OPEN

Review

Your neighbours matter – non-autonomous control ofapoptosis in development and disease

M Eroglu1,2 and WB Derry*,1,2

Traditionally, the regulation of apoptosis has been thought of as an autonomous process in which the dying cell dictates its owndemise. However, emerging studies in genetically tractable multicellular organisms, such as Caenorhabditis elegans andDrosophila, have revealed that death is often a communal event. Here, we review the current literature on non-autonomousmechanisms governing apoptosis in multiple cellular contexts. The importance of the cellular community in dictating the funeralarrangements of apoptotic cells has profound implications in development and disease.Cell Death and Differentiation (2016) 23, 1110–1118; doi:10.1038/cdd.2016.41; published online 13 May 2016

Facts

� Engulfment genes act non-autonomously to enable variousforms of programmed cell death during development.

� Cells that have initiated apoptosis can coax surroundingcells to evade or undergo apoptosis.

� Stress-induced apoptosis relies on non-autonomousfactors.

Open Questions

� How are the intercellular communication networks thatregulate non-autonomous apoptosis organised?

� Relevance of non-autonomous apoptosis regulation tocancer and other diseases.

� Are cell non-autonomous apoptosis signals stimulus-induced or constitutively on?

Development occurs through a series of finely orchestratedevents that results in the precise sculpting of tissues and organsof varying shapes and sizes. One of the most importantprocesses in animal development is programmed cell death(PCD), where specific sets of cells are eliminated from theorganism under rigorous genetic control. Apoptosis is the best

characterised form of PCD, and is critically important not only fordevelopment but also differentiation, immunity, stress response,genome stability and tissue homoeostasis in both multicellularand unicellular organisms.1–4 In general, errors in the regulationof apoptosis can lead to disastrous consequences, such asdevelopmental abnormalities, degenerative diseases, auto-immunity, susceptibility to infection and cancer.1,5

PCD via apoptosis occurs through distinct cellular signallingevents that culminate in morphological changes includingnuclear and cellular fragmentation, and eventual engulfmentof the dying cell by surrounding healthy cells. Apoptosis hastraditionally been viewed as a process in which the dying cellcontrols its own demise in response to stresses or devel-opmentally programmed cues. Historically, the first indicationthat apoptosis can be regulated by extrinsic biological factorscame from the discovery of pro-apoptotic tumour necrosisfactors (TNF) and anti-apoptotic growth factors, both ofwhich are now well-characterised.6–10 For this review, wefocus on more recently discovered non-autonomous regula-tors of apoptosis and refer readers to several excellent reviewsof Rita Levi-Montalcini’s work on growth factors, as well asreviews on the discovery and characterisation of TNF.11–14

In recent years, work in a variety of model organisms hasuncovered many novel cell non-autonomous regulators of

1Developmental and Stem Cell Biology Program, The Hospital for Sick Children, Toronto, ON, Canada and 2Department of Molecular Genetics, University of Toronto,Toronto, ON, Canada*Corresponding author: WB Derry, Developmental and Stem Cell Biology, The Hospital for Sick Children, Toronto, ON M5G 0A4, Canada. Tel: +416-813-7654;Fax: +416-813-2212; E-mail: brent.derry@sickkids.ca

Received 18.1.16; revised 14.3.16; accepted 07.4.16; Edited by E Baehrecke; published online 13.5.16

