_:b66078443 . _:b66078496 . . _:b66078417 . _:b632446522 "5"^^ . _:b66078459 _:b66078464 . _:b66078459 _:b66078465 . _:b66078459 _:b66078466 . _:b632446518 . _:b66078459 _:b66078467 . _:b632446533 "4"^^ . _:b66078459 _:b66078468 . . _:b632446532 "4"^^ . . _:b632446531 . _:b632446535 "4"^^ . _:b632446559 . _:b66078388 . _:b632446534 "4"^^ . _:b66078430 "To address this, we inhibited JNK signalling specifically in loser RpS3 +/\u2212 cells during cell competition, by overexpressing the JNK inhibitor puckered (puc) 45, >>49<<. JNK inhibition reduced competition-induced cell death, as expected23 (Supplementary Fig.\u00A02e\u2013g). However, we also noted a striking increase in the overall ability of RpS3 +/\u2212 cells to colonise the tissue (Fig.\u00A02f, g), consistent with the" . _:b66078360 _:b66078364 . _:b66078360 _:b66078365 . _:b66078360 _:b66078366 . _:b66078360 _:b66078367 . _:b632446529 "4"^^ . _:b66078360 _:b66078361 . _:b66078376 "In addition to Minute, many other mutations have been shown to induce a loser status against WT cells, such as mutations in the oncogene myc >>20<<, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless6, 25 and EGF26." . _:b66078360 _:b66078362 . _:b632446525 . . _:b66078434 . _:b632446528 "4"^^ . _:b66078360 _:b66078363 . _:b66078360 _:b66078372 . _:b66078360 _:b66078373 . _:b66078360 _:b66078374 . _:b632446524 . _:b66078360 _:b66078375 . _:b632446531 "4"^^ . _:b66078360 _:b66078368 . _:b66078406 . _:b66078365 "Cell competition was originally described in Drosophila wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo>>7<<\u20139, liver10 and heart11 and in mammalian cell culture8, 9, 12, 13." . _:b66078360 _:b66078369 . _:b66078360 _:b66078370 . _:b632446527 . . _:b632446530 "4"^^ . _:b66078360 _:b66078371 . _:b66078360 _:b66078380 . _:b66078461 . _:b66078360 _:b66078381 . _:b66078360 _:b66078382 . _:b632446541 "3"^^ . _:b632446526 . _:b632446575 . _:b66078360 _:b66078383 . _:b66078360 _:b66078376 . _:b66078404 . _:b66078360 _:b66078377 . _:b66078360 _:b66078378 . _:b632446540 "3"^^ . _:b632446521 . _:b66078360 _:b66078379 . . . _:b66078360 _:b66078388 . _:b66078360 _:b66078389 . _:b66078473 "Constitutive JNK pathway activation in loser cells has been previously reported>>27<<. However, it was thought that JNK signalling simply provides a pro-apoptotic signal during competition." . _:b66078360 _:b66078390 . _:b66078482 . _:b632446543 "3"^^ . _:b632446520 . _:b66078360 _:b66078391 . _:b66078360 _:b66078384 . _:b632446536 . _:b66078360 _:b66078385 . _:b66078360 _:b66078386 . _:b632446542 "3"^^ . _:b632446523 . . _:b66078360 _:b66078387 . _:b66078386 . _:b632446642 . _:b66078360 _:b66078396 . _:b66078364 "Cell competition was originally described in Drosophila wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues>>4<<\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture8, 9, 12, 13." . . _:b66078476 "Several reports have indicated that winner cells enhance their proliferation in various cell competition models5, 6, >>8<<, 28\u201331, including Minute competition5, 29, 31. However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates69." . . _:b66078360 _:b66078397 . _:b66078360 _:b66078398 . _:b632446522 . _:b66078360 _:b66078399 . _:b66078449 . _:b632446537 "4"^^ . _:b66078360 _:b66078392 . _:b66078448 "However, Upds are soluble proteins capable of long-range signalling and have been reported to cause non-autonomous overgrowth phenotypes61, >>62<<. In addition we recently showed that in the adult Drosophila midgut Upd ligands produced by RpS3 +/\u2212 cells promote the proliferation of WT cells during cell competition5. This suggested that their production from RpS3 +/\u2212 wing disc cells" . _:b66078360 _:b66078393 . _:b66078360 _:b66078394 . _:b66078481 . _:b66078384 . _:b632446533 . _:b632446536 "4"^^ . _:b66078360 _:b66078395 . . _:b66078392 . _:b632446539 "3"^^ . _:b632446532 . _:b632446561 . _:b66078360 _:b66078400 . _:b66078460 "The following Drosophila stocks were used: RpS3[Plac92] (BL5627), RpS3*(BL5699), M(2)53 >>1<< (BL5698), FRT42D mahj 12, TRE-16 48, UAS-puc-RNAi (B." . _:b66078360 _:b66078401 . _:b66078360 _:b66078402 . _:b66078438 . _:b632446535 . _:b632446538 "4"^^ . _:b632446549 "3"^^ . _:b66078436 "Specifically in Drosophila imaginal discs, the pathway is required for developmental patterning>>53<<, 54 and to promote proliferation under normal conditions and during regeneration52, 55." . _:b632446534 . _:b632446548 "3"^^ . _:b66078374 . _:b632446529 . _:b632446550 . _:b66078422 "2, mahj 12, 27, myc 28, APC 6 and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition>>29<<, 46. Our RNAseq data indicates that both Minute and mahj \u2212/\u2212 cells in their naive state upregulate the JNK pathway (as both JNK activators, such as Gadd45 and JNK target genes, such as scarface 47 and reaper 45 are upregulated (Fig." . _:b632446551 "3"^^ . _:b632446528 . _:b632446626 . _:b66078432 . _:b66078392 . _:b632446550 "3"^^ . _:b632446531 . . _:b632446545 "3"^^ . _:b66078435 . _:b632446530 . . _:b632446572 . _:b66078491 "It has been suggested that winner cells overproliferation stems from winner/loser recognition32, 33, or from compensatory proliferative signals emanating from dying loser cells>>31<<, 34. Our data instead demonstrates that, at least for Minute cell competition, the overproliferation of winner cells is not a consequence of and in fact is independent of winner/loser cell recognition and of loser cell death, since Upd-2" . _:b632446544 "3"^^ . _:b632446541 . _:b632446584 . _:b66078469 . _:b632446547 "3"^^ . _:b632446540 . _:b66078501 "Nrf2 canonically serves a pro-survival role, protecting cells from the negative effects of toxic/oxidising compounds64, >>65<<. This is the case also for RpS3 +/\u2212 and mahj \u2212/\u2212 cells, since silencing Nrf2 causes autonomous cell death in Minute cells." . _:b632446596 . _:b66078436 . _:b632446637 . _:b632446546 "3"^^ . _:b66078405 . _:b632446543 . _:b66078371 "imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture8, 9, 12, >>13<<. Further studies have also shown the existence of this process in several stem cell compartments14\u201317, although it is likely that in this case it happens via a different mechanism(s)." . _:b66078397 . _:b632446542 . _:b632446557 "3"^^ . _:b66078469 _:b66078496 . _:b632446537 . _:b66078469 _:b66078497 . _:b632446556 "3"^^ . _:b66078469 _:b66078498 . _:b66078469 _:b66078499 . _:b632446648 . _:b66078469 _:b66078500 . _:b66078427 . . _:b632446536 . _:b66078469 _:b66078501 . _:b632446559 "3"^^ . _:b66078469 _:b66078502 . _:b632446604 . _:b632446539 . _:b632446558 "3"^^ . _:b632446562 . _:b66078425 . . _:b632446553 "3"^^ . _:b632446538 . _:b66078469 _:b66078480 . _:b632446549 . _:b632446600 . _:b66078469 _:b66078481 . _:b632446552 "3"^^ . _:b66078469 _:b66078482 . _:b66078469 _:b66078483 . _:b66078469 _:b66078484 . _:b66078391 . _:b632446548 . _:b66078469 _:b66078485 . _:b632446555 "3"^^ . _:b66078469 _:b66078486 . _:b66078453 "To confirm activation of the Nrf2 pathway in loser cells, we used a fluorescent transcriptional reporter of Nrf2\u2014GstD1-GFP>>65<<. In line with our RNAseq data, both RpS3 +/\u2212 (Fig.\u00A05c, d and Supplementary Fig.