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. 2024 Sep 3;20(9):e1011387.
doi: 10.1371/journal.pgen.1011387. eCollection 2024 Sep.

An unscheduled switch to endocycles induces a reversible senescent arrest that impairs growth of the Drosophila wing disc

Yi-Ting Huang  1 , Lauren L Hesting  1 , Brian R Calvi  1
Affiliations

An unscheduled switch to endocycles induces a reversible senescent arrest that impairs growth of the Drosophila wing disc

Yi-Ting Huang et al. PLoS Genet. .

Abstract

A programmed developmental switch to G / S endocycles results in tissue growth through an increase in cell size. Unscheduled, induced endocycling cells (iECs) promote wound healing but also contribute to cancer. Much remains unknown, however, about how these iECs affect tissue growth. Using the D. melanogaster wing disc as model, we find that populations of iECs initially increase in size but then subsequently undergo a heterogenous arrest that causes severe tissue undergrowth. iECs acquired DNA damage and activated a Jun N-terminal kinase (JNK) pathway, but, unlike other stressed cells, were apoptosis-resistant and not eliminated from the epithelium. Instead, iECs entered a JNK-dependent and reversible senescent-like arrest. Senescent iECs promoted division of diploid neighbors, but this compensatory proliferation did not rescue tissue growth. Our study has uncovered unique attributes of iECs and their effects on tissue growth that have important implications for understanding their roles in wound healing and cancer.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Growth by iEC hypertrophy lags behind that from cell division.
(A) Experimental timeline: FLP-Out Clones of mitotic or endocycling cells were heat induced at different time points of larval development (arrows) and wing discs were dissected from late wandering 3rd (L3). (B) Examples of RFP-marked mitotic control (Ctrl) and UAS-rux expressing iEC (rux) clones at different times after induction. Scale bar = 10 μm (C-F) Mitotic control (Ctrl) and iEC (rux) FLP-Out clones were quantified for cell number per clone (C), average cell size per clone (D), fold increase in DNA content relative to diploid (E), and total clone area (F) over a period of 72 hours. ** p<0.01 **** p<0.0001.
Fig 2
Fig 2. Unscheduled endocycles cause wing disc and adult wing undergrowth.
(A) Experimental timeline: en-GAL4 activity was induced in the posterior compartment of wing discs at different times by shifting from 18°C to 29°C (arrows), the GAL80ts nonpermissive temperature, followed by dissection during late wandering 3rd instar (L3). (B) Wing discs from control (Ctrl) and UAS-rux expressing (rux) wing discs after different times of induction. The posterior compartment is marked by UAS-RFP expression. Scale bar = 80 μm (C) Quantification of the ratio of posterior compartment to total wing disc area in control (Ctrl) or UAS-rux larvae (rux). (D-E) Induction of unscheduled endocycles in the wing pouch results in undergrowth. (D) pdm2-GAL4 G-TRACE wing disc without (Ctrl) or with UAS-rux (rux) expression. Scale bar = 80 μm. (E) Quantification of the ratio of wing pouch area to total wing disc area during late 3rd instar for pdm2-GAL4 alone (Ctrl) or pdm-GAL4 UAS-rux (rux). (F) pdm2-GAL4 wing disc without (Ctrl) or with UAS-rux (rux) expression. Cell boundaries were labeled with anti-Fas3 antibody. Scale bar = 10 μm. (G) Quantification of the cross-sectional cell area for the two genotypes shown in F. (H) Two magnifications of adult fly wing phenotypes from control (Ctrl) and pdm2-GAL4; UAS-rux (rux) as shown in D. Scale bar = 500 μm and 200 μm. ** p<0.01 **** p<0.0001.
Fig 3
Fig 3. Death of iECs does not play a major role in tissue undergrowth.
(A-B”’) Wing discs without (ctrl, A-A”) or with UAS-rux (rux, B-B”’) expression in the posterior compartment for three days and labeled with anti-activated caspase Dcp-1 antibody. Scale bar = 40 μm. (C) Quantification of Dcp-1+ cells in the anterior or posterior wing disc for the genotypes shown in A-B”. (D-I) Wing disc with expression of UAS-mRFP only (D,G, Ctrl), UAS-mRFP UAS-rux (E,H, rux), or UAS-mRFP UAS-rux and the caspase inhibitor UAS-p35 (F, I, rux; p35) in the posterior compartment for 72 hours (D-F) or 96 hours (G-I). Scale bar = 80 μm. (J) Quantification of the ratio of posterior compartment to total wing disc area for the three genotypes shown in D-I. (K-L”) iECs are apoptosis resistant. Wing discs without (Ctrl, K-K”) or with UAS-rux (rux, L-L”) expression in the mRFP-marked posterior compartment for three days were irradiated (IR+) with gamma-rays and labeled with anti-activated caspase Dcp-1 antibody four hours later. Scale bar = 40 μm. (M) Quantification of anti-Dcp-1+ fluorescent volume in the same size region of interest (ROI) in anterior or posterior wing disc for the genotypes shown in K-L”. **** p<0.0001, *** p<0.001, n.s. not significant.
Fig 4
Fig 4. Arrest of iECs at different terminal ploidies causes tissue undergrowth.
