Programmed Cell Death
in Animal Development and Disease
Yaron Fuchs1and Hermann Steller1,*
1Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA
Programmed cell death (PCD) plays a fundamental role in animal development and tissue homeo-
stasis. 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
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
While naturally occurring cell death was already observed 170
years ago, it was long considered a passive phenomenon and
in Glu ¨cksmann, 1951). 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 neuro-
trophins (Lockshin and Williams, 1965; Tata, 1966). Although
neurotrophins were initially seen as a form of special ‘‘nourish-
ment’’ 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., 1993). Moreover, this mecha-
nism 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., 1997). 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., 1972). 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 develop-
ment 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.,
1997). 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 develop-
mental cell death and that backup mechanisms likely compen-
sate when it is prevented (reviewed in Yuan and Kroemer, 2010).
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., 1998; Ellis and Horvitz, 1986). 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, 2000; Thornberry and
Roles of Programmed Cell Death in Development
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, 1986). During development of the hermaphrodite, 131
esis and soon after cell division (Ellis et al., 1991). In loss of func-
tion 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., 1992).
For each of these C. elegans genes, homologs have been
identified in other organisms: CED-3 is a caspase; CED-4 is
742 Cell 147, November 11, 2011 ª2011 Elsevier Inc.
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, 2000;
Figure 1). Additional genes have been identified in C. elegans
that affect the decision of cells to die, including the transcrip-
tional regulators CES-1, CES-2, and CEH-30 (Metzstein et al.,
1996; Thellmann et al., 2003; Schwartz and Horvitz, 2007).
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., 2007).
Another important model to study PCD during development is
the fruit fly, Drosophila melanogaster. This organism is com-
prised 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 environ-
ment (reviewed in Kornbluth and White, 2005). 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 develop-
ment 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., 1994, Grether et al., 1995; Xu et al., 2005; Srivastava
et al., 2007). Many of the genes that pattern the Drosophila
embryo, including Hox genes, activate cell death by direct tran-
scriptional regulation of the proapoptotic Reaper, Hid, and Grim
(RHG) genes (see, for example, Lohmann et al., 2002). These
genes are also transcriptionally induced by many other sig-
naling 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., 2000). In contrast, ecdysone acts as a prosurvival
factor for a set of adult neurons that survive through this transi-
tion butdie soon after eclosion. Inthis case, ecdysone represses
the expression of reaper and grim (Robinow et al., 1997; Draizen
et al., 1999). 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
Figure 1. Evolutionary Conservation of the
Core Apoptotic Machinery
A comparison between the apoptotic pathways
in C. elegans, Drosophila, and mammals reveals
conservation and expansion of the apoptotic
pathway during evolution.
(A) In C. elegans, apoptotic signals regulate the
interplay between Egl-1 (BH3-only homology) and
CED-9 (Bcl-2 family homolog), liberating CED-4
(Apaf-1 homolog) to activate CED-3 (caspase-9
homolog) for programmed death of 131 cells.
(B) In Drosophila, many different signaling path-
ways regulate the IAP antagonists Reaper, Hid,
and Grim (RHG) and the apoptosome proteins Ark
(Apaf-1 homolog) and Dronc (caspase-9 homo-
log). On the one hand, this causes the ubiquitin-
mediated degradation of DIAP1 and derepression
of caspases, and on the other hand, it enables
Dronc (caspase-9 homolog) to associate with Ark,
creating active apoptosomes and activation of the
effector caspases DrICE and Dcp1. Both path-
ways are required for efficient caspase activation
and are coordinately regulated, in analogy with
driving a car with ‘‘gas’’ and ‘‘brake.’’ However,
removal of the ‘‘brakes’’ is necessary for the effi-
cient induction of apoptosis in vivo and often
initiates it. The P35 protein can specifically inhibit
the activity of Dcp-1 and DrICE.
(C) In mammals, the balance between proapo-
ptotic and antiapoptotic Bcl-2 family members is
a key factor in the commitment to apoptosis by
regulating the release of cytochrome c and IAP
antagonists from mitochondria. Binding of cyto-
chrome c to Apaf-1 promotes apoptosome as-
sembly, which recruits and activates caspase-9.
IAP antagonists liberate caspases from the inhi-
bition of IAPs, most notably X-linked inhibitor of apoptosis (XIAP), which targets bothinitiator and effector caspases. The XIAP antagonist ARTS islocalized tothe
mitochondrial outer membrane and acts prior to the release of cytochrome c, Smac, and other proteins released from the mitochondrial intermembrane space.
Homologs proteins (by either function or sequence) are similarly illustrated.
Cell 147, November 11, 2011 ª2011 Elsevier Inc. 743
fluous cells during normal development (reviewed in Ryoo and
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., 1989). 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 ex-
ample, Lindsten and Thompson, 2006; Okamoto et al., 2006).
One reason appears to be considerable redundancy within the
caspase family and the existence of multiple mechanisms for
caspase activation. For example, some effector caspase can
be activated in the absence of Apaf-1 function (Nagasaka
et al., 2010). In addition, there is evidence for alternative backup
mechanisms that eliminate cells when apoptosis is defective
(reviewed in Yuan and Kroemer, 2010). Despite the apparent
robustness of cell death mechanisms in mammals, inhibition
of apoptosis has been linked to several specific develop-
mental abnormalities and also a variety of human pathol-
ogies, including cancer and degenerative disease (Thompson,
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.
Sculpting Structures and Driving Morphogenesis
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., 2000). Although apoptosis is the major cell death mecha-
nism 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 apo-
ptosis fails (Yuan and Kroemer, 2010). 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., 2002).
Furthermore, apoptosis is also required to permit tissue rotation
that drives looping morphogenesis of male genitalia in the fly
(Kuranaga et al., 2011).
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, 1995; Weil
et al., 1997). 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 con-
struct the mammalian palate (Glu ¨cksmann, 1951). In addition,
PCD is involved in sculpting the future inner ear in chicks (Aval-
loneetal.,2002)andisessential forgenerating thefour-chamber
architecture of the heart (Abdelwahid et al., 2002).
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 re-
quired 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 Mu ¨llerian duct forms the oviducts and uterus but is deleted
in males. Conversely, the Wolffian duct that forms the vas
Figure 2. Functions of PCD during Development
(A) PCD regulates proper structure sculpting by eliminating interdigital
(B) During Drosophila metamorphosis, nearly all larval structures are de-
stroyed, such as the salivary glands (SG), muscles (M), midgut (MG), and
hindgut (HG) (depicted by purple), whereas novel structures are raised from
undifferentiated cells termed imaginal discs (depicted by various colors).
The locations and developmental fates of the imaginal discs are similarly
(C and D) PCD also controls cell number, for example, by deleting cells that fail
to partner (C) and eliminates dangerous and abnormal cells such as autor-
eactive lymphocytes (D).
744 Cell 147, November 11, 2011 ª2011 Elsevier Inc.
deferens, epididymis, and seminal vesicle in males is degraded
in females (Jacobson et al., 1997). 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, 2002).
