Programmed Cell Death in Animal Development

Programmed cell death (PCD) occurs during the development of all animals that have been studied, but only recently has its molecular basis been discovered. In this review, we briefly consider some of the main events in the history of PCD in animal development. We then summarize what has been learned about the molecular mechanism of PCD and some of the intracellular proteins that control it. We next discuss the functions of PCD in development and how PCD is regulated during development by signals from other cells. Finally, we consider what the evolutionary origins of PCD may have been. Soon after it was recognized in the middle of the last century that organisms are made of cells, it was discovered that cell death can be an important part of animal development (reviewed in10Clarke P.G.H Clarke S Nineteenth century research on naturally occurring cell death and related phenomena.Anat. Embryol. 1996; 193: 81-99Crossref PubMed Scopus (175) Google Scholar). First observed during amphibian metamorphosis (64Vogt C Untersuchungen über die Entwicklungsgeschichte der Geburtshelferkroete. >, Alytes obstetricans)1842Google Scholar), normal cell death was soon discovered to occur in many developing tissues in both invertebrates and vertebrates (reviewed in21Glucksmann A Cell deaths in normal vertebrate ontogeny.Biol. Rev. 1951; 26: 5986Crossref Scopus (864) Google Scholar, 10Clarke P.G.H Clarke S Nineteenth century research on naturally occurring cell death and related phenomena.Anat. Embryol. 1996; 193: 81-99Crossref PubMed Scopus (175) Google Scholar). Inhibitors of RNA and protein synthesis were later found to inhibit cell deaths that occurred during amphibian (60Tata J.R Requirement for RNA and protein synthesis for induced regression of the tadpole tail in organ culture.Dev. Biol. 1966; 13: 77-94Crossref PubMed Scopus (248) Google Scholar) and insect (39Lockshin R.A Programmed cell death. Activation of lysis by a mechanism involving the synthesis of protein.J. Insect Physiol. 1969; 15: 1505-1516Crossref PubMed Scopus (118) Google Scholar) metamorphosis, indicating that the deaths require macromolecular synthesis. The term programmed cell death was initially used to describe the cell deaths that occur in predictable places and at predictable times during development, to emphasize that the deaths are somehow programmed into the developmental plan of the organism (40Lockshin R.A Williams C.M Programmed cell death. II. Endocrine potentiation of the breakdown of the intersegmental muscles of silkmoths.J. Insect Physiol. 1964; 10: 643Crossref Scopus (247) Google Scholar). It was already clear, however, that some of these cell deaths can be prevented by substances released by other tissues, indicating that the deaths are not inevitable and can apparently be suppressed by signals from other cells (reviewed in53Saunders Jr., J.W Death in embryonic systems.Science. 1966; 154: 604-612Crossref PubMed Scopus (698) Google Scholar). In a seminal paper, 34Kerr J.F.R Wyllie A.H Currie A.R Apoptosis a basic biological phenomenon with wide-ranging implication in tissue kinetics.Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Scopus (12234) Google Scholar marshalled morphological evidence from studies of their own and of others to draw a clear distinction between the cell deaths that occur in both animal development and tissue homeostasis, as well as in some pathological states, and the pathological cell deaths that occur at the center of acute lesions such as trauma and ischemia. In the latter case, the cells and their organelles tend to swell and rupture in a process called cell necrosis. The leakage of the contents of the cells usually induces an inflammatory response. By contrast, when cells die during normal development or tissue homeostasis, or at the periphery of acute lesions, they usually shrink and condense, and the organelles and plasma membrane retain their integrity in a process Kerr and his colleagues named apoptosis. The dead cells or their fragments are rapidly phagocytosed by neighboring cells or macrophages before there is any leakage of the contents of the cells, and thus they do not induce an inflammatory response. Apoptotic cells in developing tissues are almost always inside other cells (Figure 1A), suggesting that dying cells are usually phagocytosed before they display the morphological changes of apoptosis. Because apoptotic cell deaths usually look so similar from tissue to tissue and animal to animal (Figure 1A), Kerr and his colleagues proposed that these deaths reflect the operation of an active, intracellular death program that can be activated or inhibited by a variety of physiological or pathological environmental stimuli. It took almost another 20 years, however, before the idea that animal cells have a built-in death, or suicide, program became generally accepted, largely through genetic studies in the nematode Caenorhabditis elegans that identified genes that seem dedicated to the death program and its control (27Horvitz H Ellis H Sternberg P Programmed cell death in nematode development.Neurosci. Comment. 