174 (A, D), nub-Gal4/RpL19K03704; UAS-CD8-PARP-Venus/+ (B, E). SgsΔ3–GFP/+ (F), SgsΔ3–GFP/FRT82B, Ubi-GFP, RpS3Plac92 (G).

    (TIF)

    Click here for additional data file. (3.2MB, tif)
    S2 Fig. Related to Fig 2. Epithelial cell-turnover is essential for robust wing development in M/+ animals.

    (A-C) Wing discs of wild-type (A), RpS3/+ (B), or RpS3/+, nub-Gal4, UAS-p35 (C) flies. Nuclei were stained with DAPI. Scale bar, 100 μm. (D and E) Adult wings of RpS17/+, nub-Gal4, UAS-p35 (D), or RpL19/+, nub-Gal4, UAS-p35 (E) fly. Scale bar, 500 μm. (F) The rate of defective wings in the genotypes shown in (D) (n = 32) and (E) (n = 31). (G and H) Time-lapse of wing eversion in RpS3/+ (G) or RpS3/+, nub-Gal4, UAS-p35 (H). Arrows indicate the direction of extension. Arrowheads indicate abnormal wrinkled structures. (I) The percentage of abnormal pupal wings in RpS3/+ (n = 53) or RpS3/+, nub-Gal4, UAS-p35 (n = 36), which were classified according to the level of severity. Genotypes are as follows: nub-Gal4/+; UAS-CD8-PARP-Venus/+ (A), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/+ (B), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/ UAS-p35 (C), nub-Gal4/+; UAS-p35/RpS174 (D), nub-Gal4/RpL19K03704; UAS-p35/+ (E), nub-Gal4/+; FRT82B, Ubi-GFP, RpS3Plac92/+ (G), and nub-Gal4/+; FRT82B, Ubi-GFP, RpS3Plac92/UAS-p35 (H).

    (TIF)

    Click here for additional data file. (2.7MB, tif)
    S3 Fig. Related to Fig 3. Aberrant Wg signal gradient is essential for the induction of massive cell-turnover.

    (A-B’) RpS3 was overexpressed in the posterior compartment of wing discs of RpS3/+, wg-lacZ/+ (A) flies using the en-Gal4 driver. wg expression was visualized by anti-β-galactosidase staining (white). Arrowheads indicate relatively lower expression of wg. Scale bar, 100 μm. (C) Quantification of wg intensity in the pale green-marked area shown in (A) (n = 6) or (B) (n = 7). The intensity was automatically or manually measured using ImageJ (Fiji) software as described in the previous manuscript [67]. (D-E’) RpS3 was overexpressed in the posterior compartment of wing discs of RpS3/+ (D) flies using the en-Gal4 driver. Wg expression was visualized by anti-Wg staining (white). Arrowheads indicate relatively lower expression of Wg. Scale bar, 100 μm. (F) Quantification of Wg intensity in the pale green-marked area shown in (D) (n = 8) or (E) (n = 7) are shown. The intensity was automatically or manually measured using ImageJ (Fiji) software as described in the previous manuscript [67]. (G-J) Dying cells were visualized by anti-cleaved PARP staining in RpS3/+, dsh3/+ (G), RpS3/+, nub-Gal4, UAS-tcf-RNAi (H), RpS3/+, wgGla-1/+ (I), or RpS3/+, nub-Gal4, UAS-ArmS10, TG80 ts7(J) wing disc. Massive cell death was significantly suppressed by reducing Wg signal gradient. Scale bar, 100 μm. (K) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (Fig 3B) (n = 12, number of wing pouches), (G) (n = 8), (H) (n = 15), (I) (n = 31), and (J) (n = 24). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. Genotypes are as follows: en-Gal4, UAS-GFP/wg17en40cP1; RpS3Plac92, UAS-RpS3/+ (A), en-Gal4, UAS-GFP/wg17en40cP1; RpS3Plac92/+ (B), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/+ (D), en-Gal4, UAS-GFP/+; RpS3Plac92/+ (E), dsh3/+; nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/+ (G), nub-Gal4/UAS-tcf-RNAi; UAS-CD8-PARP-Venus, RpS3Plac92/+ (H), nub-Gal4/wg gla-1; UAS-CD8-PARP-Venus, RpS3Plac92/+ (I), and UAS-ArmS10/+; nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/ TG80 ts7 (J).