Regulating Cell Number
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 subse-
quently 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, 2000). It
has been estimated that more than half of all neurons generated
in the mammalian CNS are eliminated by PCD (Barres and Raff,
1999). Competition for limiting amounts of survival signals en-
sures 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., 1998). Competition
also occurs between cells that proliferate at different rates. In
this case, slower dividing cells are eliminated from the popula-
tion by more rapidly dividing cells. This phenomenon was in-
itially observed in Drosophila but is also seen in mammals (Mor-
eno, 2008; Bondar and Medzhitov, 2010). 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 (Mor-
eno, 2008). ‘‘Looser cells’’ are eliminated by hid-mediated apo-
ptosis, and genes that mediate cell engulfment are also required
for this process (de la Cova et al., 2004; Li and Baker, 2007).
Although cell competition is not essential under laboratory con-
ditions, it appears to facilitate tissue repair and has been con-
nected to oncogenic pathways (Moreno, 2008; Bondar and
Medzhitov, 2010). 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
Elimination of Unwanted and Potentially Dangerous
PCD also serves as a protective process in both animal develop-
ment 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 selec-
tion, demonstrating a functional antigen receptor that is not au-
toreactive (Figure 2D; Opferman and Korsmeyer, 2003). 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
den and Prives, 2009; Malumbres and Barbacid, 2009; Koto
et al., 2011). In all of these circumstances, apoptotic cell death
serves as an important ‘‘quality control’’ mechanism for the elim-
ination of faulty cells.
Regulation of Apoptosis
Apoptosis is the most studied and best understood form of PCD
(Figure 1). A central step in the execution of apoptosis is the acti-
vation of caspases, a family of cysteine proteases that are ubiq-
uitously expressed as inactive precursors (zymogens) with little
or no protease activity (Hengartner, 2000; Thornberry and Laz-
ebnik, 1998). 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, 2000). 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, 2004).
The caspase family has been traditionally subdivided into initi-
ator and effector caspases (Hengartner, 2000; Thornberry and
Lazebnik, 1998). Effector caspases have short prodomains and
are thought to execute apoptosis after they are proteolytic pro-
cessed by initiator caspases. Initiator caspases have long pro-
domains that bind large adaptor molecules that promote multi-
merization and caspase activation. In the case of mammalian
caspase-9, the prodomain associates with the adaptor protein
Apaf-1 and cytochrome c to form the apoptosome complex
and initiate apoptosis (Rodriguez and Lazebnik, 1999). However,
it has also been suggested that the Apaf-1/caspase-9 system
amplifies rather than initiates the mammalian caspase cascade
(Adams and Cory, 2002). Among other things, this would explain
and mouse mutants lacking Apaf-1/Ark and caspase-9/Dronc
function (Xu et al., 2005; Srivastava et al., 2007; Nagasaka
et al., 2010). In addition, it would explain the requirement of
two effector caspases, caspase-3 and -7, upstream of the mito-
chondrial outer membrane permeabilization (MOMP) that re-
leases cytochrome c (Lakhani et al., 2006). Therefore, it is likely
that, in some conditions, low-level activation of effector cas-
pases, perhaps via inactivation of their inhibitors, occurs up-
stream of ‘‘initiator’’ caspase activation.
A critical event for apoptosome assembly is the release of
cytochrome c from the mitochondria, a step tightly regulated
by members of the B cell lymphoma 2 (Bcl-2) protein family
(Figure 1; Youle and Strasser, 2008). This family is comprised
of three subfamilies depending on the number of Bcl-2 homolog
(BH) domains that they present. The antiapoptotic subfamily
consists of members with BH4 domain of which BCl-2 is most
known. The two other subfamilies are proapoptotic in nature,
either lacking the BH4 domain (BAX, BAK, and BOK) or solely
displaying BH3 (BH3-only). Once the BH3-only family is acti-
vated, they overcome the inhibitory effect of the antiapoptotic
Bcl-2 family proteins, enabling the oligomerization of BAK-BAX
within the mitochondrial outer membrane (MOM). This facilitates
MOMP and allows the release of cytochrome c (and other pro-
teins from the intermembrane space) into the cytosol, resulting
in the formation of the apoptosome (Martinou and Youle,
2011). The apoptosome then promotes the cleavage and
Cell 147, November 11, 2011 ª2011 Elsevier Inc. 745
activation of executioner caspases, such as caspase-3 and -7,
which orchestrate the destruction of the cell by cleavage of
many vital proteins (Hengartner, 2000). Caspase activation can
also occur via association with death receptors, which leads
to the oligomerization and activation of an initiator caspase,
caspase-8 (Strasser et al., 2009).
An equally important layer of cell death regulation involves
negative regulation of caspases. Procaspases are widely ex-
pressed in living cells and have low but significant protease
activity. Despite this potential danger, healthy cells avoid activa-
tion of a caspase cascade and death and use effector caspases
for nonapoptotic functions. Therefore, efficient mechanisms
must exist that prevent unwanted caspase activation. One im-
portant family of caspase inhibitors is the inhibitor of apoptosis
(IAP) proteins, which can bind to and inhibit caspases via their
baculovirus inhibitory repeat (BIR) domain (Vaux and Silke,
2005). IAPs were first discovered in insect viruses, where they
prevent apoptosis of infected cells (Crook et al., 1993). Subse-
quently, a family of related proteins has been described in both
Drosophila and mammalian genomes (Vaux and Silke, 2005).
IAPs are characterized by at least one BIR domain that can
directly bind and inhibit caspases (Vaux and Silke, 2005). The
Drosophila IAP family member Diap1 is essential for preventing
inappropriate caspase activation and ubiquitous apoptosis
(Goyal et al., 2000; Ryoo et al., 2004). Diap1 functions as an E3
ubiquitin ligase, targeting caspases in living cells and promoting
self-conjugation and degradation in apoptotic cells (Ryoo et al.,
2002; Ryoo et al., 2004). The best-studied mammalian IAP is the
X-linked inhibitor of apoptosis (XIAP) protein, which is consid-
ered the most potent caspase inhibitor in vitro (Eckelman and
Salvesen, 2006). As in Drosophila, XIAP functions as an E3 ubiq-
uitin ligase to inhibit caspases in vivo (Schile et al., 2008). How-
ever, compared to Drosophila, inactivation of XIAP causes only
relatively ‘‘mild’’ phenotypes, as there is some redundancy
among mammalian IAPs (Schile et al., 2008).
In cells committed to apoptosis, IAPs are inactivated by
specific antagonists originally discovered in Drosophila (re-
viewed in Kornbluth and White, 2005). Deletion of three closely
linked genes—reaper, hid, and grim (RHG)—blocks virtually all
apoptotic cell death in Drosophila (White et al., 1994). On the
induction of apoptosis (White et al., 1996). Although the proteins
encoded by these genes share very little overall similarity, they
all contain a short N-terminal peptide motif termed IAP-binding
motif (IBM). The IBM is structurally conserved between
Drosophila and mammalian IAP antagonists and is required for
IAP binding and cell killing (Shi, 2002).
The reaper gene and, to some extent, grim and hid are
transcriptionally induced by multiple death-inducing signals,
including developmental signals mediated by segmentation
genes, steroid hormones, Dpp, Notch signaling, JNK, and
various forms of cellular stress or injury (Figure 3; Steller, 2008).
