INTRODUCTION

Cell death occurs as a normal component of the developmentof multicellular eukaryotes. Classical examples of develop-mental cell death are embryonic tissue deletions required forproper morphogenesis (Saunders, 1966; Hinchliffe, 1981;Zakeri et al., 1993; Garcia-Martinez et al., 1993), eliminationof neurons failing to establish adequate trophic connections atthe appropriate time (Oppenheim, 1991; Johnson andDeckwerth, 1993), and negative selection of lymphocytesbearing inappropriate antigen receptors (Cohen, 1991; Golsteinet al., 1991). Developmental cell death is programmed, notonly as to its occurrence in the organism, but also as to itscourse within the dying cell (Golstein et al., 1991). We shalluse throughout this paper the term programmed cell death(PCD; Lockshin and Zakeri, 1991) in its developmentalbiology sense (Schwartz and Osborne, 1993) encompassingboth programmed occurrence and programmed course of celldeath. The term apoptosis (Kerr et al., 1972) usually refers toa morphological type often observed in PCD, associating cyto-plasmic and nuclear condensation and fragmentation, DNAfragmentation (Wyllie, 1980) and sometimes a requirement formacromolecular synthesis (Cohen and Duke, 1984; Wyllie etal., 1984). In most of the models studied so far, PCD followsan apoptotic pattern; however, other PCD morphological types

have also been described (Clarke, 1990; Schwartz et al., 1993;Vaux, 1993; Schwartz and Osborne, 1993).

Which molecules directly effect cell death? We are referringhere not to molecules involved in the transduction of a celldeath signal, nor to control molecules such as those encodedby certain oncogenes or viral genes, nor to moleculesmodifying the morphological aspect of a dying cell, but to‘core death molecules’ more directly involved in the putativefirst irreversible event leading to death (Golstein et al., 1991).

ced-3 and ced-4 involved in developmental cell death in thenematode Caenorhabditis elegans (Hedgecock et al., 1983;Ellis and Horvitz, 1986; Ellis, 1992), the ced-3 equivalent ICEcysteine protease in mouse (Miura et al., 1993), the severalmolecules preferentially expressed in dying thymocytes(Owens et al., 1991), the recently described reaper moleculeinvolved in the death of a large number of cells duringembryonic development in Drosophila (Abrams et al., 1993;White et al., 1994), or inappropriately expressed cell cyclemolecules (Shi et al., 1994) are candidates for being such coredeath molecules.

Dictyostelium discoideum, which multiplies as a unicellularprotist under favourable conditions, undergoes, when starved,cell differentiation and morphogenesis leading within 24 hoursto a multicellular fungus-like structure called sorocarp (Raper,1935, 1984). The sorocarp ultimately includes two main cell

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Programmed cell death (PCD) of

Dictyostelium discoideumcells was triggered precisely and studied quantitatively inan in vitro system involving differentiation without mor-phogenesis. In temporal succession after the triggering ofdifferentiation, PCD included first an irreversible stepleading to the inability to regrow at 8 hours. At 12 hours,massive vacuolisation was best evidenced by confocalmicroscopy, and prominent cytoplasmic condensation andfocal chromatin condensation could be observed byelectron microscopy. Membrane permeabilizationoccurred only very late (at 40-60 hours) as judged bypropidium iodide staining. No early DNA fragmentationcould be detected by standard or pulsed field gel elec-trophoresis. These traits exhibit some similarity to those of

previously described non-apoptotic and apoptotic PCD,suggesting the hypothesis of a single core molecularmechanism of PCD emerging in evolution before the pos-tulated multiple emergences of multicellularity. A singlecore mechanism would underly phenotypic variations ofPCD resulting in various cells from differences inenzymatic equipment and mechanical constraints. A pre-diction is that some of the molecules involved in the corePCD mechanism of even phylogenetically very distantorganisms, e.g. Dictyostelium and vertebrates, should berelated.

Key words: cell death, Dictyostelium, evolution

SUMMARY

Programmed cell death in

Dictyostelium

Sophie Cornillon1, Colette Foa2, Jean Davoust1, Nathalie Buonavista1, Julian D. Gross3

and Pierre Golstein1,*1Centre d’Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France2Unité INSERM 387 ‘Adhésion Cellulaire’, Laboratoire d’Immunologie, Hopital Ste Marguerite, 270 Bd de Ste Marguerite, BP 29,13274 Marseille Cedex 09, France3Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK

*Author for correspondence

Journal of Cell Science 107, 2691-2704 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

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populations, on the one hand viable spores, and on the otherhand stalk cells. Differentiation to stalk cells seems to resultfrom the sequential action of at least two factors, i.e cyclicAMP promoting, in particular, cell aggregation, and a factorcalled DIF promoting, in particular, the differentiation ofstarved cAMP-subjected cells to stalk cells (Town et al., 1976;Town and Stanford, 1979; Sobolewski et al., 1983; Morris etal., 1987). Stalk cells are vacuolated (Raper and Fennell, 1952;Maeda and Takeuchi, 1969; George et al., 1972; de Chastel-lier and Ryter, 1977; Quiviger et al., 1980; Schaap et al., 1981)and are considered non-viable according to the criterium ofnon-regrowth in culture medium (Whittingham and Raper,1960).