1982; 1: 56-65Google Scholar, 16Ellis 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), and then through the finding that some of these genes were homologous to mammalian genes (69Yuan J Shaham S Ledoux S Ellis H.M Horvitz H.R The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme.Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2182) Google Scholar, 26Hengartner M.O Horvitz H.R C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2.Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1009) Google Scholar). With this acceptance, the term programmed cell death (PCD) has come to have a different meaning from its original. It now generally refers to any cell death that is mediated by the intracellular death program, no matter what triggers it and whether or not it displays all of the characteristic features of apoptosis. It is probable that all normal cell deaths that occur in developing and mature animals, as well as many pathological cell deaths, utilize this evolutionarily conserved death program. A cell that undergoes PCD in animal development is usually degraded so rapidly (often disappearing in an hour or less) that even when there is large-scale PCD, there are surprisingly few dead cells to be seen. This may help to explain why PCD was understudied for so long. It also suggests that the extent of PCD in animal development is still underestimated. As it is still not possible to measure clearance times in most animal tissues, quantitating PCD remains an unsolved problem. There has been spectacular progress in the past few years in understanding the intracellular mechanism of PCD and its control, reflected in the flood of reviews celebrating this success (see, for example,44Martin S.J Green D.R Protease activation during apoptosis death by a thousand cuts?.Cell. 1995; 82: 349-352Abstract Full Text PDF PubMed Scopus (1241) Google Scholar, 8Chinnaiyan A Dixit V The cell-death machine.Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar, 66White E Life, death, and the pursuit of apoptosis.Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1299) Google Scholar). Rather than covering this ground again, we focus on some of the evidence that supports the following four propositions concerning the intracellular death program: (1) its protein components are constitutively expressed in all nucleated animal cells, (2) its execution seems to involve a proteolytic cascade, (3) its activation is controlled by a family of dedicated intracellular regulatory proteins, and (4) its activation during development may often be controlled transcriptionally. One line of evidence that all nucleated animal cells constitutively express all of the proteins required to undergo PCD makes use of the drug staurosporine (STS), a bacterial product that inhibits many protein kinases (59Tamaoki T Nakano H Potent and specific inhibitors of protein kinase C of microbial origin.Bio/Technology. 1990; 8: 732-735Crossref PubMed Scopus (243) Google Scholar). When used at micromolar concentrations, in the presence of cycloheximide to inhibit protein synthesis, STS rapidly induces PCD in all of the many nucleated mammalian cell types that have been tested. These include all of the cells that can be dissociated from a 13 day mouse embryo (after removal of its brain and liver) (29Ishizaki Y Cheng L Mudge A.W Raff M.C Programmed cell death by default in embryonic cells, fibroblasts, and cancer cells.Mol. Biol. Cell. 1995; 6: 1443-1458Crossref PubMed Scopus (129) Google Scholar) and the cells in explant cultures of a variety of neonatal and adult rodent organs (65Weil M Jacobson M.D Coles H.S.R Davies T.J Gardener R.L Raff K.D Raff M.C Constitutive expression of the machinery for programmed cell death.J. Cell Biol. 1996; 133: 1053-1059Crossref PubMed Scopus (350) Google Scholar). The only reported exception is human red blood cells (65Weil M Jacobson M.D Coles H.S.R Davies T.J Gardener R.L Raff K.D Raff M.C Constitutive expression of the machinery for programmed cell death.J. Cell Biol. 1996; 133: 1053-1059Crossref PubMed Scopus (350) Google Scholar), which do not have a nucleus or other organelles. The nucleus, however, is not required for PCD in cells that normally have one, as anucleate cytoplasts undergo PCD when treated with STS (31Jacobson M.D Burne J.F Raff M.C Programmed cell death and Bcl-2 protection in the absence of a nucleus.EMBO J. 1994; 13: 1899-1910Crossref PubMed Scopus (525) Google Scholar). Thus, it seems that the machinery for PCD is in place and ready to run in all of our nucleated cells, beginning with the zygote (65Weil M Jacobson M.D Coles H.S.R Davies T.J Gardener R.L Raff K.D Raff M.C Constitutive expression of the machinery for programmed cell death.J. Cell Biol. 1996; 133: 1053-1059Crossref PubMed Scopus (350) Google Scholar). Genetic experiments in both C. elegans and Drosophila suggest that the death program is also expressed constitutively in invertebrate cells (58Steller H Mechanisms and genes of cellular suicide.Science. 