These genes share a very large transcriptional control region
that is the target for numerous transcription factors conveying
these signals, such as various Hox transcription factors, nuclear
hormone receptors, AP-1, Polycomb, p53, and histone-modi-
fying enzymes (Brodsky et al., 2000; Jiang et al., 2000; Christich
et al., 2002; Lohmann et al., 2002; Zhang et al., 2008; Tan et al.,
2011). Therefore, one major mechanism by which different
signaling pathways converge in Drosophila is through transcrip-
tional activation of reaper, hid, and grim.
The pro- activity of Hid is inhibited upon phosphorylation by
MAP-kinase, and its transcription is repressed by the prosurvival
the EGF-receptor/Ras-pathway in Drosophila (Bergmann et al.,
1998, 2002). RHG genes are also the target for microRNA regu-
lation (Brenneckeet al., 2003). Finally, for effective cell killing, the
Reaper protein has to localize to the MOM (Sandu et al., 2010).
This is achieved through multimerization with Hid, which local-
ing et al., 1999). Taken together, the activity of RHG proteins is
regulated by many different signaling pathways at both the tran-
scriptional and posttranscriptional level, and these proteins
serve as ‘‘integrators’’ to connect diverse signaling pathways
with the core cell death program.
Mammalian IAPantagonistscontaining anIBMhavealsobeen
discovered, including Smac/Diablo and HtrA2/Omi (Verhagen
et al., 2000; Suzuki et al., 2001). In contrast to RHG proteins,
these proteins are localized within the mitochondrial intermem-
brane space and require MOMP to access and bind cytosolic
onist is ARTS (Larisch et al., 2000; Gottfried et al., 2004).
Although ARTS does not contain a recognizable IBM, it is local-
ized, like RHG proteins, to the MOM and is a specific and phys-
iological inhibitor of XIAP (Gottfried et al., 2004; Garcı ´a-Ferna ´n-
dez et al., 2010; Edison et al., 2011). Therefore, ARTS does not
require MOMP and acts upstream of cytoC and Smac/Diablo
(Edison et al., 2011). Mice deficient for the Sept4 gene, which
encodes ARTS, have elevated XIAP protein levels, are defective
in the caspase-mediated elimination of bulk cytoplasm during
spermiogenesis, and have elevated numbers of hematopoietic
stem and progenitor cells (Kissel et al., 2005; Garcı ´a-Ferna ´ndez
Figure 3. Integration of Different Signaling Pathways by RHG
Thegenesencoding Reaper,Hid,andGrim (RHG)arethetargetsforregulation
of many signaling pathways that influence thedecision between cell death and
survival. The transcriptional control region of RHG genes, exemplified here by
reaper, contains binding sites for many different transcription factors that are
the downstream targets of different signaling pathways, including for the
steroid hormone ecdysone, patterning signals and Hox genes, and DNA
damage/p53. Examples for both activators (green) and repressors (red) have
been described. The steroid hormone ecdysone can either induce or repress
reaper transcription depending on the stage of development. RHG genes are
clustered and, to some extent, coregulated at the transcriptional level, and the
locus can be silenced by histone-modifying enzymes and Polycomb.
746 Cell 147, November 11, 2011 ª2011 Elsevier Inc.
et al., 2010). In addition, these mice demonstrate increased sus-
ceptibility toward developing malignancies (Garcı ´a-Ferna ´ndez
et al., 2010). Importantly, these phenotypes are suppressed by
inactivation of XIAP, suggesting that it is a major physiological
target for ARTS. These observations support a physiological
role of ARTS and XIAP in the regulation of stem cell apoptosis.
In the end, the activation of caspases is under dual control by
both activators and inhibitors, which in turn are subject to many
layers of regulation. In analogy with driving a car, a combination
of ‘‘gas’’ and ‘‘brakes’’ ensures that apoptosis only proceeds
when multiple checkpoints are passed. The complexity of cas-
pase regulation increases as the number of cells increases from
worms to flies to mammals. As organismal complexity and life
span increase, additional backup mechanisms are in place,
presumably due to the elevated danger that unwanted cells
pose to a long-lived animal.
The removal of apoptotic cells is the final step in the execution of
apoptosis. Historically, this process has presented a significant
hurdle in evaluating the amount of apoptosis, as the dying cells
are rapidly cleared by phagocytes, making it very difficult to
detect apoptotic cells animal tissues. The engulfment process
can be divided into the following stages: (1) sensing of the apo-
ptotic cell, (2) recognition by the phagocyte, (3) internalization of
target cell, (4) ingestion, and (5) postengulfment response of the
phagocyte (Ravichandran and Lorenz, 2007).
Dying apoptotic cells secrete ‘‘find me’’ and ‘‘eat me’’ signals
that attract and recruit phagocytes. The first ‘‘find me’’ signal to
be identified is lysophospatidylcholine (LPC). LPC is released by
apoptotic cells in a caspase-3-dependent fashion. Caspase-3
activates the calcium-independent phospholipase A2(iPLA2),
which converts phosphatidylcholine to LPC (Lauber et al.,
2003). Several other molecules have also been proposed to
portray the ‘‘find me’’ role, such as sphingosine 1 phosphate
tion, ATP and UTP nucleotides, released by the PANX1 channel,
have been suggested to act as ‘‘find me’’ signals (Elliott et al.,
2009). PANX1 is a target of effector caspase-3 and -7, in which
a specific caspase cleavage site is essential for PANX1 function
in mediating release of ‘‘find me’’ signals during apoptosis
(Chekeni et al., 2010).
The best-studied ‘‘eat me’’ signal is phosphatidylserine (PS),
a component retained exclusively on the inner leaflet of the
plasma membrane, which is only exposed to the extracellular
surface when cells apoptose (Fadok et al., 1992). The exposure
of PS on apoptotic cells is a conserved feature demonstrated by
C. elegans, Drosophila, and mammals (Nagata et al., 2010). This
process iscaspase dependent (Martin et al.,1996), but the exact
mechanism remains controversial. PS can be bound directly by
a phagocyte receptor or indirectly by binding to bridging mole-
cules that recognize PS, thus mediating the interaction with the
phagocyte receptor (Nagata et al., 2010).
Apoptotic cell engulfment is regulated by the Rho family of
GTPases such as Rac1, RhoA, and Rab5 (Nakaya et al., 2006).
Cells are then converted into their basic building blocks: amino
somes. Engulfment is not merely the concluding step of cell
death, but also plays a decision-making role in cell death (Li
and Baker, 2007). Engulfment is also required for cell competi-
tion in Drosophila, with the engulfment genes—Draper, wasp,
PS receptor, mbc/dock180, and Rac1—required for the execu-
tion of a neighboring cell.
A promising avenue for future research will be to examine the
engulfment/cell competition process in cancer (Moreno, 2008).
As tumors are comprised of cells with distinct genotypes, it is
highly attractive to speculate that they can outcompete wild-
type cells. Interestingly, in Drosophila, elimination of oncogenic
neighbors is mediated by a JNK-dependent engulfment mecha-
nism (Ohsawa et al., 2011).
Nonapoptotic Programmed Cell Death
Although apoptosis represents the most prominent and best-
studied mode of PCD in development, it is clear that alternative
mechanisms exist (Yuan and Kroemer, 2010). In C. elegans,
nearly all PCD is achieved by apoptosis, but death of the linker
cell occurs in a caspase-independent fashion (Abraham et al.,
cells often die by other mechanisms (Yuan and Kroemer, 2010).