    (TIF)

    Click here for additional data file. (3.1MB, tif)
    S4 Fig. Related to Fig 4. JNK-Dilp8-mediated developmental delay is required for massive cell-turnover.

    (A and B) Wg was overexpressed using the endogenous wg promoter (wg-Gal4). Dying cells in the wing discs of wild-type (A) or wg-Gal4, UAS-Wg (B) flies were visualized by anti-cleaved Dcp-1 staining. Scale bar, 100 μm. (C) Boxplot with dots representing cleaved-Dcp-1-positive dying cells per wing pouch of genotypes shown in (A) (n = 17, number of wing pouches) and (B) (n = 16). Error bars, SEM; n.s, not significant; non-parametric Mann-Whiteney U-test. (D) The expression of dilp8 in wing discs of RpS3/+, Dilp8::eGFP/+, nub-Gal4, UAS-Puc flies were visualized with GFP (green). Scale bar, 100 μm. (E-F’) RpS3 was overexpressed in the posterior compartment of the wing disc of TRE-DsRed/+ (E) or RpS3/+, TRE-DsRed/+ (F) flies using the en-Gal4 driver. JNK-activated cells were visualized by TRE-DsRed reporter (white). (G-H’) RpS3 was overexpressed in the posterior compartment of the wing discs of msn-lacZ/+ flies (G) or RpS3/+, msn-lacZ/+ (H) flies using the en-Gal4 driver. JNK activity was visualized by anti-β-galactosidase staining (white). Scale bar, 100 μm. (I and J) Wing disc of ecd1/ecd1 (I) or ecd1/ecd1, wg-Gal4, UAS-Wg (J) were stained with anti-phospho-histone H3 (pH3) (Ser10) antibody (white). Wing pouches were marked by pale green. ecd1 is a temperature-sensitive ecd mutant allele that blocks biosynthesis of the active-form of the hormone 20-Hydroxyecdysone at 29°C. For heat-shock treatment, fly culture was transferred to 29°C for 48 hours during the 3rd instar 1arval stage. Scale bar, 100 μm. (K) Boxplot with dots representing pH3 positive cells per pouch in genotypes shown in (I) (n = 13, number of wing pouches) and (J) (n = 13). Error bars, SEM; *, p<0.05; non-parametric Mann-Whitney U-test. (L and M) Wg was overexpressed using the endogenous wg promoter (wg-Gal4) in 3rd instar larvae fed with food containing erg-2 mutant yeast. Dying cells in the wing discs of wild-type (L) or wg-Gal4, UAS-Wg (M) flies were visualized by anti-cleaved Dcp-1 staining. Scale bar, 100 μm. (N) Boxplot with dots representing dying cells of cleaved-Dcp-1-positive cells per pouch in genotypes shown in (A) (n = 17, number of wing pouches), (B) (n = 16), (L) (n = 17), and (M) (n = 14). Error bars, SEM; ***, p<0.001; **, p<0.01; non-parametric Mann-Whitney U-test. Scale bar, 100 μm. (O and P) Dying cells in the wing disc were visualized by anti-Dcp1 antibody staining (white). Wing disc of wild-type (O) or wg-Gal4, UAS-Wg (P) shown in flies bearing the eye disc in which scrib mutant clones were generated and surrounding wild-type cells were simultaneously removed by a combination of GMR-hid and a recessive cell-lethal mutation, CL3R. Scale bar, 100 μm. (Q) Boxplot with dots representing cleaved-Dcp-1-positive dying cells per pouch in genotypes shown in (O) (n = 14, number of wing pouches) and (P) (n = 7). Error bars, SEM; *, p<0.05; non-parametric Mann-Whitney U-test. Scale bar, 100 μm. Genotypes are as follows: wg-Gal4/+ (A, L), wg-Gal4/+;UAS-Wg/+ (B, M), nub-Gal4/+; RpS3Plac92, dilp8::eGFP/UAS-Puc (D), en-Gal4, UAS-GFP/+; TRE-DsRed/UAS-RpS3 (E), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/ TRE-DsRed (F), en-Gal4, UAS-GFP/+; UAS-RpS3/msn06946 (G), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/msn06946 (H), ecd1/ecd1 (I), wg-Gal4/UAS-Wg; ecd1/ ecd1 (J), eyFLP1/+;; FRT82B,scrib1/FRT82B, GMR-hid.CL3R (O), and eyFLP1/+; wg-Gal4/UAS-Wg; FRT82B,scrib1/FRT82B, GMR-hid.CL3R (P).