(A-D”) 3rd instar wing discs with UAS-rux and UAS-RFP expression in the posterior for the indicated durations of time were dissected and incubated in EdU in vitro for one hour. Scale bar = 20 μm. (E) Fold change in DNA content (DAPI fluorescence) of mRFP-positive posterior iECs relative to diploid mRFP-negative anterior diploid cells from the same wing discs after different durations of en-GAL4 expression. (F) Fraction of EdU positive cells in the anterior and posterior compartments of wing discs after the indicated durations of en-GAL4 expression. (G) Ploidy fold change of EdU positive cells only. **** p<0.0001.
Fig 5
Fig 5. iECs express senescence-associated genes and have DNA damage.
(A-D) iECs have increased SA-β-Gal activity (A-B’) and MMP1 (C-D’) expression after 72 hours induction in posterior wing disc compartment. Scale bar = 40 μm. (E-F’) DNA damage foci in posterior iECs labeled with anti-γH2Av antibody. Scale bar = 10 μm. (G-H’) iECs have DNA damage near heterochromatin. High magnification of iECs labeled with antibodies against damage marker γH2Av and heterochromatin marker H3K9me3. Scale bar = 10 μm.
Fig 6
Fig 6. JNK signaling negatively regulates iEC growth.
(A-C’) Expression of the JNK reporter, TRE-GFP, in wing discs with pdm2-GAL4 only (A, A’), or with UAS-rux (B, B’), or UAS-rux and UAS-bskDN (C, C’). Scale bar: 40 μm. (D-E’) SA-β-Gal activity in wing discs expressing UAS-rux alone (D-D’) or UAS-rux and UAS-bskDN (E-E’) in the wing pouch. Scale bar: 100 μm. (F-G’) Wing discs expressing UAS-rux alone (F-F’) or UAS-rux and UAS-bskDN (G-G’) in the posterior compartment for 72 hours. Scale bar: 40 μm. (H) Quantification of DNA content for genotypes shown in F-G’. The DNA content of RFP+ iEC in the posterior was normalized to the average DNA content of anterior RFP-negative diploid cells. The dataset used for UAS-rux (rux) expression group is the same as the 72 hours induction in Fig 4E. (I-J’) 3rd instar wing discs with UAS-rux alone (I-I’) or UAS-rux and UAS-bskDN (J-J’) expression in the posterior for 72 hours and were labeled with EdU in vitro for an hour after dissection. Scale bar: 20 μm. (K) Quantification of S phase fraction in the anterior and posterior wing disc compartments for genotypes shown in I-J’. The dataset used for UAS-rux (rux) expression group is the same as the 72 hours induction in Fig 4F. (L-M’) Wing discs expressing UAS-rux alone (L-L’) or UAS-rux and UAS-hep (M-M’) in the wing pouch. Scale bar: 40 μm. (N) Quantification of DNA content for genotypes shown in L-M’. DNA content of RFP+ iEC in the wing pouch was normalized to the average DNA content of RFP-negative diploid cells. **** p<0.0001, n.s. not significant.
Fig 7
Fig 7. The iEC senescent-like arrest is reversible.
(A-C’) Pdm2-GAL4 wing discs expressing UAS-rux alone (A-A’) or UAS-rux and tub-Gal80ts (B-C’) were raised at 29°C for 96 hours and shifted to 18°C for 24 hours recovery. Cells were co-labeled with mitotic marker anti-pH3 antibody and senescence marker SA-β-Gal. Arrows in C’ indicate chromatin fragments off of the metaphase plate in a polyploid iEC mitosis. Scale bars: 40 μm (A-B’) and 10 μm (C-C’). (D-E’) Dividing iEC co-labeled with mitotic chromosome marker anti-pH3 and cell periphery marker anti-Fas3 (D-D’) or anti-pH3 and mitotic spindle marker anti-β-tubulin (E-E’). Arrows in E-E’ indicates microtubule organizing center. Scale bar: 10 μm. (F-I’) Wing discs expressing UAS-rux alone (F-F”, H-H’) or UAS-rux and tub-Gal80ts (G-G”, I-I’) were raised at 29°C for 96 hours and shifted to 18°C for 72 hours recovery. Cells were co-labeled with mitotic marker anti-pH3 antibody and senescence marker SA-β-Gal. (H-I’) Higher magnifications of boxed area in panels F and G. Scale bar: 40 μm (F-G”) and 10 μm (H-I’). (J-L”’) Lineage analysis indicates iEC daughter cells continue to proliferate. (J-J”’) Control pdm2-GAL4 G-TRACE wing disc with real time GAL4 expression (RFP) and historical GAL4 expression (GFP) in the wing pouch. (K-L”’) pdm2-GAL4 G-TRACE wing disc with UAS-rux alone (K-K”’) or UAS-rux and tub-Gal80ts (L-L”’) were raised at 29°C for 96 hours and shifted to 18°C for 72 hours recovery. Scale bar: 40 μm .
Fig 8
Fig 8. iECs induce proliferation of neighboring diploid cells.
(A-B”) 3rd instar wing discs expressing RFP alone (Ctrl, A-A”) or with 96 hours of UAS-rux expression (rux, B-B”) in the posterior compartment were labeled with EdU in vitro for one hour. Arrows in B-B" point to hyperplastic overgrowth of the notum. Scale bar: 80 μm. (C-D) Distribution of distances between RFP and EdU (C) and histogram of numbers of EdU+ cells at different distances from the RFP+ posterior for the genotypes shown in A-B”. (E-F”) 3rd instar wing discs expressing RFP alone (Ctrl, E-E”) or with 96 hours of UAS-rux expression (rux, F-F”) in the posterior were incubated with EdU in vitro for one hour and co-labeled with anterior marker anti-Ci and posterior marker anti-RFP. Scale bar: 40 μm. (G) Summary and model for a reversible senescent-like and JNK-dependent growth arrest of iECs and their effect on neighboring cells. See text for details.
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