JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress
- University of Freiburg, Germany
- Ludwig-Maximilians-University Munich, Germany
- University of Cologne, Germany
Figures
Figure 1 with 1 supplement
Tissue injury induces a transient G2-shift.
(A–C’) Undamaged control wing disc (A), a wing disc with surgical damage 6 hr (B) or 16 hr (C) into the recovery (R) period. Wing discs were counterstained with DAPI (cyan in A-C) and express the …
Figure 1—figure supplement 1
Tissue injury induces a transient G2-shift.
(A) Scheme of a xy-section and yz-section through a wing imaginal disc. Gray shading in xy-view defines the disc subregions of pouch, hinge and notum. Broken lines represent the Anterior-Posterior …
Figure 2 with 1 supplement
Stress-induced JNK activity correlates with G2-stalling.
(A,B) Control wing disc (A) and a wing disc at 0 hr into the recovery period, after 24 hr of egr-expression in the pouch domain (B) (see Figure 2—figure supplement 1A). Discs also express the …
Figure 2—figure supplement 1
Stress-induced JNK activity correlates with G2-stalling.
(A) Time line of development and induction of cell ablation by expression of pro-apoptotic transgenes as a function of rearing temperature. Larvae were raised at 18°C (blue) and transferred to 30°C …
Figure 3 with 2 supplements
JNK activity is necessary and sufficient for G2-stalling.
(A,A’) A wing disc expressing a constitutively active JNKK hepACT in the pouch, assayed by FUCCI reporters (A) and EdU incorporation (A’) for cycling cells. At the center of the pouch, a G2-shifted …
Figure 3—figure supplement 1
JNK activity is necessary and sufficient for G2-stalling.
(A,A’) The peripodium of a wild type disc expressing the FUCCI reporters (A) and assessed for cycling activity by EdU incorporation to reveal DNA replication activity (A’). (B–D) Quantification of …
Figure 3—figure supplement 2
DNA damage is not rate-limiting for G2 stalling.
(A–A’’) egr-expressing disc at 0 hr into the recovery period expressing the FUCCI reporters (A) to visualize G2-stalled cells (yellow) was stained for phosphorylated H2Av (γH2Av, A’, green in A’’) a …
Figure 4 with 1 supplement
Cdc25/String and Tribbles regulate G2 stalling.
(A–B’) Control wing disc (A,A’) and egr-expressing disc at 0 hr into the recovery period (B,B’) expressing a GFP trap element in the stg locus (A’,B’, cyan in A,B) and the G2-specific FUCCI reporter …
Figure 4—figure supplement 1
Cdc25/String and Tribbles regulate G2 stalling.
(A–D) A wild type control disc (A) or wing discs expressing an RNAi transgene targeting cdk1 (B), overexpressing stg (C) or expressing an RNAi transgene targeting trbl (D) under the control of rnGAL4…
Figure 5 with 1 supplement
Chronic stalling in G2 interferes with proliferative capacity.
(A–D’) X-Y view of a control wing disc (A), an egr-expressing (B), egr,stg-co-expressing (C) or hid-expressing disc (D) at 0 hr into the recovery period. Cross-sections through the tissue (A’–D’) …
Figure 5—figure supplement 1
Chronic stalling in G2 interferes with proliferative capacity.
(A–C) Representative adult wings arising from of egr-expressing (A), egr,stg-co-expressing (B) and hid-expressing discs (C) under the control of rnGAL4. (D–G) A control wing disc (D), a stg-expressin…
Figure 6 with 1 supplement
Transient stalling in G2 promotes survival by protecting cells from JNK-induced apoptosis.
(A–E) Undamaged control (A) and an undamaged disc expressing stg under the control of rnGAL4 (B). An injured control disc (C) and an injured stg-expressing disc (D), 6 hr into the recovery period. …
Figure 6—figure supplement 1
Stalling in G2 promotes survival by protecting cells from JNK-induced apoptosis.
(A–A’’’) A stg-expressing disc at 0 hr into the recovery period stained for the apoptotic marker Dcp-1 (A’’, green in A,A’) and for the HA-tag to visualize overexpressed Stg-HA (A’’’, magenta in A’).…
Figure 7 with 2 supplements
Chronic stalling in G2 promotes non-autonomous overgrowth.
(A,A’) A wing disc expressing TRE-RFP and carrying mosaic wtsx1/x1 clones marked by the absence of GFP (cyan in A). (B–D’) A surgically damaged wing disc 6 hr into the recovery period (B,B’), a wing …
Figure 7—figure supplement 1
Chronic stalling in G2 promotes non-autonomous overgrowth.