1995; 267: 1445-1449Crossref PubMed Scopus (2392) Google Scholar, 54Shaham S Horvitz H.R Developing Caenorhabditis elegans neurons may contain both cell-death protective and killer activities.Genes Dev. 1996; 10: 578-591Crossref PubMed Scopus (195) Google Scholar), making it likely that this is a basic feature of all nucleated animal cells. The most important clue to the molecular nature of the death program came initially from genetic studies in C. elegans that identified a gene called ced-3 that is required for the 131 PCDs (see Figure 1C) that occur during the development of the worm (reviewed in17Ellis R.E Yuan J.Y Horvitz H.R Mechanisms and functions of cell death.Annu. Rev. Cell Biol. 1991; 7: 663-698Crossref PubMed Scopus (1906) Google Scholar). The gene encodes a cysteine protease that is homologous to interleukin-1β-converting enzyme (ICE) (69Yuan J Shaham S Ledoux S Ellis H.M Horvitz H.R The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme.Cell. 1993; 75: 641-652Abstract Full Text PDF PubMed Scopus (2182) Google Scholar), a mammalian cysteine protease that produces the proinflammatory cytokine IL-1β from its precursor protein. At least 11 members of the Ced-3/ICE family of proteases have now been identified in humans, and a number of them have been implicated in PCD (reviewed in8Chinnaiyan A Dixit V The cell-death machine.Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar, 36Kuida K Zheng T.S Na S Kuan C.-Y Yang D Karasuyama H Rakic P Flavell R.A Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature. 1996; 384: 368-372Crossref PubMed Scopus (1655) Google Scholar). All cleave their substrates after specific aspartic acids and are themselves activated by cleavage at specific aspartic acids. They are now therefore referred to as caspases (for cysteine aspases, see2Alnemri E.S Livingston D.J Nicholson D.W Salvesen G Thornberry N.A Wong W.W Yuan J Human ICE/CED3 protease nomenclature.Cell. 1996; 87: 171Abstract Full Text Full Text PDF PubMed Scopus (2078) Google Scholar). At least in vitro, some caspases can activate themselves, and some can activate other caspases, suggesting that they probably act in a proteolytic cascade (see 71Nagata S Apoptosis by death factor.Cell, this issue. 1997; >Google Scholar). Caspases mediate PCD by cleaving selected intracellular proteins, including proteins of the nucleus, nuclear lamina, cytoskeleton, endoplasmic reticulum, and cytosol. Some of the cleaved proteins activate other destructive processes in the cell and thereby help kill the cell neatly and quickly (8Chinnaiyan A Dixit V The cell-death machine.Curr. Biol. 1996; 6: 555-562Abstract Full Text Full Text PDF PubMed Google Scholar). As specific protein or peptide caspase inhibitors can block PCD in all animal cells that have been tested, it seems likely that caspases form the core of the death program in all animal cells, although some of them, such as ICE, have other functions. At least one of the intracellular mechanisms that controls the death program in animal cells has also been conserved in evolution. The ced-9 gene, which acts to inhibit PCD in C. elegans (26Hengartner M.O Horvitz H.R C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2.Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1009) Google Scholar), is homologous to the bcl-2 gene, which acts to inhibit PCD in mammalian cells (61Vaux D.L Cory S Adams J.M Bcl-2 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; reviewed in35Korsmeyer S Regulators of cell death.Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (603) Google Scholar). The human bcl-2 gene is even able to inhibit PCD in the worm (62Vaux 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, 26Hengartner M.O Horvitz H.R C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2.Cell. 1994; 76: 665-676Abstract Full Text PDF PubMed Scopus (1009) Google Scholar). A number of Ced-9/Bcl-2 family members have been identified in mammals. Some, such as Bcl-2 and Bcl-XL, inhibit PCD, whereas others, such as Bax and Bak, promote PCD. The various family members can dimerize with one another, with one monomer antagonizing or enhancing the function of the other. In this way, the ratio of inhibitors to activators in a cell may determine the propensity of the cell to undergo PCD (35Korsmeyer S Regulators of cell death.Trends Genet. 