The choice of Dictyostelium to study PCD is appealing forseveral reasons. First, phylogenetically, this organism can beconsidered as one of the probably several (Kaiser, 1986) evo-lutionary attempts at multicellularity. If cell death is a corollaryto multicellular development, as suggested long ago(Weismann, 1890), it would seem of interest to characterise thetype of death observed in the development of this ‘transitional’organism. Also, Dictyostelium is one of the most ancientlydiverged currently surviving eukaryotes (Christen et al., 1991;Field et al., 1988): a demonstration of a common cell deathmechanism between this organism and some of the highereukaryotes would be a strong argument for the generality ofthis mechanism. The second reason is ontogenetic: the rela-tively simple pattern of development of Dictyostelium shouldfacilitate the study of cell death occurring during this devel-opment. The third reason, which builds on the previous one, isthat methods exist to trigger, in vitro, differentiation withoutmorphogenesis (Town et al., 1976; Kopachik et al., 1983;Sobolewski et al., 1983; Kay, 1987), and thus to obtain isolateddying Dictyostelium cells, potentially easier to identify andstudy. Finally, another reason is genetic: the genome of Dic-tyostelium, about 100-fold smaller than that of higher eukary-otes, is haploid, which might make it feasible to generate andselect mutants of death-associated genes, and to identify thelatter, especially using recently developed genetic tools(Loomis, 1987; Kuspa and Loomis, 1992). Because of thetemporal separation between vegetative growth and develop-ment, such developmental mutants can be propagated undervegetative conditions, thus behaving as conditional mutants(Loomis, 1987).

We describe here programmed cell death in Dictyostelium,under in vitro conditions mimicking developmental circum-stances. These in vitro conditions are more amenable thanprevious in vivo experiments on stalk cell death (Whittinghamand Raper, 1960) to direct experimental investigation and tofurther genetic manipulations. Owing to the difficulties ofunambiguously defining cell death, we made use of severaldistinct experimental end points for death: staining with fluo-rescent dyes reflecting cell membrane permeability changes,checking vacuolisation, checking the state of nuclear DNA bygel electrophoresis, checking morphological changes byelectron microscopy, and quantifying reversibility to vegeta-tive growth of surviving cells upon returning to favorablegrowth conditions. Death could thus be described as a devel-opmentally induced sequence of intracellular events, includingan early irreversible step, subsequent massive vacuolisation,cytoplasmic condensation, focal chromatin condensation, andvery late membrane lesions. No early DNA fragmentation

could be detected. These results show that, while PCD in Dic-tyostelium has some traits of its own, it is also akin to apoptoticand non-apoptotic morphological aspects of cell deathdescribed in higher eukaryote cells, a similarity leading to thehypothesis of a single core molecular mechanism of PCD.

MATERIALS AND METHODS

Cells and culture conditionsDictyostelium discoideum AX2, an axenic mutant (Watts andAshworth, 1970) derived from NC4, and obtained from M. Hilde-brandt (Pasteur Institute, Paris), was used to calibrate culture anddifferentiation conditions. HMX44, an axenic mutant derived fromV12M2, not producing DIF but responding to exogenously addedDIF, was obtained from J. G. Williams (Imperial Cancer ResearchFund Laboratories, Clare Hall). The subclone HMX44A was derivedand used in this laboratory. AX2 and HMX44A cells were culturedin HL5 medium (Sussman, 1987) with the following modifications:bactopeptone (Oxoid, Basingstoke, Hampshire, England) 14.3 g/l;yeast extract (Difco Laborarories, Detroit, MI) 7.15 g/l; maltose(Sigma Chemical Co., St Louis, MO) 18 g/l; Na2HPO4, 3.6 mM;KH2PO4, 3.6 mM; source water. Cultures were in 50 ml flasks(Falcon, Becton Dickinson Labware, Lincoln Park, NJ) kept horizon-tally, and subcultured twice a week in 10 ml of culture medium; 250ml and 750 ml flasks (Falcon) were used to provide sufficient numbersof cells for large experiments. Cultures and experiments were at 22-23°C in a water-saturated atmosphere.

DO.11.10, a murine T cell hybridoma line (Shimonkevitz et al.,1983) and mouse Balb/c thymocytes were processed and cultured inRPMI 1640 (Gibco/BRL, Grand Island, NY) plus 5% FCS (D.Deutscher, Brumath, France) for DO.11.10 cells, or 10% FCS for thy-mocytes. Incubations and culture procedures were at 37°C in a water-saturated 8% CO2 atmosphere.

Differentiation conditionsReagents and differentiation media were cAMP (adenosine 3

′-5′-cyclic monophosphate; Sigma; 30 mM sterile stock solution in water,aliquoted and stored at −20°C), DIF as DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone; Molecular Probes, Inc.,Eugene, OR; 10−4 M stock solution in absolute ethanol, kept at−20°C), and SB (Soerensen buffer; 50× stock solution: 100 mMNa2HPO4, 735 mM KH2PO4; 1×: 17 mM phosphate, final pH 6.0).Experiments were done in 50 ml plastic flasks or on 18 mm-diametercoverslips in wells of 6-well plates (Falcon). We followed a protocolby Kay (1987) with only slight modifications. Briefly, logarithmicgrowing cells were washed twice with SB and were incubated for 8hours in SB in the presence of 3 mM cAMP at a density of 105

cells/cm2 and a concentration of 106 cells/ml (2.5 ml per flask, 1.25ml per well). This first incubation was followed by one careful washwith twice the volume of SB used for the first incubation and by asecond incubation in SB alone (control) or in SB in the presence of100 nM DIF (experimental), for the indicated lengths of time. Dif-ferentiated cells were then usually examined by fluorescence staining.To ensure maximum differentiation under every experimentalcondition described in this report, we used DIF at a concentration of100 nM for the whole duration of the differentiation periods tested.At this concentration, DIF affected neither vegetatively growing Dic-tyostelium cells, nor the mouse hybridoma cell line DO.11.10, andfrom another point of view ensured differentiation at 48 hours evenwhen incubated with cells for only 15 minutes (not shown).

