Programmed Cell Death in Animal Development and Disease

Programmed cell death (PCD) plays a fundamental role in animal development and tissue homeostasis. Abnormal regulation of this process is associated with a wide variety of human diseases, including immunological and developmental disorders, neurodegeneration, and cancer. Here, we provide a brief historical overview of the field and reflect on the regulation, roles, and modes of PCD during animal development. We also discuss the function and regulation of apoptotic proteins, including caspases, the key executioners of apoptosis, and review the nonlethal functions of these proteins in diverse developmental processes, such as cell differentiation and tissue remodeling. Finally, we explore a growing body of work about the connections between apoptosis, stem cells, and cancer, focusing on how apoptotic cells release a variety of signals to communicate with their cellular environment, including factors that promote cell division, tissue regeneration, and wound healing. Programmed cell death (PCD) plays a fundamental role in animal development and tissue homeostasis. Abnormal regulation of this process is associated with a wide variety of human diseases, including immunological and developmental disorders, neurodegeneration, and cancer. Here, we provide a brief historical overview of the field and reflect on the regulation, roles, and modes of PCD during animal development. We also discuss the function and regulation of apoptotic proteins, including caspases, the key executioners of apoptosis, and review the nonlethal functions of these proteins in diverse developmental processes, such as cell differentiation and tissue remodeling. Finally, we explore a growing body of work about the connections between apoptosis, stem cells, and cancer, focusing on how apoptotic cells release a variety of signals to communicate with their cellular environment, including factors that promote cell division, tissue regeneration, and wound healing. While naturally occurring cell death was already observed 170 years ago, it was long considered a passive phenomenon and viewed as an inevitable end point of biological systems (reviewed in Glücksmann, 1951Glücksmann A. Cell deaths in normal vertebrate ontogeny.Biol. Rev. Camb. Philos. Soc. 1951; 26: 59-86Crossref Google Scholar). This view began to change with studies of developmentally timed cell death in the silkworm and tadpole. These early studies showed that cell death can be delayed with inhibitors of protein or RNA synthesis and that neuronal cell survival requires extracellular survival factors termed neurotrophins (Lockshin and Williams, 1965Lockshin R.A. Williams C.M. Programmed Cell Death–I. Cytology of Degeneration in the Intersegmental Muscles of the Pernyi Silkmoth.J. Insect Physiol. 1965; 11: 123-133Crossref PubMed Google Scholar, Tata, 1966Tata 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 Google Scholar). Although neurotrophins were initially seen as a form of special “nourishment” required for cell survival, it later became clear that these factors suppress the execution of an intrinsic cell suicide program (reviewed in Raff et al., 1993Raff M.C. Barres B.A. Burne J.F. Coles H.S. Ishizaki Y. Jacobson M.D. Programmed cell death and the control of cell survival: lessons from the nervous system.Science. 1993; 262: 695-700Crossref PubMed Google Scholar). Moreover, this mechanism is not restricted to the nervous system, and competition for a limiting supply of extracellular survival signals is a widely used general mechanism that regulates cell number in animals (reviewed in Jacobson et al., 1997Jacobson M.D. Weil M. Raff M.C. Programmed cell death in animal development.Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (1926) Google Scholar). Another major contribution to the cell death field is the ultrastructural study by Kerr, Wylie, and Currie that defined a series of distinct morphological changes in cells dying under physiological conditions (Kerr et al., 1972Kerr J.F. Wyllie A.H. Currie A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br. J. Cancer. 