However, the importance of these alternative pathways during
normal development remains to be critically examined. In addi-
tion, as many of these experiments utilize caspase inhibitors to
prevent apoptosis, it is not clear whether complete inhibition of
caspases has been achieved and to what extent low caspase
activity may compromise cell function.
logical criteria: type I cell death, known today as apoptosis
(detailed above), and type II, now often referred to as autophagic
cell death. Although necrosis has been traditionally seen as a
passive form of cell death, proteins that regulate this process
have been identified recently. This form of cell death is termed
‘‘necroptosis,’’ and it can be inhibited by a small molecule inhib-
itor necrostatin 1 (Nec-1) (Yuan and Kroemer, 2010).
Autophagy is a catabolic process that disposes of various
cytoplasmic components, including protein aggregates and
organelles. These components are marked for autophagy and
are then engulfed by autophagosomes, which fuse with lyso-
somes to be degraded. This process has been extensively
studied in the yeast Saccharomyces cerevisiae in response to
starvation. In this context, it protects the cell by recycling of its
content. This is dependent upon a large group of autophagy-
related (ATG) genes, which are conserved from yeast to human
(Nakatogawa et al., 2009). Under most conditions, autophagy
is a survival mechanism that can sustain cell viability for weeks
in response to growth factor withdrawal or nutrient deprivation
(Levine and Yuan, 2005). However, studies on the metamorphic
death of Drosophila salivary glands suggest that autophagy
contributes to the programmed death of this tissue (Berry and
Baehrecke, 2007). At the same time, several apoptotic genes,
including caspases, participate in the death of salivary glands
as well, and the exact contribution of each mechanism awaits
further clarification (Lee et al., 2003). Another system involving
both caspase activation and autophagy is the death of
Drosophila larval midgut cells (Denton et al., 2009).
In mice, some experiments suggest that autophagy can serve
as an alternate killing mechanism in situations in which the
Cell 147, November 11, 2011 ª2011 Elsevier Inc. 747
apoptotic program cannotbeexecuted. Mouse embryonicfibro-
blasts (MEFs) isolated from BAX?/?BAK?/?mice are resistant to
a variety of apoptotic inducers but can undergo cell death in an
autophagy-mediatedmanner (Maiuri etal., 2007). Knockdown of
Beclin/ATG6 reduces cell death in BAX?/?BAK?/?MEFs (Shi-
mizu et al., 2004). Together, these observations link caspases
and autophagy, and one of the important challenges in the field
is understanding the mechanistic link between apoptosis and
Nonapoptotic Roles of Caspases
The first caspase to be characterized, caspase-1 (then termed
interleukin converting enzyme [ICE]) was studied for its role in
processing of the inflammatory cytokine proIL-1b into mature
IL-1b (Thornberry et al., 1992). Therefore, it should not come as
However, what was somewhat unexpected is that apoptotic
effector caspases, together with their upstream regulators nor-
mally used in apoptotic cell death (e.g., Dronc/caspase-9,
Apaf-1, and cytochrome c), are also used to eliminate portions
of a cell, sculpting or dramatically altering cytoarchitecture.
Caspases in Immunity
The link between apoptosis and the immune response is well
conserved among metazoans. For example, when C. elegans
are fed Salmonella typhimurium, the bacteria inhabit the intes-
tine, triggering cell death in the gonad and untimely death of
the organism. Although ced-3 or ced-4 mutant worms develop
normally, they are hypersensitive to S. typhimurium-mediated
killing, suggesting that that these apoptotic proteins play an
important role in defending worms against pathogens (Aballay
and Ausubel, 2001).
In Drosophila, the innate immune system is comprised of two
signaling networks. The Toll pathway recognizes Gram-positive
bacteria and fungi, and the immune deficiency (IMD) pathway is
responsible for responding to Gram-negative bacteria (Ferran-
don et al., 2007). The IMD signaling cascade activates the NF-
kB-related transcription factor Relish, an essential regulator of
antimicrobial peptide gene induction. Activation of Relish re-
quires phosphorylation by the Drosophila IkB kinase (IKK) and
then cleavage by the death-related ced-3/Nedd2-like protein
(DREDD), an ortholog of caspase-8 (Ertu ¨rk-Hasdemir et al.,
2009). DREDD not only cleaves Relish, but also activates the
IKK complex, functioning as a key component of the defense
response (Ertu ¨rk-Hasdemir et al., 2009). It is, therefore, not
surprising that mutant dredd flies are highly susceptible to
E. coli infection failing to produce antimicrobial peptides (Leulier
et al., 2002).
In mammals, external death stimuli, such as those generated
by bacterial infections, trigger the formation of two distinct cas-
pase complexes: the death-inducing signaling complex (DISC)
and the inflammasome, which is essential for the maturation of
cytokines (Mace and Riedl, 2010). When a death ligand, such
as Fas, binds its receptor, the death receptors oligomerize and
subsequently recruit FADD and initiator procaspase-8 or -10,
forming the DISC (Yu and Shi, 2008). Originally described for
its role in mediating cell death, recent data also uncover nona-
poptotic functions for the DISC complex, demonstrating that
caspase-8 functions are conserved between mammalsand flies.
Similar to DREDD, caspase-8 associates with the IKK complex
and induces NF-kB transcriptional activity (Lemmers et al.,
2007). In addition, two independent groups demonstrate that
caspase-8 regulates the immune response, as deficient mice
were unableto clear a viralinfection or efficientlyresist livercolo-
nization of listeria monocytogenes (Salmena et al., 2003; Ben
Moshe et al., 2007).
maturation of inflammatory cytokines. Caspase-1, also known
as interleukin-1b-converting enzyme, or ICE, is the mammalian
homolog of ced-3 and the founding member of the proinflamma-
tory caspase family (Miura, 2011). Originally known for its ability
to regulate the conversion of prointerleukin1b (IL-1b) into mature
IL-1b, we now know that it regulates other cytokines, such as
IL-18 and IL-33 (Miura, 2011; Yuan et al., 1993). In addition to
caspase-1, caspase-11 and -12 act as proinflammatory cas-
pases in mice; caspase-4 and caspase-5 carry out this function
in humans (Martinon and Tschopp, 2004). Mice deficient for
either caspase-1 or caspase-11 display a remarkably similar
phenotype. They are resistant to LPS-induced shock and exhibit
impaired IL-1 production in this context (Wang et al., 1998).
two caspases can hetrodimerize (Yi and Yuan, 2009). The com-
plex that activates caspase-1 in response to pathogens is
named the inflammasome. To date, four distinct inflammasomes
have been described. The NLRP3 inflammasome, which has
been most extensively characterized, is composed of an NLRP3
scaffold, the ASC adaptor, and caspase-1 and responds to a
variety of pathogens. In this context, autocleavage of procas-
pase-1 is stimulated, leading to the production of the active
p10/p20 tetramer, which processes immature cytokines and
generate active molecules (Schroder and Tschopp, 2010).