Abbreviations: PCD, programmed cell death; TNF, tumour necrosis factor; Bcl-2, B-cell lymphoma 2; CEP-1, C. elegans p53-like 1; EGL-1, egg laying defective 1; CED-9,cell death abnormal 9; CED-3, cell death abnormal 3; CED-4, cell death abnormal 4; IAP, inhibitor of apoptosis protein; Hid, head involution defective; Rpr, reaper; Skl,sickle; Diap1, Drosophila inhibitor of apoptosis protein 1; Dronc, Drosophila Nedd2-like caspase; Drice, Drosophila interlukin-1-β converting enzyme; Dcp-1, Drosophilacaspase 1; Decay, Death executioner caspase related to Apopain/Yama; JNK, c-Jun N-terminal kinase; BH3, Bcl-2 homology 3; Apaf-1, Apoptotic protease activating factor1; Xkr8, Xk-related protein 8; CED-8, cell death defective 8; VC, ventral cord; CED-1, cell death defective 1; CED-2, cell death defective 2; CED-5, cell death defective 5;CED-6, cell death defective 6; CED-10, cell death defective 10; CED-12, cell death defective 12; PSR-1, phosphatidylserine receptor 1; SRCM-1, scrambalase 1; INA-1,integrin α 1; SRC-1, sarcoma oncogene related 1; Ras, Rat sarcoma oncogene; MAP, mitogen activated protein; vps25, vacuolar protein-sorting-associated protein 25;Hippo, Hippopotamus-like; Yorkie; LEC, larval epidermal cell; RanBP2, Ran-binding Protein 2; lin-35, abnormal cell lineage 35; kri-1, Krev interaction trapped homologue 1(KRIT1) ; CCM1, cerebral cavernous malformation 1; PI3K, phosphatidylinositol-3 kinase; IGF-1, insulin-like growth factor 1; DAF-2, abnormal dauer formation 2; AKT-1/2,RAC-α serine/threonine-protein kinase 1/2; DAF-16, abnormal dauer formation 16; FOXO, forkhead box O; HIF, Hypoxia-inducible factor; VHL, von Hippel-Lindau; tyr-2/3,tyrosinase 2/3; TRP2, L-dopachrome tautomerase; HIPK2, homeodomain-interacting protein kinase 2; IRE-1, inositol-requiring protein 1; VAB-1, variable abnormalmorphology 1; VEGF, vascular endothelial growth factor; RNAi, RNA interference

Cell Death and Differentiation (2016) 23, 1110–1118& 2016 Macmillan Publishers Limited All rights reserved 1350-9047/16

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apoptosis, where genetic or biochemical factors in onepopulation of cells can activate and fine-tune the apoptoticprogram in different populations of cells. Conceptually, newfindings demonstrate that even when apoptosis signalling isinitiated in a dying cell (and in some cases progressed veryfar), its progress and eventual completion – which have beenregarded as being largely autonomous – depends onregulatory input from neighbouring cells. In this review, weoutline several novel non-autonomous regulators of apoptosis,as well as gaps in our understanding of the intercellularcommunication during this process. Finally, we speculate onthe adaptive purpose of these control mechanisms indevelopment, physiology and disease.

Brief Overview of Apoptosis Pathways

In mammals, there are two distinct apoptosis pathways,intrinsic and extrinsic, that lead to activation of pro-apoptoticcaspases (summarised in Figure 1c). In the intrinsic pathway,

intracellular signals (e.g. p53 in response to DNA damage)result in the production of pro-apoptotic Bcl-2 family membersthat contain single Bcl-2 homology 3 domains (BH3-onlyproteins).15 Interactions between subsets of BH3-only pro-teins with anti-apoptotic Bcl-2 family members that containmultiple BH domains results in mitochondrial outer membranepermeabilization and cytochrome c efflux into the cytosol.15–17

This enables the assembly of a complex containing cyto-chrome c, Apaf-1 and caspase 9, termed as the apoptosomeholoenzyme, which activates downstream effector caspasesthat trigger cell execution.16,18 In contrast, apoptosis initiatedby leukocytes, such as natural killer or cytotoxic T cells, isextrinsic and receptor mediated. Binding of pro-apoptoticligands (e.g. cytokines of the TNF superfamily) to deathreceptors leads to the formation of a death-inducing signallingcomplex, resulting in the activation of caspases 8 and 10.19–21

Subsequently, in both pathways, cells that have undergoneapoptosis are rapidly engulfed by macrophages or other cells.For comprehensive descriptions of themolecular framework ofintrinsic and extrinsic pathways, we recommend severalrecent reviews.19,22–24

Regulation of Apoptosis by Engulfing Cells

Pioneering studies in the nematode worm C. elegansidentified the core apoptosis genes and demonstrated thatthey function in a linear pathway (Figure 1a).25,26 The majorsteps of this pathway are conserved in humans, but withdifferences in complexity and involvement of mitochondrialproteins. Although in most organisms apoptosis is necessaryfor viability, C. elegans mutants that are unable to eliminatecells by apoptosis during development are viable, making it aconvenient model organism to study genetic mechanismsgoverning this process in vivo.3,25,26 Although, transcriptionalactivation of the pro-apoptotic BH3-only gene egl-1 issufficient to induce apoptosis, which has been regarded as acell-autonomous process (Figure 1a)3 it is clear now that thereis regulatory input other than egl-1 induction alone. In fact, inC. elegans, there is now clear evidence of non-autonomousregulation of core apoptotic machinery at each of its distinctphases (i.e. specification, execution and engulfment).In mammals, cells that are undergoing apoptosis are

engulfed and degraded by macrophages in order to removecellular debris that can cause secondary necrosis of surround-ing healthy cells. In C. elegans, engulfment is carried out bynon-specialized cells surrounding the dying cell.3,27 In manycases during development, cell death does not have to beinitiated or complete before engulfment begins.28 To initiateengulfment, apoptotic cells display surface markers such asphosphatidylserine (PS, the so-called ‘eat me’ signal) thatallow recognition by engulfing cells.29,30 These signals areintegrated by two genetically distinct pathways in engulfingcells that facilitate engulfment of the dying cell.29–33