\u00A05a\u2013c) and mahj RNAi (Fig.\u00A05e, f) cells exhibited higher levels of reporter expression compared to WT cells, both before and during cell competition," . _:b66078378 . . _:b66078469 _:b66078487 . _:b66078469 _:b66078488 . _:b632446551 . _:b66078413 . _:b66078469 _:b66078489 . _:b632446554 "3"^^ . _:b66078469 _:b66078490 . _:b66078469 _:b66078491 . _:b632446565 "2"^^ . _:b66078429 . _:b66078469 _:b66078492 . _:b632446550 . _:b66078469 _:b66078493 . . _:b66078469 _:b66078494 . _:b66078469 _:b66078495 . _:b632446545 . . . _:b632446564 "3"^^ . _:b632446607 . _:b632446567 "2"^^ . _:b66078416 "JNK signalling has also been implicated in multiple cell competition systems, such as scribble 2, mahj >>12<<, 27, myc 28, APC 6 and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, 46." . _:b632446544 . . _:b66078469 _:b66078470 . _:b66078469 _:b66078471 . _:b632446566 "2"^^ . _:b66078365 . _:b66078469 _:b66078472 . _:b632446547 . _:b66078469 _:b66078473 . _:b66078469 _:b66078474 . _:b66078469 _:b66078475 . _:b632446589 . . _:b66078469 _:b66078476 . . _:b632446546 . _:b66078445 "It has long been known that during cell competition winner cells proliferate faster than they would normally do on their own5, >>28<<\u201331. This has mostly been attributed to winner\u2013loser recognition signals or to apoptosis-induced proliferation32\u201334." . _:b66078469 _:b66078477 . _:b632446561 "3"^^ . _:b66078469 _:b66078478 . _:b66078469 _:b66078479 . . _:b632446599 . _:b632446557 . _:b632446560 "3"^^ . _:b632446610 . _:b632446556 . _:b632446558 . _:b632446563 "3"^^ . _:b66078468 . _:b632446595 . _:b632446559 . _:b632446562 "3"^^ . _:b632446573 "2"^^ . _:b632446558 . _:b66078405 "Both alleles are mutations of the Minute gene M(3)95A and confer a loser status18, >>39<< (Supplementary Fig." . . _:b632446572 "2"^^ . _:b632446553 . . . _:b66078444 . _:b66078373 . _:b632446575 "2"^^ . _:b66078388 "First, loser cells commonly exhibit slower proliferation rates than their winner counterparts and this passively contributes to expansion of the winner cell population>>1<<, 19. Secondly, it has been reported that during cell competition winner cells further increase their proliferation rates over their already faster baseline5, 28\u201331. It is unclear how that is elicited; however, it has been proposed to be a" . _:b632446552 . _:b632446574 "2"^^ . _:b632446555 . _:b632446569 "2"^^ . _:b632446554 . . _:b66078452 "regulator of the oxidative stress response and is involved in the adaptation to oxidative and chemical stress through upregulation of a number of genes with anti-oxidant function or involved in the removal of harmful oxidation products>>64<<. Specifically, we found that prospective loser cells show upregulation of genes involved in glutathione synthesis (glutamate-cysteine ligase catalytic subunit\u2014Gclc), glutathione conjugation to xenobiotics (glutathione" . _:b632446568 "2"^^ . _:b632446565 . _:b632446578 . _:b66078426 "2a)), in agreement with earlier reports>>27<<. In addition we found that RpS3 +/\u2212 cells show strong activation of the JNK reporter TRE-dsRED48 (Fig." . _:b632446571 "2"^^ . _:b632446564 . . _:b632446570 "2"^^ . _:b66078448 . _:b632446567 . _:b66078466 "Perrimon), 10xSTAT-GFP 59, UAS-Dome \u0394Cyt 63, Dome g0218 (BL11953), GstD1-GFP65, UAS-mahj-RNAi (BL34912), UAS-Nrf2-RNAi>>65<<, UAS-Nrf2 65, UAS-dIAP1 (P." . _:b632446581 "2"^^ . . _:b632446566 . . _:b66078407 "1k, l); and mutants in mahj, a gene that is functionally unrelated to ribosomal genes (mahj is an interactor of polarity genes and a component of a cullin ubiquitin ligase complex>>12<<, 40), but whose mutation nevertheless confers a loser status against WT cells12 (Fig." . _:b632446580 "2"^^ . _:b66078414 . _:b632446561 . _:b66078420 . _:b66078401 "Several molecules, such as Flower32, Azot36, the Toll/IMD pathway>>37<<, and the Sas/PTP10D ligand-receptor complex38 have been implicated in triggering the apoptosis of losers." . _:b66078401 . _:b632446583 "2"^^ . . . _:b632446560 . _:b66078386 "20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless6, 25 and EGF>>26<<." . _:b66078484 . _:b632446586 . _:b632446582 "2"^^ . _:b632446563 . _:b632446520 . _:b632446605 . _:b632446577 "2"^^ . . _:b632446562 . _:b632446576 "2"^^ . _:b66078442 . _:b632446573 . _:b632446579 "2"^^ . _:b66078471 . _:b632446572 . _:b632446578 "2"^^ . _:b632446560 . _:b632446575 . _:b66078376 . _:b632446589 "2"^^ . _:b632446574 . _:b632446588 "2"^^ . _:b632446590 . _:b632446569 . . _:b632446591 "2"^^ . _:b632446568 . . _:b632446590 "2"^^ . _:b632446571 . _:b66078460 . . _:b632446585 "2"^^ . _:b632446570 . _:b632446620 . _:b66078390 "Secondly, it has been reported that during cell competition winner cells further increase their proliferation rates over their already faster baseline>>5<<, 28\u201331. It is unclear how that is elicited; however, it has been proposed to be a consequence of winner/loser recognition or simply a compensatory mechanism triggered by loser cell death28\u201334. The third and most striking aspect of cell" . _:b66078410 . _:b632446584 "2"^^ . _:b66078438 "Specifically in Drosophila imaginal discs, the pathway is required for developmental patterning53, 54 and to promote proliferation under normal conditions and during regeneration>>52<<, 55. Our RNAseq data show that both RpS3 +/\u2212 and mahj \u2212/\u2212 cells upregulate the JAK/STAT targets Socs36E and chinmo as well as all three ligands of the pathway\u2014the cytokines unpaired 1, 2 and 3 (upd, upd2, upd3)56\u201358 (Fig.\u00A04a), suggesting" . _:b66078414 . _:b632446581 . _:b66078391 "Secondly, it has been reported that during cell competition winner cells further increase their proliferation rates over their already faster baseline5, >>28<<\u201331. It is unclear how that is elicited; however, it has been proposed to be a consequence of winner/loser recognition or simply a compensatory mechanism triggered by loser cell death28\u201334. The third and most striking aspect of cell" . _:b66078440 "Our RNAseq data show that both RpS3 +/\u2212 and mahj \u2212/\u2212 cells upregulate the JAK/STAT targets Socs36E and chinmo as well as all three ligands of the pathway\u2014the cytokines unpaired 1, 2 and 3 (upd, upd2, upd3)>>56<<\u201358 (Fig.