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Autophagy 7:5, 557-559; May 2011; © 2011 Landes Bioscience

Autophagy and apoptosis are redundantly required for C. elegans embryogenesis Éva Borsos, Péter Erdélyi and Tibor Vellai* Department of Genetics; Eötvös Loránd University; Budapest, Hungary

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poptosis, the main form of regulated (or programmed) cell death, allows the organism to tightly control cell numbers and tissue size, and to protect itself from potentially damaging cells. This type of cellular self-killing has long been assumed to be essential for early development. In the nematode Caenorhabditis elegans, however, the core apoptotic cell death pathway appears to be dispensable for embryogenesis when most developmental cell deaths take place: mutant nematodes defective for apoptosis develop into adulthood, with superficially normal morphology and behavior. Accumulating evidence indicates a similar situation in mammalian systems as well. For example, apoptosis-deficient mice can grow as healthy, fertile adults. These observations raise the possibility that alternative cell death mechanisms may compensate for the lack of apoptotic machinery in developing embryos. Interestingly, C. elegans embryogenesis can also occur without autophagy, an alternative form of cellular self-destruction (also called autophagic cell death). In an upcoming paper we report that simultaneous inactivation of the autophagic and apoptotic gene cascades in C. elegans arrests development at early stages, and the affected embryos exhibit severe morphological defects. Doublemutant nematode embryos deficient in both autophagy and apoptosis are unable to undergo body elongation or to arrange several tissues correctly. This novel function of autophagy genes in morphogenesis indicates a more fundamental role for cellular self-digestion in tissue patterning than previously thought.

Of the 1,090 cells that are generated during development of the C. elegans hermaphrodite soma, 131 undergo programmed cell death. As these dying cells are highly refractile at certain stages under differential interference contrast optics, they can be readily identified; the identity of the dying cells and the time in development at which these cells die are essentially invariant among individuals.1,2 At the molecular level, in cells committed to die, the transcription of the egl-1 (egg-laying defective-1) gene coding for a BH3-only protein is first activated. EGL-1 then binds to the Bcl-2 (B cell lymphoma-2)-like anti-apoptotic protein CED-9 (cell death defective-9), which normally protects cells from undergoing apoptosis. EGL-1 binding to CED-9 leads to the subsequent activation of the pro-apoptotic proteins CED-4 and CED-3, the nematode orthologs of mammalian Apaf1 and caspases, respectively. The activity of CED-4 and CED-3 is required for executing apoptotic cell suicide. Under normal growth conditions, loss-of-function mutations in ced-4 and ced-3 allow normal development, and the homozygous single mutant adults exhibit a superficially wild-type phenotype (note that CED-4 is required for both caspasedependent and -independent cell death).3 Why does C. elegans maintain this complicated molecular machinery if the lack of developmental cell death is not detrimental for its viability, morphogenesis and behavior? A simple answer may stem from the cell lineage-specific mode of nematode development: In apoptosis-defective mutant nematodes, the remaining extra cells that would have died in the wild type do not interfere with major morphogenetic

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Key words: autophagy, apoptosis, development, viability, embryogenesis, C. elegans Submitted: 12/28/10 Revised: 12/30/10 Accepted: 12/30/10 DOI: 10.4161/auto.7.5.14685 *Correspondence to: Tibor Vellai; Email: [email protected]

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events. Alternatively, another form of programmed cell death may work in parallel with apoptosis in this organism.4 Indeed, the fate of cells that normally undergo apoptosis yet remains to be determined in an apoptosis-deficient mutant background. It is possible that at least some of these surviving cells may eventually be eliminated from ced-4 and ced-3 mutants via a non-apoptotic mechanism. Autophagy is a highly conserved lysosome-mediated self-degradation (catabolic) process of eukaryotic cells, which primarily promotes survival in terminally differentiated cells.5-7 Under certain developmental and pathological settings, however, it can promote cell death (called autophagic cell death), thereby representing an alternative form of developmentally regulated cell loss.8 For example, elimination of larval organs during Drosophila metamorphosis occurs by a caspase-independent process, which can be characterized by the intense formation of (macro) autophagic structures (i.e., double membrane-bound small intracellular vesicles).9 The C. elegans genome also encodes several conserved autophagy-related (ATG) genes, and various cell types of the worm are capable of forming typical autophagosomes and autolysosomes. Thus, autophagy is likely to operate in a conserved way in this organism too. An intriguing feature of the C. elegans autophagic system is that inactivating (hypomorphic and amorphic) mutations in atg genes do not affect embryonic development and viability. The only known exception is bec-1 that is similar to yeast ATG6 and mammalian beclin 1. Without maternal contribution, mutational inactivation of bec-1 arrests nematode development at different stages of embryogenesis.10,11 BEC-1, however, is a multifunctional protein that may also function independently of autophagy in the developing embryo. Mutational inactivation of unc-51/ATG1 (unccordinated-51), atg-11 and atg-18 each can allow the animal to develop as healthy, fertile adults (only a small portion—less than 2–4% —of these mutants die as embryos).12 Although elimination of atg-7 or lgg-1/ATG8 (LC3, GABARAP and GATE-16 family-1) activity by deletion alleles terminates development at early larval stages, embryogenesis in these mutants can happen normally.