(A) Quantification of relative TRE-RFP fluorescence intensity in mosaic wing discs carrying wtsX1 clones at 96 hr to 196 hr after egg deposition (AED) at 25°C (circles). Note that wild type larvae …
Figure 7—figure supplement 2
Chronic stalling in G2 promotes mitogenic signaling.
(A,B) A control wing disc (A) and a surgically injured disc 24 hr after injury (B). Both discs use the CasExpress and G-TRACE reporter system to drive expression of GFP in cells that had experienced …
Figure 8
Model.
(A) Transient (surgical injury), prolonged (egr-expression) and chronic (mosaic tumors) disruption of tissue homeostasis induces transient, prolonged and chronic JNK activity, thereby driving …
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Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | Diap-1-GFP.3.5 | PMID: 18258485 | ||
| Genetic reagent (D. melanogaster) | enGAL4, UAS-GFP | D.Bilder, UC Berekely | ||
| Genetic reagent (D. melanogaster) | ‘eyMARCM19A’; ‘ey-FLP, FRT19A tub-GAL80; act5c>y[+]>GAL4, UAS-GFP[S56T]’ | PMID: 29494583 | ||
| Genetic reagent (D. melanogaster) | ‘ubxMARCM82B’; ‘Ubx-FLP, tubP-GAL4, UAS-GFP; FRT82B tubP-GAL80’ | Bloomington Drosophila Stock Center | BDSC: 42734 | |
| Genetic reagent (D. melanogaster) | FRT19A dlg[G0342] | Kyoto Stock Center | DGGR: 111872 | |
| Genetic reagent (D. melanogaster) | FRT82B wts[x1] | Bloomington Drosophila Stock Center | BDSC: 44251 | |
| Genetic reagent (D. melanogaster) | ‘G-TRACE’; ‘UAS-FLP.Exel, Ubi-p63E(FRT.STOP)Stinger’ | Bloomington Drosophila Stock Center | BDSC: 28282 | |
| Genetic reagent (D. melanogaster) | gstD-GFP | PMID: 18194654 | ||
| Genetic reagent (D. melanogaster) | hep[R75] | Bloomington Drosophila Stock Center | BDSC: 6761 | |
| Genetic reagent (D. melanogaster) | ‘hid-GFP’; ‘hid[MI06452-GFSTF.1]’ | Bloomington Drosophila Stock Center | BDSC: 65331 | |
| Genetic reagent (D. melanogaster) | mei-41[RT1] | Bloomington Drosophila Stock Center | BDSC: 4169 | |
| Genetic reagent (D. melanogaster) | ‘puc-LacZ’; ‘puc[A251.1F3]’ | Bloomington Drosophila Stock Center | BDSC: 11173 | |
| Genetic reagent (D. melanogaster) | ‘rnGAL4’; ‘rn[GAL4-DeltaS]’ | Bloomington Drosophila Stock Center | BDSC: 8142 | |
| Genetic reagent (D. melanogaster) | ‘rn(ts)>‘; ‘rn[GAL4-DeltaS], tubGAL80[ts]’ | Bloomington Drosophila Stock Center | BDSC: 8142; BDSC: 7018 | recombinant |
| Genetic reagent (D. melanogaster) | ‘rn(ts)>egr’; ‘rn[GAL4-5], UAS-egr, tubP-GAL80[ts]’ | Bloomington Drosophila Stock Center | ||
| Genetic reagent (D. melanogaster) | ‘stg-GFP’; ‘stg[YD0685]’ | Bloomington Drosophila Stock Center | BDSC: 50879 | |
| Genetic reagent (D. melanogaster) | ‘Trbl-GFP’; ‘trbl[MI01025-GFSTF.2]’ | Bloomington Drosophila Stock Center | BDSC: 61654 | |
| Genetic reagent (D. melanogaster) | TRE-RFP | PMID: 22509270 | ||
| Genetic reagent (D. melanogaster) | UAS-bsk[DN] | Bloomington Drosophila Stock Center | BDSC: 6409 | |
| Genetic reagent (D. melanogaster) | UAS-Cdk1 RNAi [TRiP.HMS01531] | Bloomington Drosophila Stock Center | BDSC: 36117 | |
| Genetic reagent (D. melanogaster) | UAS-GFP S56T | BDSC: 1521 | ||
| Genetic reagent (D. melanogaster) | UAS-grp RNAi [TRiP.HMC05162] | Bloomington Drosophila Stock Center | BDSC: 62155 | |
| Genetic reagent (D. melanogaster) | UAS-hep[act] | Bloomington Drosophila Stock Center | BDSC: 9306 | |
| Genetic reagent (D. melanogaster) | UAS-hid | G. Morata, CBSMO Spain | ||