\u00A04a), suggesting pathway activation. We first validated these findings using an in vivo fluorescent reporter of STAT activity (10xSTAT-GFP59). Indeed we found that RpS3 +/\u2212 cells display reporter activation compared to WT cells" . _:b66078403 _:b66078440 . _:b632446587 "2"^^ . _:b66078410 "This served as a proof of principle of the validity of our approach, as the Toll pathway has been shown to be activated downstream of cell competition and to lead to loser cells apoptosis>>37<<. Our data suggest that the Toll pathway might already be affected in prospective loser cells before cell competition." . _:b66078403 _:b66078441 . _:b632446580 . _:b66078403 _:b66078442 . . . _:b66078403 _:b66078443 . . _:b66078403 _:b66078444 . _:b632446586 "2"^^ . _:b66078403 _:b66078445 . _:b632446583 . _:b66078502 "As oxidative stress is activated in a number of pathological conditions including cancer>>74<<, our findings suggest that cell competition might be common in these instances and potentially plays a role in disease prevention and progression." . _:b66078403 _:b66078446 . . _:b66078403 _:b66078447 . . _:b66078403 _:b66078432 . _:b66078403 _:b66078433 . _:b632446597 "2"^^ . _:b632446582 . _:b66078403 _:b66078434 . _:b632446616 . _:b632446617 . _:b66078372 "Further studies have also shown the existence of this process in several stem cell compartments>>14<<\u201317, although it is likely that in this case it happens via a different mechanism(s)." . _:b66078403 _:b66078435 . _:b632446618 . _:b66078403 _:b66078436 . _:b632446619 . _:b632446596 "2"^^ . _:b66078403 _:b66078437 . _:b632446577 . _:b66078403 _:b66078438 . _:b632446620 . _:b632446621 . _:b66078492 . _:b66078403 _:b66078439 . _:b632446622 . _:b66078403 _:b66078456 . _:b632446623 . _:b632446599 "2"^^ . _:b66078403 _:b66078457 . _:b632446576 . _:b66078403 _:b66078458 . _:b632446608 . _:b632446609 . _:b632446610 . _:b632446611 . _:b66078500 . _:b632446598 "2"^^ . _:b632446579 . _:b66078483 . _:b632446612 . . _:b632446537 . _:b632446544 . _:b632446613 . _:b632446614 . _:b66078403 _:b66078448 . _:b632446615 . _:b632446593 "2"^^ . _:b66078403 _:b66078449 . _:b632446578 . . _:b66078403 _:b66078450 . . _:b632446600 . _:b632446554 . _:b632446601 . _:b66078403 _:b66078451 . _:b632446602 . _:b66078403 _:b66078452 . _:b632446603 . _:b632446592 "2"^^ . _:b66078403 _:b66078453 . _:b632446604 . _:b632446589 . _:b66078403 _:b66078454 . _:b632446605 . _:b66078403 _:b66078455 . _:b632446606 . _:b66078403 _:b66078408 . _:b632446607 . _:b632446595 "2"^^ . _:b66078403 _:b66078409 . _:b632446592 . _:b632446588 . _:b66078403 _:b66078410 . _:b632446593 . _:b66078403 _:b66078411 . _:b632446594 . _:b66078403 _:b66078412 . _:b632446595 . _:b632446594 "2"^^ . _:b66078403 _:b66078413 . _:b632446596 . _:b632446591 . _:b66078403 _:b66078414 . _:b632446585 . _:b66078403 _:b66078415 . _:b632446597 . _:b632446598 . . _:b632446599 . _:b632446605 "2"^^ . _:b632446648 . _:b632446590 . _:b632446649 . _:b632446650 . _:b66078403 _:b66078404 . _:b66078403 _:b66078405 . _:b66078378 "In addition to Minute, many other mutations have been shown to induce a loser status against WT cells, such as mutations in the oncogene myc 20, in the polarity genes scribble 2, dlg >>21<< and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless6, 25 and EGF26." . _:b632446604 "2"^^ . _:b632446585 . _:b66078403 _:b66078406 . _:b66078403 _:b66078407 . _:b66078468 . _:b66078403 _:b66078424 . _:b66078403 _:b66078425 . _:b632446607 "2"^^ . _:b632446584 . . _:b66078403 _:b66078426 . _:b632446640 . _:b632446641 . _:b66078494 "For example cells that overexpress dMyc, lose their ability to outcompete cells if they are also Minute +/\u2212 >>28<<." . _:b66078403 _:b66078427 . _:b632446642 . _:b66078469 . _:b66078403 _:b66078428 . _:b632446643 . _:b632446633 . _:b632446606 "2"^^ . _:b66078403 _:b66078429 . _:b632446587 . _:b66078403 _:b66078430 . _:b632446644 . _:b632446645 . _:b66078502 . _:b66078470 . _:b66078456 "5a) and given that free reduced glutathione is the main cytosolic anti-oxidant and regulator of the cytosolic redox state>>66<<, we wondered whether the oxidative stress response might instead be triggered by low cytosolic levels of reduced glutathione." . _:b66078403 _:b66078431 . _:b632446646 . _:b66078423 "2, mahj 12, 27, myc 28, APC 6 and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, >>46<<. Our RNAseq data indicates that both Minute and mahj \u2212/\u2212 cells in their naive state upregulate the JNK pathway (as both JNK activators, such as Gadd45 and JNK target genes, such as scarface 47 and reaper 45 are upregulated (Fig." . _:b66078403 _:b66078416 . _:b632446647 . _:b632446601 "2"^^ . _:b66078403 _:b66078417 . _:b632446586 . _:b66078403 _:b66078418 . _:b632446632 . _:b632446633 . _:b66078471 . _:b66078403 _:b66078419 . _:b632446634 . _:b66078403 _:b66078420 . _:b632446635 . _:b632446600 "2"^^ . _:b66078403 _:b66078421 . _:b632446636 . _:b632446597 . _:b66078403 _:b66078422 . . _:b66078464 . _:b66078403 _:b66078423 . _:b632446637 . _:b632446638 . _:b632446639 . _:b66078392 . _:b632446649 . _:b632446603 "2"^^ . _:b632446624 . _:b66078392 . _:b632446596 . _:b632446625 . _:b66078465 . _:b632446626 . _:b632446627 . _:b632446602 "2"^^ . _:b632446628 . _:b632446599 . _:b632446629 . _:b66078466 . _:b66078421 "JNK signalling has also been implicated in multiple cell competition systems, such as scribble 2, mahj 12, 27, myc 28, APC 6 and Minute competition5, >>23<<, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, 46." . _:b632446634 . _:b632446630 . _:b632446631 . _:b66078495 . _:b632446613 "2"^^ . _:b632446598 . _:b66078467 . . _:b632446612 "2"^^ . . _:b66078434 "We next wondered whether the increase in clone size was due to the anti-apoptotic effect of JNK inhibition, which could inhibit background-level cell death in Minute cells>>50<<, leading to bigger clone sizes." . _:b632446593 . _:b66078476 . _:b632446615 "2"^^ . _:b66078458 . _:b632446615 . _:b632446592 . _:b66078477 . _:b632446614 "2"^^ . _:b66078424 "Our RNAseq data indicates that both Minute and mahj \u2212/\u2212 cells in their naive state upregulate the JNK pathway (as both JNK activators, such as Gadd45 and JNK target genes, such as scarface >>47<< and reaper 45 are upregulated (Fig." . _:b632446639 . _:b632446595 . _:b66078478 . . _:b66078498 "Exciting recent advances point at a role for Flower, Toll and PTP10D signalling as factors involved earmarking cells as losers and/or in their elimination32, >>37<<, 38. However what pre-existing conditions prime cells as losers and initiate cell competition is not known. Importantly, our work reveals that RpS3 +/\u2212 and mahj \u2212/\u2212 cells share a common chronic activation of the oxidative stress response" . _:b632446609 "2"^^ . . _:b66078402 . _:b632446594 . _:b632446597 . _:b66078479 . _:b66078464 "Perrimon), 10xSTAT-GFP 59, UAS-Dome \u0394Cyt >>63<<, Dome g0218 (BL11953), GstD1-GFP65, UAS-mahj-RNAi (BL34912), UAS-Nrf2-RNAi65, UAS-Nrf2 65, UAS-dIAP1 (P." . . _:b632446608 "2"^^ . _:b632446605 . _:b632446525 . _:b66078472 . . _:b632446611 "2"^^ . . _:b632446604 . _:b66078473 . _:b66078380 . _:b632446610 "2"^^ . _:b632446607 . _:b66078471 . . _:b66078474 . _:b66078372 . _:b66078364 . _:b632446621 "2"^^ . . _:b632446606 . _:b66078475 . _:b66078370 . _:b632446620 "2"^^ . _:b66078453 . _:b632446601 . _:b66078425 "Our RNAseq data indicates that both Minute and mahj \u2212/\u2212 cells in their naive state upregulate the JNK pathway (as both JNK activators, such as Gadd45 and JNK target genes, such as scarface 47 and reaper >>45<< are upregulated (Fig." . _:b66078484 . _:b632446614 . _:b66078389 . _:b632446623 "2"^^ . _:b632446600 . _:b66078485 . . _:b66078451 . _:b66078391 . _:b632446622 "2"^^ . . . _:b66078391 . _:b632446603 . . _:b66078486 . _:b632446617 "2"^^ . _:b632446602 . _:b66078474 . _:b632446601 . _:b66078487 . _:b632446616 "2"^^ . _:b632446613 . _:b66078429 "To address this, we inhibited JNK signalling specifically in loser RpS3 +/\u2212 cells during cell competition, by overexpressing the JNK inhibitor puckered (puc) >>45<<, 49. JNK inhibition reduced competition-induced cell death, as expected23 (Supplementary Fig.