1995; 11: 101-105Abstract Full Text PDF PubMed Scopus (603) Google Scholar), although the activity of some of these proteins can also be regulated by phosphorylation (reviewed in19Gajewski T.F Thompson C.B Apoptosis meets signal transduction elimination of a BAD influence.Cell. 1996; 87: 589-592Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). It is still not known how any of these proteins operate. The three-dimensional structure of Bcl-XL suggests that it may function as a pore-forming protein in the intracellular membranes where it is found (47Muchmore S.W Sattler M Liang H Meadows R.P Harlan J.E Yoon H.S Nettesheim D Chang B.S Thompson C.B Wong S.L et al.X-ray and NMR structure of human Bcl-xL, an inhibitor of programmed cell death.Nature. 1996; 381: 335-341Crossref PubMed Scopus (1255) Google Scholar). Whatever their mechanism of action turns out to be, it is clear that they play a crucial role in regulating PCD in development. If ced-9 is inactivated by mutation, for example, most cells in the developing worm undergo PCD, and the worm dies early in development (25Hengartner M.O Ellis R.E Horvitz H.R Caenorhabditis elegans gene ced-9 protects cells from programmed cell death.Nature. 1992; 356: 494-499Crossref PubMed Scopus (692) Google Scholar). It seems that Ced-9 is required to keep the death program off if a cell is to survive in the developing worm. Similarly, if either bcl-x (46Motoyama N Wang F Roth K.A Sawa H Nakayama K Negishi I Senju S Zhang Q Fujii S Loh D.Y Massive cell death of immature hematopoietic cells and neurons in Bcl-x–deficient mice.Science. 1995; 267: 1506-1510Crossref PubMed Scopus (1002) Google Scholar) or bcl-2 (63Veis D.J Sorenson C.M Shutter J.R Korsmeyer S.J Bcl-2–deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair.Cell. 1993; 75: 229-240Abstract Full Text PDF PubMed Scopus (1406) Google Scholar) is disrupted in mice, the animals die as embryos or postnatally, respectively, as the result of excessive PCD in particular organs. Conversely, if bax is disrupted, some normal PCDs fail to occur (14Deckwerth T.L Elliott J.L Knudsen C.M E.M. Johnson J Snider W.D Korsmeyer S.J BAX is required for neuronal death after trophic factor deprivation and during development.Neuron. 1996; 17: 401-411Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Although all of the proteins required to execute the death program seem to be constitutively expressed in animal cells, inhibitors of RNA or protein synthesis often inhibit PCD, indicating that transcription and translation are often required to activate the program. The strongest evidence that new gene transcription is involved in activating PCD during normal development comes from experiments in Drosophila, where linked genes (reaper, hid, and grim) required for triggering PCD are transcriptionally activated 1–2 hours before the cells die. If these genes are inactivated, none of the normal cell deaths occur and the flies die early in development (67White K Grether M.E Abrams J.M Young L Farrell K Steller H Genetic control of programmed cell death in Drosophila.Science. 1994; 264: 677-683Crossref PubMed Scopus (872) Google Scholar). It is not known how the products of these genes activate the caspase-dependent death program in developing fly cells. Although homologous genes have not yet been found in other organisms, it seems likely that many of the PCDs that occur during the development of other animals are also transcriptionally controlled (60Tata J.R Requirement for RNA and protein synthesis for induced regression of the tadpole tail in organ culture.Dev. Biol. 1966; 13: 77-94Crossref PubMed Scopus (248) Google Scholar, 39Lockshin R.A Programmed cell death. Activation of lysis by a mechanism involving the synthesis of protein.J. Insect Physiol. 1969; 15: 1505-1516Crossref PubMed Scopus (118) Google Scholar, 43Martin D.P Schmidt R.E DiStefano P.S Lowry O.H Carter J.G Johnson E.M Inhibitors of protein synthesis and RNA synthesis prevent neuronal death caused by nerve growth factor deprivation.J. Cell Biol. 1988; 106: 829-844Crossref PubMed Scopus (795) Google Scholar, 51Oppenheim R.W Prevette D Tytell M Homma S Naturally occurring and induced neuronal death in the chick embryo in vivo requires protein and RNA synthesis evidence for the role of cell death genes.Dev. Biol. 1990; 138: 104-113Crossref PubMed Scopus (306) Google Scholar). How important is PCD in animal development? Mutant nematodes that are PCD-deficient can have a normal lifespan in the laboratory, even though they have about 15% more cells than normal and function less well than wild-type worms (17Ellis R.E Yuan J.Y Horvitz H.R Mechanisms and functions of cell death.Annu. Rev. Cell Biol. 1991; 7: 663-698Crossref PubMed Scopus (1906) Google Scholar). PCD-deficient flies, by contrast, die early in development (67White K Grether M.E Abrams J.M Young L Farrell K Steller H Genetic control of programmed cell death in Drosophila.Science. 