1972; 26: 239-257Crossref PubMed Google Scholar). When cells die in response to overwhelming stress or injury, they swell and rupture in a process termed “necrosis.” In contrast, the majority of cells that die during normal development and homeostasis shrink, have condensed nuclei, retain membrane integrity, and are rapidly eliminated by phagocytosis in a process termed apoptosis (reviewed in Jacobson et al., 1997Jacobson M.D. Weil M. Raff M.C. Programmed cell death in animal development.Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (1926) Google Scholar). As we will discuss in more detail below, more recent studies have uncovered other forms of programmed cell death (PCD), revealing that apoptosis is not the only form of developmental cell death and that backup mechanisms likely compensate when it is prevented (reviewed in Yuan and Kroemer, 2010Yuan J. Kroemer G. Alternative cell death mechanisms in development and beyond.Genes Dev. 2010; 24: 2592-2602Crossref PubMed Scopus (82) Google Scholar). A breakthrough in elucidating the mechanism by which cells undergo PCD came from genetic studies in the nematode C. elegans. The identification of mutations with specific effects on programmed cell death and their ordering into a genetic pathway demonstrated that cell death is a developmental fate, with specific genes acting to initiate a program of cell suicide (Metzstein et al., 1998Metzstein M.M. Stanfield G.M. Horvitz H.R. Genetics of programmed cell death in C. elegans: past, present and future.Trends Genet. 1998; 14: 410-416Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, Ellis and Horvitz, 1986Ellis 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 Google Scholar). The subsequent molecular characterization of the corresponding genes led to the identification of a core cell death machinery that has been conserved in evolution and centers around a family of cysteine proteases, termed caspases, as key executioners of apoptosis (Figure 1) (reviewed in Hengartner, 2000Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (4173) Google Scholar, Thornberry and Lazebnik, 1998Thornberry N.A. Lazebnik Y. Caspases: enemies within.Science. 1998; 281: 1312-1316Crossref PubMed Google Scholar). Most of our knowledge regarding the role and regulation of PCD has come primarily from three model systems, the nematode C. elegans, the fruit fly Drosophila melanogaster, and the mouse. In C. elegans, the programmed death of somatic cells is an invariant fate that is strictly controlled by cell lineage (Ellis and Horvitz, 1986Ellis 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 Google Scholar). During development of the hermaphrodite, 131 out of the total 1090 somatic cells die, mostly during embryogenesis and soon after cell division (Ellis et al., 1991Ellis R.E. Yuan J.Y. Horvitz H.R. Mechanisms and functions of cell death.Annu. Rev. Cell Biol. 1991; 7: 663-698Crossref PubMed Google Scholar). In loss of function mutants for egl-1, ced-3, or ced-4, cell death is blocked, leading to the survival of all 131 cells (Figure 1). Despite the long-term persistence of undead cells, development proceeds normally, and the life span, behavior, and appearance of cell death-defective mutants are similar to wild-type worms. In contrast, loss of ced-9 function results in developmental lethality due to widespread ectopic cell death (Hengartner et al., 1992Hengartner 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 (470) Google Scholar). For each of these C. elegans genes, homologs have been identified in other organisms: CED-3 is a caspase; CED-4 is a homolog of the adaptor protein apoptosis activating factor 1 (Apaf-1), which promotes assembly and activation of caspases; CED-9 is a multidomain Bcl-2 family member; and EGL-1 is similar to proapoptotic BH3-only proteins (Hengartner, 2000Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (4173) Google Scholar; Figure 1). Additional genes have been identified in C. elegans that affect the decision of cells to die, including the transcriptional regulators CES-1, CES-2, and CEH-30 (Metzstein et al., 1996Metzstein M.M. Hengartner M.O. Tsung N. Ellis R.E. Horvitz H.R. Transcriptional regulator of programmed cell death encoded by Caenorhabditis elegans gene ces-2.Nature. 