Cellular Remodeling by Caspases
In mammals, caspase-1 regulates the maturation and release of
thioredoxin (Keller et al., 2008). Secretion of these caspase-1-
mediated paracrinic and autocrinic factors can increase cell pro-
liferation, indicating that caspase-1 might have a crucial role in
homeostasis and reconstruction of tissues under stress or
damage conditions. In this regard, it is noteworthy that caspases
can induce mitogenic pathways, thereby facilitating proper
wound repair and regeneration. During apoptosis, caspase
activity causes rapid and complete destruction of the entire
cell. This lethal activity can be refocused and restrained for the
specific demolition of particular cellular structures during cellular
remodeling and differentiation. Although distinct from apoptotic
cell death, there are striking morphological and biochemical
similarities, and this process can be viewed as subcellular
nucleus is also critical for the terminal differentiation of certain
cell types, including megakaryocytes, erythrocytes keratino-
cytes, and epithelial cells in the lens. The vertebrate lens func-
tions to focus incoming light, which is possible by virtue of its
748 Cell 147, November 11, 2011 ª2011 Elsevier Inc.
transparency. During embryonic development, lens fibers
achieve transparency by degrading all membrane-bound organ-
elles in the center of the lens (Feinstein-Rotkopf and Arama,
2009). The morphological features of this terminal differentiation
process highly resemble that of apoptosis, demonstrating chro-
matin condensation, nuclear pore clustering, and staining for
TUNEL (Feinstein-Rotkopf and Arama, 2009). The enucleation
process can be blocked with caspase inhibitors, and overex-
pression of Bcl-2 in both mice and chicken lenses resulted in
retention of the nuclei (Feinstein-Rotkopf and Arama, 2009).
However, mice deficient for caspase-3, -6, or -7, as well as
mice lacking both caspase-3 and -6, do not demonstrate lens
defects, indicating that no single executioner caspase (nor a
combination of caspase-3 and -6) is required for organelle loss
(Zandy et al., 2005). Interestingly, caspase-3 activity has been
found to be required for the maintenance of lens transparency,
as caspase-3 null mice get cataracts (Zandy et al., 2005). Given
the functional redundancy between caspase-3 and -7, it will be
interesting to investigate lens differentiation in these animals.
Limiting this type of analysis, however, the inactivation of both
of these caspases prevents mitochondrial and death receptor-
mediated apoptosis, and these double-mutant animals die
immediately after birth due to defective cardiac development
(Lakhani et al., 2006).
Another epithelial cell type that relies on a caspase-mediated
process for enucleation is the keratinocyte, which forms the
major component of the skin epidermis. The epidermis under-
goes constant cell turnover, with the keratinocytes in the basal
layer being continuously pushed away from the basement
membrane toward the outer differentiated layers. Upon detach-
ment from the basement membrane, keratinocytes withdraw
from the cell cycle and undergo a cornification, a specialized
form of differentiation. This process is typified by expression
and processing of caspase-14, which in contrast to ubiquitously
expressed caspases, is only expressed in the differentiating and
cornifying layers of the epidermis. Caspase-14 has been found
to localize to the nuclear remnants of corneocytes and is associ-
ated with the nucleus in the precursor granular layer. Although
these findings support the hypothesis that caspase-14 is re-
quired for nuclear degradation, caspase-14-deficient mice
show no defects in cornification (Denecker et al., 2007).
Red blood cells and megakaryocytes also lose their nuclei as
they mature. Erythroblasts mature into anucleate red blood cells
in a process termed erythropoiesis, which also requires caspase
activity. Caspase-2, -3, and -9 have been shown to be active
fere with the maturation of erythroid progenitors (Lamkanfi et al.,
2007). These caspases cleave nuclear proteins Lamin B, PARP,
Similarly, the differentiation of megakaryocytes into mature
enucleate platelets requires activation of caspase-3 and -9 (De
Botton et al., 2002).
In insects and mammals, spermatogenesis yields mature (yet
short-lived) germ cells with limited cytoplasm and very few
organelles. In Drosophila, this process is associated with the
appearance of several apoptotic markers and caspase activa-
tion (Arama et al., 2003). Inhibition of caspase activity interferes
with the removal of cytoplasm and causes sterility (Arama et al.,
2003; Huh et al., 2004), and caspase activation in this system
strictly depends on a testis-specific cytochrome c gene, cyt-
c-d. Mutations in cyt-c-d cause male sterility due to impaired
Dronc activation (Arama et al., 2006). Other components of the
apoptosome complex, ark and dronc, are also involved in cas-
pase activationduringspermatogenesis (Huhetal., 2004;Arama
et al., 2006). Finally, mutations in the giant IAP-like protein,
dBruce, cause nuclear degeneration spermatid death, indicating
that this protein plays a role in protecting the nucleus from
unwanted caspase activity (Arama et al., 2003).
During spermatogenesis, regulation of apoptosis and control
of nonapoptotic functions of caspases relies on the UPS (Bader
sisting of Cullin-3, Roc1b, and Klhl10, regulates caspase activa-
tion in spermatids by targeting dBruce for local degradation
(Arama et al., 2007; Kaplan et al., 2010). A recently identified
inhibitor of this Cullin-3 complex called Soti competes with
dBruce for biding to Klhl10, and Soti mutants exhibit elevated
levels of active effector caspase. Interestingly, Soti protein is
distributed as a subcellular gradient in the inverse direction of
caspase activation. This provides a mechanism to explain how
spatial regulation of caspases can be achieved to drive differen-
tiation instead of cell death execution (Kaplan et al., 2010).
Another ubiquitin E3 ligase complex critical for caspase acti-
vation in spermatids contains the Cullin-1 and the F box protein,
Nutcracker (Bader et al., 2010). Surprisingly, Nutcracker is not
only required for caspase activation, but also regulates protea-
some activity (Bader et al., 2011). In particular, Nutcracker
promotes the stability of DmPI31, which in turnserves asan acti-
vator of the 26S proteasome. Proteasome function is required
for sperm differentiation and has been previously linked to the
apoptotic pathway (Zhong and Belote, 2007). Taken together,
these findings reveal the coordinate use of two major proteolytic
systems—caspases and the 26S proteasome—to localized cell
demolition and the removal of unwanted organelles.
In mammalian spermatogenesis,
caspase-3 is also activated during late stages of spermatogen-
esis (Kissel et al., 2005). Targeted deletion of the Sept4 locus,
which encodes the IAP antagonist ARTS protein, causes male
infertility and structural abnormalities in sperm, including defects
as in Drosophila, mammalian KLHL10 interacts with Cullin-3 and
is exclusively expressed in spermatids (Wang et al., 2006; Yan
et al., 2004). Inactivation of KLHL10 in the mouse results in
complete sterility, with spermatogenesis arrested during the
elongating stage. Further supporting a conserved function for
in KLHL10 is associated with human male infertility (Yan et al.,
2004; Yatsenko et al., 2006). A direct role for KLHL10 in caspase
regulation remains to be established in mammals. Therefore, it
that has been conserved in evolution from flies to humans.