Fluorescence stainingFor fluorescence microscopy, fluorescein diacetate (FDA) andpropidium iodide (PI) (both from Sigma; stock solutions at 10 mg/mlin acetone and 8×10−5 M in sterile water, respectively) were added

S. Cornillon and others

2693Programmed cell death in

Dictyostelium

directly to Dictyostelium-containing flasks to final concentrations of0.05 mg/ml and 4 µM, respectively. After a 10 minute incubation, theflasks were carefully washed with twice the volume of SB used indifferentiation and were ready for observation using an invertedAxiovert 35 microscope (C. Zeiss, Oberkochen, Germany).

For confocal observation, Dictyostelium-bearing coverslips wereoverlaid with 400 µl of a mixture of FDA and PI solutions in SB atthe final concentrations indicated above. After a 10 minute incuba-tion, the coverslips were carefully washed in 5 ml of SB and mountedcells-down in a home-made 400 µl chamber accommodating livingcells. These were observed with a TCS 4 D confocal microscopebased on a Leica DMR stand and equiped with a mixed gasargon/krypton laser and 3 detectors (Leica Laser Technik, Heidelberg,Germany; Humbert et al., 1993). FDA and PI were excited simulta-neously using the argon 488 nm line and the krypton 568 nm line.Simultaneous confocal excitation and detection were performedthrough a double dichroic mirror and appropriate band-pass filtersallowing the recording of the green FDA fluorescence from 520 to540 nm and the red PI fluorescence above 590 nm. The correspond-ing electronic images were merged and printed as described (Humbertet al., 1993).

Electron microscopyCultured cells were rinsed in PBS, fixed in 2.5% glutaraldehyde in2.5% sucrose in 0.2 M cacodylate buffer, pH 7.4, at room tempera-ture, rinsed twice and postfixed with 1% OsO4. After dehydration ingraded ethanols and embedding in araldite (Fluka), sections wereobtained with a Reichert Ultracut E ultramicrotome, stained withuranyl acetate and lead citrate and examined with a JEOL 200Celectron microscope.

Re-growth experiments‘Re-growth’ experiments checked the ability of differentiating cellsto revert to a vegetative state. First, a differentiation experiment wasdone in flasks as described above. Second, at various times after DIFaddition, per flask a volume of 7.5 ml of HL5 culture medium wasadded to the 2.5 ml of DIF-containing SB. Cells were counted after2 days of further incubation.

Checking the state of DNAAgarose plugs containing about 5×106 vegetative or 5-10×106 differ-entiating HMX44A cells were prepared as described (Cox et al., 1990)except that LMP agarose (Gibco/BRL) and 100 µl rectangular moldswere used instead of 25 µl ones, with the corresponding adjustmentsas to final cell concentrations and amounts of solutions. Agarose plugscontaining 5×106 Balb/c thymocytes were prepared as follows. Cellswere washed twice in PBS and mixed with an equal volume oflukewarm 2% LMP agarose/PBS. Aliquots were pipetted on ice intomolds. After cooling, the plugs were incubated in 0.5 M EDTA, pH8.0/1% sarcosyl/2mg/ml proteinase K (Appligene, Pleasanton, CA) ata concentration of 8 plugs per 2.5 ml, at 50°C for 2 days and then at4°C. For some plugs, the cells had been treated previously withneomycin (for HMX44A plugs, 20 µg/ml neomycin for 3 or 5 days)or dexamethasone (Sigma; for thymocyte plugs, 10−6 M for 7 hours).Before insertion into a gel, plugs were washed for 1 hour in 5-10 mlof TBE running buffer (COX90). The electrophoresis conditions wereas previously described (Cox et al., 1990), except that migration inpulsed field gel electrophoresis used an LKB 2015 Pulsaphor elec-trophoresis unit for 30 hours at 6°C, 115 mA pulses for 25 seconds.After migration, gels were stained with ethidium bromide, pho-tographed, and alkali-treated (0.5 M NaOH, 1.5 M NaCl) for 1 hourbefore capillary transfer to nylon membranes (GeneScreen, Dupontde Nemours) in the same alkali solution. Membranes were then neu-tralized in 50 mM NaHPO4 buffer, pH 6.5 (prepared by mixing 1 MNa2HPO4 and 1 M NaH2PO4 solutions) and UV-irradiated. They wererapidly soaked in 1 mM EDTA/0.1% SDS before a 1 hour prehy-bridization at 65°C in 1 mM EDTA, 0.5 M NaHPO4, pH 7.2 (prepared

