Patterning of tissue stress responses by JNK and JAK/STAT

The restoration of tissue homeostasis upon injury relies on the spatial and temporal coordination of multiple processes, including damage-induced apoptosis and compensatory proliferation of the surviving neighboring cells. In Drosophila, the JNK pathway plays a central role in tissue stress responses and promotes both apoptosis and proliferation. How activation of the same signaling cascade results in these alternative outcomes and how these responses are spatially patterned is still unclear. We address these questions by studying two distinct regions around the site of injury in Drosophila imaginal discs. Cells at the wound margin, which experience high JNK levels, undergo a JNK-dependent extension of the G2 cell cycle phase. While acute stress conditions induce a transient and reversible G2 stalling that protects from JNK-induced apoptosis, chronic stress results in G2 arrest and promotes paracrine proliferation. We also show that, in cells located at the periphery of the injury site, activation of JAK/STAT plays an essential role by promoting survival of JNK-signaling cells, thereby allowing the execution of JNK-dependent compensatory proliferation. Together, we propose that the apoptotic and proliferative outputs of JNK signaling are balanced in domains proximal to the wound site via G2 stalling and in more distal areas via activation of JAK/STAT.Die Wiederherstellung der Gewebehomöostase nach einer Verwundung beruht auf räumlicher und zeitlicher Koordination mehrerer Prozesse, einschließlich der verletzungsinduzierten Apoptose und der kompensatorischen Proliferation der überlebenden Nachbarzellen. In Drosophila Imaginalscheiben, ein epitheles Modellsystem, spielt der JNK Signalweg eine zentrale Rolle in der Wundantwort und fördert sowohl Apoptose, als auch Proliferation. Wie die Aktivierung einer Signalkaskade zu unterschiedlichen zellulären Antworten führt und wie diese Antworten räumlich und zeitlich koordiniert werden, ist unklar. Wir haben diese Frage untersucht, indem wir zwei unterschiedliche Regionen in der Umgebung einer in der Imaginalscheibe induzierten Verletzung untersuchten. Zellen, welche sich am Rand der Wunde befinden und hohe JNK Aktivität aufweisen, reagieren mit einer JNK-abhängigen Verlängerung der G2-Phase des Zellzyklus. Während akuter Stress zu einem transienten und reversiblen Anhalten in der G2-Phase führt, welches vor JNK-induzierter Apoptose schützt, führt chronischer Stress zu einem Stillstand in der G2-Phase und einer Überaktivierung parakriner Signale, die die Proliferation der umliegenden Zellen anregt. Zusätzlich konnten wir nachweisen, dass in diesen wund-peripheren, proliferierenden Zellen JAK/STAT Aktivität eine essentielle Rolle spielt, indem es das Überleben der JNK-signalübertragenden Zellen fördert und damit die Ausführung der JNK-abhängigen kompensatorischen Proliferation erlaubt. Wir schlagen ein Modell vor, in dem apoptotische und proliferative Reaktionen auf JNK in wund-proximalen Regionen durch ein Anhalten der G2-Phase balanciert werden und in wund-distalen Regionen durch Aktivierung der JAK/STAT Signalkaskade

Patterning of tissue stress responses by JNK and JAK/STAT

The restoration of tissue homeostasis upon injury relies on the spatial and temporal coordination of multiple processes, including damage-induced apoptosis and compensatory proliferation of the surviving neighboring cells. In Drosophila, the JNK pathway plays a central role in tissue stress responses and promotes both apoptosis and proliferation. How activation of the same signaling cascade results in these alternative outcomes and how these responses are spatially patterned is still unclear. We address these questions by studying two distinct regions around the site of injury in Drosophila imaginal discs. Cells at the wound margin, which experience high JNK levels, undergo a JNK-dependent extension of the G2 cell cycle phase. While acute stress conditions induce a transient and reversible G2 stalling that protects from JNK-induced apoptosis, chronic stress results in G2 arrest and promotes paracrine proliferation. We also show that, in cells located at the periphery of the injury site, activation of JAK/STAT plays an essential role by promoting survival of JNK-signaling cells, thereby allowing the execution of JNK-dependent compensatory proliferation. Together, we propose that the apoptotic and proliferative outputs of JNK signaling are balanced in domains proximal to the wound site via G2 stalling and in more distal areas via activation of JAK/STAT.Die Wiederherstellung der Gewebehomöostase nach einer Verwundung beruht auf räumlicher und zeitlicher Koordination mehrerer Prozesse, einschließlich der verletzungsinduzierten Apoptose und der kompensatorischen Proliferation der überlebenden Nachbarzellen. In Drosophila Imaginalscheiben, ein epitheles Modellsystem, spielt der JNK Signalweg eine zentrale Rolle in der Wundantwort und fördert sowohl Apoptose, als auch Proliferation. Wie die Aktivierung einer Signalkaskade zu unterschiedlichen zellulären Antworten führt und wie diese Antworten räumlich und zeitlich koordiniert werden, ist unklar. Wir haben diese Frage untersucht, indem wir zwei unterschiedliche Regionen in der Umgebung einer in der Imaginalscheibe induzierten Verletzung untersuchten. Zellen, welche sich am Rand der Wunde befinden und hohe JNK Aktivität aufweisen, reagieren mit einer JNK-abhängigen Verlängerung der G2-Phase des Zellzyklus. Während akuter Stress zu einem transienten und reversiblen Anhalten in der G2-Phase führt, welches vor JNK-induzierter Apoptose schützt, führt chronischer Stress zu einem Stillstand in der G2-Phase und einer Überaktivierung parakriner Signale, die die Proliferation der umliegenden Zellen anregt. Zusätzlich konnten wir nachweisen, dass in diesen wund-peripheren, proliferierenden Zellen JAK/STAT Aktivität eine essentielle Rolle spielt, indem es das Überleben der JNK-signalübertragenden Zellen fördert und damit die Ausführung der JNK-abhängigen kompensatorischen Proliferation erlaubt. Wir schlagen ein Modell vor, in dem apoptotische und proliferative Reaktionen auf JNK in wund-proximalen Regionen durch ein Anhalten der G2-Phase balanciert werden und in wund-distalen Regionen durch Aktivierung der JAK/STAT Signalkaskade