Pruning of axons and dendrites is a crucial process that sculpts
neuronal connections during development, as it removes
Cell 147, November 11, 2011 ª2011 Elsevier Inc. 749
of the cell (Luo and O’Leary, 2005). During insect metamor-
phosis, larval neurons undergo massive pruning via a multistep
process that includes destabilization of the cytoskeleton, frag-
mentation, and then clearance of cellular remnants (Awasaki
and Ito,2004).Severalgroupshavedemonstrated thatcaspases
promote the removal of dendrites during Drosophila metamor-
phosis. In Dronc-deficient flies, pruning of dendrites is sup-
pressed, with ectopic branches present after puparium forma-
tion (Williams et al., 2006). Furthermore, overexpression of
DIAP1, p35, or a dominant-negative form of Dronc resulted in
suppression of pruning (Williams et al., 2006). Modulation of
this process requires the ubiquitin proteasome system (UPS).
UPS promotes the degradation of Diap1 to unleash Dronc
activity (Kuo et al., 2006). In addition, a ubiquitin-selective chap-
erone, VCP, binds Diap1 in a ubiquitin- and BIR domain-depen-
dent manner, facilitating Diap1 degradation (Rumpf et al., 2011).
Inhibition of VCP leads to elevated Diap1 levels and reduced
caspase activation and impairs the dendritic pruning process.
Finally, inactivation of the Drosophila effector caspase Drice
can also result in suppression of branch removal (Schoenmann
et al., 2010).
In the mouse, effector caspases are also used for neurite
pruning during development (Nikolaev et al., 2009). The death
receptor DR6 protein is localized to both the cell bodies and
axon of many neurons. Significantly, blocking DR6 function
delays pruning of sensory axons in vitro and retinocollicular
down of caspase-6 were also shown to inhibit sensory axon
degeneration in Campenot chambers (Nikolaev et al., 2009).
Remarkably, this study also showed that the amyloid precursor
protein (APP), a protein widely studied for its role in Alzheimer’s
disease, serves as a regulated ligand of DR6 in this system. In
this context, it collaborates with DR6 to initiate caspase activa-
tion, which may differentially trigger axonal degeneration and
neuronal cell death. These findings reveal a mechanism that
may contribute to shaping neuronal circuits during develop-
ment and in adult plasticity, and they raise the possibility that
abnormal regulation of this process may contribute to Alz-
Caspases in Learning and Memory
There is also emerging evidence that caspases serve a role in
learning and memory. In the zebra-finch auditory forebrain, the
concentration of active caspase-3 is highly induced within
minutes after exposure to recorded birdsong. Furthermore,
caspase-3 is present in the dendritic spines in an inactive state
through interaction with XIAPand isrequired formemoryconsol-
idation. Birds were repetitively trained with a song stimulus,
testing the habitual response the following day, employing a
habituation marker. In the control placebo group, birds acknowl-
edged the trained song. In striking contrast, the experimental
group that wastreated with acell-permeable caspase-3 inhibitor
did not recognize the song, reacting as if it were novel (Hues-
mann and Clayton, 2006). These results, combined with the
aforementioned studies on neuronal remodeling, lead to the
obvious hypothesis that caspase activity may be important for
the structural modifications required for memory.
Caspases may facilitate learning and memory by influencing
the trafficking and internalization of neurotransmitter receptors.
Many forms of learning and memory require experience-depen-
dent synaptic adjustments in the hippocampus (Bateup and
Sabatini, 2010). For example, the NMDA receptor-dependent
synaptic modifications such as long-term potentiation (LTP)
and long-term depression (LTD) correlate with an increase or
decrease in the number of receptors, respectively. LTD occurs
mainly by removal of AMPA receptors from the postsynaptic
membrane. In neurons of the hippocampus, an area of the brain
critical for learning and memory, the activity of initiator and
executioner caspases is essential for LTD and AMPA receptor
internalization (Li et al., 2010b). Blocking caspase activity with
caspase inhibitors, overexpression of XIAP or Bcl-2 inhibits
LTD and AMPA receptor internalization. Furthermore, LTD is
abolished in the hippocampus of caspase-3-deficient mice (Li
et al., 2010b). In a subsequent study, caspase-3 was shown to
be activated by BAD and BAX, but not Bid. BAD is activated in
a transient and moderate fashion, and BAX does not translocate
to the mitochondria. This appears to fine-tune the activation
(Jiao and Li, 2011). Further studies are needed to establish the
molecular targets linking caspases with AMPA receptor inter-
nalization, and it remains to be seen whether this caspase-
dependent mechanism is responsible for the apparent effect
on learning and memory in zebra-finches.
Role of Caspases in Cell Differentiation
Besides promoting the destruction of specific cellular compart-
ments, caspases also promote cell differentiation in more subtle
ways. One example is for the specification of external sensory
organs in Drosophila. Flies mutant for Ark, Dronc, or cyto-c-d
contain a number of additional bristles that are part of a mecha-
et al., 2006). In this system, caspases promote activation of
Sgg46, the Drosophila ortholog of gsk3-b, an antagonist of the
Wnt pathway (Kanuka et al., 2005). Activation of sgg46 is re-
quired for negative regulation of Wg signaling, indicating that
caspases can regulate neural development by antagonizing Wg
signaling. In a screen for caspase suppressors, dmIKKε, the
Drosophila ortholog of IKKε or NAK, was identified (Kuranaga
et al., 2006). dmIKKε was found to regulate Dronc activity by
promoting phosphorylation and subsequent degradation of
Diap1. Silencing of dmIKKε resulted in attenuation of caspase-3
activity in SOP cells and the appearance of additional macro-
chaetes (Kuranaga et al., 2006). Furthermore, mammalian IKKε
plays a conserved role in phosphorylation-dependent degrada-
tion of XIAP, indicating a similar role in caspase regulation in
mammals (Kuranaga et al., 2006).
Caspases have also been implicated in skewing monocyte
differentiation toward to macrophages rather than dendritic cells
(Sordet et al., 2002). As monocytes differentiate into macro-
phages, they activate both caspase-9 and -3 but do not exhibit
apoptotic features. Inhibition of caspase activity with p35 or
zVAD or overexpression of Bcl-2 inhibited the macrophage dif-
ferentiation (Sordet et al., 2002). Furthermore, a screen for cas-
pase substrates during cell differentiation processes uncovers
38 differentially expressed caspase targets (Cathelin et al.,
750 Cell 147, November 11, 2011 ª2011 Elsevier Inc.
tion leads to arrest of differentiation into macrophages (Kang
et al., 2004). Moreover, caspase-8 binds and cleaves RIP1, pre-
venting sustained activation of NF-kB in monocytes undergoing
macrophagic differentiation (Re ´be ´ et al., 2007).
Caspases alsoplay crucial nonapoptoticroles in the differenti-
crucial role in osteogenic differentiation of bone marrow stromal
SCs, neural SC differentiation, and differentiation of embryonic
SCs, as well as in regulating the differentiation and proliferation
in hematopoietic SCs (Feinstein-Rotkopf and Arama, 2009).
