cell-death alternative model organisms - Nature

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CELL-DEATH ALTERNATIVE MODEL ORGANISMS: WHY AND WHICH? Pierre Golstein*, Laurence Aubry ‡ and Jean-Pierre Levraud § Classical model organisms have helped greatly in our understanding of cell death but, at the same time, might have constrained it. The use of other, non-classical model organisms from all biological kingdoms could reveal undetected molecular pathways and better-defined morphological types of cell death. Here we discuss what is known and what might be learned from these alternative model systems. CASPASE

A subfamily of cysteine proteases. Caspases have a catalytic cysteine residue and recognize a tetrapeptide on their substrate that always includes aspartic acid at position 1.

*Pierre Golstein, Centre d’Immunologie de Marseille-Luminy, CNRS-INSERMl'Université de la Mediteranée, Parc Scientifique de Luminy, Case 906, 13288 Marseille cedex 9, France. ‡ Laurence Aubry, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 09, France. § Jean-Pierre Levraud, Unité Macrophages et Développement de l’Immunité, Institut Pasteur, 25, rue du Dr Roux, 75724 Paris cedex 15, France. Correspondence to P.G. e-mail: golstein@ciml. univ-mrs.fr doi:10.1038/nrm1224

In many instances, scientists have gained only a partial insight into a given biological phenomenon. The degree of insight achieved is closely dependent on the biological model(s) that are used. The use of too few models could give a scattered and distorted view, whereas increasing the number and the variety of models might help correct this limitation. This is also likely to be the case in studies on cell death. Although the recent accumulation of knowledge in this field has indeed been remarkable, many questions still remain unanswered. So far, few biological models have been used to investigate cell death. Despite their huge contribution to our current understanding of cell death, further progress in the resolution of a number of pending questions is likely to result from the use of alternative biological models, in parallel with the classical models. We discuss why this might be so, and we present some examples of alternative models. Classical model organisms and apoptosis

Programmed cell death (PCD) refers to the programmed occurrence of cell death in the organism during development, as well as to the programmed course of events in the dying cell. PCD is phenomenologically polymorphic, as it can be apoptotic, vacuolar or necrotic, with intermediate aspects, as described below. Mechanistically, it is both complex — CASPASE-dependent PCD, for example, includes many regulatory and effector molecules — and multi-faceted, as there are not only caspase-dependent but also caspase-independent mechanisms. Later in this review, we consider some aspects of these

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mechanisms, and evaluate the suitability of current or alternative model organisms for their study. Our perception of PCD has been greatly enhanced and, at the same time, biased by the initial description of apoptosis.Apoptosis1–3 is characterized by early condensation (of chromatin, leading to characteristic perinuclear chromatin masses, and of cytoplasm, resulting in cell shrinkage), followed by fragmentation, leading to apoptotic bodies still surrounded by an intact membrane. Apoptosis includes early DNA fragmentation, which can be detected as a ladder pattern in gel electrophoresis of DNA from dying cell populations4 or at the dying-cell level through a terminal-transferase-dependent labelling procedure known as TUNEL (TdT-mediated dUTP-biotin nick end labelling)5. The apoptotic cell finally disappears through the systematic internal caspase-mediated dismantling of the main cell structures, and engulfment of cell remnants by neighbouring cells. Apoptosis is usually mediated by a mechanism that involves caspases. The activation of effector caspases, such as caspase-3, contributes to cell death and leads to cell dismantling (FIG. 1). The role of caspases in cell death was first discovered in Caenorhabditis elegans through observations of cell-death mutants6. The initial description of these caspase-deficient and other cell-death mutants in C. elegans was an important breakthrough, as it provided a molecular basis for PCD. The availability of molecular databases favoured the preferential discovery of similar pathways in other organisms through the search for homologues. So, both apoptotic cell death and caspase-dependent mechanisms benefited from an anteriority bias, and, as

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Cell death receptors Nucleus Casp-8 Receptors Casp-3 Reaper Grim Hid Sickle

Dismantling

Casp-9 IAPs Apaf1

Cyt c

ATP

Bcl2 family members

Smac/Diablo Omi/HtrA2

AIF EndoG

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Other organelles ?