\u00A02e\u2013g). However, we also noted a striking increase in the overall ability of RpS3 +/\u2212 cells to colonise the tissue (Fig.\u00A02f, g), consistent with" . _:b66078480 . . . _:b632446619 "2"^^ . _:b632446612 . _:b66078461 "The following Drosophila stocks were used: RpS3[Plac92] (BL5627), RpS3*(BL5699), M(2)53 1 (BL5698), FRT42D mahj >>12<<, TRE-16 48, UAS-puc-RNAi (B." . _:b66078445 . _:b66078481 . _:b632446618 "2"^^ . _:b632446615 . _:b632446629 . _:b66078482 . _:b66078360 "introduction" . _:b66078400 . . _:b632446629 "2"^^ . . . _:b66078478 . _:b632446614 . _:b66078483 . _:b632446628 "2"^^ . . _:b632446609 . _:b632446539 . _:b66078492 . _:b66078387 . _:b632446631 "2"^^ . _:b632446608 . _:b66078493 . _:b632446630 "2"^^ . _:b66078394 . _:b632446611 . _:b66078494 . _:b66078470 "While it is increasingly recognised that cells can compete via multiple unrelated mechanisms>>13<<, 23\u201325, 36\u201338, 67, 68 our understanding of even the best characterised and prototypical models of cell competition, such as Minute cell competition, is still rather fragmented." . _:b632446625 "2"^^ . _:b66078374 "While Minute heterozygous cells and animals are viable, in mosaic tissues Minute heterozygous cells behave as losers and are killed when confronted with wild-type (WT) cells, allowing the healthy WT population to expand efficiently>>1<<, 19. In addition to Minute, many other mutations have been shown to induce a loser status against WT cells, such as mutations in the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the" . _:b632446610 . . _:b66078495 . . _:b66078369 . _:b632446621 . . _:b66078403 . _:b632446624 "2"^^ . _:b632446621 . _:b66078485 . _:b632446541 . _:b66078488 . _:b632446557 . . _:b632446627 "2"^^ . _:b632446620 . _:b66078413 "Interestingly, however, p53 activation in Minute cells has been shown not to be required for their autonomous cell survival or for their elimination during competition>>39<<." . _:b66078489 . . _:b632446626 "2"^^ . _:b632446623 . _:b66078490 . _:b632446637 "2"^^ . _:b66078375 . . _:b632446622 . _:b632446520 . _:b632446521 . _:b66078491 . _:b632446522 . . _:b632446636 "2"^^ . _:b632446523 . _:b632446524 . _:b632446617 . _:b632446525 . _:b66078500 . _:b66078446 . _:b632446526 . . _:b632446639 "2"^^ . _:b632446527 . _:b632446616 . _:b66078501 . _:b632446638 "2"^^ . _:b632446619 . _:b632446517 . _:b66078502 . _:b632446518 . _:b632446519 . _:b632446633 "2"^^ . _:b632446618 . _:b632446632 "2"^^ . . _:b632446629 . _:b66078496 . . _:b632446635 "2"^^ . _:b632446624 . _:b632446628 . _:b66078366 . _:b66078497 . _:b66078459 . _:b632446634 "2"^^ . _:b632446631 . . _:b66078498 . . _:b632446645 "2"^^ . _:b632446552 . _:b632446630 . _:b632446553 . _:b66078499 . _:b66078472 . _:b632446554 . . _:b632446644 "2"^^ . _:b632446555 . _:b632446556 . _:b632446625 . . _:b632446611 . . _:b632446557 . _:b632446558 . _:b632446559 . _:b632446647 "2"^^ . _:b632446544 . _:b632446624 . _:b632446545 . _:b66078435 "The JAK/STAT pathway is a cytokine signalling pathway involved in the proliferative, immune and inflammatory responses51, >>52<<. Specifically in Drosophila imaginal discs, the pathway is required for developmental patterning53, 54 and to promote proliferation under normal conditions and during regeneration52, 55." . _:b632446546 . _:b632446547 . _:b632446646 "2"^^ . _:b632446548 . _:b632446627 . _:b632446549 . _:b632446550 . _:b632446551 . _:b66078393 "The third and most striking aspect of cell competition is that loser cells are eliminated in the presence of their fitter neighbours>>1<<, 19, mostly (but not exclusively) via induction of apoptosis5, 23, 31, 35." . _:b632446641 "2"^^ . _:b632446536 . _:b632446626 . . _:b632446537 . _:b632446538 . _:b66078462 "The following Drosophila stocks were used: RpS3[Plac92] (BL5627), RpS3*(BL5699), M(2)53 1 (BL5698), FRT42D mahj 12, TRE-16 >>48<<, UAS-puc-RNAi (B." . _:b632446539 . _:b632446640 "2"^^ . _:b632446540 . _:b632446637 . _:b632446541 . _:b632446542 . _:b632446543 . _:b66078490 "It has been suggested that winner cells overproliferation stems from winner/loser recognition32, >>33<<, or from compensatory proliferative signals emanating from dying loser cells31, 34." . _:b632446643 "2"^^ . _:b632446528 . _:b632446636 . _:b66078360 . _:b632446523 . _:b632446529 . _:b632446530 . _:b632446531 . _:b632446642 "2"^^ . _:b632446532 . _:b632446639 . _:b632446533 . _:b66078412 "Since the DDR is often associated with p53 activation, we cross-compared our DDR signature genes with previously identified p53 targets in Drosophila that are upregulated following DNA damage41, >>42<<. Indeed, we found that 29 of our DDR-related genes were also proposed p53 targets (Supplementary Fig.\u00A01o, p). This suggests that the p53 pathway is activated in prospective losers. Interestingly, however, p53 activation in Minute cells" . . _:b632446534 . _:b632446535 . _:b66078366 "competition was originally described in Drosophila wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver>>10<< and heart11 and in mammalian cell culture8, 9, 12, 13. Further studies have also shown the existence of this process in several stem cell compartments14\u201317, although it is likely that in this case it happens via a different mechanism(s)." . _:b632446584 . _:b66078483 "Furthermore our results are consistent with our earlier observation that in the adult posterior midgut WT clones overgrow during Minute cell competition in a JAK-STAT-dependent manner>>5<<. This might have been a peculiarity of the midgut, where JAK-STAT signalling is commonly activated as part of the gut homeostasis/repair70. Our new data indicate instead that this is a general phenomenon integral to Minute cell" . _:b66078418 . _:b632446638 . _:b632446585 . _:b632446540 . _:b632446586 . _:b632446587 . _:b632446588 . _:b632446633 . _:b632446589 . _:b66078475 . _:b632446517 . _:b632446590 . _:b632446591 . _:b66078367 "was originally described in Drosophila wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart>>11<< and in mammalian cell culture8, 9, 12, 13. Further studies have also shown the existence of this process in several stem cell compartments14\u201317, although it is likely that in this case it happens via a different mechanism(s)." . _:b632446576 . _:b632446632 . _:b632446577 . _:b66078459 _:b66078460 . _:b632446578 . _:b66078459 _:b66078461 . _:b632446579 . _:b66078459 _:b66078462 . _:b632446635 . _:b632446580 . _:b66078459 _:b66078463 . _:b66078369 "wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture8, >>9<<, 12, 13. Further studies have also shown the existence of this process in several stem cell compartments14\u201317, although it is likely that in this case it happens via a different mechanism(s)." . _:b632446581 . . _:b66078464 . . _:b632446582 . . _:b66078440 . _:b66078422 . _:b632446649 "2"^^ . _:b632446583 . _:b632446568 . _:b66078500 "Nrf2 canonically serves a pro-survival role, protecting cells from the negative effects of toxic/oxidising compounds>>64<<, 65. This is the case also for RpS3 +/\u2212 and mahj \u2212/\u2212 cells, since silencing Nrf2 causes autonomous cell death in Minute cells." . _:b632446634 . _:b632446569 . _:b632446570 . _:b632446571 . _:b66078447 "However, Upds are soluble proteins capable of long-range signalling and have been reported to cause non-autonomous overgrowth phenotypes>>61<<, 62. In addition we recently showed that in the adult Drosophila midgut Upd ligands produced by RpS3 +/\u2212 cells promote the proliferation of WT cells during cell competition5. This suggested that their production from RpS3 +/\u2212 wing disc" . _:b632446648 "2"^^ . _:b632446572 . _:b632446645 . _:b632446573 . _:b632446574 . _:b632446575 . _:b632446622 . _:b632446560 . _:b632446644 . _:b632446561 . _:b632446562 . _:b632446563 . _:b632446650 "2"^^ . _:b632446564 . _:b66078367 . _:b632446647 . _:b632446565 . _:b632446581 . _:b632446566 . _:b632446567 . _:b632446646 . . . _:b66078492 "It has been suggested that winner cells overproliferation stems from winner/loser recognition32, 33, or from compensatory proliferative signals emanating from dying loser cells31, >>34<<. Our data instead demonstrates that, at least for Minute cell competition, the overproliferation of winner cells is not a consequence of and in fact is independent of winner/loser cell recognition and of loser cell death, since Upd-2 and" . _:b66078439 . _:b66078445 . _:b632446641 . _:b66078445 . _:b632446646 . _:b632446640 . . _:b632446643 . _:b66078487 . _:b632446642 . _:b66078468 "Clone areas were measured on a medial section of the pouch region either manually or using a custom script in Fiji>>75<<. For cell death quantifications, cells that were positive for cleaved caspase-3 (by immunostaining) and within the pouch region were counted. The total number of dying cells was normalised to the respective clone area/perimeter." . . _:b66078490 . _:b66078471 "While it is increasingly recognised that cells can compete via multiple unrelated mechanisms13, >>23<<\u201325, 36\u201338, 67, 68 our understanding of even the best characterised and prototypical models of cell competition, such as Minute cell competition, is still rather fragmented." . _:b632446529 . _:b632446598 . _:b66078403 "results" . _:b66078432 "JNK overactivation leads to autonomous cell death>>45<<; however, after carefully optimising JNK activation levels (by partial silencing of the JNK inhibitor puc using tub-Gal80 TS, see Supplementary Table\u00A01), we obtained large clones with active JNK, so as to assess whether they would behave" . _:b632446649 . _:b632446524 . . _:b632446648 . _:b66078392 . _:b66078394 "The third and most striking aspect of cell competition is that loser cells are eliminated in the presence of their fitter neighbours1, >>19<<, mostly (but not exclusively) via induction of apoptosis5, 23, 31, 35." . _:b66078485 "Indeed, while some of the signals involved in loser cell elimination have now been identified>>23<<, 32, 36\u201338, 71, no signal involved in the overproliferation of fitter cells during cell competition had been found so far." . _:b632446650 . _:b66078373 "The discovery of cell competition emerged from Drosophila studies of heterozygous mutations in ribosomal genes known as Minute mutations>>18<<. While Minute heterozygous cells and animals are viable, in mosaic tissues Minute heterozygous cells behave as losers and are killed when confronted with wild-type (WT) cells, allowing the healthy WT population to expand efficiently1, 19." . . . . . _:b632446647 . _:b66078363 . . _:b66078409 "mahj, a gene that is functionally unrelated to ribosomal genes (mahj is an interactor of polarity genes and a component of a cullin ubiquitin ligase complex12, 40), but whose mutation nevertheless confers a loser status against WT cells>>12<< (Fig.\u00A01a). Interestingly, there was little overlap between loser RpS3 +/\u2212 and non-loser RpS15 +/\u2212 cells (Fig." . _:b632446551 . . . _:b66078363 "Cell competition was originally described in Drosophila wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, >>3<< including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture8, 9, 12, 13." . . _:b66078382 "such as mutations in the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, >>23<<, JAK/STAT24, Wingless6, 25 and EGF26." . _:b66078479 "Several reports have indicated that winner cells enhance their proliferation in various cell competition models5, 6, 8, 28\u201331, including Minute competition5, >>29<<, 31. However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates69." . _:b632446546 . _:b66078452 . . _:b632446569 . . _:b632446547 . . _:b66078414 "JNK (Basket (Bsk) in Drosophila) is a major stress response factor and an important regulator of cell growth and death in many Drosophila tissues>>43<<\u201345. JNK signalling has also been implicated in multiple cell competition systems, such as scribble 2, mahj 12, 27, myc 28, APC 6 and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell" . _:b66078391 . _:b66078419 "JNK signalling has also been implicated in multiple cell competition systems, such as scribble 2, mahj 12, 27, myc 28, APC >>6<< and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, 46." . . _:b632446606 . _:b632446612 . . _:b632446573 . _:b66078377 "In addition to Minute, many other mutations have been shown to induce a loser status against WT cells, such as mutations in the oncogene myc 20, in the polarity genes scribble >>2<<, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless6, 25 and EGF26." . _:b632446587 . _:b66078365 . _:b66078450 . . . . . . . . _:b66078437 "Specifically in Drosophila imaginal discs, the pathway is required for developmental patterning53, >>54<< and to promote proliferation under normal conditions and during regeneration52, 55." . _:b66078470 . _:b632446552 . _:b66078457 . . . _:b66078379 . . _:b632446594 . . _:b66078499 "Exciting recent advances point at a role for Flower, Toll and PTP10D signalling as factors involved earmarking cells as losers and/or in their elimination32, 37, >>38<<. However what pre-existing conditions prime cells as losers and initiate cell competition is not known. Importantly, our work reveals that RpS3 +/\u2212 and mahj \u2212/\u2212 cells share a common chronic activation of the oxidative stress response and" . . _:b632446645 . _:b66078481 "However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates>>69<<. This is in apparent contradiction with our finding that during cell competition Minute cells promote the proliferation of WT cells above their intrinsic rate by stimulating JAK/STAT signalling. We think that this apparent discrepancy can" . . _:b66078450 "These include multiple known target genes of the mammalian transcription factor Nrf>>264<< (CncC in Drosophila 65), although neither the CncC gene itself nor the canonical inhibitor of the pathway Keap1 are transcriptionally affected (Supplementary Data\u00A01)." . _:b632446630 . _:b66078415 . . _:b66078446 "This has mostly been attributed to winner\u2013loser recognition signals or to apoptosis-induced proliferation>>32<<\u201334. However, Upds are soluble proteins capable of long-range signalling and have been reported to cause non-autonomous overgrowth phenotypes61, 62. In addition we recently showed that in the adult Drosophila midgut Upd ligands produced by" . _:b66078417 "JNK signalling has also been implicated