Is C. elegans a specific organism that can develop without any major form of programmed cell death? To address this issue, we generated double-mutant nematodes that are deficient in both apoptosis and autophagy.13 We first crossed ced-4(n1162) and atg18(gk378) genetic null (loss-of-function) mutant animals (the vast majority of either homozygous single mutants develop into adulthood, and the mutant adults are not distinguishable from the wild type under a dissecting microscope) to produce ced-4; atg-18 homozygous double mutants [genotype: ced-4(-/-); atg-18(-/-)]. The first generation of homozygous double mutants (they were generated by a hermaphrodite of genotype ced-4(-/-); atg-18(+/-); i.e., being heterozygous for the atg-18 mutation) was able to grow as adults, but produce dead embryos only. The arrested embryos (the second generation of homozygous double mutants) displayed severe defects in morphology as they were unable to undergo body elongation and could not pattern several tissues correctly (Fig. 1A). Body malformation in these embryos was partially due to the formation of large vacuoles at various positions. Viability of the first double-mutant generation is likely to result from maternal effect, which characterizes the inheritance of atg genes in C. elegans. Using a hypomorphic (reduction-of-function) mutation in ced4, n2273, we obtained a similar effect, but with a partially penetrant embryonic lethal phenotype in the second generation of homozygous double mutants. We also used mutations in other atg genes, including unc-51/ATG1, atg-7 and lgg-1/ATG8, to inactivate the autophagic pathway in an apoptosis defective background and ced-3 mutations to downregulate apoptosis in an autophagy-deficient mutant background. Both combinations were able to recapitulate embryonic lethality and morphological malformations in the second generation of homozygous double mutant (i.e., autophagy-apoptosis defective) animals. These results indicate that the apoptotic and autophagic gene cascades share essential developmental functions during C. elegans embryogenesis. This may explain why single mutants defective for either of the two processes can pass embryonic development.

The relationship between autophagy and apoptosis is well established, but how the two processes are related to each other during animal development is still largely unknown. In C. elegans, BEC-1 is able to bind to CED-9 and inactivation of bec-1 leads to an increase in apoptotic cell death in the early embryo. Although in some bec-1 mutant embryos even though nearly all cells exhibit apoptotic (refractile) features,10 one can argue that the elevated number of apoptotic cell corpses observed in this autophagy-deficient system is simply a consequence of failure in the heterophagic elimination of dying cells,5 a process that also requires autophagy gene function. Besides this problem, it would be important to study whether defects in apoptosis can also enhance autophagic activity. In our upcoming paper we showed that excessive accumulation of autophagy proteins is accompanied with ectopic cell death and obvious tissue degeneration in C. elegans embryos.13 Moreover, we found that refractive apoptotic cell corpses often display intense expression of bec-1. Thus, whether autophagy proteins protect against (at basal levels) or mediate (at elevated levels) apoptosis is still a question that should be addressed in the near future. How can we explain the synthetic embryonic lethal phenotype of doublemutant nematodes defective for both autophagy and apoptosis? An explanation could be that at least a portion of cells that would normally undergo apoptosis may be eliminated by autophagic cell death in the apoptosis-deficient mutant background.4 However, when autophagy is also blocked, the remaining extra cells may disrupt key morphogenetic events, thereby causing the death of the developing organism. A careful cell lineage analysis of ced-4 null mutants will certainly clarify this problem. This alternative predicts redundant (shared) roles for the autophagic and apoptotic gene cascades during embryonic development (Fig. 1B). Another possibility is that regulated loss of certain (unnecessary) somatic cells may provide cytoplasmic components and energy for those remaining cells to survive. In the absence of apoptotic cell death, autophagy may become overactivated in order to compensate for this deficit in the cells’

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energetic status. Parallel inactivation of autophagy may trigger an alternative (e.g., necrotic) cell death program in “starving” cells, which eventually leads to a catastrophe in certain cell lineages. As a third alternative, it is also possible that a combination of the above-mentioned options operates in autophagy-apoptosis double-mutant embryos. Nevertheless, the potential answers will substantially move forward the field of cell death research and developmental biology. Acknowledgements

This work was supported by grants from the Ministry of Health (ETT 142/2009), the Hungarian Scientific Research Funds (OTKA: K68372, K75843 and NK78012), and the National Office for Research and Technology (TECH_08_ A1/2-2008-0106). T.V. is a grantee of the János Bolyai scholarship.

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Figure 1. The apoptotic and autophagic gene cascades interact during C. elegans development. (A) Simultaneous inactivation of autophagy and apoptosis arrests development at various stages of embryogenesis. Upper parts: apoptosis-deficient ced-4(n1162) and autophagy-deficient atg18(gk378) single mutant embryos at different stages. Gross morphology of these mutant embryos appears to be normal. Elongation of their body occurs normally. Bottom parts: ced-4(n1162); atg-18(gk378) double-mutant embryos defective for both apoptosis and autophagy at different stages. Obvious body malformations (e.g., defects in body elongation) characterize these specimens. Bars indicate 20 μm. (B) Models (two alternatives) for the regulation of programmed cell death by apoptosis and autophagy in C. elegans. Arrows indicate activations, bars represent inhibitory interactions. Excessive cell death or the loss of programmed cell death (dotted grey lines) can lead to morphological malformations and embryonic lethality. On the bottom, the lack of apoptosis and autophagy triggers the induction of an alternative cell death mechanism(s), such as necrosis.

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