Figure 1 | A simplified view of the main pathways of caspase-dependent cell death. Caspase activation can occur through two main pathways. A receptor pathway (light-blue arrows) involves ‘cell-death receptors’, such as Fas, which, when associated with its ligand, leads to the activation of apical caspase-8 (Casp-8). A mitochondrial pathway (dark-blue arrows) involves the activation of apical caspase-9, which is helped by released mitochondrial products such as cytochrome c (Cyt c). The mitochondrial pathway can be controlled by Bcl2 family members and by molecules released from other organelles. Caspase-9 activation can also be controlled by IAP (inhibitors of apoptosis) family members, which are themselves modulated by mitochondrially released or transcriptionally regulated molecules. In Drosophila, the latter include Reaper and related molecules. Both pathways converge on effector caspases, such as caspase-3, which they activate. The activation of effector caspases leads to the dismantling of many key subcellular structures. Other, apparently mitochondria- and death-receptor-independent pathways can also lead to the activation of apical caspases. Details can be found in a recent review (REF. 21). AIF, apoptosis-inducing factor; Apaf1, apoptotic protease activating factor; Bcl2, B-cell leukaemia 2; EndoG, Endonuclease G; HtrA2, high-temperature-requirement protein A2; Omi, Omi stressregulated endopeptidase; Smac, second mitochondria-derived activator of caspase.

TUNEL

A technique that enables the detection of DNA fragmentation at the cellular level. Fragmented DNA generates many free DNA ends and, using terminal transferase, these can be labelled with tagged nucleotides, which can be secondarily detected using appropriate reagents. SPHINGOLIPID

A derivative of the long-chain amino diol sphingosine. Its structure is similar to a glycerolbased phospholipid, with a polar head group as well as two hydrophobic hydrocarbon chains (one is the sphingosine and the other is a fatty acid chain). PARA-CASPASE

Protein that shows only weak homology to caspases, and is found in metazoans and Dictyostelium.

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a result, tended to be preferentially seen. As most of the easily accessible models of animal cell death indeed displayed caspase-dependent apoptosis, other co-occurring types of cell death might have been overlooked. Much of what is known in the cell-death field stems from the use of two invertebrate (C. elegans and Drosophila) and two vertebrate (human and mouse) animal models. The role of caspases and functionally connected molecules in cell death was discovered in C. elegans 6. The mammalian biological models greatly benefited from the efficiency of genetic screening in humans. For example, a pathological chromosomal translocation led to the early discovery of BCL2 and its anti-apoptotic function7. The accumulated knowledge, experimental work and possibilities for genetic manipulation in the mouse led to functional studies of the caspase and Apaf1 homologues of C. elegans ced-3 and ced-4 using gene inactivation, and also to early investigations of the receptor pathway (FIG. 1) and its components, including Fas and related molecules (reviewed in REF. 8). Whereas the simplicity of C. elegans helped to delineate the caspase-dependent core apoptosis machinery, the mammalian systems showed that within this conserved death machinery the cell-death pathways have evolved to become much more complex and varied.

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Similarly, studies in Drosophila led to the discovery of a group of inducible molecules, exemplified by Reaper9, which indirectly control caspase activation. These molecules have no homologues in mammals but do have functional equivalents10. For caspase-independent mechanisms, most of our limited knowledge stems from studies in mammals, although invertebrate models have now begun to make important contributions. Obviously, C. elegans, Drosophila, mice and humans represent a tiny fraction of all living organisms (FIG. 2), even if they do span a considerable distance in animal evolution11. Apoptotic cell death and bona fide caspases are only found in the animal kingdom, whereas outside the animal kingdom only caspase-independent, nonapoptotic cell death occurs. In the animal kingdom, both caspase-independent and caspase-dependent deaths can occur. The caspase-independent mechanisms in the animal and other kingdoms might be phylogenetically related. Therefore, studying non-animal species might contribute to our understanding of caspaseindependent cell death in animals. Pending questions