1996; 382: 545-547Crossref PubMed Scopus (112) Google Scholar, Thellmann et al., 2003Thellmann M. Hatzold J. Conradt B. The Snail-like CES-1 protein of C. elegans can block the expression of the BH3-only cell-death activator gene egl-1 by antagonizing the function of bHLH proteins.Development. 2003; 130: 4057-4071Crossref PubMed Scopus (52) Google Scholar, Schwartz and Horvitz, 2007Schwartz H.T. Horvitz H.R. The C. elegans protein CEH-30 protects male-specific neurons from apoptosis independently of the Bcl-2 homolog CED-9.Genes Dev. 2007; 21: 3181-3194Crossref PubMed Scopus (25) Google Scholar). Finally, a nonapoptotic form of cell-autonomous PCD that is not mediated by caspases is responsible for the death of a specialized linker cell during the larval/adult transition (Abraham et al., 2007Abraham M.C. Lu Y. Shaham S. A morphologically conserved nonapoptotic program promotes linker cell death in Caenorhabditis elegans.Dev. Cell. 2007; 12: 73-86Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Another important model to study PCD during development is the fruit fly, Drosophila melanogaster. This organism is comprised of > 1,000-fold more cells than C. elegans, and its total number of cells depends on environmental factors, including nutrient availability, DNA damage, and environmental stress. In Drosophila, PCD is not an invariant fate specified by cell lineage, but like in vertebrates, it is regulated by a wide variety of stimuli originating from both within a cell, as well as from the environment (reviewed in Kornbluth and White, 2005Kornbluth S. White K. Apoptosis in Drosophila: neither fish nor fowl (nor man, nor worm).J. Cell Sci. 2005; 118: 1779-1787Crossref PubMed Scopus (99) Google Scholar). Drosophila has a well-defined mechanism of development and relatively simple and accessible anatomy and is amenable to powerful genetics and molecular biology techniques. Therefore, it provides an important system for studying the role of PCD during development and its regulation by different signaling pathways. Unlike the situation in C. elegans, PCD is required for the successful completion of development, and inhibition of PCD results in severe developmental defects and organismal lethality (White et al., 1994White 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 Google Scholar, Grether et al., 1995Grether M.E. Abrams J.M. Agapite J. White K. Steller H. The head involution defective gene of Drosophila melanogaster functions in programmed cell death.Genes Dev. 1995; 9: 1694-1708Crossref PubMed Google Scholar, Xu et al., 2005Xu D. Li Y. Arcaro M. Lackey M. Bergmann A. The CARD-carrying caspase Dronc is essential for most, but not all, developmental cell death in Drosophila.Development. 2005; 132: 2125-2134Crossref PubMed Scopus (105) Google Scholar, Srivastava et al., 2007Srivastava M. Scherr H. Lackey M. Xu D. Chen Z. Lu J. Bergmann A. ARK, the Apaf-1 related killer in Drosophila, requires diverse domains for its apoptotic activity.Cell Death Differ. 2007; 14: 92-102Crossref PubMed Scopus (35) Google Scholar). Many of the genes that pattern the Drosophila embryo, including Hox genes, activate cell death by direct transcriptional regulation of the proapoptotic Reaper, Hid, and Grim (RHG) genes (see, for example, Lohmann et al., 2002Lohmann I. McGinnis N. Bodmer M. McGinnis W. The Drosophila Hox gene deformed sculpts head morphology via direct regulation of the apoptosis activator reaper.Cell. 2002; 110: 457-466Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). These genes are also transcriptionally induced by many other signaling pathways, including the steroid hormone ecdysone. During metamorphosis, ecdysone induces the rapid destruction of larval tissues by activating transcriptional cascades that culminate in expression of RHG genes and caspase activation (Jiang et al., 2000Jiang C. Lamblin A.F. Steller H. Thummel C.S. A steroid-triggered transcriptional hierarchy controls salivary gland cell death