Cell Death in Development

Physiological mechanisms of cell death are used by multicellular organisms for development and morphogenesis, to control cell number, and as a defensive strategy to remove infected, mutated, or damaged cells. Cell death is required for the normal development of almost all multicellular organisms and commonly involves the production of excess cells and removal of those that are superfluous. Frequently in plants, and occasionally in other organisms, cytoplasmic or structural components of the dead cell serve important functions. The process of cell death used by metazoans for development is highly conserved and is morphologically recognizable as apoptosis. The evolution of multicellularity and cell specialization brought with it a need for the regulation of cell death by intercellular signaling. The mechanisms that implement developmental apoptosis may have originated from cell-autonomous cell death processes used for defense. While the apoptosis effector mechanisms have been extensively characterized, understanding of the pathways that signal and control developmental cell death is far from complete. When a cell in an organism dies due to a process encoded by that organism for the purpose of killing its own cells, that death can be considered to be a physiological process. The great majority of our cells are destined to die by just such a mechanism; relatively few die through injury or inability to sustain their own viability. In a human about a hundred thousand cells are produced every second by mitosis, and a similar number die by a physiological suicide process known as apoptosis. Most of the cells produced during mammalian embryonic development undergo physiological cell death before the end of the perinatal period. During our life span, over 99.9% of our cells undergo the same fate. That cell death occurs in a predictable “programmed” fashion in physiological circumstances was first recognized by Carl Vogt who saw dying cells in the neuronal system of developing toad embryos (85Vogt C Untersuchungen über die Entwicklungsgeschichte der Geburtshelferkroete (Alytes obstertricians). Jent und Gassman, Solothurn, Switzerland1842Google Scholar). (However, the very first cells described, those from cork and named “cells” by Hooke in 1665, were also corpses that had died physiologically.) Developmental biologists were also quick to realize that cell death was involved in the process of metamorphosis, both in insects and mammals. For example, Lockshin coined the phrase “programmed cell death” in 1965 to describe cell death in insect metamorphosis (53Lockshin R Williams C Programmed cell death. II. endocrine potentiation of the breakdown of the intersegmental muscles of silkworms.J. Insect Physiol. 1965; 11: 803-809Crossref PubMed Scopus (67) Google Scholar), and a year later Tata showed that loss of the tadpole’s tail involved cell death that could be blocked by cycloheximide and therefore required expression of endogenous genes (1966). When Kerr, Wyllie, and Currie saw that the morphology of liver cells exposed to toxins, and lymphocytes treated with hormones, was the same as that described by the embryologist Glucksmann earlier in the century, they coined a new term, “apoptosis,” for cell deaths with this distinct morphology—whether they be in response to physiologic or pathologic stimuli (29Glucksmann A Cell deaths in normal vertebrate ontogeny.Biol. Rev. 1951; 26: 59-86Crossref Scopus (864) Google Scholar, 44Kerr J.F Wyllie A.H Currie A.R Apoptosis a basic biological phenomenon with wide-ranging implications in tissue kinetics.Brit. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12238) Google Scholar). Although the first component of a cell death mechanism to be recognized was the mammalian gene Bcl2 (82Vaux D.L Cory S Adams J.M Bcl2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells.Nature. 1988; 335: 440-442Crossref PubMed Scopus (2634) Google Scholar), the first evidence that a genetic program existed purely for physiological cell death came from studying development in C. elegans (38Horvitz H.R Ellis H.M Sternberg P.W Programmed cell death in nematode development.Neurosci. Commentaries. 