Caspase-dependent apoptosis. Caspase-dependent apoptosis itself is incompletely explored; for example, with regard to the consequences of the association of celldeath receptors with other cell-surface receptors (Fas with the Met receptor), the role of cell-death receptors in non-cell-death functions and their existence and functions in invertebrates, and the importance of ‘dependence’ receptors that signal death when they are not engaged by their ligands. Also, there are still gaps in our knowledge concerning the complex role of SPHINGOLIPIDS, the role of DAXX and other cell-death molecules in the nucleus, the relationship between the classical cell-death machinery and mitochondrial fission, and the many roles of death-associated protein kinase (DAPk), which might represent an example of a molecular bridge between apoptosis and autophagic cell death. There is also much to be learnt about the existence of non-receptor, non-mitochondrial, death-inducing pathways that involve, for example, caspase-2 and possibly several cell organelles; the role of certain caspases in functions other than cell death; the elusive role of PARAand META-CASPASES; the involvement of some non-caspase proteases in certain cases of cell death; the impact of some effector molecules of animal cell death on non-animal cell death; and indeed the precise role of some of the classical ‘cell-death molecules’. For example, although BCL2 family members have generally been attributed a mainly mitochondrial location and function, they can also control Ca2+ concentration in the endoplasmic reticulum, which only indirectly governs mitochondrial induction of apoptosis12. This is just one example of organelle-specific death responses that cause a mitochondrial alteration or caspase activation, both (or either) of which can induce cell death13. How organelles such as the Golgi, lysosomes and the endoplasmic reticulum are affected in the first place remains to be determined. Also, because inactivation of caspase and Apaf1 genes does not seem to prevent certain instances of cell

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Plants

Fungi

Arabidopsis Volvox

Podospora Yeast

Animals Vertebrates Invertebrates Mus musculus Homo sapiens Ciona

Xenopus

Drosophila

Zebrafish

C. elegans Hydra

– 0.7 × 109 years

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Dictyostelium

Leishmania Trypanosoma Trichomonas

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–1.5 × 109 years

Eubacteria and archaebacteria

Figure 2 | Phylogenetic tree of model organisms used to study cell death. The tree indicates the relative phylogenetic positions of some current and alternative model organisms to study cell death (timescale not accurate). The branching order of nematodes (Caenorhabditis elegans), arthropods (Drosophila) and deuterostomes (Ciona and the vertebrates) is still a matter of debate. The order shown here is that suggested in REF. 11.

death during embryonic development in the mouse, another outstanding question is which pathways mediate embryonic developmental cell death in vertebrates? Caspase-independent, non-apoptotic pathways. In spite of the unresolved issues mentioned earlier, apoptotic cell death is relatively well understood compared with the poorly defined non-apoptotic cell-death pathways. Numerous examples of necrotic or autophagic cell death have been described14–16 and reviewed17–21. These types of cell death can occur during development, for example after insect metamorphosis of intersegmental muscle cells22–24 or of salivary gland cells25, or during mammalian development, at least when caspase activation is genetically or pharmaceutically prevented26, under several pathological conditions, and certainly not only accidentally as once thought. Necrotic and autophagic cell death are best distinguished using electron microscopy, although the identification of cell death as autophagic or necrotic is often difficult, particularly with respect to previously published work.Also, there are many intermediate types, and both necrotic and autophagic cell death can be accompanied by vacuolization. Perhaps the most recognizable aspect is that of autophagy with vacuoles. One approach could be to study this type of cell death in more detail in appropriate biological models and to identify corresponding biochemical markers, which would in turn facilitate a genetic screen for the molecules involved. META-CASPASE

Protein that shows only weak homology to caspases, and is found in plants, fungi and protozoa.

Autophagic cell death. Macro-autophagy (referred to here as autophagy) involves the inducible sequestration of cytoplasm and organelles, or organelle fragments, in a double-membrane structure, known as an