as above), 7% SDS, 250 µg/ml herring sperm DNA. HMX44A orDO.11.10 probes were random-labeled (Feinberg and Vogelstein,1984) using 32P-labeled dT. For probing HMX44A Southern blots,probes were prepared with HMX44A total DNA extracted aspublished (Welker et al., 1985) with some modifications: nuclei werelysed at room temperature in 20 ml of a 0.2 M EDTA, pH 7.2/2%sarcosyl solution, and after a cesium chloride gradient centrifugationthe isolated DNA band was diluted with 1 volume of low TE buffer(10 mM Tris-HCl, pH 7.5/0.1 mM EDTA) before ethanol precipita-tion. A labeled probe was made with 100 ng of total DNA. Forthymocyte Southern blots, probes were prepared from one DO.11.10agarose plug. Briefly, a 100 µl plug containing 5×106 DO.11.10 cellswas allowed to melt at 65°C and mixed with 300 µl of distilled water.Probes were prepared using 25 µl of this mix according to standardprotocols (Feinberg and Vogelstein, 1984) except that annealing wasat 37°C instead of 4°C. Labelled probes were added to membranes in40 ml of prehybridization buffer. After overnight hybridization at65°C, 3 or 4 washes were as follows: (1) 2× SSC, 5 minutes, RT, (2-3) 2× SSC, 0.1% SDS, 15 minutes, 65°C, (4) 0.2× SSC, 0.1% SDS,15 minutes, 65°C. Filters were autoradiographed at −80°C for 5-14days.

RESULTS

We wished to establish a system in which isolated Dic-tyostelium cells could be triggered to differentiate into stalkcells, facilitating a quantitative study of the death process.Dictyostelium strain V12M2 could be induced by DIF to dif-ferentiate in vitro to stalk cells much more efficiently thanstrain NC4 (Town et al., 1976), probably because of less inter-ference of cAMP with DIF-induced effects (Berks and Kay,1988). HM44, a derivative of V12M2 producing little or noDIF but still sensitive to it (Kopachik et al., 1983, 1985),allows precise timing of initiation of differentiation uponaddition of exogenous DIF. HMX44 (kindly provided by J. G.Williams, ICRF, Clare Hall) is an axenic derivative of HM44.We derived from HMX44 by serial limiting dilution cloning(with a cloning efficiency close to 100%, not shown) thesubstrain HMX44A, used in all subsequent experiments. Thedoubling time of HMX44A under vegetative growth con-ditions was 8 hours (not shown). The sequence of starva-tion/DIF addition used in previously described in vitrosystems (Kay, 1987) led to the differentiation of theseHMX44A cells within loose clumps and as isolated cells,allowing individual cell examination. Accordingly, ourstandard procedure included, starting from vegetatively-growing cells, a first incubation in SB in the presence of anexcess of cAMP for 8 hours, followed by one wash and by asecond incubation in SB in the presence of DIF for variouslengths of time. This addition of DIF triggered the chain ofcell death events studied in this report.

Massive vacuolisation and late membrane lesionsFluorescein diacetate (FDA), a hydrophobic molecule, entersthe cell in a passive way and is then cleaved by cytoplasmicenzymes, becoming as a result both fluorescent in the greenspectrum and charged, unable to leave the cell. This stain thuslabels cells with an intact plasma membrane. Propidium iodide(PI) can only enter cells with an altered plasma membrane andthen stains, in particular, nucleic acids, re-emitting mostly redfluorescence. Vegetative HMX44A cells were all labelled byFDA and not by PI.

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Cells were allowed to differentiate according to the standardprotocol and were treated with both FDA and PI at varioustimes after addition of DIF. As shown in the photographs ofFig. 1 and quantitatively in Fig. 2, at 8 hours almost all thecells were FDA-labelled, fluorescing green. Only a few vac-uolated cells could already be seen among these green cells.With time, the percentage of vacuolated cells increased,reached 50% at about 12 hours, peaked at 16-24 hours and thendecreased, while vacuolated PI-labelled red cells appeared andincreased (Fig. 2). Most of these red cells contained a smalldense red mass, most probably the nucleus, which was notobviously altered at this scale even at 48 hours. Only very fewnon-vacuolated PI-labelled cells could be seen. In threeseparate experiments, 50% of the cells were vacuolized after

11-14 hours in DIF, and 50% were PI+ after 40-60 hours inDIF.

Confocal microscope fluorescence micrographs of Dic-tyostelium cells subjected to DIF for 24 hours and stained withboth FDA and PI are shown in Fig. 3. There are significantmorphological differences between even successive opticalslices of the same cells. Also of note is the massive vacuoli-sation of most cells and the presence of a number of heavilyvacuolated PI-stained cells. Isolated red-labeled apparentlyintact nuclei could be seen (Fig. 3), and RNase-treated PI-labeled cells contained morphologically intact nuclei (notshown). No cell was detected which, immediately afterstaining, fluoresced red and green at the same time. However,some ‘non-specific’ red fluorescence, limited to the cell

S. Cornillon and others

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Fig. 1. Kinetics of HMX44Acell differentiation asvisualized by FDA/PIstaining. After 8 hours ofincubation in SB in thepresence of an excess ofcAMP, the cells wereincubated in either SB plus100 nM DIF (DIF, rightpanel) or SB with no DIF(control SB, left panel) for theindicated lengths of time. Foreach time, in each panel, threepictures are shown of thesame group of FDA/PI-stained cells, either in phase-contrast or using UV light andthe appropriate filters forvisualizing green (FDA+) orred (PI+) fluorescentlylabelled cells.

2695Programmed cell death in Dictyostelium

surface, was sometimes seen on green cells. Also, lengthymicroscopic examination often led to some PI labelling ofinitially ‘green’ cells (unpublished experiments), resulting in aproportion of green-yellow cells by confocal microscopy (Fig.3).

The control groups in these experiments were incubated inSB without DIF. Compared to the experimental groups withDIF, most of the cells fluoresced green throughout the experi-ment, with only a few cells fluorescing red at all times; moststrikingly, only very few vacuolated cells appeared (Figs 1, 2).These patterns in control groups and in groups with DIF werevery reproducible. In more than 50 differentiation experiments,only one gave results at variance with the description above.This could be traced to an alteration of the quality of the water

used to prepare the SB solution,leading to the use of source water forthe preparation of all media.