1982; 1: 56-65Google Scholar, 21Ellis H.M Horvitz H.R Genetic control of programmed cell death in the nematode C. elegans.Cell. 1986; 44: 817-829Abstract Full Text PDF PubMed Scopus (1302) Google Scholar). The ability of human Bcl2 to prevent programmed cell death in C. elegans showed that apoptosis in mammalian cells and programmed cell death in the nematode were the same highly conserved process (83Vaux D.L Weissman I.L Kim S.K Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2.Science. 1992; 258: 1955-1957Crossref PubMed Scopus (502) Google Scholar). While most developmental cell deaths in metazoans are mediated by a family of cysteine proteases termed caspases (1Alnemri E.S Mammalian cell death proteases—a family of highly conserved aspartate specific cysteine proteases.J. Cell. Biochem. 1997; 64: 33-42Crossref PubMed Scopus (280) Google Scholar), there are a number of other ways, some of which are physiological, in which cells can die. For example, if the gene for the caspase CED-3 is mutated in C. elegans, death of the 131 somatic cells that are normally programmed to die during development does not occur (21Ellis H.M Horvitz H.R Genetic control of programmed cell death in the nematode C. elegans.Cell. 1986; 44: 817-829Abstract Full Text PDF PubMed Scopus (1302) Google Scholar), but cells in these animals can still die if a metabolic process required for their survival is blocked. This may be due to an external event, such as a scientist’s laser beam, addition of a poison, or lack of a nutrient, or due to an intrinsic defect such as mutation of an enzyme needed to generate ATP, or expression of an altered gene product that is toxic to the cell. Thus, gain-of-function mutations in components of the sodium channels MEC-4 and DEG-1 cause death of cells in C. elegans that are independent of CED-3, but due to malfunctioning of the plasma membrane (34Hall D.H Gu G.Q Garciaanoveros J Gong L Chalfie M Driscoll M Neuropathology of degenerative cell death in Caenorhabditis elegans.J. Neurosci. 1997; 17: 1033-1045Crossref PubMed Google Scholar). Examples of the various forms of physiological and nonphysiological cell death are listed in Table 1.Table 1Physiological and Nonphysiological Cell Death MechanismsType of Cell DeathExamplesImplicated MechanismsUsesApoptosisPCD in worm, death of mammalian cells due to removal of growth factorsCaspase effectors, signaling by homophilic adaptors using DDs, DEDs, and CARDsDevelopment, defense, homeostasisPhysiological killingC. elegans male linker cell?DevelopmentCTL killinggranzyme B perforinDefenseAgingAging in animals, death of plants in winter?Removal of parental generationDeath of somatic cells in Volvox?Terminal differentiationMammalian erythrocytes, keratinocytes?Allows recycling of DNAPlant defense against microorganismsHypersensititive responseSignaling by Toll/IL-1R like proteins (e.g., downy mildew resistance protein RPP5), or S/T kinases (e.g., PTO, FEN)DefenseDevelopmental cell death in plantsFormation of the xylem, holes in Monstera leaves?DevelopmentToxic/nonphysiologicalMEC-4, DEG-1 mutantsL.o.f. of essential metabolic components; g.o.f. mutation yielding toxic productse.g., diphtheria toxin, methotrexate, antibiotics, azideBlock to vital metabolic pathway (e.g., transcription, ATP generation, DNA synthesis) Open table in a new tab While interruption of a vital metabolic process will inevitably lead to cell death irrespective of whether physiological cell suicide processes are activated, in many instances analysis of the mechanisms of cell death is complicated because cells experiencing potentially lethal metabolic changes often react by activating their physiological death mechanisms, to commit suicide before they are killed (81Vaux D.L Hacker G Hypothesis—apoptosis caused by cytotoxins represents a defensive response that evolved to combat intracellular pathogens.Clin. Exp. Pharmacol. Physiol. 