autophagosome. The autophagosome delivers its sequestered cargo to a lysosome by a fusion process that results in an autophagolysosome. In yeast, the autophagosome fuses with the vacuole (which is thought to be equivalent to lysosomes in animal cells), where its cargo is released as an autophagic body and degraded27. Autophagy is induced by starvation27–29 and helps ensure, at least for some time, the survival of cells that are deprived of extracellular nutrients. Autophagic cell death implies primary autophagy (unlike autophagy that is secondary to other mechanisms, such as in some cases of late necrosis). Operationally, cell death is often said to be autophagic when dying cells contain autophagic bodies or autophagosomes, with or without large vacuoles. However, the relationship between autophagy and autophagic cell death is not entirely clear. For example, how can we reconcile the role of autophagy in ensuring the survival of starved cells and its apparent role as a mechanism of cell death? Also, is autophagy causing this cell death, or is it only accompanying it? A possibility is that autophagy leads to the disappearance of crucial cellular organelles, resulting in secondary cell death. So, when cells were induced to undergo cell death in the presence of caspase inhibitors and then returned to nonadverse culture conditions, they survived for several days. However, during this period their mitochondria disappeared — a process that could be blocked by BCL2 overexpression30. Perhaps related to this, autophagic stimulation of rat hepatocytes by serum deprivation and glucagon caused an increase in spontaneously depolarizing mitochondria that moved into acidic vacuoles31. Alternatively, autophagy plus another step might be required for cell death. This other step might entail vacuolization (leading to autophagic vacuolar cell death; for an example, see REF. 32), or some other lesion process in the case of non-vacuolar autophagic death. This question could be explored in some of the model systems described below. These models might also be useful in helping to delineate which molecules are involved in autophagic cell death. Although about twenty aut and apg mutants have been isolated, which define a series of molecules that are involved in autophagy and conserved throughout the eukaryotes33,34, it is not yet clear whether these molecules are required for autophagic cell death. Interestingly, in this caspase-independent cell-death pathway, genome-wide analyses recently pointed at a possible role for molecules, including caspases, that are involved in apoptosis in autophagic cell death in Drosophila25,35, indicating a degree of overlap between molecules that are involved in autophagic cell death and apoptosis. Necrotic cell death. As indicated previously, necrotic cell death is defined in part by negative criteria (non-apoptotic, non-autophagic), as the corresponding cellular lesions are less characteristic than in other types of cell death. Necrotic cell death includes mild ‘mottled’ chromatin condensation, mitochondrial swelling and moderate cell swelling. Interestingly, whereas the initial data about caspase-dependent apoptosis came mainly from



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* Figure 3 | Zebrafish. a | An adult male. b | Three 28-hour-old embryos. c | An in vivo Nomarski microscopic view of a macrophage in one of the embryos. The macrophage contains a phagocytosed apoptotic erythroblast (asterisk) and contacts two healthy erythroblasts.

C. elegans, and were confirmed later in Drosophila, these organisms are now also providing informative examples of caspase-independent cell death. For example, in C. elegans, necrotic vacuolar neuronal cell death is induced by gainof-function mutations in genes that encode proteins that are similar to subunits of a vertebrate epithelial sodium channel. Infolding of the plasma membrane and production of small whorls is followed by the emergence of cytoplasmic vacuoles and cell swelling36. Curiously, similar whorls were observed in starved Dictyostelium autophagy mutants37. This C. elegans cell death is caspase independent, and requires an increase in the cytoplasmic Ca2+ concentration and the activity of Ca2+-regulated calpain proteases and aspartyl proteases38. Interestingly, similar findings have been made in models of neurodegenerative diseases in humans39. Altogether, apoptosis is relatively well characterized, but it is complex and not completely understood. The non-apoptotic types of cell death — perhaps because they have often been defined as such, rather than as distinct detailed types — can be difficult to distinguish unambiguously and are mechanistically poorly defined. Alternative model organisms might provide ways to clarify some of these issues. Death decisions. Whichever cell-death mechanism is at play, another important and not completely solved issue is why cells die at all in some situations. Cell death might be part of a constant turnover of cells, for example, in skin or gut, where the necessity for renewal would seem to be a justification in itself. Also, in some situations, cell death is part of a selection process, for example, for cells with the most appropriate receptors (in the immune system) or the most appropriate connections (in the nervous system).