In C. elegans, engulfment was traditionally viewed as theend stage of apoptosis and dispensable for its activation ascell corpses are readily observed in engulfment defectivemutants.34,35 However, the first evidence of non-autonomousapoptosis regulation in the worm was shown to be actingduring the engulfment phase. The caspase CED-3 is essentialfor activation of apoptosis, and ced-3 partial loss-of-function

Figure 1 Apoptosis pathways in various organisms. (a) In C. elegans, a stimulus(e.g. CEP-1/p53 in response to DNA damage) activates the core apoptosis pathwaythrough transcriptional induction of EGL-1, leading to a suppression of CED-9.Suppression of CED-9 results in the release of CED-3 and formation of a complexwith CED-4. This complex leads to apoptosis. (b) Apoptosis in D. melanogaster canbe initiated autonomously or through receptor-mediated pathways. Activation ofantagonists of inhibitors of apoptosis (IAP) including Hid, Rpr, Grim or Skl leads toinhibition of Diap1. Consequently, initiator caspases (Dronc and Dredd) are activatedand lead to activation of effector caspases (Drice, Dcp-1 and Decay) and apoptosis.This pathway can be influenced by extrinsic factors including Eiger, upstream of JNK.(c). In mammals, apoptosis can be initiated intrinsically or extrinsically. The intrinsicpathway is similar to C. elegans and D. melanogaster pathways. In the extrinsicpathway, activation of a ‘death’ receptor leads to formation of the death-inducingsignalling complex (DISC) and activation of caspases 8 and 10, leading to apoptosis

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mutants (hypomorphs) have reduced levels of apoptosisduring embryonic development.36 Intriguingly, enhancerscreens performed in these hypomorphic ced-3 mutantsuncovered mutations in engulfment genes that enhanced cellsurvival.34 Engulfment defective and hypomorphic ced-3double mutants exhibit a three- to fourfold increase in cellsurvival compared to ced-3 single mutants, indicating thatelimination of cells by apoptosis is somehow assisted byengulfment genes.34,35 Interestingly, loss-of-function muta-tions in engulfment genes alone can increase survival ofneuroblast and progenitor daughter cells normally pro-grammed to die by apoptosis.34 These surviving cells areable to initiate apoptosis and undergo morphological changesassociated with CED-3 activation, such as nuclear andcytoplasmic condensation, but can occasionally reverse theseeffects.34 This does not appear to involve regulation of the anti-apoptotic protein CED-9 or the Xkr8-like protein CED-8;perhaps acting via CED-3 through an unknown mechanism.34

Undead neural progenitors can differentiate into VC motorneurons, although the penetrance and number of survivingcells in engulfment defectivemutants is low compared to ced-3mutants.Whereas expression of engulfment genes specifically in

engulfing cells is sufficient to rescue apoptosis defects,ablation of engulfing cells promotes survival and differentiationof cells normally programmed to undergo apoptosis.34,35

Combined, these observations established that the regulationof apoptosis by engulfment proteins is a cell non-autonomousprocess (Figure 2a). However, a major question that remainsconcerns the mechanistic basis by which engulfment genesassist the apoptotic death of their neighbours. Very recently, itwas shown that the engulfment receptor CED-1 can stimulateformation of a CED-3 caspase gradient in adjacent dividingcells, resulting in its unequal distribution, and consequently,differential apoptotic potential in the daughter cells(Figure 2b).37 More work needs to be done to determineexactly how CED-1 establishes a CED-3 gradient in the dyingcell and whether this is a general phenomenon by whichengulfment promotes apoptosis.In many other organisms, perturbation of engulfment can

lead to defects in tissue remodelling and survival of cellsnormally programmed to die.38–40 For instance, geneticablation of macrophages in the mouse eye or inhibition ofmacrophages in the tadpole tail results in persistence oftissues that normally should regress.38,39 In addition, in theDrosophila ovary, engulfment machinery in follicle cells isrequired for death of nurse cells by a non-apoptotic processduring development.40 However, in all of these cases it is notentirely clear which factors contribute to communicationbetween engulfing cells and dying cells. Determining thesefactors is fundamental to understanding PCD as a dynamiccell–cell communication process, and may shed new light ondiseases involving its misregulation.Another stage at which engulfing cells influence apoptosis is