in multiple cell competition systems, such as scribble 2, mahj 12, >>27<<, myc 28, APC 6 and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, 46." . _:b66078473 . . . _:b632446576 . . . _:b632446530 . _:b632446579 . _:b632446613 . _:b66078398 "The third and most striking aspect of cell competition is that loser cells are eliminated in the presence of their fitter neighbours1, 19, mostly (but not exclusively) via induction of apoptosis5, 23, 31, >>35<<. Collectively, the combination of these three processes, results in cell competition and in the effective colonisation of tissues by winners at the expense of losers. Several molecules, such as Flower32, Azot36, the Toll/IMD pathway37," . _:b632446535 . . _:b66078449 "In addition we recently showed that in the adult Drosophila midgut Upd ligands produced by RpS3 +/\u2212 cells promote the proliferation of WT cells during cell competition>>5<<. This suggested that their production from RpS3 +/\u2212 wing disc cells could have a similar effect on winner cell proliferation in this tissue. Indeed, we found that just removing one copy of the pathway receptor Dome was sufficient to" . _:b632446635 . _:b66078399 "Several molecules, such as Flower>>32<<, Azot36, the Toll/IMD pathway37, and the Sas/PTP10D ligand-receptor complex38 have been implicated in triggering the apoptosis of losers." . . _:b632446640 . _:b66078379 "In addition to Minute, many other mutations have been shown to induce a loser status against WT cells, such as mutations in the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl >>22<<, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless6, 25 and EGF26." . . _:b66078491 . _:b66078484 "This might have been a peculiarity of the midgut, where JAK-STAT signalling is commonly activated as part of the gut homeostasis/repair>>70<<. Our new data indicate instead that this is a general phenomenon integral to Minute cell competition." . _:b66078385 . _:b66078392 . . . . _:b66078423 . _:b66078364 . _:b632446528 . _:b66078399 . . _:b66078480 "Several reports have indicated that winner cells enhance their proliferation in various cell competition models5, 6, 8, 28\u201331, including Minute competition5, 29, >>31<<. However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates69." . _:b66078480 . _:b66078365 . _:b66078402 "Several molecules, such as Flower32, Azot36, the Toll/IMD pathway37, and the Sas/PTP10D ligand-receptor complex>>38<< have been implicated in triggering the apoptosis of losers." . . _:b66078458 "Nrf2 is generally considered a stress adaptation factor and typically serves a pro-survival role64, >>65<<. Indeed we found that knockdown of Nrf2 by Nrf2 RNAi causes increased cell death in RpS3 +/\u2212 cells, but not in WT cells (Fig." . _:b66078366 . _:b66078383 . _:b66078367 . . _:b632446565 . _:b66078360 . _:b632446577 . _:b632446555 . _:b66078361 . _:b66078487 "Indeed, while some of the signals involved in loser cell elimination have now been identified23, 32, >>36<<\u201338, 71, no signal involved in the overproliferation of fitter cells during cell competition had been found so far." . _:b66078362 . _:b632446563 . . _:b66078363 . . . _:b66078474 "Several reports have indicated that winner cells enhance their proliferation in various cell competition models>>5<<, 6, 8, 28\u201331, including Minute competition5, 29, 31. However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates69." . _:b632446574 . _:b66078372 . _:b66078372 . _:b632446548 . . . _:b632446583 . _:b66078373 . _:b632446644 . _:b66078374 . . _:b632446571 . . . _:b632446588 . _:b66078375 . _:b632446602 . _:b632446564 . _:b66078368 . . . . _:b66078369 . . _:b66078380 "mutations have been shown to induce a loser status against WT cells, such as mutations in the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)>>12<< and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless6, 25 and EGF26." . _:b66078477 . _:b632446542 . _:b66078370 . _:b632446643 . _:b66078428 . _:b66078497 "Exciting recent advances point at a role for Flower, Toll and PTP10D signalling as factors involved earmarking cells as losers and/or in their elimination>>32<<, 37, 38. However what pre-existing conditions prime cells as losers and initiate cell competition is not known. Importantly, our work reveals that RpS3 +/\u2212 and mahj \u2212/\u2212 cells share a common chronic activation of the oxidative stress" . _:b66078371 . _:b66078472 . _:b66078368 "wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture>>8<<, 9, 12, 13. Further studies have also shown the existence of this process in several stem cell compartments14\u201317, although it is likely that in this case it happens via a different mechanism(s)." . _:b632446538 . _:b66078380 . _:b66078381 . . _:b66078381 "such as mutations in the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP>>8<<, 23, JAK/STAT24, Wingless6, 25 and EGF26." . _:b66078370 "wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture8, 9, >>12<<, 13. Further studies have also shown the existence of this process in several stem cell compartments14\u201317, although it is likely that in this case it happens via a different mechanism(s)." . _:b66078382 . _:b66078383 . . . . _:b66078454 . _:b66078389 "First, loser cells commonly exhibit slower proliferation rates than their winner counterparts and this passively contributes to expansion of the winner cell population1, >>19<<. Secondly, it has been reported that during cell competition winner cells further increase their proliferation rates over their already faster baseline5, 28\u201331. It is unclear how that is elicited; however, it has been proposed to be a" . _:b66078459 "methods" . _:b66078376 . _:b66078455 . _:b66078377 . . . _:b66078378 . . _:b66078384 "the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless>>6<<, 25 and EGF26." . . _:b632446526 . _:b66078379 . _:b66078419 . . _:b66078442 "We next asked whether JNK signalling, which has been shown to activate Upd ligands in this and other tissues>>60<<\u201362, was responsible for the production of Upd ligands in RpS3 +/\u2212 wing discs." . . _:b66078388 . _:b66078496 "Given that inflammation plays a key role in promoting cancer>>73<< our findings that cells with ribosomal mutations activate a chronic inflammatory/pro-proliferative response could provide a molecular explanation for this predisposition." . _:b66078385 "oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT24, Wingless6, >>25<< and EGF26." . _:b632446545 . _:b66078389 . _:b66078362 . . _:b66078390 . . _:b66078391 . _:b66078384 . _:b66078487 . _:b66078446 . _:b66078456 . _:b66078400 "Several molecules, such as Flower32, Azot>>36<<, the Toll/IMD pathway37, and the Sas/PTP10D ligand-receptor complex38 have been implicated in triggering the apoptosis of losers." . _:b66078385 . _:b66078451 "These include multiple known target genes of the mammalian transcription factor Nrf264 (CncC in Drosophila >>65<<), although neither the CncC gene itself nor the canonical inhibitor of the pathway Keap1 are transcriptionally affected (Supplementary Data\u00A01)." . _:b66078487 . _:b66078386 . . . _:b66078472 . . _:b66078387 . . _:b66078469 "discussion" . _:b632446632 . _:b66078396 . _:b66078383 "in the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1 complex component mahjong (mahj)12 and in genes associated with many signalling pathways such as BMP8, 23, JAK/STAT>>24<<, Wingless6, 25 and EGF26." . . _:b632446619 . _:b66078397 . . _:b66078398 . _:b66078428 "2b,d,e) and of active phosphorylated-JNK>>6<< (pJNK; Supplementary Fig.