    (TIF)

    Click here for additional data file. (3.6MB, tif)
    S5 Fig. The activation pattern of JAK/STAT signaling in the M/+ wing disc is indistinguishable from that in the wild-type wing disc.

    (A-B’) The wild-type (A) or RpS3/+ (B) wing disc bearing the 10xSTAT-GFP reporter (green). The nuclei were visualized by DAPI staining (blue) Scale bar, 100 μm. Genotypes are as follows: 10xSTAT-GFP/+ (A) and 10xSTAT-GFP/ RpS3Plac92 (B).

    (TIF)

    Click here for additional data file. (1MB, tif)
    S1 Movie. Live imaging of wing discs with the cell-cycle probe S/G2/M-Green.

    The S/G2/M-Green probe was expressed in the wing pouches of wild-type larvae. Wing discs were dissected and a part of the wing pouch was subjected to time-lapse imaging at 10-min intervals for 7 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: nub-Gal4/+; UAS-S/G2/M-Green/+. Each experiment was replicated at least 3 times.

    (MOV)

    Click here for additional data file. (11.5MB, mov)
    S2 Movie. Live imaging of wing discs with the cell-cycle probe S/G2/M-Green.

    The S/G2/M-Green probe was expressed in the wing pouches of or RpS3/+ larvae. Wing discs were dissected and a part of the wing pouch was subjected to time-lapse imaging at 10-min intervals for 7 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: nub-Gal4/+; UAS-S/G2/M-Green/RpS3Plac92. Each experiment was replicated at least 3 times.

    (MOV)

    Click here for additional data file. (11.8MB, mov)
    S3 Movie. Live imaging of wing disc eversion.

    GFP-expressing wing discs of wild-type larvae were dissected and subjected to time-lapse imaging at 10-min intervals for over 12 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: FRT82B, Ubi-GFP/+. Each experiment was replicated at least 3 times.

    (MOV)

    Click here for additional data file. (2MB, mov)
    S4 Movie. Live imaging of wing disc eversion.

    GFP-expressing wing discs of RpS3/+ larvae were dissected and subjected to time-lapse imaging at 10-min intervals for over 12 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: FRT82B, Ubi-GFP, RpS3Plac92/+. Each experiment was replicated at least 3 times.

    (MOV)

    Click here for additional data file. (3.4MB, mov)
    S5 Movie. Live imaging of wing disc eversion.

    GFP-expressing wing discs of RpS3/+, nub-Gal4, UAS-p35 larvae were dissected and subjected to time-lapse imaging at 10-min intervals for over 12 hours with a Leica SP8 confocal microscope. See Methods for details. Genotypes are as follows: nub-Gal4/+; FRT82B, Ubi-GFP, RpS3Plac92/UAS-p35. Each experiment was replicated at least 3 times.

    (MOV)

    Click here for additional data file. (2.6MB, mov)
    Attachment

    Submitted filename: Response to reviewrs.Akai et al.pdf

    Click here for additional data file. (5.1MB, pdf)

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