during DNA degradation. In mammals, apoptotic cells that aredeficient in autonomous caspase-activated DNases areunable to degrade their own DNA.41 However, once thesecells are engulfed by macrophages, DNase II from macro-phage lysosomes promotes degradation of engulfed-cellDNA, which can push apoptosis to completion in a non-

autonomous manner.41 In fact, caspase-activated DNases-deficient mice are fertile, whereas mice deficient in DNase IIdie at birth and contain many engulfed cells with undigestedDNA.41,42 As there is conflicting evidence fromC. elegans andother model organisms that DNase II may also have cell-autonomous roles, this is still somewhat controversial.43–45 Itwill be interesting to know whether loss of macrophage-specific nucleases allows dying cells to reverse initiation ofapoptosis and undergo differentiation in a similar manner toengulfment defective mutants in C. elegans. Overall, in manycases engulfing cells that neighbour dying cells appear tohave an integral role in the regulation of apoptosis.

Communal Suicide and Herd Mentality in Apoptotic Cells

In multicellular organisms, proper coordination of cell pro-liferation and apoptosis is a critical determinant of tissueshape, size and homoeostasis. In Drosophila, apoptosis isnormally prevented by the inhibitor of apoptosis (IAP) proteinDIAP1.46–50 In response to pro-apoptotic signals, DIAP1antagonists such as Grim, Reaper and Hid, inhibit DIAP1and relax inhibition of the caspase Dronc, leading to activation

Figure 2 Engulfment pathways regulate core apoptosis machinery in C. elegans.(a) ‘Eat-me’ signals from the dying cell signals to phagocytic cell in order to initiateengulfment. Engulfment factors from engulfing cells can act to permit completion ofapoptosis during development. (b) CED-1 in engulfing cells can cause CED-3caspase gradients in dividing cells, which leads to its unequal distribution in daughtercells. This results in differential apoptotic potential

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of effector caspases such as DrICE and DCP-1 to triggerapoptosis (Figure 1b).50,51

The connection between proliferation and apoptosis is wellestablished in Drosophila, where activation of apoptosis insome tissues can trigger a process called compensatoryproliferation in nearby cells, a dynamic non-autonomousprocess required for tissue development, remodelling andresponse to injury.48,52–58 Non-autonomous inhibition ofapoptosis by mitogens and survival signals throughRas/MAP kinases has been known to converge at suppres-sion of IAP antagonists (namely Hid) through phosphory-lation.50 However, factors have recently been identified thatdirectly influence DIAP1 transcription in novel ways. Dyingcells can inhibit apoptosis of surrounding cells through vps25,a component of the endosomal sorting complex requiredfor transport, which non-autonomously induces DIAP1 andpromotes proliferation.59 Notch signalling from vps25 mutantdying cells activates the Hippo signalling in neighbouringcells, leading to Yorkie-mediated induction of DIAP1.60

Furthermore, activation of Notch alone is sufficient to induceYorkie and DIAP1 in neighbouring cells.60 In addition, hyper-activation of hedgehog signalling also makes neighbouringcells resistant to apoptosis through induction of DIAP1.61

Thus, in Drosophila different non-autonomous signals thatcan inhibit apoptosis appear to converge on DIAP1.The processes that control tissue remodelling – prolifera-

tion, migration, and apoptosis – must be coordinated at themulticellular level. During Drosophila abdominal epithelialreplacement in the pupal stage, larval epidermal cells (LECs)undergo apoptosis and are replaced by abdominal histoblaststhat proliferate and migrate.62 Interactions between proliferat-ing abdominal histoblasts and LECs were recently shown tobe critical for induction of LEC apoptosis.63 These histoblastsare normally arrested at G2 before pupariation, but enter thecell cycle once the pupal stage begins.64,65 When histoblasts

are forced to arrest at S/G2 phase as they are migrating, theadjacent LECs do not undergo apoptosis.63 Their transitioninto the cell cycle is necessary for coordinating apoptosis ofneighbouring LECs.63 Although proximity between histoblastsand LECs is necessary, themechanism of apoptosis activationin LECs by cell-cycle transition in histoblasts is not known. Thefact that apoptosis can be regulated non-autonomously bysuch a fundamental process as the cell cycle highlights theimportance of coordinating life and death decisions betweentissues and cells during development.Interestingly, in both Drosophila and vertebrate develop-

ment, there are also many instances of communal death, orgroup suicide behaviour, where a number of adjacent cellsundergo apoptosis rapidly and in synchrony (Figure 3).66–71