Apoptotic Cells Can Stimulate Proliferation, Wound
Healing, and Tissue Regeneration
Regeneration, a process of regrowth or repair, equips animals
with the capacity to maintain homeostasis, even after severe
injury. Remarkable examples for regeneration can be seen in
of a new head, and planaria have the ability to regenerate com-
plete individuals from small body fragments (Birnbaum and Sa ´n-
chez Alvarado, 2008). In Drosophila, imaginal discs can give rise
regenerative capacity of the liver enables restoration of full organ
mass within a very short time frame, even after 70% of the liver
has been removed (Taub, 2004). Depending on the type of tissue
damage, the regenerative process includes several steps, in-
cluding wound repair, formation of highly proliferative blastema
cells, differentiation, and patterning (Gurtner et al., 2008). Work
force behind regeneration. Specifically, the proliferative aspect
of regeneration, including blastema formation, is stimulated
by signals from apoptotic cells. This phenomenon is termed
apoptosis-induced compensatory proliferation (Bergmann and
Cells undergoing apoptosis in response to stress or injury can
stimulate the proliferation of neighboring cells. A series of exper-
iments in Drosophila first demonstrated this phenomenon
(Pe ´rez-Garijo et al., 2004; Ryoo et al., 2004). These studies had
to overcome a fundamental problem in studying apoptotic-stim-
ance. To circumvent this problem, the potent caspase inhibitor
p35 was used to block the execution of apoptosis and sustain
program is induced but cannot be executed. Because p35
specifically inhibits effector caspases, Dronc is active in ‘‘un-
dead cells’’ and is able to perform nonapoptotic functions. This
system allowed the identification of genes and mechanisms
that govern compensatory proliferation. p53 and Dronc collabo-
rate to induce compensatory proliferation and stimulate blas-
tema formation (Wells et al., 2006). Because the proapoptotic
genes reaper and hid are direct transcriptional targets of p53, a
positive feedback loop involving Dronc, Reaper, and Hid pro-
teins may operate in this system. Such a mechanism under-
scores the similarities between compensatory proliferation and
JNK signaling plays a critical role in compensatory prolifera-
tion and wound healing in Drosophila (Ryoo et al., 2004; Bosch
et al., 2005). Different reports have indicated that JNK is either
a downstream target of Dronc or that it acts independently
from the apoptosis program (Ryoo et al., 2004; Pe ´rez-Garijo
et al., 2009). A recent study reconciles these different observa-
stream of the proapoptotic RHG genes and the initiator caspase
Dronc, establishing a positive feedback loop that amplifies initial
apoptotic stimuli and facilitates the apoptotic stress response
(Shlevkov and Morata, 2011).
In addition to the p53 and JNK pathways, compensatory
proliferation requires mitogenic signals. ‘‘Undead cells’’ secrete
mitogens/morphogens, such as Wingless (Wg, Wnt ortholog)
ation of surrounding cells (Figure 4A) (Ryoo et al., 2004). These
factors play key signaling roles during early development, pro-
mote the self-renewal of stem cells, and stimulate tissue regen-
eration in vertebrates and insects (Bergmann and Steller, 2010).
These mechanisms are not an artifact of p35 overexpression:
active JNK signaling induces transcription of wingless in ‘‘nor-
mal’’ apoptotic cells (Ryoo et al., 2004), and Wg and JNK are
requiredfor regeneration in
(Figure 4B) (Bergantin ˜os et al., 2010; Smith-Bolton et al., 2009).
For example, in the differentiating Drosophila retina, Hedgehog
(Hh) signaling is required for apoptosis-induced compensatory
proliferation. In response to stress, postmitotic photoreceptor
neurons secrete Hh, stimulating the proliferation of nearby cells
(Figure 4C; Fan and Bergmann, 2008). Contrary to the Wg,
Dpp, and JNK pathways, which require p53 and Dronc, hh acti-
vation occurs downstream of the effector caspases DrICE and
Compensatory proliferation also occurs in other organisms,
including Hydra, Xenopus, Planaria, newts, and mice. In the
freshwater polyp Hydra, apoptosis is both necessary and suffi-
cient for head regeneration post midgastric bisection (Chera
et al., 2009). Similar to Drosophila, caspases stimulate release
of Wnt3, promoting proliferation and facilitating regeneration
(Figure 4D). Although planarians and newts also demonstrate
massive apoptosis at the site of amputation, the molecular
connections with regeneration await discovery (Figure 4E). In
the Xenopus tadpole, a large number of apoptotic cells are
seen in the nascent regeneration bud within 12 hr postamputa-
tion (Tseng et al., 2007), and apoptosis is important for the initi-
ation of tail regeneration. Although Wnt signaling is crucial for
regeneration in Xenopus, it remains to be seen whether it is
secreted by the dying cells (Figure 4F). Studies in mice also
support a link between apoptosis and regeneration. For ex-
ample, mice lacking either caspase-3 or -7 have impaired skin
wound healing and liver regeneration. Interestingly, a down-
stream target of these caspases is Prostaglandin E2, known to
tenance and tissue regeneration (Figure 4G; Li et al., 2010a;
Goessling et al., 2009).
These studies of apoptosis-induced compensatory prolifera-
tion illustrate that both initiator and executioner caspases influ-
ence the release of mitogens from stressed or injured cells,
thereby promoting regeneration. This body of work also reveals
striking similarities between tissue regeneration and cancer.
Malignant tumors often develop at sites of chronic injury, and
Cell 147, November 11, 2011 ª2011 Elsevier Inc. 751
tissue injury has an important role in the pathogenesis of malig-
nant disease (Scha ¨fer and Werner, 2008). Ifcompensatory prolif-
eration operates in tumor cells, inducing apoptosis in cancer
cells may actually stimulate proliferation of surviving cells.
Even if cancer therapies kill the majority of cells and decrease
tumor size, mitogenic signaling may contribute to relapse, sec-
ondary tumors, or metastases by stimulating proliferation of
of this scenario, cancer cells demonstrate some level of apo-
ptotic resistance, a feature that they share with artificially gener-
ated ‘‘undead’’ cells.
Figure 4. Apoptosis-Induced Compensa-
tory Proliferation in Various Organisms
In different model organisms, apoptosis and
caspase activity have been observed to induce
secretion of mitogenic factors, thereby promoting
hyperplastic overgrowth or tissue regeneration.
Caspase targets and mitogenic factors are
indicated in pink, and question marks indicate
(A) In Drosophila, inhibition of caspases by P35
renders cells in an ‘‘undead’’ state unable to
complete apoptosis. This results in the activation
of p53 and JNK, triggering the release of the Wg
and Dpp mitogens and thereby promoting hyper-
(B) In Drosophila, temporal and spatial apoptosis
via compensatory proliferation (CP) by secretion
of Wg. A dP53/JNK positive feedback loop is
essential for the apoptotic response.
(C) In Drosophila, differentiating neurons induce a
different compensatory proliferation pathway, via
hedgehog (Hh), in a manner requiring both DrICE
and Dcp-1. Hh stimulates the proliferation of
(D) In Hydra, head regeneration post midgut
bisection is dependent upon caspase activity,
during which apoptotic cells secrete Wnt3 pro-
moting compensatory proliferation.
(E) In newts and planaria, amputation is charac-
terized by apoptosis and caspase activity in the
wound site. However, it still unknown whether this
apoptotic response is responsible for the release
of Wnt and Hh.
(F) In Xenopus, amputation of the tail results in
caspase activity, whereas inhibition of caspase-9
and -3 prohibits cell proliferation and the regen-
erative process. It remains to established whether
this form of CP is mediated by Wnt signaling.