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A classical explanation for certain developmental cell deaths is the optimization of organ structure. In some organisms, however, the re-allocation of essential nutrients or energy can be the main purpose of certain types of cell death. When an organism needs to re-allocate resources from ‘superfluous’ cells to centres of proliferation, death and recycling of the excess cells is one option, but transdifferentiation followed by migration of those cells is another. In the animal kingdom, the first option is often relied on. Energy considerations might also explain cell death in other circumstances. For example, in Dictyostelium, cells in the stalk die while fulfilling their structural role of building a cellulose-based stalk, and it has been shown that their death is not a consequence of being cellulose encased40. It seems unlikely that these cells die to provide nutrients to non-dying cells, as it is not clear how these nutrients in the stalk would reach cells elsewhere. It is possible, however, that stalk cell death provides energy for the building of the stalk. The existence of a mechanism for the efficient recycling of dead cells might have been of great importance in evolution. It could be argued that new organs or functions that involve extensive cell death might not have emerged were it not for such an efficient process. For example, in the adaptive immune system the generation of B and T lymphocytes in primary lymphoid organs involves extensive proliferation and death of a considerable fraction (>90%) of the precursor cells. This is a wasteful process, which must involve a significant metabolic effort. When this system first evolved in primitive fishes, the selective advantage it brought had to balance the selective disadvantage linked to this energy demand. The more efficient the recycling of dead cells, however, the more easily this selective disadvantage could be counterbalanced. So, an economically efficient cell-death process, such as apoptosis (which evolved in animals at least 200 million years before adaptive immunity), can be thought of as a prerequisite for the evolution of adaptive immunity as we know it. Building on this argument, the recycling of energy and nutrients might be more efficient in apoptotic than in autophagic death in animals. Apoptosis is phylogenetically more recent, being known only in animals, whereas autophagic death dates back at least to slime moulds. Both types of cell death coexist in animals, in which apoptosis seems to be more common. Apoptotic cell death might have been selected in evolution, in part, because of its superior economic efficiency. However, there are also other advantages to apoptosis, such as the rapid and complete disappearance of dead cells through internal dismantling and phagocytosis. To our knowledge, a systematic experimental study of the energy balance of cell death has not been performed. The feasibility of such a study depends on finding an appropriate model organism. Alternative model organisms

Many ‘non-classical’ organisms could be used as possible model organisms to study cell death. An encouraging thought is that C. elegans or Drosophila themselves were once considered non-classical cell-death models.



REVIEWS Among unicellular eukaryotes, both phylogenetic and medical considerations could justify the use of parasitic protists, such as Leishmania41, Trypanosoma42,43 and the amitochondrial species Trichomonas vaginalis44. The yeast Saccharomyces cerevisiae has been used to investigate the role of mammalian molecules in adoptive death and shows, in some genetic configurations, apoptosislike cell death45,46 in which a meta-caspase might have a role47. Although yeast is in principle unicellular, it can be induced to build amazing stalk-like structures with probable vacuolar death48. Still, the question has been raised as to whether programmed cell death exists in wild-type S. cerevisiae under non-experimental conditions49. Shifting to multicellular eukaryotes, several model plant species, for which powerful genetic tools are now available, are the subject of intensive studies on cell death in development50 and resistance to pathogens. The best established model plant species is Arabidopsis. Among animals, sponges, which belong to the extant metazoan phylum that was the earliest to emerge in evolution, show cell death and possess at least one caspase gene51. A long-known source of synchronously dying cells are metamorphosing animals, for example, insects52, other invertebrates such as Ciona intestinalis that show caspasedependent apoptosis53, and Xenopus laevis, recent studies of which indicate cell-autonomous vacuolar death54. Which criteria should we use to choose an alternative model organism? In the cell-death field, some criteria derive from the pending questions discussed above and from the suitability of these models to answer these questions. Among the examples proposed below, there are two animal models: zebrafish, which might be useful to study embryonic cell death, and Hydra, which might help to answer questions related to the energy balance in apoptosis. The fungus Podospora and the protist Dictyostelium seem to provide an apparently similar (in spite of their evolutionary distance) model of vacuolar cell death, as both possess genetic advantages that should facilitate the further study of this caspase-independent cell death. Volvox is an example of a plant which is likely to be used in the future because of its synchronous cell death and transparency. However, the choice of a model organism also relies on more than just cell-death-related considerations, such as the ease of manipulation and genetic tractability. Therefore, for each model we briefly provide not only cell-death characteristics, but also some more general and practical considerations.

MORPHOLINO

A chemically modified oligonucleotide that behaves as an antisense RNA analogue and which is used to interfere with gene function.

Zebrafish. The zebrafish (Danio rerio) is a small (~4 cm) carnivorous freshwater fish (FIG. 3) and one of the easiest fish to rear. Zebrafish reproduce exclusively sexually, fertilization is external and, because the embryo is transparent, its fast development can be followed in exquisite detail using Nomarski (differential interference contrast) optics. Initially chosen by embryologists because of the embryo’s transparency and the ease of care, the zebrafish soon proved to be well-suited for certain genetic approaches. The high efficiency of ENU (N-ethyl-N-nitrosourea) mutagenesis, together with the high fertility of females (they can lay several hundred


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Figure 4 | Hydra. a | A Hydra polyp from which a new polyp is budding. Insert, a polyp stalk, which is nutrient-starved to increase cell death and labelled with acridine orange to visualize dead cells. b | Phase-contrast image of an epithelial cell dissociated from a Hydra pre-treated with wortmannin to increase cell death. This epithelial cell contains three phagocytosed apoptotic cells. c | 4,6-diamidino-2-phenylindole (DAPI) staining showing the three condensed apoptotic nuclei and the normal nucleus of the epithelial cell. Images courtesy of Angelika Böttger, University of Munich, Germany.

eggs per week), allowed for fast screening of developmental mutants in nearly 2,000 loci55,56, and further efforts are under way to reach saturation. A notable advantage of the ectothermic zebrafish over the mouse, as far as mutagenesis is concerned, is the possibility of selecting for thermosensitive mutations, allowing for the temporal dissection of gene function. The Sanger Center is due to release the complete sequence of the 1,700 Mb zebrafish genome at the end of 2003. Many genetic manipulations are routinely practised in zebrafish, including transgenesis or transient gene overexpression following injection of DNA or messenger RNA in the egg. Functional reverse genetics can be easily performed through transient knockdown of protein expression with MORPHOLINO oligonucleotides57. Gene knockout is not an option because of a lack of suitable embryonic stem-cell lines, but the identification of mutants in a gene of interest by sequencing a library of ENU-mutagenized sperm is a valuable alternative58. Patterns of gene expression are routinely determined through whole-mount in situ hybridization. Biochemical screening in vivo is possible, because the embryos seem readily permeable to small compounds of less than 400 daltons59. The zebrafish offers incomparable access to embryonic cell death, thanks to its transparent, readily accessible embryo and the power of mutagenesis in this model. Apoptotic and non-apoptotic cell death can be viewed dynamically in vivo during zebrafish embryonic development (FIG. 3). The overall pattern of apoptosis, as shown by TUNEL staining, has been established for the first four days of development 60. In live embryos, acridine orange fluorescence (which labels dead cells) or Nomarski optics can be used to identify apoptotic cells in any tissue. Not surprisingly, many of the already



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Figure 5 | Podospora anserina. a | The barrage as an indicator of programmed cell death (PCD) in Podospora incompatibility. Four mycelia of 2 cm diameter were grown on rich medium in agar in a Petri dish. Somatic fusion of cells in the contact area between two incompatible mycelia leads to PCD, which can be revealed at the macroscopic level by the formation of a barrage (the horizontal dark line in the contact area), as shown on the left. On the right, this line is absent from the contact area of two compatible strains. b | Nomarski highpower microscopy of two mycelium-constituting filaments that are 3–5 µm thick and made of multinucleated articles that are separated by septa. The filaments are derived from a thermosensitive auto-incompatible strain. In the upper image, the filament was kept at 32°C and shows living articles. The image below shows the filament 4.5 hours after a shift to 26°C. The filament contains several vacuoles per article, which will fuse into a huge vacuole and collapse, as shown on the right. Images courtesy of Corinne Clavé, University of Bordeaux, France.

described developmental mutants have abnormalities in the timing of apoptosis. Phagocytosis of apoptotic cells can be studied in great detail with video-enhanced Nomarski optics61. Types of cell death other than apoptosis have not yet been studied in the zebrafish; however, there is little doubt that they occur, and that they might help solve the problem of which mechanism(s) are responsible for animal embryonic cell death — for example, no clear sign of apoptosis can be seen during involution of the hatching gland, between three and four days post-fertilization, at which point massive cell disappearance occurs (P. Herbomel, personal communication). So far, the molecular mechanisms of PCD in the zebrafish have not been studied in great detail — one reason might be that this model is not well suited for biochemical approaches, because of its small size and the relative lack of in vitro cell lines. However, as can be expected from a vertebrate, many genes involved in cell death in mammals have orthologues in zebrafish62, which, as far as is known, perform very similar functions63. Candidate gene mutants, especially if temperature-sensitive, can help unravel the molecular mechanisms that are at play. NEMATOCYTE

Stinging cell found in Hydra that is used for capturing prey and for defence. Nematocytes differentiate from interstitial cells and are mainly found in the tentacles.