The mechanisms that govern this are best understood inDrosophila, where signals emanating from dying cells are atleast partially sufficient to stimulate apoptosis of theirneighbours. Expression of the viral caspase inhibitor p35leads to survival of cells programmed to undergo apoptosis.72

When p35 and the pro-apoptotic gene hid are overexpressedin the posterior wing imaginal discs, the resulting undead cellsare able to coax large numbers of neighbouring anterior disccells to commit suicide (Figure 3a).73 Moreover, the coaxedcells fully undergo apoptosis, whereas the undead cells showno biochemical markers of apoptosis such as caspaseactivation and fragmented DNA.73 This phenomenon is alsoobserved in the haltere and leg discs, but not in the eye-antennal discs, and is dependent on the amount of apoptoticstimulus.73

In each of the discs tested, the posterior parts with ectopicapoptosis were enlarged as a consequence of compensatoryproliferation.73 It is possible that some sort of secondarycompensatory effect is responsible for the resulting abnormalapoptosis in the anterior disc. Harbouring a large number ofabnormal undead cells for an extended period of time may

Figure 3 Non-autonomous induction of apoptosis by other apoptotic cells. (a) During D. melanogaster development, apoptotic cells secrete Eiger to induce apoptosis of othercells through JNK-mediated pathways. Persistence of apoptotic cells can coax groups of cells that normally survive to undergo apoptosis. (b) Mammalian hair follicle cells undergocoordinated apoptosis through secretion of TNF-α by apoptotic cells. Treatment of follicle with a TNF-α neutralising antibody is sufficient to disrupt apoptosis

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result in altered secretion of morphogens that triggerapoptosis. However, correcting for size, morphogen gradientsor other developmental effects, does not inhibit activation ofnon-autonomous apoptosis.73 Only overexpressing hid or rpr(without p35) in posterior disc cells is sufficient to induceapoptosis in anterior disc cells.73 Altogether, these observa-tions suggest that a secreted factor from bona fide apoptoticcells is sufficient to induce the apoptotic death of theirneighbours.How do apoptotic cells coax non-apoptotic cells to commit

suicide? It turns out that the c-Jun N-terminal kinase (JNK)pathway, a MAP kinase pathway that regulates stressresponse in many organisms, has a role.48,74 Flies withundead cells in the posterior compartment, and with upregu-lated JNK signalling through ablation of its inhibitor puckered,show much higher levels of non-autonomous apoptosis.73

Conversely, downregulation of JNK signalling in the anteriordisc alone is sufficient to abrogate induction of non-autonomous apoptosis, suggesting that the JNK pathwayintegrates some pro-apoptotic signal.73 What is that signal?The Drosophila TNF ortholog, Eiger, is known to activate JNKdependent apoptosis in Drosophila (Figure 3a).75–77 In fact,Eiger is upregulated in undead cells, and its depletion in thesecells significantly reduces apoptosis in different cells of theanterior disc, consistent with the idea that its secretionactivates apoptosis of neighbouring cells.73 This may repre-sent an ancestral developmental mechanism by which tissuessculpt themselves into distinct shapes and sizes.Inmice there is evidence that dying cells can induce death of

their neighbouring cells in a manner similar to the Drosophilaimaginal wing disc (Figure 3b). During hair follicle cycleprogression, a number of follicle cells undergo group suicide,which is dependent on TNF signalling.67,78,79 Interestingly,only apoptotic cells were found to express TNF-α, whichconfirmed that the source of the pro-apoptotic signal wasdying cells themselves, and not other tissues.73 Injection ofmice with TNF-α antibodies is sufficient to disrupt synchroni-zation of apoptosis, or completely inhibit it, in hair follicles.73 In