1995; 22: 861-863Crossref PubMed Scopus (20) Google Scholar). That cell suicide frequently occurs as a response to stress may explain why so many agents (several thousand) with diverse biochemical activities have been found to induce apoptosis. Thus, while many cancer chemotherapeutic agents will elicit an apoptotic response from cells, induction of apoptosis is often not their primary mechanism. In several organisms, including vertebrates, some physiological cell deaths occur independently of caspases. Keratinocytes extrude their nuclei as part of their normal program of differentiation and are eventually shed from the skin. Mammalian red blood cells survive for 120 days after they have lost their nuclei to finally succumb when they are phagocytosed. Caspases do not appear to be involved in the eventual death of these enucleated cells and may not be involved in the death of cells with inactive nuclei such as sperm and chicken erythrocytes (88Weil M Jacobson M.D Raff M.C Are caspases involved in the death of cells with a transcriptionally inactive nucleus? Sperm and chicken erythrocytes.J. Cell Sci. 1998; 111: 2707-2715PubMed Google Scholar). In C. elegans the male linker cell dies not by suicide, but non–cell autonomously (21Ellis H.M Horvitz H.R Genetic control of programmed cell death in the nematode C. elegans.Cell. 1986; 44: 817-829Abstract Full Text PDF PubMed Scopus (1302) Google Scholar). Like the targets of vertebrate cytotoxic “killer” T lymphocytes (71Russell J.H Dubois C.B Mechanisms of immune lysis. II. CTL-induced nuclear disintegration of the target begins within minutes of cell contact.J. Immunol. 1980; 125: 1256-1261PubMed Google Scholar), it is “murdered” by another cell. As these deaths occur due to a host process that has evolved for the killing of its own cells, these too are physiological cell deaths. In some circumstances, normal deaths of cells may be by a mixture of direct cell suicide and indirect nonphysiological cell deaths. Loss of leaves by plants in winter, loss of antlers by deer, or loss of a lizard’s tail to distract a predator are physiological processes that involve the death of large numbers of cells. While some cells at the line of abscission may die cell autonomously, other cells may die indirectly following loss of their supply of nutrients. Sometimes it is hard to classify a protein as a “cell death” protein or not. Even though some caspases have roles unrelated to apoptosis, such as activation of cytokines (47Kronheim S.R Mumma A Greenstreet T Glackin P.J Van Ness K March C.J Black R.A Purification of interleukin-1 beta converting enzyme, the protease that cleaves the interleukin-1 beta precursor.Arch. Biochem. Biophys. 1992; 296: 698-703Crossref PubMed Scopus (39) Google Scholar, 78Thornberry N.A Bull H.G Calaycay J.R Chapman K.T Howard A.D Kostura M.J Miller D.K Molineaux S.M Weidner J.R Aunins J et al.A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes.Nature. 1992; 356: 768-774Crossref PubMed Scopus (2095) Google Scholar), most seem to have no role other than in cell death, so it is clear they are cell death proteins. On the other hand, the signal transduction pathways that regulate cell death often have important roles in other cellular processes. Is every molecule that can transmit signals influencing cell survival a cell death protein? What if cell death results from inactivation of a nutritional pathway? While it has not yet been observed, it is plausible that cells may die by starving themselves to death, for instance, by turning off their glucose transporters. While this kind of cell death must be considered to be physiological if it were genetically programmed, the primary role of the protein may be intended for another process. The finding that cell death probably occurs in all multicellular organisms, and the same mechanisms operate throughout the metazoa, raises the questions of how these mechanisms evolved, whether plants and animals use cell death mechanisms with a common origin, and whether these mechanisms evolved in single-celled organisms. The answer to these questions will require identification of the gene products responsible for cell death in each case. If plants and animals do turn out to share a cell death effector mechanism, it must have evolved in their common ancestor, which presumably had only one cell. Is it possible for cell suicide to have evolved in single-celled organisms? While it is true that only a multicellular organism would have cell death as an obligatory program, single-celled organisms could use suicide as a contingent, defensive strategy. For example, to avoid starvation or spread of infection, death of some (but not all) single-celled organisms within a gene pool could be advantageous to the group as a whole. Currently there is no strong evidence that effector mechanisms related to those that mediate apoptosis in mammalian cells exist in single-celled organisms or in plants, but there are a number of examples of physiological cell deaths in these organisms, suggesting that cell suicide may have evolved as an adaptive strategy on a number of occasions. In considering the physiological cell death among unicellular organisms, it is important to recognize that even the definition of “single-celled” is not trivial. During sporulation the bacterium Bacillus subtilus forms two cells, but only the spore cell survives (25Errington J Determination of cell fate in Bacillus subtilis.Trends Genet. 