\u00A02a, c, d) compared to WT cells, whereas naive RpS15 +/\u2212 cells, which do not behave as losers, show a markedly lower level of reporter activation (Fig." . _:b66078399 . . _:b66078392 . . _:b66078393 . _:b66078394 . . _:b66078395 . _:b66078457 "Nrf2 is generally considered a stress adaptation factor and typically serves a pro-survival role>>64<<, 65. Indeed we found that knockdown of Nrf2 by Nrf2 RNAi causes increased cell death in RpS3 +/\u2212 cells, but not in WT cells (Fig." . _:b632446608 . _:b66078501 . _:b66078442 . _:b66078404 . . _:b66078476 . _:b66078454 "Reactive oxygen species (ROS), are powerful activators of Nrf2 and have been shown to be produced under some metabolic imbalance conditions>>61<<. However, RpS3 +/\u2212 cells showed only a negligible increase in dihydroethidium (DHE) staining (Supplementary Fig.\u00A05d, e) and a decrease in CM-H2DCFDA staining (Supplementary Fig.\u00A05f, g), two widely used fluorescent dyes sensitive to" . _:b66078408 . . _:b66078405 . _:b66078498 . _:b632446566 . _:b66078406 . _:b632446609 . _:b66078407 . . _:b66078398 . _:b66078400 . _:b632446603 . _:b66078455 "5f, g), two widely used fluorescent dyes sensitive to different types of ROS>>61<<. Given that prospective loser cells display the upregulation of several enzymes involved in glutathione synthesis or utilisation (Fig." . _:b66078447 . _:b66078401 . _:b66078446 . _:b66078439 "Specifically in Drosophila imaginal discs, the pathway is required for developmental patterning53, 54 and to promote proliferation under normal conditions and during regeneration52, >>55<<. Our RNAseq data show that both RpS3 +/\u2212 and mahj \u2212/\u2212 cells upregulate the JAK/STAT targets Socs36E and chinmo as well as all three ligands of the pathway\u2014the cytokines unpaired 1, 2 and 3 (upd, upd2, upd3)56\u201358 (Fig.\u00A04a), suggesting" . _:b66078402 . _:b66078408 "1k, l); and mutants in mahj, a gene that is functionally unrelated to ribosomal genes (mahj is an interactor of polarity genes and a component of a cullin ubiquitin ligase complex12, >>40<<), but whose mutation nevertheless confers a loser status against WT cells12 (Fig." . _:b66078403 . _:b632446533 . _:b66078412 . _:b66078427 "In addition we found that RpS3 +/\u2212 cells show strong activation of the JNK reporter TRE-dsRED>>48<< (Fig.\u00A02b,d,e) and of active phosphorylated-JNK6 (pJNK; Supplementary Fig.\u00A02a, c, d) compared to WT cells, whereas naive RpS15 +/\u2212 cells, which do not behave as losers, show a markedly lower level of reporter activation (Fig.\u00A02b,c,e and" . _:b66078441 "We first validated these findings using an in vivo fluorescent reporter of STAT activity (10xSTAT-GFP>>59<<). Indeed we found that RpS3 +/\u2212 cells display reporter activation compared to WT cells (Figs.\u00A04b, c) and upregulate upd3 expression as detected by in situ (Supplementary Fig.\u00A04a, b), by upd3-Gal4, UAS-GFP expression (Fig.\u00A04d, e and" . _:b66078413 . _:b66078372 . _:b632446616 . _:b66078414 . . _:b66078415 . _:b66078489 . . _:b66078424 . _:b66078431 . _:b66078408 . _:b66078420 "JNK signalling has also been implicated in multiple cell competition systems, such as scribble 2, mahj 12, 27, myc 28, APC 6 and Minute competition>>5<<, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, 46." . . _:b66078409 . _:b66078477 . . _:b66078486 "Indeed, while some of the signals involved in loser cell elimination have now been identified23, >>32<<, 36\u201338, 71, no signal involved in the overproliferation of fitter cells during cell competition had been found so far." . _:b66078477 . _:b66078463 "Perrimon), 10xSTAT-GFP >>59<<, UAS-Dome \u0394Cyt 63, Dome g0218 (BL11953), GstD1-GFP65, UAS-mahj-RNAi (BL34912), UAS-Nrf2-RNAi65, UAS-Nrf2 65, UAS-dIAP1 (P." . _:b66078396 "The third and most striking aspect of cell competition is that loser cells are eliminated in the presence of their fitter neighbours1, 19, mostly (but not exclusively) via induction of apoptosis5, >>23<<, 31, 35. Collectively, the combination of these three processes, results in cell competition and in the effective colonisation of tissues by winners at the expense of losers. Several molecules, such as Flower32, Azot36, the Toll/IMD" . _:b66078410 . _:b66078477 "Several reports have indicated that winner cells enhance their proliferation in various cell competition models5, 6, 8, >>28<<\u201331, including Minute competition5, 29, 31. However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates69." . _:b632446527 . _:b66078411 . _:b66078479 . _:b66078420 . _:b66078421 . . _:b66078493 "Interestingly, it has previously been shown that high STAT activity can induce the winner status>>24<<. Thus it seems counterintuitive that Minute cells, which have high stat levels (most likely as a result of autocrine signalling), are losers against WT cells with lower STAT signalling." . _:b66078365 . _:b632446518 . _:b66078421 . . _:b66078422 . _:b66078362 "Cell competition was originally described in Drosophila wing imaginal discs1; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues>>2<<, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture8, 9, 12, 13." . . _:b632446556 . _:b632446543 . _:b66078423 . _:b66078475 "Several reports have indicated that winner cells enhance their proliferation in various cell competition models5, >>6<<, 8, 28\u201331, including Minute competition5, 29, 31. However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates69." . _:b66078430 . _:b66078416 . _:b66078478 "Several reports have indicated that winner cells enhance their proliferation in various cell competition models5, 6, 8, 28\u201331, including Minute competition>>5<<, 29, 31. However, it has more recently been reported that during Minute cell competition WT cells grow at their normal cell-autonomous rates69." . _:b66078390 . _:b66078415 "JNK signalling has also been implicated in multiple cell competition systems, such as scribble >>2<<, mahj 12, 27, myc 28, APC 6 and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, 46." . _:b66078417 . _:b632446522 . _:b66078433 "These clones displayed some residual apoptosis, however unlike during Minute cell competition, where cell death is observed within 1\u20132 cell diameters of the clone boundary>>31<< (Supplementary Fig." . _:b66078418 . _:b66078431 "JNK inhibition reduced competition-induced cell death, as expected>>23<< (Supplementary Fig." . _:b66078395 "The third and most striking aspect of cell competition is that loser cells are eliminated in the presence of their fitter neighbours1, 19, mostly (but not exclusively) via induction of apoptosis>>5<<, 23, 31, 35." . _:b66078482 "We think that this apparent discrepancy can be resolved, if considering the experimental design in Martin and Morata>>69<<. Their conclusion was based on the observation that clones of WT cells, allowed to grow for the same amount of time (e.g., 48\u2009h) before they are analysed at the end of third instar, reach similar sizes irrespective of whether they were in" . _:b66078419 . _:b66078406 "To this aim, we compared the RpS3 +/\u2212 mutant transcriptomes to those of two additional mutants: RpS15 ribosomal gene mutants (M(2)53), which exhibits the Minute phenotype>>18<< but do not behave as losers against WT cells (Supplementary Fig." . _:b66078395 . _:b66078428 . _:b66078486 . "PMC0" . . . . _:b66078429 . _:b66078411 "Since the DDR is often associated with p53 activation, we cross-compared our DDR signature genes with previously identified p53 targets in Drosophila that are upregulated following DNA damage>>41<<, 42. Indeed, we found that 29 of our DDR-related genes were also proposed p53 targets (Supplementary Fig.