addition, in the mammalian eye, genetic ablation of the Ran-binding protein (RanBP2) in cone photoreceptors causesthem to die by a non-apoptotic mechanism; however, the dyingcone photoreceptors stimulate apoptotic death of neighbour-ing rod photoreceptors.80 Together, these observationsindicate that dying cells in vertebrates can secrete pro-apoptotic signals that initiate apoptosis of neighbouringcells in a controlled manner. It will be interesting to know thefactor(s) responsible for activating the apoptotic death of rodphotoreceptors. Perhaps this occurs through TNF or anothersecreted molecule, such as tyrosinase (see below)?Although secretion of Eiger to activate JNK and subsequentlyapoptosis in Drosophila may explain how an apoptotic signalpropagates from one cell to another in other organisms, it isstill not clear what establishes the borders of these apoptoticcells. Why are some cells more sensitive than others to thepro-apoptotic signal?Why is it that only anterior wing disc cellsundergo non-autonomous apoptosis when undead cells aregenerated in the posterior? These questions are critical toexplaining tissue and organ development, and are currentlyunanswered.It is also interesting to note that transmission of protein

aggregates between cells are able to induce non-autonomousapoptosis in the developing fly.81,82 Aggregates of thehuntingtin protein, known for causing Huntington disease,are able to induce widespread apoptosis of nearby neuronswhen mutant protein is expressed in olfactory neurons.81,82

Interestingly, this is dependent on uptake of the aggregates asinhibition of endocytosis prevented the abnormal apoptosis.81

It will be important to determine exactly how huntingtinaggregates activate the apoptotic machinery in dying cells,which could help in the development of therapies that reverseor slow Huntington disease.

Assisted Suicide From Worms to Humans

Cells in the adult C. elegans hermaphrodite germline arecompetent to undergo apoptosis in response to a variety

Figure 4 Non-autonomous regulation of germ cell apoptosis in C. elegans. CEP-1/p53 is activated in response to DNA damage and initiates apoptosis. Permissive signalfrom intestinal cells (KRI-1) is required for progression of apoptotic cascade in germ cells. In contrast, accumulation of neuronal HIF-1 results in suppression of the apoptosiscascade, probably through inhibition of CEP-1. Other somatic factors such as insulin/IGF-1 signalling and the RB protein LIN-35 also contribute to apoptosis

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of cellular stresses including DNA damage, antimitoticcompounds and pathogenic infection (reviewed in refs83,84). DNA damage-induced germ cell apoptosis requiresthe p53-like protein CEP-1 in C. elegans.85,86 Tumoursuppressor p53 has a central role in mediating the cellularresponse to stress and it is themost frequentlymutated gene inhuman cancer.87,88 In C. elegans, genotoxic stress createsvarious forms of DNA damage that are recognised by a set ofcheckpoint proteins that transduce signals leading to thephosphorylation and stabilisation of CEP-1.89,90 CEP-1 acti-vates the core apoptosis pathway in the germline bytranscriptionally upregulating the BH3-only gene egl-1, whichleads to increased EGL-1 protein that binds and inhibits theanti-apoptotic protein CED-9 (Figure 1a).89,91–93 Interestingly,apoptosis in response to DNA damage is not regulated entirelyby the cells fated to die; communication with the neighbouringsomatic cells is also essential. Several recent studieshave identified factors produced in somatic cells that assistCEP-1 in promoting apoptosis of C. elegans germ cells(Figure 4).83,84,94,95

Somatic Factors Permit C. elegans Germ Cell Apoptosis

In addition to CEP-1-dependent transcriptional activation ofegl-1, germ cells also require input from the soma to promoteapoptosis in response to DNA damaging agents, such asionising radiation. CEP-1-induced apoptosis is at leastpartially dependent on functional lin-35, the C. elegansorthologue of the retinoblastoma susceptibility gene, in boththe somatic gonad and germline.84 Rescuing arrays in lin-35loss-of-function mutants fail to restore apoptosis whenexpressed either in the somatic gonad or the germline,indicating that at least some aspect of lin-35 dependentapoptosis is non-autonomous.84 Furthermore, loss-of-functionmutations in the kri-1 gene, which encodes a scaffold proteinorthologous to human KRIT1/CCM1, completely preventionising radiation-induced germ cell apoptosis despite havingno defects in physiological germ cell apoptosis or the DNAdamage checkpoint (Figure 4).94 Although kri-1 does notregulate developmental apoptosis in the soma, its expressionis required in the soma (intestine) to permit DNA damage-induced apoptosis in the germline by a mechanism that isindependent or downstream of cep-1.94

Since loss of Krit1/CCM1 is implicated in the formation ofcerebral cavernous malformations (CCM) in the human brain,it will be important to determine the mechanism by whichKRI-1 feeds into the core apoptosis pathway in the germline,and which signalling pathways it engages in the soma to assistthe suicide of damaged germ cells.96 Understanding howKRI-1/CCM1 modulates cross-tissue signalling should alsohelp understand the pathobiology of CCM in humans, andpossibly identify non-invasive ways to treat these patients inthe clinic. Many questions remain. For example, does kri-1mediate the secretion of a pro-apoptotic factor from intestinalcells that permits apoptosis in the germline or do intestinalcells send anti-death signals to the germline in the absence ofkri-1? Is the kri-1 signal constitutive or is it induced in responseto genotoxic stress? Are these mechanisms conserved andrelevant to CCM disease in humans?