1994; 264: 677-683Crossref PubMed Scopus (872) Google Scholar). At least in terms of complexity, vertebrates are more similar to flies than to worms, and it seems likely that they would die early in development if their cells could not undergo PCD. Consistent with this view, mice in which CPP32 (caspase-3) has been deleted by targeted gene disruption die perinatally with a vast excess of cells in their central nervous system, apparently as a result of decreased PCD in neuroepithelial cells, although PCD in other organs seems to occur normally (36Kuida K Zheng T.S Na S Kuan C.-Y Yang D Karasuyama H Rakic P Flavell R.A Decreased apoptosis in the brain and premature lethality in CPP32-deficient mice.Nature. 1996; 384: 368-372Crossref PubMed Scopus (1655) Google Scholar). PCD serves many functions in animal development, and these have been classified in different ways (see, for example,21Glucksmann A Cell deaths in normal vertebrate ontogeny.Biol. Rev. 1951; 26: 5986Crossref Scopus (864) Google Scholar, 27Horvitz H Ellis H Sternberg P Programmed cell death in nematode development.Neurosci. Comment. 1982; 1: 56-65Google Scholar). We consider five functions, most of which involve eliminating unwanted cells: (1) sculpting structures; (2) deleting unneeded structures; (3) controlling cell numbers; (4) eliminating abnormal, misplaced, nonfunctional, or harmful cells; and (5) producing differentiated cells without organelles. PCD plays an essential role in sculpting parts of the body. The formation of digits in some higher vertebrates is a well-studied example, where PCD eliminates the cells between developing digits (Figure 2A). If the cell death is inhibited by treatment with a peptide caspase inhibitor, digit formation is blocked (45Milligan C.E Prevette D Yaginuma H Homma S Cardwell C Fritz L.C Tomaselli K.J Oppenheim R.W Schwartz L.M Peptide inhibitors of the ICE protease family arrest programmed cell death of motoneurons in vivo and in vitro.Neuron. 1995; 15: 385-393Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 32Jacobson M.D Weil M Raff M.C Role of Ced-3/ICE family proteases in stuarosporine-induced programmed cell death.J. Cell Biol. 1996; 133: 1041-1051Crossref PubMed Scopus (358) Google Scholar). Similarly, PCD is involved in hollowing out solid structures to create lumina (Figure 2B). In early mouse embryos, for example, the preamniotic cavity is formed by the death of the ectodermal cells in the core of the developing embryo (13Coucouvanis E Martin G.R Signals for death and survival a two-step mechanism for cavitation in the vertebrate embryo.Cell. 1995; 83: 279-287Abstract Full Text PDF PubMed Scopus (481) Google Scholar). PCD also occurs wherever epithelial sheets invaginate and pinch off to form tubes or vesicles, as in the formation of the vertebrate neural tube or lens, and it is observed when two epithelial sheets come together and fuse, as in the formation of the mammalian palate (21Glucksmann A Cell deaths in normal vertebrate ontogeny.Biol. Rev. 1951; 26: 5986Crossref Scopus (864) Google Scholar). It is not known, however, if cell death is required for any of these processes, although it seems likely that it plays an important part. In the course of animal development, various structures are formed that are later removed by PCD. These include vestigial structures that were required in an ancestral species but not in the descendant, structures that are needed at one stage of development but not later (Figure 2C), and structures that are required in one sex but not in the other. Pronephric tubules, for example, form functioning kidneys in fish and amphibian larvae, but they are not used in mammals and are eliminated by PCD. Subplate neurons are required transiently during the development of the mammalian cerebral cortex and are subsequently removed by PCD. The Müllerian duct forms the uterus and oviducts in female mammals, but it is not needed in males and is thought to be lost by PCD. Conversely, the Wolffian duct forms the vas deferens, epididymis, and seminal vesicle in males, but it is not needed in females and is eliminated by PCD (Figure 2D). In many organs, cells are overproduced and then culled by PCD to adjust their numbers (Figure 2E). In the vertebrate nervous system, for example, both neurons and oligodendrocytes are generated in excess, and up to half or more are eliminated by PCD, apparently to match their numbers to the number of target cells they innervate (4Barde Y.A Trophic factors and neuronal survival.Neuron. 1989; 2: 1525-1534Abstract Full Text PDF PubMed Scopus (1426) Google Scholar, 50Oppenheim R.W Cell death during development of the nervous system.Annu. Rev. Neurosci. 