during Drosophila metamorphosis.Mol. Cell. 2000; 5: 445-455Abstract Full Text Full Text PDF PubMed Google Scholar). In contrast, ecdysone acts as a prosurvival factor for a set of adult neurons that survive through this transition but die soon after eclosion. In this case, ecdysone represses the expression of reaper and grim (Robinow et al., 1997Robinow S. Draizen T.A. Truman J.W. Genes that induce apoptosis: transcriptional regulation in identified, doomed neurons of the Drosophila CNS.Dev. Biol. 1997; 190: 206-213Crossref PubMed Scopus (57) Google Scholar, Draizen et al., 1999Draizen T.A. Ewer J. Robinow S. Genetic and hormonal regulation of the death of peptidergic neurons in the Drosophila central nervous system.J. Neurobiol. 1999; 38: 455-465Crossref PubMed Scopus (46) Google Scholar). As discussed in greater detail below, PCD by apoptosis contributes to the patterning and normal development of virtually all adult structures in the fly, including legs, wings, eyes, genitalia, digestive system, and the nervous system. Also, defects in cell division, specification, or differentiation almost invariantly cause apoptotic death, revealing a stringent quality control that removes defective and useless cells during development. In addition to apoptosis, other forms of PCD have been described in Drosophila as well, and several studies suggest that autophagy also contributes to the removal of superfluous cells during normal development (reviewed in Ryoo and Baehrecke, 2010Ryoo H.D. Baehrecke E.H. Distinct death mechanisms in Drosophila development.Curr. Opin. Cell Biol. 2010; 22: 889-895Crossref PubMed Scopus (25) Google Scholar). As one may expect, the regulation of PCD in vertebrates appears considerably more complex, and vast numbers of cells undergo PCD throughout development, from as early as inner cell mass differentiation in blastocysts to maintenance of tissue homeostasis in adulthood (Hardy et al., 1989Hardy K. Handyside A.H. Winston R.M. The human blastocyst: cell number, death and allocation during late preimplantation development in vitro.Development. 1989; 107: 597-604PubMed Google Scholar). Therefore, it is somewhat surprising that the inactivation of mouse cell death genes leads to only relatively minor developmental defects and can often survive embryonic development (see, for example, Lindsten and Thompson, 2006Lindsten T. Thompson C.B. Cell death in the absence of Bax and Bak.Cell Death Differ. 2006; 13: 1272-1276Crossref PubMed Scopus (32) Google Scholar, Okamoto et al., 2006Okamoto H. Shiraishi H. Yoshida H. Histological analyses of normally grown, fertile Apaf1-deficient mice.Cell Death Differ. 2006; 13: 668-671Crossref PubMed Scopus (10) Google Scholar). One reason appears to be considerable redundancy within the caspase family and the existence of multiple mechanisms for caspase activation. For example, some effector caspases can be activated in the absence of Apaf-1 function (Nagasaka et al., 2010Nagasaka A. Kawane K. Yoshida H. Nagata S. Apaf-1-independent programmed cell death in mouse development.Cell Death Differ. 2010; 17: 931-941Crossref PubMed Scopus (14) Google Scholar). In addition, there is evidence for alternative backup mechanisms that eliminate cells when apoptosis is defective (reviewed in Yuan and Kroemer, 2010Yuan J. Kroemer G. Alternative cell death mechanisms in development and beyond.Genes Dev. 2010; 24: 2592-2602Crossref PubMed Scopus (82) Google Scholar). Despite the apparent robustness of cell death mechanisms in mammals, inhibition of apoptosis has been linked to several specific developmental abnormalities and also a variety of human pathologies, including cancer and degenerative diseases (Thompson, 1995Thompson C.B. Apoptosis in the pathogenesis and treatment of disease.Science. 