1996; 12: 31-34Abstract Full Text PDF PubMed Scopus (44) Google Scholar). Is this an example of cell death in a single-celled organism, or is it cell death in a two-celled organism? The yeast S. cerevisiae buds asymmetrically, and the mother cell appears to be programmed to die after giving rise to twenty or so daughter cells. Is this kind of cell death restricted to asymmetrical or asexual divisions? Many yeasts can form pseudohyphae, so are they multicellular after all? Cilliates have two nuclei, a micronucleus and a macronucleus, so are they strictly unicellular? After mating, when cells exchange haploid micronuclei, the old macronucleus is destroyed, and its DNA is degraded (65Prescott D The DNA of ciliated protozoa.Microbiol. Rev. 1994; 58: 233-267Crossref PubMed Google Scholar). Is this murder, suicide, or something else? There is evidence for cell suicide among bacteria that are physiological in some sense. E. coli can harbor plasmids encoding both toxins and their antidotes. If a bacterium is “cured” of the plasmid, the antidote decays faster than the toxin, and the cell dies (41Jensen R.B Gerdes K Programmed cell death in bacteria—proteic plasmid stabilization systems.Mol. Microbiol. 1995; 17: 205-210Crossref PubMed Scopus (267) Google Scholar). While this may be more properly thought of as a host–parasite relationship, what if the plasmid is integrated into the host’s genome? Phage exclusion is an altruistic response that limits multiplication of a phage and their spread through a bacterial population. For example, the e14-encoded Lit protein in many E. coli strains allows exclusion of T4 phage. A region of the major head coat protein gene of the phage triggers proteolysis of translation elongation factor Tu (EF-Tu), blocking translation, thereby preventing multiplication of the phage, and causing death of the bacterium (28Georgiou T Yu Y Ekunwe S Buttner M.J Zuurmond A.M Kraal B Kleanthous C Snyder L Specific peptide-activated proteolytic cleavage of Escherichia coli elongation factor tu.Proc. Natl. Acad. Sci. USA. 1998; 95: 2891-2895Crossref PubMed Scopus (36) Google Scholar). When food is plentiful, the slime mould Dictyostelium discoideum multiplies as a unicellular organism, but when starved the individual cells aggregate to form a multicellular slug that produces a spore body on top of a stalk consisting of dead cells. As caspase inhibitors were not able to affect stalk cell death, Dictyostelium has presumably evolved some other effector mechanism for cell death (63Olie R.A Durrieu F Cornillon S Loughran G Gross J Earnshaw W.C Golstein P Apparent caspase independence of programmed cell death in Dictyostelium.Curr. Biol. 1998; 8: 955-958Abstract Full Text Full Text PDF PubMed Google Scholar). Like animals, plants use physiological cell death for development and for defense (30Greenberg J.T Programmed cell death—a way of life for plants.Proc. Natl. Acad. Sci. USA. 1996; 93: 12094-12097Crossref PubMed Scopus (486) Google Scholar), so it may be that any complex multicellular organism will find cell death a useful process to have in its repertoire. There are many examples of programmed cell death during plant development, such as death of cells that comprise the vascular system, the xylem and phloem. Programmed cell death also occurs in the leaves of several species to form holes or to allow fronds to separate. As in metazoans, death of gametes occurs commonly and in physiological circumstances (2Bell P.R Megaspore abortion—a consequence of selective apoptosis.Int. J. Plant Sci. 1996; 157: 1-7Crossref Scopus (60) Google Scholar, 13Christensen C.A Subramanian S Drews G.N Identification of gametophytic mutations affecting female gametophyte development in Arabidopsis.Dev. Biol. 1998; 202: 136-151Crossref PubMed Scopus (128) Google Scholar). Both