JNK-dependent cell cycle stalling in G2 promotes survival and senescence-like phenotypes in tissue stress

The restoration of homeostasis after tissue damage relies on proper spatial-temporal control of damage-induced apoptosis and compensatory proliferation. In Drosophila imaginal discs these processes are coordinated by the stress response pathway JNK. We demonstrate that JNK signaling induces a dose-dependent extension of G2 in tissue damage and tumors, resulting in either transient stalling or a prolonged but reversible cell cycle arrest. G2-stalling is mediated by downregulation of the G2/M-specific phosphatase String(Stg)/Cdc25. Ectopic expression of stg is sufficient to suppress G2-stalling and reveals roles for stalling in survival, proliferation and paracrine signaling. G2-stalling protects cells from JNK-induced apoptosis, but under chronic conditions, reduces proliferative potential of JNK-signaling cells while promoting non-autonomous proliferation. Thus, transient cell cycle stalling in G2 has key roles in wound healing but becomes detrimental upon chronic JNK overstimulation, with important implications for chronic wound healing pathologies or tumorigenic transformation

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration.

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, we investigate models of compensatory proliferation in Drosophila imaginal discs. We find that compensatory proliferation is associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models

DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis

Background: Tracking dynamic protein–chromatin interactions in vivo is key to unravel transcriptional and epigenetic transitions in development and disease. However, limited availability and heterogeneous tissue composition of in vivo source material impose challenges on many experimental approaches. Results: Here we adapt cell-type-specific DamID-seq profiling for use in Drosophila imaginal discs and make FLP/FRT-based induction accessible to GAL driver-mediated targeting of specific cell lineages. In a proof-of-principle approach, we utilize ubiquitous DamID expression to describe dynamic transitions of Polycomb-binding sites during wing imaginal disc development and in a scrib tumorigenesis model. We identify Atf3 and Ets21C as novel Polycomb target genes involved in scrib tumorigenesis and suggest that target gene regulation by Atf3 and AP-1 transcription factors, as well as modulation of insulator function, plays crucial roles in dynamic Polycomb-binding at target sites. We establish these findings by DamID-seq analysis of wing imaginal disc samples derived from 10 larvae. Conclusions: Our study opens avenues for robust profiling of small cell population in imaginal discs in vivo and provides insights into epigenetic changes underlying transcriptional responses to tumorigenic transformation.ISSN:1756-893

Correction to: DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis

Unfortunately, the original version of this article contained a typographical error in one of the author names. The name of the author Alexey Pindyurin was incorrectly spelt as Alexey Pinduyrin. The correct spelling is included here and has been updated in the original article

EdU incorporation is not sensitive to tissue architecture defects.

(A) Wing disc after 24 h of egr-expression (E) in the pouch domain. Discs also express the JNK-reporter TRE-RFP (A‘, red in A”“). The disc was stained with DAPI to visualize nuclei (A, red in A”“‘), for the mitotic marker phospho-His3 to visualize M-phase cells (A”‘, green) and was assessed for DNA replication activity by EdU incorporation (grey). Compare number of phospho-His3 positive events to the number of EdU labelled nuclei to estimate relatively low frequency of M-phase cells in discs. (B-E) Nota of control wing disc (B,D) and and wing disc after 24 h of hid-expression (C) and after 24 h of egr-expression (E) in the pouch domain. Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation (B’-E’). Cyan star in (E) marks small domain of frequent transdetermination as described in M. I. Worley, L. A. Alexander and I. K. Hariharan, CtBP impedes JNK- and Upd/STAT-driven cell fate misspecifications in regenerating Drosophila imaginal discs, Elife 2018 Vol. 7. Cells in this patch undergo compensatory-like proliferation as part of the transdetermination program and therefore incorporate more EdU. Scale bars: 50 μm. (PDF)</p

Yki and ERK cooperate to drive compensatory proliferation in response to non-inflammatory damage.