\u00A01o, p). This suggests that the p53 pathway is activated in prospective losers. Interestingly, however, p53 activation in Minute" . . _:b632446627 . . _:b66078430 . . _:b66078431 . _:b66078471 . . _:b632446570 . . _:b66078424 . _:b632446638 . _:b66078382 . _:b632446582 . . _:b66078425 . . _:b66078495 "In addition this observation might help explain the elevated cancer risk associated with patients affected by ribosomopathies>>72<<, genetic diseases caused by mutations in ribosome genes." . _:b66078361 . _:b632446631 . _:b66078414 . _:b66078426 . _:b66078377 . . _:b66078427 . _:b66078404 "Both alleles are mutations of the Minute gene M(3)95A and confer a loser status>>18<<, 39 (Supplementary Fig." . _:b66078397 "The third and most striking aspect of cell competition is that loser cells are eliminated in the presence of their fitter neighbours1, 19, mostly (but not exclusively) via induction of apoptosis5, 23, >>31<<, 35. Collectively, the combination of these three processes, results in cell competition and in the effective colonisation of tissues by winners at the expense of losers. Several molecules, such as Flower32, Azot36, the Toll/IMD" . . _:b632446625 . . _:b66078443 "Notably JAK-STAT inhibition in the P compartment, by expression of a dominant-negative version of the receptor Dome (Dome\u0394Cyt)>>63<< (whose activity we confirmed by monitoring the activity of the 10xSTAT-GFP reporter; Supplementary Fig." . _:b66078371 . _:b66078436 . _:b66078466 . _:b66078494 . _:b632446553 . _:b66078437 . _:b66078467 . _:b66078499 . _:b66078489 "It has been suggested that winner cells overproliferation stems from winner/loser recognition>>32<<, 33, or from compensatory proliferative signals emanating from dying loser cells31, 34." . _:b66078472 "While it is increasingly recognised that cells can compete via multiple unrelated mechanisms13, 23\u201325, >>36<<\u201338, 67, 68 our understanding of even the best characterised and prototypical models of cell competition, such as Minute cell competition, is still rather fragmented." . . . _:b66078438 . . _:b66078392 . _:b66078439 . _:b66078497 . . _:b66078465 . _:b66078432 . _:b66078387 "architecture (like mutations in polarity genes) or cell-fate specification (e.g., mutations in BMP, JAK/STAT and Wingless components) and cells harbouring some of these defects show signs of stress, such as activation of the JNK pathway>>27<<. It is therefore likely that cell competition prevents the accumulation of stressed or mis-specified cells, which could compromise tissue robustness/health or contribute to developmental defects." . _:b66078467 "Perrimon), 10xSTAT-GFP 59, UAS-Dome \u0394Cyt 63, Dome g0218 (BL11953), GstD1-GFP65, UAS-mahj-RNAi (BL34912), UAS-Nrf2-RNAi65, UAS-Nrf2 >>65<<, UAS-dIAP1 (P." . _:b66078433 . _:b66078493 . _:b66078488 . . _:b632446593 . _:b66078434 . _:b66078364 . _:b66078418 "JNK signalling has also been implicated in multiple cell competition systems, such as scribble 2, mahj 12, 27, myc >>28<<, APC 6 and Minute competition5, 23, where it has been proposed to play a role in the late stage induction of cell death, although this is controversial in the case of Minute and myc-induced cell competition29, 46." . _:b66078435 . _:b632446591 . _:b66078437 . . _:b66078444 . _:b66078444 "It has long been known that during cell competition winner cells proliferate faster than they would normally do on their own>>5<<, 28\u201331. This has mostly been attributed to winner\u2013loser recognition signals or to apoptosis-induced proliferation32\u201334." . _:b66078440 . _:b66078445 . _:b66078446 . _:b66078396 . _:b66078447 . _:b632446521 . _:b66078440 . _:b66078375 "While Minute heterozygous cells and animals are viable, in mosaic tissues Minute heterozygous cells behave as losers and are killed when confronted with wild-type (WT) cells, allowing the healthy WT population to expand efficiently1, >>19<<. In addition to Minute, many other mutations have been shown to induce a loser status against WT cells, such as mutations in the oncogene myc 20, in the polarity genes scribble 2, dlg 21 and lgl 22, in the polarity-associated/Cul4-DDB1" . _:b632446568 . _:b66078445 . _:b66078441 . _:b66078368 . _:b632446549 . . _:b66078442 . _:b632446641 . _:b66078443 . _:b66078441 . _:b66078416 . _:b66078452 . _:b66078433 . _:b66078453 . . _:b66078440 . _:b66078411 . _:b66078454 . . _:b632446617 . . _:b66078455 . _:b66078372 . _:b632446628 . . _:b66078448 . _:b66078392 "It is unclear how that is elicited; however, it has been proposed to be a consequence of winner/loser recognition or simply a compensatory mechanism triggered by loser cell death>>28<<\u201334. The third and most striking aspect of cell competition is that loser cells are eliminated in the presence of their fitter neighbours1, 19, mostly (but not exclusively) via induction of apoptosis5, 23, 31, 35. Collectively, the" . _:b66078449 . _:b66078477 . . _:b632446518 "8"^^ . _:b66078450 . . _:b632446519 "7"^^ . . _:b66078451 . _:b632446580 . _:b632446517 "11"^^ . . _:b66078460 . _:b66078381 . _:b66078409 . . _:b66078461 . _:b632446623 . _:b66078488 "Indeed, while some of the signals involved in loser cell elimination have now been identified23, 32, 36\u201338, >>71<<, no signal involved in the overproliferation of fitter cells during cell competition had been found so far." . _:b66078462 . _:b66078462 . . . _:b66078463 . . _:b632446519 . _:b632446650 . . _:b66078456 . . _:b632446618 . _:b66078457 . _:b632446592 . _:b66078393 . . _:b632446525 "5"^^ . _:b632446534 . _:b66078458 . _:b66078364 . _:b632446524 "5"^^ . . _:b66078361 "Cell competition was originally described in Drosophila wing imaginal discs>>1<<; however, it is now clear that it is a universal process, which also occurs in other Drosophila tissues2, 3 including adult tissues4\u20136, in the mouse embryo7\u20139, liver10 and heart11 and in mammalian cell culture8, 9, 12, 13." . . _:b66078459 . _:b632446567 . _:b632446527 "4"^^ . _:b66078426 . . _:b632446636 . _:b66078465 "Perrimon), 10xSTAT-GFP 59, UAS-Dome \u0394Cyt 63, Dome g0218 (BL11953), GstD1-GFP>>65<<, UAS-mahj-RNAi (BL34912), UAS-Nrf2-RNAi65, UAS-Nrf2 65, UAS-dIAP1 (P." . _:b66078412 . _:b632446526 "5"^^ . _:b66078463 . "10.1038%2Fs41467-017-00145-y" . _:b632446521 "5"^^ . _:b632446532 . _:b632446517 . _:b66078407 . _:b632446520 "6"^^ . _:b632446523 "5"^^ . _:b632446519 .