loss of leaves in autumn and formation of bark are physiological cell death processes. The hypersensitive response in plants is used as a defense against invasion by microorganisms and involves production of antimicrobial products and death of host cells where the threat is detected. When a leaf on a tomato plant detects the product of the avrPTO gene of the bacterium Pseudomonas syringae, a signaling pathway is activated, beginning with the serine/threonine protein kinase PTO (99Zhou J.M Tang X.Y Frederick R Martin G Pathogen recognition and signal transduction by the pto kinase.J. Plant Res. 1998; 111: 353-356Crossref Google Scholar). Although proteins such as PTO resemble metazoan protein kinases, to date no caspases or Bcl2 homologs have been cloned from plants, so they may have evolved their cell death mechanisms independently. Nevertheless, peptides designed to inhibit caspases can influence the hypersensitivity response of tobacco leaves (18Delpozo O Lam E Caspases and programmed cell death in the hypersensitive response of plants to pathogens.Curr. Biol. 1998; 8: 1129-1132Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar), but it is not yet clear whether they are targeting proteases that are evolutionarily related to metazoan caspases. Just as plants use cell death as a defense against infection, so do metazoans. Since viruses are obligate intracellular parasites that need to use the host cell’s synthetic machinery in order to replicate, early death of the host cell is an ideal way to limit viral replication and spread. Metazoans have evolved cell suicide mechanisms to counter viruses, and viruses have evolved strategies to ensure their survival. Study of viruses that infect insects and vertebrates has shown that they carry inhibitors of many steps of the apoptosis process. These include homologs of Bcl2, direct inhibitors of activated caspases, molecules that prevent caspase activation by adaptors, and inhibitors of p53. The fact that these viral cell death inhibitors target the same mechanisms that are used for developmental cell death strongly argues that the defensive and developmental apoptotic processes have a common origin, and it also questions the prevailing dogma that inflammation is not associated with apoptosis. Indeed, inflammation is associated with apoptosis triggered by viruses and cytokines such as TNF and IFNγ, and apoptosis triggered during graft rejection. As inflammation does not occur with developmental or homeostatic cell death, apoptosis may have initially evolved as a defense mechanism, but later, perhaps with the advent of multicellularity, it was adopted for use during development, in which case inflammation was not required. Aging can also be considered to be a physiological cell death process. For many plants, and most animals, life span is genetically predetermined. For example, tomato plants senesce and die before they are 1 year of age, mice live for no more than 3 years, no matter how well they are treated, and humans rarely live beyond 100 years. In the life cycle of the algae Volvox, adult somatic cells synchronously die to release the daughter colonies (45Kirk, D.L. (1994). Germ cell specification in Volvox carteri. Ciba Foundation Symposium 182, 2–15.Google Scholar). Is this an example of programmed cell death, aging, or both? The life span of C. elegans (3 weeks) is not determined by the genes that implement apoptosis, since ced-3 mutant worms senesce and die on cue (21Ellis H.M Horvitz H.R Genetic control of programmed cell death in the nematode C. elegans.Cell. 1986; 44: 817-829Abstract Full Text PDF PubMed Scopus (1302) Google Scholar). Presumably the mechanisms that determine life span evolved independently, after the evolution of multicellular organisms with separate germ and somatic cell lineages. If this is true, it is unlikely that life span in plants and animals will be determined by related mechanisms. The eusocial hymenopterans (ants, bees, and wasps) display a particularly striking dimorphism in their life span. Although embryonic honey bees have equivalent potential when laid, those reared as workers have a life span of 30–40 days, whereas those reared as queens can live for several decades (91Wilson E.O The insect societies. Belknap Press, Cambridge, MA1971Google Scholar). Clearly cell death is involved in aging