In conclusion for this type ofexperiment, the addition of DIF ledto an early and marked increase in theproportion of vacuolated cells, andlater to PI-labelling of a significantproportion of these cells. Theseresults are consistent with the inter-pretation that DIF induces within afew hours vacuolisation in almost allcells, of which only some (within 4days) will, significantly later,develop alterations of their plasmamembrane.

Early irreversible impairmentof re-growth In experiments on in vivo differen-tiation of D. purpureum, Whitting-ham and Raper (1960) studied as anindicator of cell death the inability ofcells released from stalks to grow invegetative culture conditions. Wetook advantage of in vitro cultureconditions to reassess the ability togrow after differentiation, with moreprecise time kinetics and in conjunc-tion with other indicators of celldeath. After incubation in SB andcAMP, Dictyostelium discoideumcells were incubated in the presenceof DIF for variable lengths of time,then received an excess of HL5medium and were incubated for afurther 48 hour period. These exper-imental cells and also control cellswere then counted. Vegetative cellsgrown in the same proportions of SBand HL5 in the presence of DIF didnot differentiate and went on multi-plying at their usual rate (not shown).

The flasks with cells incubated for24 hours in DIF, and furtherincubated for 48 hours in thepresence of HL5 medium, contained

about 10-20% of the cells in control flasks not subjected to DIF(Fig. 4). These regrowing cells were vegetative-looking andmultiplying (not shown). Assuming a similar doubling time forvegetative cells that had never been subjected to differentiationconditions and for cells having survived these conditions, theseresults indicated that 80-90% of the cells subjected to differ-entiation conditions for 24 hours were irreversibly altered andunable to re-grow. Longer incubation times in the presence ofDIF (48-96 hours) did not significantly change the proportionsof regrowing cells (not shown). Of note, the duration of incu-bation with DIF required for the non-regrowth of 50% of thecells was about 8 hours (shown in three distinct kinetics exper-iments, Fig. 4A-C), thus a few hours less than that required forvacuolisation of the cells (Fig. 2). A direct comparison in the

SB + DIF

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same experiment of the kinetics of induction of non-regrowthon the one hand and of vacuolisation on the other hand isshown in Fig. 4C. If non-regrowth means that an irreversibleevent has occurred in the differentiating cell, then clearly thisirreversible event occurred significantly before any morpho-logical alteration detectable by the techniques above.

Absence of early DNA fragmentationApoptotic cell DNA shows fragments of 50-300 kbp and oftenof 200 bp and multiples thereof (see references in the Discus-sion). We wondered whether DNA in differentiating Dic-tyostelium cells would be similarly fragmented. Cells incubatedwith or without DIF were harvested at different times, insertedin agarose plugs, proteinase K-treated, and subjected toclassical electrophoresis to assess the existence of smallfragments of DNA (200 bp), and to pulsed field electrophor-

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A B Fig. 2. Kinetics of HMX44A cell differentiation quantified on a percell basis. After FDA/PI staining, cells were examined forfluorescence and the presence of large vacuoles. Three areas of eachflask were scored for each point of the graphs. At least 100 cellswere examined per area. The results were pooled and expressed aspercentages. (A) in SB alone. (B) in SB plus DIF.

j, Non-vacuolated FDA+ cells. h, Vacuolated FDA+ cells. Initially all cellswere FDA+, without vacuoles; with time, almost all of these cellsbecame vacuolated, and some became PI+. s, PI+ cells. Almost allPI+ cells were non-vacuolated in SB alone and vacuolated in SB plusDIF (in 4 experiments the average percentage of non-vacuolatedcells in SB plus DIF at 48 hours of differentiation was around 3%).

Fig. 3. Confocal fluorescence micrographs of Dictyostelium cells after 24 hours of incubation in DIF. Three pictures are shown for each of twogroups of cells, above and below, respectively. For each group, two views 2.8 µm apart (middle and right) and 8 stacked views (maximumdetections) over a total thickness of 19.5 µm (left) are shown. Individual FDA and PI images were superimposed numerically. Pseudocoloursmimic FDA or PI staining, as previously described (Humbert et al., 1993).

2697Programmed cell death in Dictyostelium

esis (PFGE) to detect larger fragments (i.e. 50-300 kbp). As apositive control for fragmentation, dexamethasone-treated thy-mocytes fragmented their DNA as expected, showing bands of200 bp and multiples thereof (Fig. 5A) and a smear around 50kbp (Fig. 5C). All Dictyostelium DNA samples showed the 88kbp band corresponding to extrachromosomal nuclear rDNA(Cockburn et al., 1978). The presence of this band in the exper-imental groups indicated that the method used allowed releaseof DNA from the nuclei, and its degradation after treatment with20 µg/ml neomycin for 3 or 5 days (Fig. 5C) provided an addi-tional positive control. DNA from Dictyostelium cells subjectedto DIF for 12 or 24 hours showed, compared to the same cellsincubated in SB alone without DIF, no sign of massive degra-dation, i.e. neither small (Fig. 5A) nor large (Fig. 5B) DNAfragments, nor marked smears. Another experiment includingcells at 8 hours of differentiation gave similar results (notshown). The more marked smear obtained with 66 hour cellsalso occurred in the control group without DIF. All gels wereblotted onto membranes, which were probed with 32P-labelledtotal genomic probes to increase the sensitivity of detection offragmented DNA. The results thus obtained (not shown) did notchange the conclusions above as to the absence of early massiveDNA degradation in Dictyostelium PCD.