1995; 267: 1456-1462Crossref PubMed Google Scholar). Studies in worms, flies, and mice have been complemented and extended by work in many other systems, including Hydra, Manduca, Xenopus, zebrafish, chicken, and the analysis of human patients. Collectively, this work has illustrated why cells need to be eliminated in different physiological contexts: (1) sculpting and (2) deleting structures, (3) regulating cell number, and (4) eliminating defective cells. PCD plays a crucial role in organogenesis and tissue remodeling. Perhaps the best-known example is the formation of digits in higher vertebrates in which PCD eliminates the interdigital webs primarily via the apoptotic machinery (Figure 2A ; Lindsten et al., 2000Lindsten T. Ross A.J. King A. Zong W.X. Rathmell J.C. Shiels H.A. Ulrich E. Waymire K.G. Mahar P. Frauwirth K. et al.The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues.Mol. Cell. 2000; 6: 1389-1399Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). Although apoptosis is the major cell death mechanism in developing limbs, inactivation of proapoptotic genes in the mouse only partially prevents the removal of the interdigital tissue, suggesting that backup mechanisms exist when apoptosis fails (Yuan and Kroemer, 2010Yuan J. Kroemer G. Alternative cell death mechanisms in development and beyond.Genes Dev. 2010; 24: 2592-2602Crossref PubMed Scopus (82) Google Scholar). In Drosophila, apoptosis plays a critical role in the formation of leg joints and for the morphogenesis of segments, in particular of the head, which all require RHG-mediated apoptosis (Lohmann et al., 2002Lohmann I. McGinnis N. Bodmer M. McGinnis W. The Drosophila Hox gene deformed sculpts head morphology via direct regulation of the apoptosis activator reaper.Cell. 2002; 110: 457-466Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Furthermore, apoptosis is also required to permit tissue rotation that drives looping morphogenesis of male genitalia in the fly (Kuranaga et al., 2011Kuranaga E. Matsunuma T. Kanuka H. Takemoto K. Koto A. Kimura K. Miura M. Apoptosis controls the speed of looping morphogenesis in Drosophila male terminalia.Development. 2011; 138: 1493-1499Crossref PubMed Scopus (10) Google Scholar, Suzanne et al., 2010Suzanne M. Petzoldt A.G. Spéder P. Coutelis J.B. Steller H. Noselli S. Coupling of apoptosis and L/R patterning controls stepwise organ looping.Curr. Biol. 2010; 20: 1773-1778Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). PCD is also involved in the conversion of solid structures to hollow tubes, thereby yielding lumina such as in the creation of the proamniotic cavity (Coucouvanis and Martin, 1995Coucouvanis 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 Google Scholar, Weil et al., 1997Weil M. Jacobson M.D. Raff M.C. Is programmed cell death required for neural tube closure?.Curr. Biol. 1997; 7: 281-284Abstract Full Text Full Text PDF PubMed Google Scholar). It is observed when epithelial sheets invaginate, forming tubes or vesicles, for example, in the establishment of the neural tube or lens and when epithelial sheets fuse to construct the mammalian palate (Glücksmann, 1951Glücksmann A. Cell deaths in normal vertebrate ontogeny.Biol. Rev. Camb. Philos. Soc. 1951; 26: 59-86Crossref Google Scholar). In addition, PCD is involved in sculpting the future inner ear in chicks (Avallone et al., 2002Avallone B. Balsamo G. Trapani S. Marmo F. Apoptosis during chick inner ear development: some observations by TEM and TUNEL techniques.Eur. J. Histochem. 2002; 46: 53-59PubMed Google Scholar) and is essential for generating the four-chamber architecture of the heart (Abdelwahid et al., 2002Abdelwahid E. Pelliniemi L.J. Jokinen E. Cell death and differentiation in the development of the endocardial cushion of the embryonic heart.Microsc. Res. Tech. 2002; 58: 395-403Crossref PubMed Scopus (27) Google Scholar). During development, various structures that serve a transient function are removed by PCD when they are no longer required. Examples include evolutionary relics, structures that are required in only one sex, or structures that are transiently required. In fish and amphibians, pronerphric tubules form functioning kidneys; however, they are not utilized in mammals and are hence eliminated during embryogenesis. In female mammals, the Müllerian duct forms the oviducts and uterus but is deleted in males. Conversely, the Wolffian duct that forms the vas deferens, epididymis, and seminal vesicle in males is degraded in females (Jacobson et al., 1997Jacobson M.D. Weil M. Raff M.C. Programmed cell death in animal development.Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (1926) Google Scholar). In metamorphosis, juvenile structures are removed by PCD. In amphibians, the tadpole tail and intestine are deleted, and in insects, most larval tissues are eliminated by PCD (Figure 2B, Baehrecke, 2002Baehrecke E.H. How death shapes life during development.Nat. Rev. Mol. Cell Biol. 2002; 3: 779-787Crossref PubMed Scopus (229) Google Scholar). Developing tissues and organs rely heavily on an intricate balance between cell division and PCD to achieve appropriate cell numbers. In many organs, such as the nervous, immune, and reproductive system, cells are overproduced and subsequently removed by PCD (Figure 2C). In human females, PCD is responsible for culling nearly 80% oocytes prior to birth, and in almost all instances, these eliminated cells are typified by apoptotic morphology (Reynaud and Driancourt, 2000Reynaud K. Driancourt M.A. Oocyte attrition.Mol. Cell. Endocrinol. 2000; 163: 101-108Crossref PubMed Scopus (68) Google Scholar). It has been estimated that more than half of all neurons generated in the mammalian CNS are eliminated by PCD (Barres and Raff, 1999Barres B.A. Raff M.C. Axonal control of oligodendrocyte development.J. Cell Biol. 1999; 147: 1123-1128Crossref PubMed Scopus (175) Google Scholar). Competition for limiting amounts of survival signals ensures proper matching of the numbers of different cell types in a tissue. This strategy is used in Drosophila, in which survival signaling via the Ras/EGFR pathway prevents activity of the proapoptotic hid gene (Bergmann et al., 1998Bergmann A. Agapite J. McCall K. Steller H. The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling.Cell. 1998; 95: 331-341Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar). Competition also occurs between cells that proliferate at different rates. In this case, slower dividing cells are eliminated from the population by more rapidly dividing cells. This phenomenon was initially observed in Drosophila but is also seen in mammals (Moreno, 2008Moreno E. Is cell competition relevant to cancer?.Nat. Rev. Cancer. 2008; 8: 141-147Crossref PubMed Scopus (57) Google Scholar, Bondar and Medzhitov, 2010Bondar T. Medzhitov R. p53-mediated hematopoietic stem and progenitor cell competition.Cell Stem Cell. 2010; 6: 309-322Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Cell competition is thought to contribute to growth homeostasis by adjusting for variations that might occur during normal growth, and selecting for the “fittest” cells is thought to optimize organ function (Moreno, 2008Moreno E. Is cell competition relevant to cancer?.Nat. Rev. Cancer. 2008; 8: 141-147Crossref PubMed Scopus (57) Google Scholar). “Loser cells” are eliminated by hid-mediated apoptosis, and genes that mediate cell engulfment are also required for this process (de la Cova et al., 2004de la Cova C. Abril M. Bellosta P. Gallant P. Johnston L.A. Drosophila myc regulates organ size by inducing cell competition.Cell. 2004; 117: 107-116Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, Li and Baker, 2007Li W. Baker N.E. Engulfment is required for cell competition.Cell. 2007; 129: 1215-1225Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Although cell competition is not essential under laboratory conditions, it appears to facilitate tissue repair and has been connected to oncogenic pathways (Moreno, 2008Moreno E. Is cell competition relevant to cancer?.Nat. Rev. Cancer. 