| Genetic reagent (D. melanogaster) | UAS-mei-41 RNAi [TRiP.GL00284] | Bloomington Drosophila Stock Center | BDSC: 35371 | |
| Genetic reagent (D. melanogaster) | UAS-stg | Bloomington Drosophila Stock Center | BDSC: 4777 | |
| Genetic reagent (D. melanogaster) | UAS-stg.HA | Bloomington Drosophila Stock Center | BDSC: 56562 | |
| Genetic reagent (D. melanogaster) | UAS-Xbp1-GFP.HG | Bloomington Drosophila Stock Center | BDSC: 60731 | |
| Genetic reagent (D. melanogaster) | ‘E2F1-FUCCI, CycB-FUCCI’; ‘Ubi-GFP.E2f1.1–230, Ubi-mRFP1.NLS.CycB.1–266’ | Bloomington Drosophila Stock Center | BDSC: 55123 | |
| Genetic reagent (D. melanogaster) | ‘CycB-FUCCI’; ‘Ubi-mRFP1.NLS.CycB.1–266’ | PMID: 24726363 | ||
| Genetic reagent (D. melanogaster) | ‘ubx-flp;; FRT82B ubi-GFP’ | I. Hariharan, UC Berkeley | ||
| Genetic reagent (D. melanogaster) | upd-lacZ (PD) | PMID: 8582614 | ||
| Genetic reagent (D. melanogaster) | UAS-trbl RNAi [TRiP.HMJ02089] | Bloomington Drosophila Stock Center | BDSC: 42523 | |
| Genetic reagent (D. melanogaster) | UAS-P35 | Bloomington Drosophila Stock Center | BDSC: 5072 | |
| Genetic reagent (D. melanogaster) | GH146-QF, QUAS-mtdTomato-3xHA | Bloomington Drosophila Stock Center | BDSC: 30037 | |
| Genetic reagent (D. melanogaster) | Ubi-CasExpress | Bloomington Drosophila Stock Center | BDSC: 65419 | |
| Antibody | Rabbit anti-cleaved Dcp-1 | Cell Signaling | Cat. #: 9578 | (1:200) |
| Antibody | Mouse monoclonal anti-CycB | Developmental Studies Hybridoma Bank | Cat. #: F2F4 | (1:20) |
| Antibody | Rat monoclonal anti-DE-cadherin | Developmental Studies Hybridoma Bank | Cat. #: DCAD2 | (1:100) |
| Antibody | Mouse monoclonal anti-discs large | Developmental Studies Hybridoma Bank | Cat. #: 4F3 | (1:100) |
| Antibody | Mouse anti-β-Galactosidase | Promega | Cat. #: Z3782 | (1:1000) |
| Antibody | Chicken anti-GFP | Abcam | Cat. #: ab13970 | (1:1000) |
| Antibody | Rabbit monoclonal anti-GFP | Invitrogen | Cat. #: G10362 | (1:200) |
| Antibody | Rabbit anti-H2Av-pS137 | Rockland | Cat. #: 600-401-914 | (1:500) |
| Antibody | Mouse anti-H3-pS10 | Abcam | Cat. #: ab14955 | (1:2000) |
| Antibody | Rat monoclonal anti-HA | Monoclonal Antibody Core Facillity at the Helmholtz Zentrum München | Clone #: 3F10 | (1:20) |
| Antibody | Mouse monoclonal anti-MMP1 | Developmental Studies Hybridoma Bank | Cat. #: 3A6B4 | (1:30) |
| Antibody | Mouse monoclonal anti-MMP1 | Developmental Studies Hybridoma Bank | Cat. #: 3B8D12 | (1:30) |
| Antibody | Mouse monoclonal anti-MMP1 | Developmental Studies Hybridoma Bank | Cat. #: 5H7B11 | (1:30) |
| Antibody | Mouse monoclonal anti-RFP | Abcam | Cat. #: ab65856 | (1:100) |
| Antibody | Rat monoclonal anti-RFP | Monoclonal Antibody Core Facillity at the Helmholtz Zentrum München | Clone #: 5F8 | (1:20) |
| Antibody | Mouse monoclonal anti-Ultrabithorax | Developmental Studies Hybridoma Bank | Cat. #: Ubx FP3.38 | (1:10) |
| Commercial assay or kit | Click-iT Plus EdU Alexa Fluor 647 Imaging Kit | Invitrogen | Cat. #: C10640 |
Additional files
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Supplementary file 1
Genotypes and experimental conditions.
This table lists detailed genotypes and temperature conditions used to generate the data for each of the main and supplementary figures in this study.
- https://doi.org/10.7554/eLife.41036.019
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Transparent reporting form
- https://doi.org/10.7554/eLife.41036.020