Cytoplasmic condensation and focal chromatincondensationBy electron microscopy, vegetatively growing Dictyostelium

cells showed, in particular, a highly developed cytoplasmicsystem of small vacuoles, and a nucleus with a large nucleolusassociated with the nuclear membrane and homogeneouslydispersed chromatin without condensation (Fig. 6A). After 8-12 hours of culture in SB without DIF, spots of condensedchromatin were visible within the nucleus, with significantcytoplasmic condensation (Fig. 6B and C). Cytoplasmicvacuoles were neither more abundant nor more developed thanin vegetative cells. After 8-12 hours of culture in the presenceof DIF (Fig. 7), the cell contours were smoothened, with lossof microvilli. The cytoplasm was dense and the nucleuscontained patches or foci of dense chromatin, however, notassociated with the nuclear membrane. The most obvious dif-ference with groups in SB without DIF was the presence oflarge cytoplasmic vacuoles, either appearing empty or con-taining residual material (Fig. 7).

Thus, perhaps surprisingly, cells incubated in SB with orwithout DIF showed similar aspects of chromatin and cyto-plasmic condensation, different from those of vegetative cells.Also, cellulose walls were prominent after incubation for 24hours in SB with, as well as without, added DIF in studiesusing Calcofluor (not shown). Since HMX44A cells do notimmediately die in SB without DIF, the results above indicatedthat neither the observed levels of cytoplasmic and chromatincondensation, nor cellulose walls, are enough to ensure PCDin Dictyostelium.

Programmed induction of Dictyostelium cell deathCultures of HMX44A Dictyostelium cells were subjected toserial additions of cAMP or DIF (not shown). cAMP, knownto be necessary in the Dictyostelium differentiation pathway,is produced by the cells themselves. We, however, systemat-ically added an excess of cAMP to reduce aggregation. Nodevelopmental death was observed in complete HL5 mediumor in the absence of DIF. Both starvation and the presence ofDIF were required for cell death. Since DIF did not inducethe death of vegetative cells, it follows that DIF can induceDictyostelium death only when added to cells that havealready reached a certain level of differentiation. Moregenerally, in agreement with previous work (Kay, 1987),under our experimental conditions cell death occurredonly when a given program of successive extra-cellularsignals, i.e. starvation and DIF, was applied to the cells (notshown).

DISCUSSION

We have studied cell death in Dictyostelium discoideum, whichis phylogenetically distant from currently used modelorganisms, has a relatively simple developmental pattern, andlends itself well to genetic analysis. In an in vitro system inwhich death could be induced without inducing morphogene-sis, thus facilitating quantitative studies, Dictyostelium celldeath was shown to be programmed as to both circumstancesof occurrence and course within the dying cell (Fig. 8). ThisPCD in Dictyostelium, occurring in vitro upon addition ofphysiological inducers, seems equivalent to developmental celldeath in vivo. The programmed course of death in vitroincludes the appearance in dying cells of very large vacuoles,while in vivo all stalk cells are similarly vacuolated. The terms

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%)

Fig. 4. Re-growth experiments. At various times after addition ofDIF, culture medium (HL5) was added to the flasks to giveproportions of 75% HL5/25% DIF-containing differentiationmedium. To evaluate the number of cells able at each time point tostart dividing again, the number of cells resulting from such divisionsat 48 hours after the addition of culture medium was determined andgraphically plotted. (A, B and C) Three separate experiments,involving two, four and three flasks per time point, respectively. Forthe experiment in C, to obtain the combined kinetics of regrowth andcell alterations in the same experiment, at each time after DIFaddition parallel flasks either received complete medium or wereFDA/PI stained, counted and percentages established as for Fig. 2.j, FDA+ non-vacuolated cells. m, number of cells after a first 8 hourcAMP incubation in SB, followed by SB plus 100 nM DIF for theindicated times, and by a 48 hour incubation in complete medium.Controls yielded for A, 17.0×106 cells, for B, 14.7×106 cells, and forC, 11.9×106 cells when the incubation period with DIF was replacedwith no incubation at all; and for A, 14.0×106 cells, for B, 8.0×106

cells and for C, 7.1×106 cells when the same period was replacedwith a 24 hour incubation in SB without DIF.

2698 S. Cornillon and others

Fig. 5. Absence of massive early DNA fragmentation in Dictyostelium PCD. Cells were included in agarose plugs, which were treated torelease DNA and inserted in gels run as classical gel electrophoresis (A) or as pulsed field gel electrophoresis (B and C). Cells were vegetativeHMX44A Dictyostelium cells (Veget.), vegetative HMX44A cells treated for 3 or 5 days with 20 µg/ml neomycin (Neo 3d or 5d), HMX44Acells incubated for the indicated number of hours either in SB alone as a control, or in SB plus DIF to induce PCD (SB or DIF), and thymocyteseither untreated or treated for 6 hours with 10−6 M dexamethasone (Thym. and Thym.+ Dex). Size markers were λ phage cut with HindIII(λHindIII), phage λ multimers (polyλ) and yeast chromosomes (yeast).