2008; 8: 141-147Crossref PubMed Scopus (57) Google Scholar, Bondar and Medzhitov, 2010Bondar T. Medzhitov R. p53-mediated hematopoietic stem and progenitor cell competition.Cell Stem Cell. 2010; 6: 309-322Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). All of these observations reveal an intimate association among the processes of cell division, differentiation, and death, with mistakes often resolved through the induction of apoptosis. PCD also serves as a protective process in both animal development and adult life by eliminating cells that are abnormal and potentially dangerous. One example is the human immune system, in which a highly stringent selection process dictates the survival of lymphocytes. In order to evade cell death, B and T lymphocytes have to pass both positive and negative selection, demonstrating a functional antigen receptor that is not autoreactive (Figure 2D; Opferman and Korsmeyer, 2003Opferman J.T. Korsmeyer S.J. Apoptosis in the development and maintenance of the immune system.Nat. Immunol. 2003; 4: 410-415Crossref PubMed Scopus (284) Google Scholar). In this manner, PCD eliminates self-reactive cells that potentially could lead to autoimmunity. Other examples include the elimination of cells in response to viral infection, unrepaired DNA damage, cell-cycle perturbations, and fate and differentiation defects (Abrams et al., 1993Abrams J.M. White K. Fessler L.I. Steller H. Programmed cell death during Drosophila embryogenesis.Development. 1993; 117: 29-43Crossref PubMed Google Scholar, Rossel and Capecchi, 1999Rossel M. Capecchi M.R. Mice mutant for both Hoxa1 and Hoxb1 show extensive remodeling of the hindbrain and defects in craniofacial development.Development. 1999; 126: 5027-5040Crossref PubMed Google Scholar, Derry et al., 2001Derry W.B. Putzke A.P. Rothman J.H. Caenorhabditis elegans p53: role in apoptosis, meiosis, and stress resistance.Science. 2001; 294: 591-595Crossref PubMed Scopus (237) Google Scholar, Vousden and Prives, 2009Vousden K.H. Prives C. Blinded by the Light: The Growing Complexity of p53.Cell. 2009; 137: 413-431Abstract Full Text Full Text PDF PubMed Scopus (783) Google Scholar, Malumbres and Barbacid, 2009Malumbres M. Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm.Nat. Rev. Cancer. 2009; 9: 153-166Crossref PubMed Scopus (631) Google Scholar, Koto et al., 2011Koto A. Kuranaga E. Miura M. Apoptosis ensures spacing pattern formation of Drosophila sensory organs.Curr. Biol. 2011; 21: 278-287Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar). In all of these circumstances, apoptotic cell death serves as an important “quality control” mechanism for the elimination of faulty cells. Apoptosis is the most studied and best understood form of PCD (Figure 1). A central step in the execution of apoptosis is the activation of caspases, a family of cysteine proteases that are ubiquitously expressed as inactive precursors (zymogens) with little or no protease activity (Hengartner, 2000Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (4173) Google Scholar, Thornberry and Lazebnik, 1998Thornberry N.A. Lazebnik Y. Caspases: enemies within.Science. 1998; 281: 1312-1316Crossref PubMed Google Scholar). In response to death-inducing stimuli, caspases are activated by cleavage at specific aspartic residues, resulting in removal of an inhibitory N-terminal domain and production of a large and a small subunit. Heterotetramers of these subunits form the active protease, leading to the demolition phase of apoptosis (Hengartner, 2000Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (4173) Google Scholar). Importantly, not all mammalian caspases participate in apoptosis; some are activated during innate immunity and function to regulate cytokine processing and maturation (Martinon and Tschopp, 2004Martinon F. Tschopp J. Inflammatory caspases: linking an intracellular innate immune system to autoinflammatory diseases.Cell. 2004; 117: 561-574Abstract Full Text Full Text PDF PubMed Scopus (493) Google Scholar). The caspase family has been traditionally subdivided into initiator and effector caspases (Hengartner, 2000Hengartner M.O. The biochemistry of apoptosis.Nature. 2000; 407: 770-776Crossref PubMed Scopus (4173) Google Scholar, Thornberry and Lazebnik, 1998Thornberry N.A. Lazebnik Y. Caspases: enemies within.Science. 1998; 281: 1312-1316Crossref PubMed Google Scholar). Effector caspases have short prodomains and are thought to execute apoptosis after they are proteolytic processed by initiator caspases. Initiator caspases have long prodomains that bind large adaptor molecules that promote multimerizatio