(A,B) A wing disc expressing the act-GAL4 ‘flip-out’ system controlling the mosaic expression of GFP (A’,B’, or green) and UAS-Warts-RNAi (A), or UAS-Hippo-RNAi (B). Discs were stained with DAPI to visualize nuclei (A,B) and were assessed for DNA replication activity by EdU incorporation (A”,B”, or magenta). (C,D) Control wing disc (C), and a wing disc heterozygous for ykiB5 (D). Discs were stained with DAPI to visualize nuclei (C,D) and were assessed for DNA replication activity by EdU incorporation (C’,D’). (E-H) Control wing disc (E), and a control wing disc after 24 h of Egfr.DN-expression in the pouch domain (F). A control wing disc after 24 h of hid-expression (G) and a wing disc after 24 h of hid- and Egfr.DN-co-expression in the pouch domain (H). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. Egfr.DN does not change EdU incorporation dynamics in wild type and hid-expressing discs. (I-L) Control wing disc (I) and a control wing disc heterozygous for Ras1 (J). A control wing disc after 24 h of hid-expression (K) and a wing disc after 24 h of hid-expression and heterozygous for Ras1 (L). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. Heterozygosity for Ras1 does not change EdU incorporation dynamics in wild type and hid-expressing discs. (M) Quantification of the percentage of DAPI areas that were positive for incorporated EdU in hid-expressing discs and hid-expressing discs heterozygous for Ras1. This serves as a proxy for the number of nuclei undergoing DNA replication. Mean and 95% CI are shown. Welch’s test was performed to test for statistical significance. (Hid, n = 8 discs; Hid, Ras1/+, n = 7 discs, ns, p = 0.266). (N) Quantification of incorporated EdU, measured as the mean EdU intensity in the EdU area within the pouch of hid-expressing discs and hid-expressing discs heterozygous for Ras1. This serves as a proxy for the dynamics of nucleotide incorporation. A Welch’s test was performed to test for statistical significance. (Hid, n = 8 discs, Hid, Ras1/+, n = 7 discs, ns, p = 0.255). Single sections are shown in (A-F,I-L). Maximum projections of multiple confocal sections are shown in (G,H). Scale bars: 50 μm. (PDF)</p

Fly strains used in this study.

(DOCX)</p

Yki and ERK cooperate to drive compensatory proliferation in response to non-inflammatory damage.

(A) A wing disc expressing the act-GAL4 ‘flip-out’ system controlling the mosaic expression of GFP and UAS-yki.S168A (green in A”‘). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation (magenta). (B, C) Control wing disc (B), wing disc after 24 h of UAS-yki expression in the pouch domain (C). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. (D) A wing disc expressing the act-GAL4 ‘flip-out’ system controlling the mosaic expression of GFP and UAS-RasV12 (green in D”‘). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation (magenta). (E,F) Control wing disc (E) and wing disc after 24 h of UAS-RasV12 expression in the pouch domain (F). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. (G,H) Wing disc after 24 h of hid-expression (G) and a wing disc heterozygous for yki B5 after 24 h of hid-expression (H). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. White frame marks the magnified view of the pouch domain shown in (G”-H”‘). (I,J) Control wing disc (I) and a wing disc after 24 h of UAS-yki-GFP and UAS-RasV12 expression in the pouch domain (J). Discs were stained with DAPI to visualize nuclei and were assessed for DNA replication activity by EdU incorporation. (K) Quantification of the percentage of DAPI area in the pouch domain of the wing disc that was positive for incorporated EdU in control wing discs, or Yki and RasV12 expressing wing discs. Mean and 95% CI are shown. Welch’s test was performed to test for statistical significance. (WT, n = 8 discs, UAS-yki-GFP, UAS-RasV12, n = 7 discs, **p(L) Quantification of incorporated EdU intensity, measured as the mean EdU intensity within the EdU area of the pouch in control wing discs and wing disc after 24 h of Yki and RasV12 expression. A Welch’s test was performed to test for statistical significance. (WT, n = 8 discs, UAS-yki-GFP, UAS-RasV12, n = 7 discs, ****p = (M,N) Control wing disc (M) and a wing disc after 24 h of UAS-yki-GFP and UAS-RasV12 expression in the pouch domain (N). Discs were stained for cleaved Dcp1, a marker of apoptosis. Graphs display mean and 95% CI. Maximum projections of multiple confocal sections are shown in (G,H,I,J). Single sections are shown in (A-F). Scale bars: 50 μm. Dotted lines (red) outline stereotypic folds in the wing discs.</p