(G) In mice, wound repair and liver regeneration
are dependent upon caspase-3 and –7, which are
necessary for proper induction of these pro-
cesses. Caspase-3 mediates the proteolytic pro-
cessing of iPLA2, which in turn produces archi-
donic acid, the precursor of PGE2, a known
stimulator of stem cell proliferation, tissue regen-
eration, and wound repair.
Apoptosis and Stem Cells
Stem cells are defined by their virtually
unlimited proliferative potential and multi-
lineage differentiation capacity and are
In tissues, SCs are maintained for long
periods of time, undergoing multiple
rounds of self-renewal. As such, they
are at risk of accumulating potentially deleterious mutations.
The combined consequence of their longevity and proliferation
potential enables the propagation of these mutations to down-
stream progeny in a manner that could lead to oncogenesis
(Rossi et al., 2008).
The hematopoietic system provides clear examples of how
changes in programmed cell death in a small number of hemato-
poietic stem cells (HSCs) can lead to differentiation defects and
increased cancer risk. Overexpression of BCL-2 increases
HSC number and enhancement in HSC repopulation potential
while displaying resistance to various chemotherapeutic agents
752 Cell 147, November 11, 2011 ª2011 Elsevier Inc.
(Domen et al., 2000; Domen and Weissman, 2003). Another
member of the BCL-2 family, Mcl-1, is important for HSC regula-
tion. Silencing of Mcl-1 reduced the self-renewal of human HSC
in vivo, whereas knockdown of Mcl-1 in human pluripotent SC
almost completely ablated SC self-renewal (Campbell et al.,
2010a). Furthermore, mice overexpressing Mcl-1 in the hema-
topoietic compartment develop stem/progenitor cell tumors
(Campbell et al., 2010b). BCL-2 proteins protect other SCs
from apoptosis. In human embryonic SCs (ESCs), BCL-2 en-
hances survival, and ESCs overexpressing BCL-2 displayed
normal growth in the absence of serum (Ardehali et al., 2011).
HSC survival alsorequires additional layersof regulation. Mice
lacking the IAP antagonist, ARTS, develop spontaneous hema-
topoietic malignancies and have an increased number of func-
tional hematopoietic stem and progenitor cells (HSPCs) in the
bone marrow. These phenotypes can be suppressed by inacti-
vation of XIAP, indicating that XIAP is a physiological target for
the proapoptotic activity of ARTS (Garcı ´a-Ferna ´ndez et al.,
2010). p53 is also required for HSC survival and homeostasis.
Upon irradiation, HSPCs lacking p53 have a selective advan-
tage, which leads to long-term clone expansion and lymphoma
development (Marusyk et al., 2010). Interestingly, p53-mediated
HSPC cell competition depends on the relative, not absolute,
level of p53 in competing cells (Bondar and Medzhitov, 2010).
Given the potentially dire consequences of accumulating
to limit DNA damage or respond rapidly to such damage (Seita
et al., 2010). One protective feature may be the quiescent state
in which many SCs are retained, minimizing the chance for repli-
cation errors and endogenous ROS-mediated DNA damage
(Yamazaki et al.,2006; Tothova etal., 2007). This quiescent state
also raises a challenge for adult SCs, as the majority of DNA
repair pathways are cell cycle dependent (Mandal et al., 2011).
When mice are subjected to ionizing radiation (IR), their rapidly
proliferating short-lived progenitors (SLPs) are quickly elimi-
nated by apoptosis, but their HSPCs are resistant. This differen-
tial resistance requires the ATM kinase, which induces cell-cycle
arrest and DNA repair in HSPCs (Mohrin et al., 2010). Interest-
ingly, both quiescent and proliferating HSPCs demonstrate
equal radio resistance but employ different types of DNA repair
mechanisms. Quiescent HSPCs preferentially utilize the error-
prone nonhomologous end-joining (NHEJ) DNA repair pathway,
which renders them intrinsically vulnerable to mutagenesis,
whereas proliferating HSPCs use the high-fidelity homologous
recombination mechanism and have significantly decreased
risk of acquiring mutations (Mohrin et al., 2010). This demon-
strates that HSPC quiescence serves as a double-edged sword
that protects against endogenous stress but also renders
HSPCs susceptible to genomic instability, thus contributing to
In striking contrast, irradiated human HSPCs isolated from
umbilical cord blood exhibit significantly increased apoptosis
and delayed DNA repair compared to downstream progeny.
IR-induced apoptosis is blocked by knockdown of p53 (p53KD)
or overexpression of BCL-2 (BCL-2OE), suggesting that the
p53-BCL-2 pathway regulates SC response to radiation and
DNA damage. Despite providing similar protection from irradia-
tion-induced apoptosis, only BCL-2OE favors self-renewal
(Milyavsky et al., 2010). It remains to be seen whether these con-
trasting conclusions about control of apoptosis in HSCs in mice
and humans are due to differences between species or the
anatomical location from which the cells were isolated. Future
work in this area will provide important new insights into how
apoptosis regulates SC homeostasis and differentiation and
how perturbations in survival and cell death signals transform
SCs into tumor SCs. Experiments that address these questions
will yield a better understanding of basic biology and may have
practical implications in the areas of regenerative medicine and
It has been almost 40 years since Kerr, Wyllie, and Currie stated:
‘‘We should now like to speculate that hyperplasia might some-
times result from decreased apoptosis rather than increased
mitosis, although we emphasize that we know of no definitive
studies that support such a hypothesis.’’ Since then, tremen-
dous progress has been made in identifying the core mechanism
underlying apoptotic cell death. At the same time, it has become
clear that other forms of cell death occur under physiological
conditions, and much remains to be learned about their molec-
ular mechanisms and connections to apoptosis. Also, apart
from a few specific models, we still know very little about pre-
cisely how cells are selected for death in vivo and how different
signaling pathways with sometimes opposing functions are inte-
grated in the decision between cell death and survival under
One area of surprising complexity in the cell death field is the
rich variety of signals released by apoptotic cells. In addition to
signals mediating the attraction and recognition of phagocytes,
apoptotic cells also secrete factors that stimulate cell prolifera-
tion, differentiation, and response of competent neighboring
cells. Core cell death proteins, including apoptotic effector cas-
pases, also function in diverse nonapoptotic processes, in-
cluding the elimination of unwanted cellular structures and
organelles (Figure 5). A major unresolved question is how the
potentially lethal activity of effector caspases is directed to
specific subcellular compartments without triggering a full-
blown apoptotic response. Finally, a better understanding of
cell death regulation is likely to provide the basis for treating
Figure 5. Nonapoptotic Function of Caspases
In addition to their central role in apoptosis, caspases are involved in many
other vital process, including: differentiation, enucleation, pruning of axons
and dendrites, sperm differentiation, immunity, compensatory proliferation,
and even learning and memory.
Cell 147, November 11, 2011 ª2011 Elsevier Inc. 753
a variety of human disorders associated with perturbations in
programmed cell death, such as cancer, autoimmunity, viral
infection, sepsis, ischemia, neurodegeneration, impaired heal-
ing, and tissue regeneration.
We would like to apologize to all our colleagues whose work we could not
adequately present here due to space constraints. We would like to thank
Yaniv Fuchs for the illustrations. H.S. is an Investigator of the Howard Hughes
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