2699Programmed cell death in Dictyostelium

Fig. 6. Electron microscopy of HMX44A Dictyostelium cells, either vegetative or after incubation in SB. (A) A cell during vegetative growth,showing an irregular shape with a highly developed vacuolar system (V); the nucleus (N) contains a large nucleolus (n) associated with thenuclear membrane; the chromatin is homogeneously dispersed. (A′) Detail of the nucleus in A. (B and C) After 12 hours of incubation in SBwithout added DIF, some spots of condensed chromatin can be seen within the nucleus; vacuoles are neither more abundant nor moredeveloped than in vegetative cells. (C′) Detail of the nucleus in C. Bars: 1 µm for A, 1.5 µm for B, and 0.5 µm for A′, C and C′.

2700 S. Cornillon and others

Fig. 7. Electron microscopy of HMX44A Dictyostelium cells after incubation in SB with added DIF. After 8 (A,B) or 12 (C,D) hours in thepresence of DIF, cell contours are smoothened with loss of microvilli, the cytoplasm is dense with large vacuoles including some residualmaterial, and the nucleus contains marked patches or foci of dense chromatin. Arrows, cellulose walls. Bars, 0.5 µm. V, vacuolar system; N,nucleus; n, nucleolus.

2701Programmed cell death in Dictyostelium

‘stalk’ or ‘vacuolated’ cells would thus seem to correspond todead or dying cells.

Dictyostelium cell death, apoptotic and non-apoptotic morphological aspects of cell deathIn Dictyostelium PCD, the first detectable irreversible eventdefining cell death, strictly speaking, was the inability toregrow. Using this criterium, under our experimental con-ditions about 50% of the cells die within 8 hours and 90% ofthe cells die within 24 hours after addition of DIF. Death,defined by inability to regrow, did not coincide in time withdeath defined with fluorescent dyes, or with any othercriterium. For instance, membrane lesions, as detected by per-meability to PI, occurred very late compared to regrowthinability. Thus, the irreversible lesion (‘death’) detectedthrough inability to regrow leads to detectable morphologicalalterations only with significant delay. Such an irreversibleevent must exist also in bona fide apoptosis (see discussion inGolstein et al., 1991), where it has not been better molecularlycharacterized so far.

A major component of cell death in Dictyostelium is vac-uolisation. Vacuolisation could be triggered independently ofcell death by the same sequence of extracellular events, andmight then be related to differentiation towards an aspect rem-iniscent of that of plant cells or fungi. Alternatively, vacuoli-sation may be directly related to cell death, either as a causeor as a consequence; the latter seems more likely, consideringthat the inability to regrow occurs on average a few hoursearlier than detectable vacuolisation. Electron microscopystudies indicated that in developing Dictyostelium vacuolesmay be related to autophagy (Maeda and Takeuchi, 1969;George et al., 1972; de Chastellier and Ryter, 1977; Quivigeret al., 1980; Schaap et al., 1981). The presence of largevacuoles is not just a reflection of an autophagic process

secondary to the incubation in starvation medium (as observedin some mutant strains of yeast cells; Takeshige et al., 1992),since HMX44 cells in SB without DIF do not show largevacuoles.

Although vacuolisation seems a peculiar trait of Dic-tyostelium cell death, it may not be that different from eventsoccurring in PCD in other organisms. Vacuolisation has beenreported in cell death in C. elegans (Robertson and Thomson,1982) and in higher eukaryotes in at least some cell types(Wyllie et al., 1980; Clarke, 1990). Also, vacuolisation in Dic-tyostelium may be related to the cytoplasmic condensationobserved in all types of PCD studied in detail so far. In thepresent studies of Dictyostelium cell death, cytoplasmic con-densation was prominent. A pronounced decrease in the cyto-plasmic volume of differentiating Dictyostelium cells has beendescribed previously (de Chastellier and Ryter, 1977). Cyto-plasmic condensation may result from a loss of cytoplasmicwater. This could manifest itself differently in various highereukaryote cells or in Dictyostelium due to the individual con-straints in each cell type, leading to spherical surface protu-berances in apoptosis (Kerr et al., 1972) and to membranewrinkling in non-apoptotic death of Manduca sexta musclecells (Schwartz et al., 1993). While many cell types haveminimal membrane constraints, Manduca muscle cells areattached to the segmental boundaries of the abdomen(Schwartz et al., 1993). In the case of Dictyostelium andperhaps of plant cells, the early synthesis of cellulose duringdifferentiation is a major limitation to the modification of shapeand the surface ‘boiling’ seen in other cells losing water, andmight favor displacement of water into a vacuole localisedwithin the cell.

In apoptosis, early ultrastructural nuclear lesions at a highlevel of chromatin organisation lead to the appearance of largeDNA fragments (300 and/or 50 kbp) revealed by pulsed fieldgel electrophoresis (Walker et al., 1991; Brown et al., 1993;Oberhammer et al., 1993; Tomei et al., 1993). This is oftenfollowed with lower level DNA fragmentation (Wyllie, 1980)resulting in a gel electrophoresis ladder pattern of fragments of180-200 base pairs and multiples thereof. No clear-cut ladderpattern of DNA fragmentation was to be expected in Dic-tyostelium death, since the nucleosomal organisation of Dic-tyostelium chromatin is irregular (Blumberg et al., 1991). IfDNA fragmentation occurred in Dictyostelium PCD, assuminga similar chromatin organisation in Dictyostelium and in highereukaryotes one would expect smears in low molecular massregions and/or in the 50 kb region, both in ethidium bromide-stained agarose gels and in autoradiograms of the correspond-ing blots probed with radiolabelled total genomic DNA. Nosuch result was obtained with DIF-treated dying Dictyosteliumcells, while, in control groups, smears appeared in Dic-tyostelium preparations treated with neomycin, and theclassical ladder pattern plus a 50 kb band were obtained withthymocytes treated with dexamethasone. Thus, we found noevidence for early alteration of DNA in Dictyostelium PCD.