Secreted Factors Regulate C. elegans Germ CellApoptosis

Recently, several secreted factors have been identified thatpromote apoptosis in the C. elegans germline. Across manyspecies, activated phosphatidylinositol 3-kinase (PI3K) signal-ling is antagonistic to apoptosis.97 In C. elegans, PI3K can beactivated by the insulin/insulin-like growth factor 1 (IGF-1)receptor DAF-2. This normally leads to activation of AKT-1 andAKT-2, which are partially redundant for inhibition of theDAF-16/FOXO transcription factor.98,99 Curiously, whereasAKT-1 autonomously antagonises CEP-1 dependent apopto-sis, DAF-2 selectively engages AKT-2 to promote apoptosis inresponse to DNA damage, which is independent or down-stream of CEP-1.95 Selective knockdown of DAF-2 in eitherthe soma or germline is not sufficient to suppress DNAdamage-induced germline apoptosis, which indicates thatDAF-2 is likely functions in both tissues through a combinationof cell-autonomous and non-autonomousmechanisms.95Morework is needed to fully understand the mechanism by whichDAF-2 regulates stress-induced apoptosis, which appears toconverge on the MAPK pathway in the germline. 95

Neuronal factors can also regulate germ cell apoptosis.Hypoxia-inducible factor (HIF) is a key regulator of oxygenhomoeostasis that is conserved in all animals, includingC. elegans.100,101 HIF is normally hydroxylated and targetedfor degradation through ubiquitylation by the von Hippel-Lindau tumour suppressor (VHL) under normal physiologicaloxygen levels.102–104 Mainly two factors lead to accumulationof HIF: reduced oxygen (hypoxia) and loss-of-function in VHL,which occurs in some forms of cancer.105–107 Tumours withaccumulated HIF generally have poor prognoses and areresistant to standard therapies.108 InC. elegans, accumulationof neuronal HIF-1 (the alpha subunit of mammalian HIF)through loss-of-function mutations in the VHL gene (vhl-1) orhypoxia treatment, causes resistance of germ cells to DNAdamage-induced apoptosis (Figure 4).83 There is evidencethat HIF-1 regulates the core apoptotic pathway at the level of(or downstream of) CEP-1 through post-translational modifi-cations including phosphorylation, which is known tomodulateits stability and activation.83 An RNAi screen of HIF-1transcriptional targets revealed that the tyrosinase genestyr-2 and tyr-3 were responsible for conferring resistance togermline apoptosis in vhl-1 mutants.83 Intriguingly, loss-of-function in vhl-1 leads to increased HIF-1-dependent expres-sion of TYR-2 in neurons, which is secreted into the germlineto inhibit CEP-1-dependent apoptosis.83 TYR-2 is homolo-gous to human TRP2, which both seem to function asL-dopachrome tautomerases, and knockdown of TRP2 sensi-tises cancer cells to p53-dependent apoptosis.83 As thisstrongly suggests conservation fromC. elegans to human, it isnot clear whether TRP2 functions through a non-autonomousmechanism or how it stabilizes p53 in human cells. HIF-1α canalso induce degradation of HIPK2, a homeodomain interactingprotein kinase that can phosphorylate p53, but the relevanceof this in vivo is not clear.109

In addition to the non-autonomous effects of neuronallysecreted TYR-2, it has also been reported that endoplasmicreticulum stress in a set of amphid sensory neurons causesincreased germline apoptosis.110 This appears to bemediated by

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the ribonuclease inositol requiring protein-1 (IRE-1), and inde-pendently of KRI-1, but whether it functions in a similar manner toTYR-2 remains to be determined.110 As TYR-2 is also secretedfromamphid sensory neurons, it appears that the nervous systemmay have numerous influences on the health of the germline.Perhaps this is the worm version of the mind–body connection?