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Hydra. Hydra — for example, Hydra vulgaris — is a small freshwater polyp, about 1 cm in height, and has a simple morphology (FIG. 4). Hydra comprises a tube with an adherent foot at one end and, at the other end, a head with a ring of tentacles that surrounds the only


opening of the animal. The tube walls have only two epithelial cell layers, the ectoderm and the endoderm, with a gelatinous matrix in between them. Some specialized cells, such as nerve cells or the stinging NEMATOCYTES, are embedded into the epithelia. Hydra is quite easy to rear in the laboratory. It reproduces mostly by a vegetative process, with new polyps budding at a regular rate (every two days in optimal conditions) from the main body column. Hydra does not seem to age64, and, like its mythical counterpart, has an amazing capacity for regeneration. If the head or the foot is removed, the remaining polyp will rebuild the missing parts in just a few days. This repatterning is associated with a transient loss of nematocytes65. Hydra tissues are in continual flux, with the death of some cells being counterbalanced by the continuous proliferation of others. A remarkable feature of this constitutive cell turnover in Hydra is that it slows down, but does not stop, if the animal is starved. This is probably related to the extraordinary ability of Hydra to withstand starvation. A starved Hydra can survive for months, slowly decreasing in size. Importantly, this shrinkage is due to a decrease in cell numbers, not in cell size 66. Genetically speaking, Hydra is not a tractable organism. Its genome is fairly large (1,600 Mb) for such a simple animal; it is therefore not considered for genome sequencing, but there is an EST (expressed sequence tag) sequencing programme under way. Mutagenesis is also not an easy option, for the time being. However, prospects are becoming brighter, as protocols have been proposed for RNA interference-mediated gene silencing67 or transient transfection68. In Hydra, cell death exhibits some of the classical hallmarks of apoptosis, including chromatin condensation and DNA laddering. It is also associated with caspase activation69. Dead cells, which are easily visualized with acridine orange, are always seen in phagocytic vacuoles of neighbouring epithelial cells (FIG. 4). This phagocytosis of apoptotic cells allows the recycling of energy and other cell constituents, and is therefore linked to the regenerative and starvation-resistance abilities of Hydra. It should be possible, in Hydra, to measure the economic efficiency of apoptotic cell death, and perhaps even to compare it with non-apoptotic death if apoptosis can be inhibited with suitable drugs. This is because starvation offers a situation in which the external energy inputs can be precisely controlled and the numbers of dying and proliferating cells in the whole organism can be measured. Another question that can be addressed is how decisions are made as to which cells are to die for the sake of others, and which must be spared. Using the regeneration-induced transient loss of nematocytes as a model, we can take advantage of the simple cellular organization of a polyp and of the possibility of quantifying the number of cells of each cell type. Podospora. Questions regarding the mechanisms of autophagic vacuolar cell death could be explored in the non-animal model organisms Dictyostelium and



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ARTICLE

A multinucleated segment of a filament limited by septa in filamentous fungi. PROTOPLAST

For a cell with an external cell wall, the protoplast is what is left (protoplasm and plasma membrane) after the cell wall has been removed. HETEROKARYON

A cell with two or more genetically different nuclei, produced by the fusion of genetically different cells.

Podospora. Podospora anserina, like all filamentous fungi, consists of a network (known as mycelium) of filaments (FIG. 5). Each of these filaments, which are 2–4 µm thick, consists of aligned multinucleated cells (known as ARTICLES) that are separated by incomplete septa. The cell that is located at the end of a filament elongates, divides and thereby ensures growth of the filament. A mycelium can be cut in small pieces, each of which can regenerate new mycelium when placed on growth medium in a Petri dish. Podospora anserina, like all filamentous fungi, has a small genome (34 Mb). As Podospora has been used for genetic studies for many years, a detailed genetic map is available. Genetic analysis, mutagenesis, complementation studies, segregation tests and many types of molecular-biology approaches are feasible. Plasmids that carry different selective markers are available for transforming PROTOPLASTS. A programme has been initiated to determine the complete sequence of the Podospora anserina genome. Different individuals can undergo filament fusion with each other, forming a vegetative HETEROKARYON. However, the fusion of filaments from some genetically different strains can lead to cell death of the resulting heterokaryons. Cell death of many heterokaryons, when two mycelia are confronted in a Petri dish, leads to ‘barrage’ formation70. This vegetative incompatibility involves a number of genes71,72 that have been intensively studied and that control incompatibility and/or are induced by incompatibility. Interestingly, some of the latter include homologues of genes that are involved in autophagy in yeast (such as pspA, which is similar to the yeast PRB1 gene, which encodes the vacuolar protease B that is involved in the degradation of autophagic bodies; and idi-7, which is orthologous to AUT7/apg8), thereby supporting the involvement of autophagy in Podospora cell death73. Similar vacuolar cell death associated with heterokaryon incompatibility has been described in other fungi, such as Neurospora crassa 74. The cytological alterations that occur during the incompatibility reaction are similar to those that are observed during starvation or rapamycin treatment (rapamycin mimics starvation by indirectly interfering with the protein kinase TOR (target of rapamycin)), all indicating autophagic vacuolar cell death73,75. The knowledge of genes that control vegetative incompatibility and the genetic manipulations that are possible in Podospora have enabled the construction of useful genetic tools, such as the thermosensitive auto-incompatible strain het-R het-V76, which, when the temperature is shifted from 32°C to 26°C, undergoes generalized vacuolar cell death (FIG. 5). Dictyostelium. Dictyostelium discoideum is a protist — a slime mould — which emerged in evolution after divergence of the kingdom Plantae, and before individualization of the kingdoms Animalia and Fungi77 (FIG. 2). When grown in rich medium, Dictyostelium multiplies vegetatively as a unicellular organism. Starvation triggers multicellular development, as isolated Dictyostelium cells aggregate, differentiate and develop into 1–2-mm high multicellular fruiting bodies. Each of these contains a mass of spores that are supported by a stalk. Cells in the stalk are


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Figure 6 | Dictyostelium discoideum. a | A plaque of 0.5 cm diameter that results from the centrifugal growth of the descendants of one Dictyostelium cell feeding on a bacterial layer on agar. The peripheral fold is the growing Dictyostelium front. This approach, which is similar to that for making phage plaques in bacteria, can be used, for example, to enumerate living Dictyostelium cells. Moreover, as a result of Dictyostelium feeding, the centre of such plaques becomes depleted of bacteria, leaving Dictyostelium cells in starvation conditions, which induce them to develop into multicellular structures. Such developing plaques can be easily screened, for example, for developmental mutants. b | A complete fruiting body, which represents an end-point of Dictyostelium development, as shown by scanning electron microscopy. This fruiting body, which is 1–2 mm in length, includes a spheroidal mass of spores at the top of a stalk that is made of dead or dying cells. c | In a monolayer system that mimics stalk-cell development, vegetative cells (top) are induced to differentiate, through a sequence of events, into vacuolated dead cells (bottom). The scanning electron micrograph is courtesy of Mireille Bof and Michel Satre, Commissariat à l’Energie Atomique, Grenoble, France.

considered to be dead on the basis of non-regrowth when these cells are incubated in rich medium78. The genome of Dictyostelium is small (~34 Mb), almost completely sequenced79 and haploid. Genome haploidy makes it relatively easy to generate and select mutants of function-associated genes and to identify the latter 80–82. Because of the temporal separation between vegetative growth and development, developmental mutants (such as those related to cell death) can be propagated under vegetative conditions, and which therefore behave like conditional mutants80. More generally, Dictyostelium has been intensively used as a model system to study basic cellular and developmental processes, both at the unicellular stage (chemotaxis and phagocytosis) and at the multicellular level (pattern formation and



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Figure 7 | Volvox. a | The parent spheroid is a translucent sphere of