By electron microscopy, we found that PCD in Dic-tyostelium included only limited and focal chromatin conden-sation. This was strikingly similar to the focal condensationfound in the nuclei of Manduca sexta muscle cells (Schwartzet al., 1993) and of some nerve cells (quoted by Schwartz etal., 1993) considered undergoing non-apoptotic death. Inter-estingly, no early DNA fragmentation was detected during

Fig. 8. Steps in programmed occurrence and programmed course ofDictyostelium HMX44A cells death. The times indicated afteraddition of DIF are the average times in hours at which 50% of thecells present the described characteristic.

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death of Manduca sexta muscle cells (Zakeri et al., 1993). Inonly partial contrast, apoptosis includes as one of its most char-acteristic traits (Duvall and Wyllie, 1986; Kerr et al., 1987;Wyllie, 1988) the condensation of chromatin and its marginal-isation, leading to the formation of chromatin masses withsharply defined edges lining the nuclear membrane. Perhapssimilarly, by electron microscopy, differentiating protophloemsieve elements in roots of wheat were shown to undergonuclear degeneration, with chromatin condensing as massesunder the nuclear membrane, followed by nuclear fragmenta-tion (Eleftheriou, 1986). It is tempting to relate chromatin con-densation to the changes in cell cycle-control molecules suchas cdc2 in at least some occurrences of apoptosis (Shi et al.,1994). A cdc2 homologue has been identified in Dictyostelium(Michaelis and Weeks, 1992). In our experiments, the occur-rence of cytoplasmic and chromatin condensation in SBwithout DIF indicated that at least in Dictyostelium theseevents do not necessarily lead to cell death.

Altogether, the most remarkable conclusion to us from thisand other studies comparing various cell death morphologicalpatterns is perhaps the absence of sharp boundaries betweenthese patterns. An irreversible event must happen in all cases.DNA fragmentation occurs neither in non-apoptotic death norin Dictyostelium, but neither is it found in some instances ofapoptosis. Chromatin condensation is prominent in apoptosisbut there is some focal condensation in both non-apoptoticdeath and in Dictyostelium PCD. Membrane alteration is a lateevent in all cases.

Evolutionary considerationsWhen in evolution did developmental cell death emerge?Developmental cell death has apparently been found each timeit was looked for in multicellular eukaryotes. This mightindicate that its emergence is at least as ancient as theemergence of multicellularity, more than 0.7 billion years ago(Field et al., 1988). Interestingly, however, ‘the evolutionarystep from single to multicellular organisms appears to havebeen taken many times’ (Kaiser, 1986 and references therein).This raises the question of whether developmental cell deathemerged before multicellularity, presumably leading to onlyone core molecular mechanism of PCD in present-day livingorganisms, or whether it emerged after multicellularity,possibly leading to more than one such mechanism.

PCD can apparently be typically apoptotic (in most ver-tebrate cells) or non-apoptotic (for instance vacuolisation inDictyostelium, PCD of invertebrate muscle cells). These appar-ently distinct cell death aspects suggest two interpretationsrelated to the evolutionary argument above. The first interpre-tation is that there is a plurality of cell death core mechanismsamong multicellular organisms. PCD would result fromdifferent basic molecular mechanisms, which may haveappeared at distinct and multiple times during evolution,perhaps paralleling or closely following the repetitiveemergence of multicellularity. Alternatively, PCD may followthe same core molecular mechanism in all cases. Elements ofit might have appeared perhaps only once in evolution, beforethe emergences of multicellularity, in, say, an early unicellu-lar eukaryote, and have been used to achieve PCD in emergingmulticellular organisms. To this core mechanism successivelayers of complexity might have been added during evolution.Cell death morphological pattern, though not its core molecular

mechanism, might then differ somewhat, as a function of theorganism (e.g. mouse vs Dictyostelium) and/or as a function ofthe cell within a given organism (e.g. thymocyte vs liver cell,or perhaps muscle cell), or more generally as a function of theenzymatic equipment of, or the mechanical constraints on, thecell that dies.

We are thus led to the hypothesis of a single and thereforeancient core molecular mechanism of PCD. A prediction ofthis hypothesis is that some genes at play in PCD should behomologous in very diverse organisms. This may be testedthrough the analysis of molecules at play in phylogeneticallyremote organisms. Already consistent with this hypothesis, thePCD-controlling ced-3 and ced-9 molecules isolated from C.elegans both have structural and functional counterparts in ver-tebrate cells (Vaux et al., 1992; Miura et al., 1993). In Dic-tyostelium, the identification of some of the genes involvedmight take advantage of the small haploid genome and of inser-tional mutagenesis procedures.

We thank J. G. Williams (ICRF Laboratories, Clare Hill) and M.Hildebrandt and M. Veron (Pasteur Institute, Paris) for Dictyosteliumstrains and for very helpful discussions, M. Fathallah for help withthe confocal microscope, M. Fraterno for help with electronmicroscopy and J.-F. Brunet and A. H. Greenberg for comments onthe manuscript. This work was supported by institutional grants fromINSERM and CNRS, and by an additional grant from the Associationpour la Recherche contre le Cancer.

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(Received 23 March 1994 - Accepted 2 June 1994)

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