Looking Forward: Death, Disease and New Life

In this review, we have outlined recently reported cases ofnon-autonomous mechanisms governing apoptosis in ani-mals. It has become clear now that many diverse mechanismsexist to control apoptosis at many of its stages as opposed tothe few that have been historically recognised as required forits initiation. Furthermore, not only do extracellular signalsregulate apoptosis, dying cells can also influence develop-mental decisions of surrounding cells, which is reviewed indetail in ref. 111. Together, these phenomena emphasise theimportance of the multicellular community in making life anddeath decisions of individual or groups of cells. This is notentirely surprising given that cell death exerts constanthomoeostatic pressures on tissues.112,113 To speculate, non-autonomous regulation of apoptosis may have evolved toensure that cell death is properly orchestrated duringdevelopment and in response to stress. The importance ofmaintaining tissue homoeostasis may explain why it arose inmulticellular organisms, particularly during phases of rapidtissue growth and remodelling.Because proper regulation of apoptosis is critical for

suppressing many diseases, understanding the mechanismsby which surrounding cells assist the suicide of theirneighbours may have critical implications in the treatment ofpathologies such as cancer, where cells eventually becomeresistant to apoptosis-inducing therapies. Aside from stress-induced and developmental apoptosis in C. elegans, there isalso evidence that the ephrin receptor VAB-1 in the gonadalsheath cells regulates physiological germ cell apoptosis,suggesting that non-autonomous mechanisms may be ageneral principle of apoptosis control in this organism.114 Animportant question is whether signals generated by genessuch as kri-1 act constitutively to permit apoptosis, similar towhat was observed for VAB-1 in physiological germ cellapoptosis, or are the signals induced by stress to fine-tuneapoptotic thresholds?Insights from tractable model organisms such asC. elegans

and Drosophila provide testable hypotheses to addresswhether abnormal non-autonomous apoptosis regulation is amajor contributing factor in human disease. For instance, it isknown that evasion of apoptosis is a hallmark feature of cancercells.115 Hypothetically, loss of apoptotic regulators in adjacenttissues may act to increase resistance to apoptosis by similarnon-autonomous mechanisms observed in kri-1 or vhl-1mutant worms. In humans, endothelial cells of the stromahave strong effects on the survival of irradiated tumour cells,conceivably through hypoxia effects or secretion of factorssuch as VEGF.116 In addition, as upregulated hedgehogsignalling is a common hallmark of many human cancers, itmay be that secretion of anti-apoptotic factors contributes tothe aggressiveness and growth of these tumours, similar towhat has been observed in Drosophila. In humans, mutations

in CCM1 (the human orthologue of C. elegans kri-1) lead tocerebral cavernous malformations, which are abnormalvascular structures, which frequently involve loss of surround-ing smooth muscle. As the only therapeutic option currentlyavailable for CCM patients is invasive neurosurgery, elucidat-ing the kri-1 pathway in C. elegans may uncover druggabletargets that are conserved in humans. Another importantclinical problem is the development of CCM lesions in braincancer patients who have undergone radiotherapy.117 Dothese radiation-induced lesions arise through known CCMsignalling pathways? Going forward, identifying the secretedfactors that transduce pro- and/or anti-apoptosis signalsacross tissue boundaries, and defining themolecular mechan-isms by which they engage core apoptotic machinery, is likelyto yield profound insights into the understanding and treatmentof many diseases.Although many novel examples of non-autonomous apop-

tosis regulation have been identified, more work needs to bedone to define the mechanistic basis of intercellular commu-nication between dying cells and their neighbours. It wasshown recently that ablation of RNAi processing gene Dicer inmouse astroglia leads to widespread non-autonomous neu-ronal apoptosis and neurodegeneration.118 Whether thismodulation of p53, through HIF-1α and Ranbp2-inducedapoptosis, all involve the TNF-regulated extrinsic apoptosispathway or feed into the intrinsic pathway through some othermechanism, remains to be determined. Regardless, whetherapoptosis is initiated intrinsically or extrinsically its completionoften relies on signalling input from neighbouring cells.As non-autonomous regulation of apoptosis has been

shown to be important in many different organisms, this islikely not a specialised process specific to a small subset oftissues and organisms but a general phenomenon of animaldevelopment, systemic stress response and maintenance oftissue homoeostasis. Looking ahead, the power of geneticallytractable model organisms holds great promise for gaining acomprehensive understanding of how communities of cellsand tissues regulate apoptosis of their neighbours. Forhumans, understanding the spatiotemporal patterns by whichpro- and anti-apoptotic factors are secreted and learning howto manipulate them will not only help in the development ofnew treatments for a variety of diseases, but perhaps also aidin the effort to synthesise artificial tissues and organs inthe lab.

Conflict of InterestThe authors declare no conflict of interest.

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