1991; 14: 453-501Crossref PubMed Scopus (2700) Google Scholar) or the number of axons they myelinate (5Barres B.A Hart I.K Coles H.C Burne J.F Voyvodic J.T Richardson W.D Raff M.C Cell death and control of cell survival in the oligodendrocyte lineage.Cell. 1992; 70: 31-46Abstract Full Text PDF PubMed Scopus (1139) Google Scholar), respectively. Although the influence of cell proliferation in controlling cell numbers in animal development has received more attention than the influence of PCD, PCD can be the dominant mechanism. In well-fed hydra, for example, cell proliferation greatly outstrips cell death, and new hydra continually bud off from the parent animal; when hydra are starved, growth stops, mainly because cell death greatly increases, while the rate of cell proliferation changes very little (7Bosch T.C David C.N Growth regulation in Hydra relationship between epithelial cell cycle length and growth rate.Dev. Biol. 1984; 104: 161-171Crossref PubMed Scopus (110) Google Scholar). PCD also functions as part of a quality-control process in animal development, eliminating cells that are abnormal, misplaced, nonfunctional, or potentially dangerous to the organism. Striking examples are seen in the vertebrate immune system, where developing T and B lymphocytes that either fail to produce potentially useful antigen-specific receptors or produce self-reactive receptors that make the cells potentially dangerous are eliminated by PCD (Figure 2F). Animal cells have poorly understood ways of recognizing when they are damaged and will undergo PCD if the damage is great enough (Figure 2G). If DNA is damaged sufficiently in a mammalian cell, for example, the cell can activate its death program by various mechanisms, one of which depends on the p53 tumor-suppressor protein (9Clarke A.R Purdie C.A Harrison D.J Morris R.G Bird C.C Hooper M.L Wyllie A.H Thymocyte apoptosis induced by p53-dependent and independent pathways.Nature. 1993; 362: 849-852Crossref PubMed Scopus (2259) Google Scholar, 42Lowe S.W Schmitt E.M Smith S.W Osborne B.A Jacks T p53 is required for radiation-induced apoptosis in mouse thymocytes.Nature. 1993; 362: 847-849Crossref PubMed Scopus (2721) Google Scholar). This response not only serves as an anticancer mechanism, but also seems to help prevent the birth of defective offspring. If pregnant mice are irradiated, the number of offspring produced decreases, as many of the irradiated embryos die, but there is surprisingly little increase in the occurrence of birth defects among those mice that are born. Mouse embryos lacking both copies of the p53 gene, however, tend not to die, and many are instead born with abnormalities (49Norimura T Nomoto S Katsuki M Gondo Y Kondo S p53-dependent apoptosis suppresses radiation-induced teratogenesis.Nat. Med. 1996; 2: 577-580Crossref PubMed Scopus (199) Google Scholar). The death program may be involved in producing specialized differentiated cells without organelles. Certain cell types, including skin keratinocytes, lens epithelial cells, and mammalian red blood cells, lose their nucleus and other organelles in the process of terminal differentiation. The differentiated lens cells and red blood cells continue to live in the sense that they continue to metabolize, whereas differentiated keratinocytes die and form a layer of corpses (squames) on the surface of the skin. There are hints that these highly specialized differentiation processes may be modified forms of PCD. The nuclei become pyknotic, the DNA becomes fragmented, and, in the case of human keratinocytes in culture, overexpression of a bcl-2 transgene inhibits terminal differentiation (48Nataraj A Pathak S Hopwood V McDonnell T Ananthaswamy H bcl-2 oncogene blocks differentiation and extends viability but does not immortalize normal human keratinocytes.Int. J. Oncology. 1994; 4: 1211-1218PubMed Google Scholar). It remains to be determined, however, whether caspases are involved in any of these differentiation processes. There are many cell deaths that occur during animal development where the function is unknown. This is the case for the cell deaths that occur in the inner cell mass of early mammalian embryos (15El Shershaby A.M Hinchliffe J.R Cell redundancy in the zona-intact preimplantation mouse blastocyst a light and electron microscope study of dead cells and their fate.J. Embryol. Exp. Morphol. 1974; 31: 643-654PubMed Google Scholar), in developing mammalian spermatogonia (1Allan D.J Harmon B.V Kerr J.F.R Cell death in spermatogenesis.in: Potten C.S Perspectives on Mammalian Cell Death. Oxford Univ. Press, London1987: 229-258Google Scholar), and among undifferentiated proliferating cells in many developing organs. Why should these cells die before they have had a chance to function? It is possible that some of these deaths reflect a continuing competition between developing cells for a limited supply of extracellular survival signals, which may serve to constantly adjust cell numbers and may, at the same time, select for the “best” cells (5