and is a genetically determined process that in some organisms can be radically altered by the environment, but the genes involved and the nature of their action are still largely mysterious (see the review by 43Johnson B.F Sinclair D.A Guarente L Molecular biology of aging.Cell. 1999; 96 (this issue,): 291-302Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar in this issue of Cell). The physiological cell death process that is best understood at the molecular level is the process of apoptosis, which was initially described on morphological grounds in vertebrate cells (44Kerr J.F Wyllie A.H Currie A.R Apoptosis a basic biological phenomenon with wide-ranging implications in tissue kinetics.Brit. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12238) Google Scholar) and was later linked to the genetic pathways of programmed cell death among invertebrate metazoans (14Clem R.J Fechheimer M Miller L.K Prevention of apoptosis by a baculovirus gene during infection of insect cells.Science. 1991; 254: 1388-1390Crossref PubMed Scopus (687) Google Scholar, 83Vaux D.L Weissman I.L Kim S.K Prevention of programmed cell death in Caenorhabditis elegans by human bcl-2.Science. 1992; 258: 1955-1957Crossref PubMed Scopus (502) Google Scholar). Genetic and, more recently, biochemical studies have revealed much of the effector mechanisms of apoptosis, but the details of most of the signaling pathways that can trigger apoptosis are poorly understood. The basic scheme of the apoptosis effector mechanism is depicted in Figure 1, which compares homologous proteins in nematode and vertebrate systems. The key effector components of apoptosis are caspases, a family of cysteine proteases that cleave their substrates after aspartate residues. There are about a dozen mammalian caspases that exist in cells as inactive zymogens. Once caspases are activated, they cleave a large number of proteins within the cell, causing its demise and resulting in the morphological changes of apoptosis (61Nicholson D.W Thornberry N.A Caspases—killer proteases.Trends Biochem. Sci. 1997; 22: 299-306Abstract Full Text PDF PubMed Scopus (2134) Google Scholar). 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For example, Caspase 8 is activated when death effector domains (DEDs) in its prodomain bind to the C-terminal DED in the adaptor FADD (5Boldin M.P Goncharov T.M Goltsev Y.V Wallach D Involvement of Mach, a novel Mort1/FADD-interacting protease, in Fas/Apo-1- and TNF receptor-induced cell death.Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2057) Google Scholar, 58Muzio M Chinnaiyan A.M Kischkel F.C O’Rourke K Shevchenko A Ni J Scaffidi C Bretz J.D Zhang M Gentz R et al.FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/Apo-1) death-inducing signaling complex.Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2657) Google Scholar). Caspase 9 is activated after association of the caspase recruitment domain (CARD) in its prodomain with the CARD in another adaptor protein, Apaf1 (37Hofmann K Bucher P Tschopp J The CARD domain—a new apoptotic signaling motif.Trends Biochem. 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In this way death activating signals can be passed from the plasma membrane via FADD to Caspase 8 (5Boldin M.P Goncharov T.M Goltsev Y.V Wallach D Involvement of Mach, a novel Mort1/FADD-interacting protease, in Fas/Apo-1- and TNF receptor-induced cell death.Cell. 1996; 85: 803-815Abstract Full Text Full Text PDF PubMed Scopus (2057) Google Scholar, 58Muzio M Chinnaiyan A.M Kischkel F.C O’Rourke K Shevchenko A Ni J Scaffidi C Bretz J.D Zhang M Gentz R et al.FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/Apo-1) death-inducing signaling complex.Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2657) Google Scholar). The structures of the homotypic interaction motifs DEDs, CARDs, and DDs are similar, consisting of six α helices, suggesting that they have a common evolutionary origin (37Hofmann K Bucher P Tschopp J The CARD domain—a new apoptotic signaling motif.Trends Biochem. 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For example, FLIP can inhibit caspase activation by binding to FADD (39Irmler M Thome M Hahne M Schneider P Hofmann B Steiner V Bodmer J.L Schroter M Burns K Mattmann C et al.Inhibition of death receptor signals by cellular FLIP.Nature. 1997; 388: