The Genetics of Hiding the Corpse: Engulfment and Degradation of Apoptotic Cells in C. elegans and D. melanogaster

4

____________________________________________________________________________

The Genetics of Hiding the Corpse: Engulfment and Degradation of Apoptotic Cells in C. elegans and D. melanogaster Zheng Zhou,*,{ Paolo M. Mangahas,{ and Xiaomeng Yu* *Verna and Marrs McLean Department of Biochemistry and Molecular Biology { Program in Developmental Biology Baylor College of Medicine Houston, Texas 77030

I. Introduction II. Studies of Cell Corpse Engulfment in C. elegans A. Morphological and Physiological Studies of the Engulfment Process B. The Genetics of Cell Corpse Engulfment C. Molecular Studies of the Engulfment Genes and Identification of the Signaling Pathways D. The Engulfment of Cells That Die by Necrosis Requires the Same Genes That Act to Engulf Apoptotic Cells E. The Process of Engulfment Assists in the Execution of Apoptosis F. The Cases of Murder: Programmed Cell Deaths That Are Critically Dependent on Engulfing Cells III. The Degradation of Nuclear DNA During Programmed Cell Death in C. elegans A. nuc-1 Encodes a DNaseII Homolog Critical for DNA Degradation During Cell Death B. cps-6 Regulates Both DNA Degradation and the Timing of Cell Death C. Multiple Pathways Are Involved in DNA Degradation in C. elegans IV. Study of Engulfment and DNA Degradation in Drosophila A. Hemocytes Act as Engulfing Cells B. croquemort Encodes a Phagocytic Receptor in Drosophila C. Engulfment of Apoptotic Cells Is Required for Proper Patterning of the CNS D. Caspase-Independent Engulfment E. DNA Degradation in Drosophila Proceeds in Two Steps V. Concluding Remarks Acknowledgments References

I. Introduction During an animal’s life, a large number of unwanted cells undergo programmed cell death, or apoptosis, a genetically controlled cell suicide process. These dying cells are rapidly removed from the body: they are Current Topics in Developmental Biology, Vol. 63 Copyright 2004, Elsevier Inc. All rights reserved. 0070-2153/04 $35.00

91

92

Zhou et al.

Figure 1 A cell that undergoes apoptosis is rapidly removed through the engulfment by phagocytic cells and is degraded inside the phagocyte.

internalized by other cells through the process of phagocytosis and then are degraded inside the phagocytes (Fig. 1) (reviewed in Platt et al., 1998). Phagocytic removal is a common fate for cells undergoing apoptosis in metazoans. It has been observed and characterized in hydra, nematodes, insects including the fruit fly Drosophila melanogaster and the tobacco hornworm Manduca sexta, the zebrafish Danio rerio, the African clawed toad Xenopus laevis, the chicken Gallus gallus, and mammals (reviewed in Ellis et al., 1991b; Golstein et al., 2003; Tepass et al., 1994). The process of engulfing apoptotic cells is strikingly similar in organisms ranging from the very simple to the most complex. In diVerent organisms, diVerent types of cells act as engulfing cells. In simpler organisms such as hydra or the nematode Caenorhabditis elegans, there are no designated phagocytes; rather, neighboring cells engulf and degrade apoptotic cells (reviewed in Golstein et al., 2003 and Section IIA.2). Vertebrates develop specialized cells called macrophages that are mobile and that act as ‘‘professional phagocytes’’ for most apoptotic cells (Platt et al., 1998). In addition, a number of other cell types can act, possibly less eYciently, as ‘‘nonprofessional phagocytes’’ in vertebrates (Platt et al., 1998). The fruit fly uses blood cells, or hemocytes, as its major phagocytes for the removal of apoptotic cells (Tepass et al., 1994). After hemocytes ingest apoptotic cells and/or cell fragments, they are defined as macrophages (Tepass et al., 1994). Because it eliminates cells generated in excess, apoptosis is important for the establishment and maintenance of tissue architecture (reviewed in Jacobson et al., 1997). Apoptosis also acts as part of a quality-control mechanism by getting rid of cells that are infected, abnormal, tumorigenic, or in any way harmful to the body (Jacobson et al., 1997). Phagocytic

4. Engulfment and Degradation of Apoptotic Cells

93

removal of apoptotic cells ensures the elimination of dying cells before they release harmful cellular contents. This process actively prevents tissue injury, inflammatory response and autoimmunity, and facilitates organ sculpture and tissue remodeling (reviewed in Savill and Fadok, 2000). Furthermore, studies have shown that macrophages, after ingestion of apoptotic cells, secrete antiinflammatory cytokines and thus actively participate in the resolution of inflammation and the repair of tissue injury (Savill and Fadok, 2000). IneYcient removal of apoptotic cells has been related to a number of human inflammatory and autoimmune diseases. The studies of one autoimmune disease, systemic lupus erythematosus (SLE), both in humans and in animal models, strongly indicate that lack of phagocytosis of apoptotic cells is closely linked to the appearance of autoantibodies in the body and the development of SLE (Botto et al., 1998; Mevorach et al., 1998). Studying the phagocytic removal of apoptotic cells is thus important for understanding animal development and homeostasis and will have an important impact in medicine. Programmed cell death has been extensively studied in the nematode C. elegans, a small free-living round-worm, due to its simple anatomy, described invariant cell lineage, well-established genetics, optical transparency, and easily distinguishable apoptotic cell morphology. Genetic studies in C. elegans have identified genes essential for the control of cell death, including the first genes required for programmed cell death (reviewed in Horvitz, 2003). Studies in C. elegans and other organisms have demonstrated that the mechanisms that regulate apoptosis are conserved throughout the animal kingdom (reviewed in Metzstein et al., 1998). Similarly, genetic analyses probing the mechanisms controlling the engulfment of apoptotic cells were pioneered in C. elegans. This line of research has identified at least seven genes required for the recognition and engulfment of apoptotic cells, ordered these genes in two partially redundant genetic pathways, and led to the finding that similar genes act to control the same process in other organisms, including mammals. Another genetic system well suited for the study of programmed cell death and the fate of the dying cells is the fruit fly Drosophila melanogaster (reviewed in Song and Steller, 1999). Studies of how apoptotic cells are engulfed and degraded in Drosophila, which have begun to shed light on the molecular mechanisms underlying this process, are described in later sections. In this chapter, we review the logic and methods employed to study how apoptotic cells are recognized, internalized, and degraded by engulfing cells in C. elegans and Drosophila and describe exciting findings in these two systems resulting from a combination of genetic and molecular studies of gene function. We also discuss the implications of these results for the understanding of the clearance of apoptotic cells in mammals.

94

Zhou et al.

II. Studies of Cell Corpse Engulfment in C. elegans A. Morphological and Physiological Studies of the Engulfment Process 1. Cell Corpse Engulfment Is a Highly Efficient Process Of the 1090 somatic cells generated during the development of a C. elegans hermaphrodite, 131 undergo programmed cell death (Kimble and Hirsh, 1979; Sulston and Horvitz, 1977; Sulston et al., 1983). Of the cells destined to die, 113 die during embryogenesis, mainly during midembryogenesis, between 250 and 450 min after fertilization (Sulston et al., 1983); the remaining 18 cells are generated, and subsequently die, during larval development (Sulston and Horvitz, 1977). Cells undergoing programmed cell death can be distinguished in live animals, using Nomarski diVerential interference contrast microscopy, by their highly refractile, button-like appearance and are commonly referred to as ‘‘cell corpses’’ (Fig. 2A) (Sulston, 1976; Sulston and Horvitz, 1977; Sulston et al., 1983). Lineage studies showed that, like most other C. elegans development events, these somatic cell deaths are essentially invariant among individuals regarding the identities of the cells that die and the timing of their deaths (Sulston, 1976; Sulston and Horvitz, 1977; Sulston et al., 1983). In C. elegans, like in other metazoan organisms, cells undergoing programmed cell death proceed through four stages: (1) specification of the dying fate; (2) execution of cell death, when the killing machinery is activated inside the cells that are determined to die and multiple morphological changes specific to apoptosis are observed; (3) engulfment by neighboring cells; and (4) degradation inside engulfing cells (Fig. 1) (reviewed in Ellis et al., 1991b). Engulfment and degradation of cells undergoing programmed cell death is a swift process, so swift that very few cell corpses can be seen in wild-type animals in the late embryonic stage and thereafter (Ellis et al., 1991a). The majority of embryonic deaths occur within 30 min after the cell divisions that generate the cells destined to die (Sulston et al., 1983). Observed using Nomarski microscopy, the progression from the first increase in refractility of the dying cell to the disappearance of the dying cell takes about an hour (Sulston and Horvitz, 1977; Sulston et al., 1983). Electron microscopic studies detected pseudopods extended from engulfing cells to surround dying cells, which are highly electron dense, at an early stage of cell death (Robertson and Thomson, 1982). In some cases, engulfing cells recognize and extend pseudopods around the cells destined to die even before the mitotic divisions that generate those cells have completed (Robertson and Thomson, 1982). This phenomenon indicates that dying cells present on their surfaces at least one specific signal that distinguishes them from living cells, the so-called ‘‘eat me’’ signal, at an early stage of death.

4. Engulfment and Degradation of Apoptotic Cells

95

Figure 2 Engulfment-defective ced mutants have persistent cell corpses. (A, C) Images of late embryos prior to hatching under Nomarski microscope. Cell corpses appear as refractile objects and are indicated by arrows. (A) In the ced-1 mutant, many cell corpses persist through embryogenesis, whereas in wild-type, most dying cells have been eliminated by the late embryonic stage. (B) Transmission electron micrographs (EMs) of cell corpses. The membranes of neighboring cells surrounding the cell corpse are indicated by arrows. Cell corpses are internalized by their neighboring cells in wild-type (a) but are left unengulfed in ced-1 mutant (b, c). (d–f ) Traces of membranes in the corresponding upper EM picture. Reproduced from Zhou et al., 2001b. (C) Some unengulfed cell corpses drift away from the embryo soma into surrounding fluid, as shown in the ced-1 mutant.

In addition to the cell deaths that occur in the soma, a large number of germ cells (estimated at between 300 and 500 germ cells per animal) undergo programmed cell death in the hermaphrodite gonad (Gumienny et al., 1999). During the adulthood of hermaphrodites, dying nuclei, which are at the pachytene stage of meiosis I and are restricted to a particular region of the germ line syncytium, quickly cellularize and are rapidly engulfed by the gonadal sheath cells (Gumienny et al., 1999). Like dying somatic cells, dying germ cells can also be distinguished, using Nomarski microscopy, by their highly refractile, disklike appearance. Due to the high eYciency of

96

Zhou et al.

engulfment and degradation, fewer than 10 germ cell corpses are found per gonad arm in the average wild-type adult hermaphrodite at any given time, despite the large number of germ cell deaths that are occurring (Gumienny et al., 1999). 2. Several Types of Cells Can Act as Engulfing Cells for Apoptotic Cells There are no ‘‘macrophage-like’’ specialized phagocytes for dying cells in C. elegans. Most cell corpses are engulfed by neighboring cells. At early stages of embryogenesis, dying cells are mostly engulfed by their sisters, which usually will go through at least one more round of cell division and thus have not become fully diVerentiated (Hoeppner et al., 2001; Sulston et al., 1983). At later stages of embryonic development and during larval development, several types of cells play major roles in engulfment. Among them are hypodermal cells, which fuse and form an outer monolayer of the animal’s body and which are the predominant engulfing cells for somatic cell corpses. Pharyngeal muscle cells and intestinal cells have also been observed to engulf cell corpses (Sulston and Horvitz, 1977; Sulston et al., 1983; Zhou et al., 2001b). Unlike the identities of the cells fated to die, the identity of the cell engulfing a particular dying cell during embryogenesis is not absolutely invariant. Hoeppner and co-workers (2001) reported that although some dying cells were observed to be repeatedly engulfed by the same engulfing cell in multiple embryos, other dying cells were observed to be engulfed by one of the several diVerent neighboring cells in diVerent embryos. Zhou and co-workers (2001b) captured transmission electron microscopy images in which two neighboring cells residing on the opposite sides of one dying cell both extend pseudopods to the same dying cell. These observations indicate that multiple neighboring cells are capable of recognizing the dying cells and initiating engulfment. What determines which cell will win as an engulfing cell remains unclear. Dying germ cells in the germ line are exclusively engulfed by gonadal sheath cells, which are part of the somatic gonad and which form a tube wrapping around the germ line (Gumienny et al., 1999; Zhou et al., 2001b). In addition to the engulfment of dying cells, gonadal sheath cells also play important roles in germ line development, oocyte maturation, and ovulation (Hubbard and Greenstein, 2000). 3. The Fate of the Engulfed Cell Corpses Electron microscopic observations showed that the entire engulfed dying cell is confined in the phagosome structure, an internalized plasma membrane structure derived from the engulfing cell, and isolated from the engulfing cell

4. Engulfment and Degradation of Apoptotic Cells

97

cytoplasm (Fig. 2B) (Ellis et al., 1991a; Robertson and Thomson, 1982; Zhou et al., 2001b). Within an engulfing cell, the dying cell shrinks in size, a process accompanied by the breakdown of the nuclear membrane and the persistent existence of condensed chromatin-like fragments (Robertson and Thomson, 1982). The last recognizable appearance of the dying cell is one or more whorls of membranes within vacuoles of the engulfing cells (Robertson and Thomson, 1982). These observations clearly indicate that the engulfed cell corpses are degraded inside the phagosomes, although very little is known about what triggers and regulates the degradation events. 4. The Fate of Unengulfed Cell Corpses Some dying cells may disappear without engulfment. In mutant embryos in which engulfment was perturbed (see Section IIB.1), a number of cell corpses were observed to have separated from the developing embryos and drifted into the extraembryonic fluid within the eggshell (Fig. 2C) (Hedgecock et al., 1983). Some embryonic cell corpses that persist for many hours to late larval stages in engulfment-defective mutants were shown to contain vacuoles and seemed to be slowly disintegrating, suggesting that these dying cells undergo secondary necrosis (Ellis et al., 1991a). In adult hermaphrodites with mutations in engulfment genes, many unengulfed germ cell corpses also seem to undergo secondary necrosis: they swell and eventually break open (Gumienny et al., 1999). This phenomenon is similar to what has been observed for mammalian apoptotic cells that escape phagocytosis.

B. The Genetics of Cell Corpse Engulfment 1. Multiple Genetic Screens Led to the Identification of at Least Seven Engulfment Genes Genetic screens have been performed to systematically identify genes whose functions are required for the engulfment of cells undergoing programmed cell death. In those screens, chemical mutagens were used to induce mutations, and second- or third-generation progeny descended from the mutated gametes that bore a large number of persistent cell corpses in the bodies were identified using Nomarski microscopy (Ellis et al., 1991a; Hedgecock et al., 1983; Zhou et al., 2001a). The lineage and morphological studies described previously laid the groundwork for these genetic screens. Three features made it possible to identify engulfment defects this way: (1) most somatic cell deaths occur within a relatively short period in embryogenesis, (2) cell corpses are distinguishable in living animals using Nomarski microscopy, and (3) engulfment and degradation is a swift process. Additional mutants in

98

Zhou et al.

engulfment genes were recovered from screens for defects in embryogenesis, in cell migration, in the removal of necrotic cells, and for enhancers of weak defects in the execution of programmed cell death (Chung et al., 2000; Gumienny et al., 2001; Kodama et al., 2002; Nishiwaki, 1999; Reddien et al., 2001; Wu et al., 2001). Hedgecock and co-workers (1983) isolated the first eight recessive mutants containing persistent cell corpses. In the mutants, cells that undergo apoptosis during embryogenesis persist for many hours, sometimes throughout larval development. These eight mutations define two genetic loci, ced-1 and ced-2 (cell death abnormal). The fact that the cells that are destined to die still die as programmed in ced-1 and ced-2 mutants despite a lack of eYcient engulfment indicated, for the first time, that engulfment is not necessary for most programmed cell deaths (Hedgecock et al., 1983). This finding is consistent with the results from mosaic analysis and transgenic expression studies for the cell death execution genes ced-3 and ced-4, which demonstrate that the key process leading to the execution of programmed cell death takes place within the dying cell (Shaham and Horvitz, 1996; Yuan and Horvitz, 1990). Programmed cell death thus is considered a cell suicide process. Although engulfment is definitely not required for most cell deaths in C. elegans, recent discoveries indicate that engulfing cells do enhance the eYciency of cell death execution. Furthermore, in C. elegans, there are a few documented examples in which cell deaths are caused by engulfment (murder) (see later). The ced-2 mutant phenotype is subject to maternal-eVect rescue: homozygous ced-2 progeny of homozygous ced-2 mothers have persistent cell corpses, but homozygous ced-2 progeny of heterozygous ced-2/+ mothers appear wild-type for engulfment (Ellis et al., 1991a). Based on this observation, Ellis and co-workers (1991a) designed a visual screen strategy that allowed the isolation of both maternal-eVect and zygotic mutant animals with engulfment defects (Fig. 3). To obtain mutants in the absence of possible maternal-eVect rescue, Ellis and co-workers (1991a) screened F3 (3rd generation from mutagenesis) progeny instead of F2 progeny for the presence of persistent cell corpses. This screen was performed in a sem-4 (sex muscles defective) mutant background in which worms could not lay eggs (Basson and Horvitz, 1996). The eggs retained in the mothers then develop and hatch internally. F2 mothers, half of which were homozygous for a mutagenized allele at any given locus, formed transparent ‘‘bags-of-worms’’ that held a brood of F3 self-progeny consisting of embryos and young larvae suitable for screening using Nomarski microscopy and that were easy to recover from the microscopic slides. Using this strategy, 24 additional recessive mutants bearing persistent cell corpses were isolated, among which were new alleles of ced-1 and ced-2. In addition, five new genes, ced-5, ced-6, ced-7, ced-8, and ced-10, were identified from mutants recovered from this screen

4. Engulfment and Degradation of Apoptotic Cells

99

Figure 3 The design for the two screens for mutations aVecting the removal of cell corpses in C. elegans as performed by Ellis et al. (1991a) and Zhou and Horvitz (unpublished). Three generations after sem-4 animals (P0) were mutagenized with EMS, F3 animals, which were held within each F2 mother, were examined under a Nomarski microscope for persistent cell corpses. F2 animals are generated either by þ/þ F1 mothers or by rare ced/þ F1 animals carrying a mutation in one of the ced genes. F2 generated in the latter case have genotypes of ced/ced, ced/þ, or þ/þ. The phenotypes of F3 animals that are held within these F2 mothers will depend on not only the F2 mother genotype but also whether the mutation is zygotic or subject to maternal-eVect rescue. As illustrated in the table, if the mutation is subject to maternal-eVect rescue, the ced/ced F2 animals will generate 100% Ced F3 animals, whereas ced/þ F2 animals will generate 0% Ced F3 animals. If the mutation is zygotic, both ced/ced and ced/þ F2 will generate Ced F3, in proportions of 100% and 25%, respectively.

(Ellis et al., 1991a). Of these genes, ced-8 was later found to be a gene controlling the timing of cell death execution rather than regulating engulfment (Stanfield and Horvitz, 2000). ced-5, ced-6, ced-7, and ced-10 represent new genes genuinely required for eYcient engulfment (see later discussion).

100

Zhou et al.

Maternal eVect rescue appears to be the rule rather than the exception among the engulfment genes. Loss-of-function mutations of six out of seven engulfment genes (including ced-12, an engulfment gene identified later) are subject to maternal eVect rescue for the engulfment of somatic cell corpses generated during embryonic and larval development (Table I) (Ellis et al., 1991a). The only zygotically required gene is ced-1 (Table I) (Ellis et al., 1991a). This phenomenon demonstrates that the maternally provided wild-type products of these six genes must be suYcient for the engulfment of dying cells. The screen performed by Ellis and co-workers (1991a) did not reach saturation. Zhou and Horvitz later performed an additional screen for engulfment

Table I Genes Functioning in the Engulfment of Cell Corpses in C. elegans

Gene

Mammalian Homologs

ced-1

1. SREC 2. CD91/LRP 3. mEGF10

ced-2 ced-5

Crk-II DOCK180

ced-6 ced-7

hCED-6/hGULP ABC transporters

ced-10 ced-12

Rac1 1. ELMO1 2. ELMO2/hCED-12A 3. Human BAB14712

a

Hedgecock et al., 1983. Zhou et al., 2001b. c Ellis et al., 1991a. d Reddien and Horvitz, 2000. e Wu and Horvitz, 1998b. f Liu and Hengartner, 1998. g Wu and Horvitz, 1998a. h Lundquist et al., 2001. i Chung et al., 2000. j Gumienny et al., 2001. k Zhou et al., 2001a. l Wu et al., 2001. b

Mutant Alleles e1735 a,b, e1754 a,b, e1797 a,b, e1798 a,b, e1799 a,b, e1800b, e1801 a,b, e1814 a,b, n691 b,n1951 b, n1995 b,c, n2000 b,c, n2089 b,c, n2091 b,c, n2092 b,c e1752 a,d, n1994 c,d, n3238 d mu57 e, n1812 c,e, n2002 c,e, n2098 e, n2099 e, n2691 e n1813 c, f, n2095 c, f n1892 c,g, n1996 c,g, n1997 c,g, n1998 c,g, n2001 c,g, n2094 c,g, n2690 g, n3072 g, n3073 g n1993 c,d, n3246 d, n3417 h bz187 i,j, k145 j, k149 j, k156 j, k158 j, n3261 k, oz167 i, j, tp2 l

Subject to Maternal-EVect Rescue? NO

Yes Yes Yes Yes

Yes Yes

4. Engulfment and Degradation of Apoptotic Cells

101

mutants using similar strategy but at a larger scale (Zhou and Horvitz, unpublished results). In addition to new alleles of ced-1, -2, -5, -6, -7, and -10, a new engulfment gene, ced-12, was identified from this screen (Zhou et al., 2001a). ced-12 was independently identified by other groups in screens seeking defects other than the failure to remove cells dying by programmed cell death (Chung et al., 2000; Nishiwaki, 1999; Wu et al., 2001). Mutants strongly defective in the engulfment of cell corpses, including null mutants of several of the engulfment genes, are fully viable, suggesting that engulfment per se is not essential for C. elegans development (Wu and Horvitz, 1998a, Zhou et al., 2001b). However, in the screens performed by Zhou and Horvitz, a new phenotypic class of mutants that displays both persistent cell corpses and embryonic lethality in late-stage embryos has been isolated (Zhou and Horvitz, unpublished results). Genetic analyses have shown that both phenotypes result from a single gene mutation. Because mutants strongly defective in engulfment are fully viable, the existence of these mutations that cause both engulfment defects and embryonic lethality suggests that the corresponding genes control processes other than engulfment and are required for viability. Thus, these mutations are likely to define a new class of genes missed in previous screens that focused on viable engulfment mutants. 2. Loss-of-Function Mutants of the Engulfment Genes Are Truly Defective in Cell Corpse Engulfment The presence of persistent cell corpses in the engulfment ced mutants listed previously could result from a lack of engulfment or from perturbation in the degradation of the engulfed cell corpses. To distinguish between these two possibilities, transmission electron microscopy was used to examine cell corpses in these mutants. In animals bearing representative mutant alleles of each of the seven engulfment genes, the observed cell corpses remained unengulfed (for example, see Fig. 2B [b, c, e, f]) (Ellis et al., 1991a; Hedgecock et al., 1983; Zhou et al., 2001a,b). In contrast, the corpses found in wild-type animals were engulfed (for example, see Fig. 2B [a, d]) (Ellis et al., 1991a; Zhou et al., 2001b). These results indicate that the seven ced genes control the process of engulfment rather than a later step of degradation. 3. Two Partially Redundant Genetic Pathways Contribute to Efficient Engulfment Ellis and co-workers (1991a) and others observed that none of the single engulfment mutants completely abolished the engulfment of cell corpses. For example, in the head region of a wild-type hermaphrodite, 93 cells undergo programmed cell death. Of these, 92 die during embryogenesis and one dies during the early L1 larval stage (Sulston et al., 1983). However,

102

Zhou et al.

in ced-1(e1735) or ced-5(n1812) mutant animals, which represent null mutants of each corresponding gene, an average of 27.4 and 33.3 corpses, respectively, were observed in the heads of L1 larvae (Zhou et al., 2001a). These persistent cell corpses represent less than 36% of the total number of dying cells in this region. This phenomenon suggests that many cell corpses have probably been engulfed in addition to those that have been shed into the extraembryonic fluid and raises the possibility that the known engulfment genes might have mutually redundant functions. To test this idea, Ellis and co-workers (1991a) generated double mutant combinations among the engulfment mutants. By scoring the number of persistent cell corpses in the pharynges of the double mutant L1 larvae, they identified two functional groups of genes, the ced-1, -6, and -7 group and the ced-2, -5, and -10 group (Fig. 4). Double mutants built within each group contain no more persistent cell corpses than the strongest single mutant in the group. However, double mutants built between the two groups contain many more persistent cell corpses than seen in any single mutant in either group (Ellis et al., 1991a). For example, the ced-1(e1735); ced-5(n1812) double mutant L1 larvae have 44 persistent cell corpses in the head. Furthermore, none of the triple mutants built among the engulfment mutants display a stronger phenotype than that seen for the strongest double mutants. Thus, none of the six genes seems to act in a third pathway (Ellis et al., 1991a). ced-12 was later placed in the ced-2, -5, -10 group by similar analyses (Gumienny et al., 2001; Wu et al., 2001; Zhou et al., 2001a). These two groups do not seem to act on distinct groups of dying cells. Mutations in all seven genes aVect the eYciency of the engulfment of somatic cell corpses generated in embryos and in larvae and of germ-cell corpses generated in the adult hermaphrodite gonad (Ellis et al., 1991a; Gumienny et al., 1999, 2001; Wu et al., 2001; Zhou et al., 2001a,b). However, in some cases it was observed that mutations in diVerent engulfment genes appear to have diVerent relative eVects on diVerent cell corpses (Ellis et al., 1991a; Gumienny et al., 1999). Further molecular and biochemical analyses revealed that these two distinct functional groups represent two

Figure 4 The known components of the two parallel and partially redundant genetic pathways that control the engulfment of cell corpses in C. elegans.

4. Engulfment and Degradation of Apoptotic Cells

103

partially redundant signaling pathways that control the initiation and execution of engulfment. Still, none of the double mutants retain all 93 cell corpses in the head. There are three probable explanations: the shedding of unengulfed cell corpses from the developing embryo (see earlier discussion), the degeneration and disappearance of unengulfed cell corpses (see earlier discussion), and possibly a third, unidentified pathway for the engulfment of cell corpses (Ellis et al., 1991a). C. Molecular Studies of the Engulfment Genes and Identification of the Signaling Pathways 1. How Are Dying Cells Recognized by Engulfing Cells? The Story of CED-1 and Its Partners a. CED-1 Is a Phagocytic Receptor. It was proposed that cells undergoing programmed cell death expose or release special substances on their outer surfaces to activate neighboring cells to engulf them through a receptor-mediated signaling pathway (Ellis et al., 1991a). Prior to the cloning of CED-1, no such receptor had been identified in C. elegans. However, a number of mammalian cell surface proteins have been implicated, mainly by in vitro studies, in the recognition of apoptotic cells and the promotion of their phagocytosis. These include members of the integrin family, members of the scavenger receptor family, lectins, MER (novel human kinase expressed in monocytes, epithelial, and reproductive tissues), CD14, and PSR (phosphatidylserine receptor) (reviewed in Henson et al., 2001). At the time of the identification of CED-1, however, the in vivo contribution of any of these proteins to the clearance of apoptotic cells was not clear. Zhou and co-workers (2001b) cloned C. elegans ced-1 using standard positional cloning techniques and found that ced-1 encodes a single-pass transmembrane protein with a long N-terminal domain predicted to be an extracellular domain based on protein topology (Fig. 5). The predicted extracellular domain of CED-1 contains an N-terminal signal peptide and 16 tandem copies of an atypical form of EGF-like repeat. An EGF-like repeat is a cysteine-rich motif found in the extracellular domains of many proteins functioning in adhesive or ligand-receptor interactions (reviewed in Campbell and Bork, 1993). N-terminal to the EGF-like repeats there is an EMI domain, another cysteine-rich domain found in many extracellular proteins including emilins and multimerin (Callebaut et al., 2003). The C-terminal, presumable intracellular domain contains two small motifs known for their functions in signal transduction pathways: putative binding motifs (NPXY and YXXL) for a PTB (phosphotyrosine-binding) domain and an SH2 (Src homology 2) domain, respectively (Songyang and Cantley,

Figure 5 The domain structures of the seven proteins involved in the engulfment of cell corpses in C. elegans (Brugnera et al., 2002; Callebaut et al., 2003; Reddien et al., 2000; Wu et al., 1998a,b; Zhou et al., 2001a,b).

4. Engulfment and Degradation of Apoptotic Cells

105

1995). The overall structure and sequence features of CED-1 suggest that it could act as a transmembrane receptor. The extracellular domain of CED-1, CED-1Ex, is similar in sequence to a number of proteins in the database that contain the CED-1 type of atypical EGF-like repeats. These include proteins with unknown functions and a protein called SREC (scavenger receptor from endothelial cells) (Adachi et al., 1997). The similarity between CED-1Ex and SREC is particularly interesting, as scavenger receptors mediate the endocytosis of a variety of anionic substrates, including lipoproteins and phospholipids (Krieger and Herz, 1994). Several scavenger receptors, including SR-A, CLA-1, CD36, SR-BI, and CD68, have been implicated in mediating the phagocytosis of apoptotic cells in mammalian cell culture studies (Platt et al., 1998). CED-1Ex may possess ligand-binding specificity scavenger receptors. Several lines of evidence indicate that CED-1 recognizes apoptotic cells in vivo (Zhou et al., 2001b). By following the expression pattern of a CED1::GFP (green fluorescence protein) fusion protein produced under the control of the ced-1 promoter, CED-1 was found to be expressed at high levels in cell types that can function as engulfing cells, such as hypodermal cells, intestinal cells, and gonadal sheath cells, but is not expressed in cells destined to die (Zhou et al., 2001b). Consistent with its expression pattern, CED-1’s function is required only in engulfing cells, but not in dying cells, for the engulfment of dying cells. Furthermore, the CED-1::GFP fusion protein is present on cell surfaces, and the CED-1::GFP on the surface of an engulfing cell accumulates at a higher concentration within the region of the plasma membrane that is contacting the dying cell, the so-called phagocytic cup (Zhou et al., 2001b). In some engulfment-mutant (e.g., ced-6 mutant) larvae, pseudopodia from engulfing cells were observed to extend around dying cells but did not fully enclose. In those mutants, CED-1::GFP was observed to form a partial green circle around the dying cells (Fig. 6A) (Zhou et al., 2001b). These observations indicate that CED-1 clusters in response to dying cells, which presumably expose extracellular signals, and that the clustering of CED-1 is not a consequence of the completion of engulfment but could actually precede the initiation of engulfment. Deletion analysis of CED-1 showed that a truncated form of CED-1 missing its intracellular domain could still localize to the plasma membrane of engulfing cells and cluster around dying cells. However, its engulfment activity, which could be scored as the ability to rescue the engulfment defects of a ced-1 null mutant, was lost (Zhou et al., 2001b). Thus, the intracellular domain of CED-1 is not needed for CED-1 to recognize dying cells; rather, it is needed for a downstream step, probably the activation of engulfing cells. This observation reinforces the hypothesis that CED-1 acts as a receptor; CED-1 not only receives the signal, but also relays the signal.

106

Zhou et al.

Figure 6 The signaling pathway led by CED-1, CED-6, and CED-7 promotes engulfing cells to recognize apoptotic cells. (A) CED-1::GFP clusters around cell corpse in ced-6 but not ced-7 mutant animals. ced-6(n2095) (upper panels) and ced-7(n1996) (lower panels) mutant larvae were induced to express Phsp ced-1::gfp. CED-1::GFP green circles around cell corpses are indicated with white arrows. Cell corpses are indicated with black arrows. Reproduced from Zhou et al., 2001b. (B) The current model elucidating the signaling pathway led by CED-1, CED-6, and CED-7. See text for details.

The clustering of CED-1 could be a consequence of a CED-1–ligand interaction and may play an important role in activating downstream signaling. Receptor clustering in response to extracellular signals has been observed for many transmembrane receptors. For example, Fc receptor (Fc R), the mammalian phagocytic receptor for opsonized foreign particles, clusters around opsonized particles and triggers downstream phosphorylation events that lead to polarized cell surface extension (reviewed in Kwiatkowska and Sobota, 1999). Overexpression of a truncated form of CED-1 that retains only its extracellular and transmembrane domains in wild-type C. elegans embryos results in the presence of a few persistent cell corpses, despite the fact that this truncated protein retains the ability to

4. Engulfment and Degradation of Apoptotic Cells

107

cluster around cell corpses (Zhou et al., 2001b). This weak dominantnegative eVect suggests that this truncated form of CED-1 may act to compete for extracellular ligand(s) and/or to disrupt the assembly or function of a putative multisubunit protein complex organized by wildtype CED-1. These results are consistent with a model in which clustering of CED-1 in response to ligand(s) is important for its signaling function. b. Tyrosine Phosphorylation Events and Adaptor Molecules May Participate in CED-1 Signaling. How does CED-1 initiate downstream signaling inside engulfing cells? Mutational analyses have shown that the two putative adaptor-binding motifs (NPLY and YASL) located within the intracellular domain of CED-1 are important for the engulfment activity of CED-1, and that they are partially redundant with each other for CED-1 function (Zhou et al., 2001b). Furthermore, mutating the tyrosine in the SH2-binding motif (YASL) to phenylalanine, a nonphosphorylatable residue structurally similar to tyrosine, completely abolishes the function of this motif in regards to engulfment activity (Z. Zhou, unpublished observations), suggesting that phosphorylation of this tyrosine is essential for function. This is consistent with previous observations that SH2 domains bind to the YXXL motif in a tyrosine phosphorylation-dependent manner (Songyang and Cantley, 1995). Mutating the tyrosine residue in the PTB-binding motif (NPLY) to phenylalanine only weakly decreases the function of this motif, although its mutation to alanine strongly aVects CED-1 function, indicating that tyrosine phosphorylation at this site is not essential (Z. Zhou, unpublished observations). This is also consistent with the knowledge that the recognition by certain PTB domains of their binding motifs is not dependent on tyrosine phosphorylation (Pawson and Scott, 1997). The identities of the putative tyrosine kinase(s) that phosphorylate the YASL motif and the SH2-containing adaptor(s) that bind to this motif remain unknown. However, CED-6, one of the two other engulfment proteins acting in the CED-1 functional group, stands out as a candidate adaptor for CED-1 that may bind to the NPLY motif with its PTB domain. ced-6 was cloned by Liu and Hengartner (1998). Its protein structure is composed of an N-terminal PTB domain with similarity to that of Shc, Numb, and Disabled, followed by a leucine zipper domain and a prolinerich C-terminal region (Fig. 5) (Liu and Hengartner, 1998; Su et al., 2000). Like the function of ced-1, the function of ced-6 is required only in engulfing but not in dying cells, based on genetic mosaic analysis (Liu and Hengartner, 1998). In addition, the CED-1::GFP clustering around persistent cell corpses is not aVected in ced-6 mutant embryo or larvae (Fig. 6A), indicating that mutations in ced-6 do not block CED-1’s recognition of the ‘‘eat me’’ signal; rather, they block a signaling pathway downstream of CED-1 (Fig. 6B) (Zhou et al., 2001b). Furthermore, Su and co-workers (2002) reported that

108

Zhou et al.

a GST-CED-1C (C-terminal intracellular domain) fusion protein could pull down the PTB domain of CED-6 when both were expressed in COS-7 cells, indicating a possible interaction between CED-1C and CED-6-PTB. This interaction was reproduced in a Sos-based yeast two-hybrid protein interaction assay and was shown to require the NPLY motif of CED-1C (Su et al., 2002). This interaction, as detected in heterologous systems, suggests that in engulfing cells in C. elegans CED-6 might be recruited to bind to the intracellular domain of CED-1. Because the interaction between CED-1C and full-length CED-6 has not been detected in vitro or in vivo, it is unclear if CED-6 might associate with CED-1 constitutively or might be recruited to CED-1 only when CED-1 is associated with its extracellular ligand(s). What are the events regulated by the CED-1 signaling pathway? Multiple events need to be initiated and coordinated inside engulfing cells for engulfment to occur. These events include the reorganization of the actin cytoskeleton, which is the driving force for cell surface extension, membrane remodeling and extension, membrane closure, and the initiation of cell corpse degradation inside the phagosomes. The CED-1 pathway could control one or several of these events. Identifying downstream targets of this pathway would help resolve this question. c. What Substance(s) Do Apoptotic Cells Expose on Their Outer Surfaces and What Does CED-1 Recognize? A number of changes have been detected on the surfaces of mammalian cells when they undergo apoptosis. One such change is the exposure of phosphatidylserine (PS), a phospholipid normally kept in the inner leaflet of the lipid bilayer in living cells (reviewed in Henson et al., 2001). This event has been observed in Drosophila, X. laevis, the chicken G. gallus, and mammals and thus represents an evolutionarily conserved feature of apoptosis (Nera et al., 2000; Schlegel and Williamson, 2001; van den Eijnde et al., 1998). Changes in cell surface carbohydrates and ionic charge have also been observed (reviewed in Savill et al., 1993). These changes may result in distinct features of apoptotic cells recognizable by phagocytes. Indeed, PS exposure on the outer surface of apoptotic mammalian cells has been implicated as a predominant ‘‘eat me’’ signal recognizable by phagocytic cells (Henson et al., 2001). In C. elegans, the events of programmed cell death and engulfment have thus far been examined only in living animals covered with either cuticles or chitin-based egg shells. Consequently, very little is known about the surfaces of apoptotic cells in C. elegans. The identity of CED-1 has provided a hint. As previously proposed, CED-1 may possess a ligand-binding specificity similar to that of scavenger receptors and, in particular, may be able to bind anionic phospholipids such as PS (Zhou et al., 2001b). The proof of this hypothesis and the identification of the C. elegans ‘‘eat me’’ signal thus await the identification of CED-1 ligand(s).

4. Engulfment and Degradation of Apoptotic Cells

109

The studies of ced-7 have provided supportive evidence that the ‘‘eat me’’ signal detected by CED-1 is likely to be PS. In ced-7 mutant animals, although CED-1::GFP is expressed and is localized to cell surfaces as in wild-type animals, GFP-positive cell corpses are nearly absent (Fig. 6A) (Zhou et al., 2001b). This result indicates that the function of CED-7 is required for CED-1 to cluster around cell corpses. In contrast, although engulfment is defective in ced-2, -5, -6, -10, and -12 mutants, CED-1::GFP still clusters around cell corpses in these mutants, indicating the block in CED-1 clustering is a defect specific to ced-7 mutants and not a result of failure to engulf cell corpses (Zhou et al., 2001b). It is unclear how CED-7 acts to promote CED-1 clustering around dying cells. ced-7 was cloned and characterized by Wu and Horvitz (1998a) and was shown to encode a member of the family of ABC (ATP-binding cassette) transporters (Fig. 5). ABC transporters have been known to actively transport a variety of substances, including sugars, ions, lipids, peptides, proteins, and lipoproteins, into and out of cells (reviewed in Klein et al., 1999). Some members of the ABC transporter family have been shown to influence the distribution of lipid species across the membrane bilayer. Of potential relevance to CED-7 function is the ability of ABC1, the closest mammalian homolog of CED-7, to promote the exposure of PS onto the outer layer of plasma membrane (Hamon et al., 2000). It is thus possible that CED-7 promotes the exposure of PS or some other ‘‘eat me’’ signal on the surface of apoptotic cells for CED-1 to recognize. CED-7 is broadly expressed in C. elegans embryos and is localized to cell surfaces (Wu and Horvitz, 1998a). Genetic mosaic analysis indicates that the function of CED-7 is needed in both dying and engulfing cells for engulfment (Wu and Horvitz, 1998a). This model predicts CED-7 functions in dying cells but is not suYcient to explain its function in engulfing cells. CED-7 may perform multiple functions: in dying cells, it may act to present the ‘‘eat me’’ signal(s); in engulfing cells, it may act to assist CED-1 to recognize the signal(s) or it may act downstream of CED-1. It is equally possible that CED-7 may act to present an adhesive molecule or molecules on the surface of both cell types to facilitate their adhesion. CED-7 is the only protein known to be required in apoptotic cells for their engulfment, and it may provide a link between the cell death execution pathway and phagocytosis pathway. It is also possible that the direct ligand of CED-1 is not the ‘‘eat me’’ signal itself. Studies in mammalian cell cultures indicated that in many cases phagocytic receptors may require ‘‘bridging molecules’’ to interact with apoptotic cells. Known bridging molecules include complement pathway components, the serum adhesive protein thrombospondin (TSP), and the secreted glycoprotein MFG-E8 (Hanayama et al., 2002; Mevorach et al., 1998; Savill et al., 1992). If the association of engulfing and dying cells also requires bridging molecules in C. elegans, identifying the ligand(s) of CED-1

110

Zhou et al.

will lead to the identification of the bridging molecule(s). Human annexin I, a calcium-dependent PS-binding protein, has been implicated as a new bridging molecule between apoptotic and engulfing cells (Arur et al., 2003). Arur and co-workers (2003) also suggested that nex-1, one of the C. elegans homologs of human annexin I, may provide a bridging function that contributes to engulfment in C. elegans. In summary, the molecular characterization of CED-1, CED-6, and CED7 indicates that this functional group actually represents a signal transduction pathway leading engulfing cells to recognize dying cells for their engulfment (Fig. 6B). In this pathway, CED-7 acts upstream of CED-1 and may be involved in the presentation and/or recognition of the ‘‘eat me’’ signals. CED-1 acts as the phagocytic receptor that activates engulfing cells in response to neighboring dying cells, and CED-6, a candidate adaptor, is a strong candidate to relay CED-1 signal to downstream cellular machinery. It is unknown whether nex-1 is part of this pathway. d. The Functions of Mammalian Homologs of CED-1, CED-6, and CED-7 in the Engulfment of Apoptotic Cells. Humans and mice have one close homolog of CED-6 named hCED-6 or hGULP (engulfment adapter protein) and mGULP, respectively (Liu and Hengartner, 1999; Smits et al., 1999; Su et al., 2002). These proteins have structural features similar to those of CED-6, although they are approximately 150 amino acids shorter at the C termini (Su et al., 2002). hGULP partially rescued the engulfment defect of ced-6 mutants when expressed in C. elegans, suggesting that hGULP and CED-6 might be conserved in function (Liu and Hengartner, 1999). In addition, overexpression of hGULP in a macrophage cell line promotes phagocytosis (Smits et al., 1999), and the PTB domain of mGULP can interact with the intracellular domain of CED-1 when both are transiently expressed in mammalian cells (Su et al., 2002), again suggesting that GULP may be involved in phagocytosis in mammals. Similarly, ABC1, the closest mouse homolog of CED-7, acts in macrophages to promote the engulfment of apoptotic cells (Hamon et al., 2000). As discussed previously, ABC1 promotes Ca2+-induced exposure of phosphatidylserine to the outer membrane, an event believed to act as a trigger for the engulfment of the PS-presenting cell (Hamon et al., 2000). As mentioned previously, CED-1 and the scavenger receptor SREC display strong structural and sequence similarity to each other in their extracellular domains and therefore may possess similar ligand-binding specificity (Zhou et al., 2001b). However, whether SREC and CED-1 can replace each other’s function is not known. SREC lacks the NPXY or YXXL motifs that are critical for CED-1 function (Adachi et al., 1997; Zhou et al., 2001b). CD91/LRP, a human protein reported to play a role in the phagocytosis of apoptotic cells, was proposed to be a functional ortholog of CED-1 (Su et al.,

4. Engulfment and Degradation of Apoptotic Cells

111

2002). The intracellular domain of CD91/LRP contains both the NPXY and YXXL motifs. However, its extracellular domain, although cysteine rich, possesses an architecture very diVerent from CED-1, and it is unknown whether CD91 shares CED-1’s ligand-binding specificity (Callebaut et al., 2003). MEGF10, a protein predicted from a human cDNA isolated from brain (Nagase et al., 2001), has been shown to display extensive structural and sequence similarity to CED-1 throughout its entire length (Callebaut et al., 2003). In particular, human MEGF10 contains the same number of tandem copies (16) of the CED-1 type of atypical EGF-like repeats in its putative extracellular domain and an NPXY motif in its C-terminal putative intracellular domain. In addition, both human MEGF10 and CED-1 were shown to bear an EMI domain at the very N terminus, N-terminal to the EGF-like repeats (Callebaut et al., 2003). MEGF10 is a protein of unknown function (Callebaut et al., 2003; Nagase et al., 2001). It will be interesting to find out whether MEGF10, CD91/LRP, and SREC have any functional relationship with CED-1. 2. What Promotes the Polarized Extension of Engulfing Cell Surfaces? CED-10 and Its Upstream Regulators a. CED-10 Is a Rac GTPase That Regulates Cytoskeletal Reorganization. It has long been established that polarized cell surface extension, which is observed during many cellular processes such as cell migration, axonal outgrowth, and phagocytosis of foreign objects, requires a driving force that comes from the reorganization of the actin cytoskeleton underneath the plasma membrane (reviewed in Hall, 1998). The Rho/Rac/Cdc42 family of small GTPases has been found to act as molecular switches that regulate cytoskeleton reorganization (reviewed in Hall and Nobes, 2000). Reddien and Horvitz (2000) cloned ced-10 and found that it encodes a C. elegans homolog of human Rac1, a member of the Rho/Rac/Cdc42 family (Fig. 5). Two recessive viable alleles of ced-10, n3246 and n1993, bear missense mutations in the conserved residues required for GTP binding and membrane targeting, respectively, suggesting that, like other members of this family, both GTPase activity and membrane localization are necessary for CED-10 function (Reddien and Horvitz, 2000). Several lines of evidence support the notion that CED-10 is critical for the reorganization of the actin cytoskeleton. It was observed that in addition to engulfment defects, ced-10 mutant animals also suVer from multiple cell migration defects, in particular in the migration of the distal tip cells (DTCs) (Kishore and Sundaram, 2002; Reddien and Horvitz, 2000; Soto et al., 2002). Accumulating evidence also indicates that ced-10 mutants are defective in axon pathfinding (Gitai et al., 2003; Lundquist et al., 2001; Wu et al., 2002). As the processes of both cell migration and axon pathfinding require

112

Zhou et al.

polarized cell surface extension, the fact that ced-10 function is required for these processes in addition to engulfment strongly suggests that it regulates cytoskeletal reorganization in response to multiple extracellular cues (Lundquist et al., 2001; Reddien and Horvitz, 2000). Additionally, ced-10 controls multiple essential cellular and developmental processes because a complete loss-of-function mutation of ced-10 results in maternal-eVect embryonic lethality (Lundquist et al., 2001; Soto et al., 2002). The direct evidence that CED-10 acts on cytoskeletal reorganization came from experiments performed in Swiss 3T3 cells (Zhou et al., 2001a). Serumstarved Swiss 3T3 fibroblasts lost almost all recognizable actin structures, including actin stress fibers, lamellipodia, and membrane ruZes (Ridley et al., 1992). Injection of DNA constructs driving the expression of the active forms of Rho family GTPases can induce the formation of distinct polymerized actin in serum-starved fibroblasts (Ridley and Hall, 1992; Ridley et al., 1992). Injection of CED-10(G12V), a presumptive active form of CED-10, resulted in the formation of membrane ruZes similar to those resulting from the injection of active human Rac1 (Fig. 7A) (Zhou et al., 2001a). This result indicated that CED-10 and Rac1 could result in similar changes to the actin cytoskeleton, indicating that the in vivo activities of CED-10 and Rac1 must be similar. b. CED-10 Is Not Alone—CED-2, CED-5, and CED-12 Form a Protein Complex That Activates CED-10. Gene interaction studies placed ced-2, -5, and -12 and ced-10 in one functional group. Strikingly, like ced-10 mutants, but unlike ced-1, -6, or -7 mutants, loss-of-function mutants of ced-2, -5, and -12 all display similar DTC migration defects. The abnormal migration patterns of DTCs in these mutants are identical to those observed in ced10 mutants, suggesting that ced-2, -5, and -12 may act in the cytoskeletal reorganization events regulated by ced-10 (Gumienny et al., 2001; Reddien and Horvitz, 2000; Wu and Horvitz, 1998b; Wu et al., 2001; Zhou et al., 2001a). ced-2, -5, -10, and -12 all act in engulfing, but not dying, cells (Gumienny et al., 2001; Reddien and Horvitz, 2000; Wu and Horvitz, 1998b; Wu et al., 2001; Zhou et al., 2001a). Furthermore, overexpression of CED-10 under the control of C. elegans heat-shock promoters was suYcient to bypass the requirement for ced-2, ced-5, or ced-12 in the engulfment of cell corpses (Gumienny et al., 2001; Reddien and Horvitz, 2000; Wu et al., 2001; Zhou et al., 2001a). This result indicates that the main functions of CED-2, CED-5, and CED-12 are to activate CED-10 for the reorganization of actin cytoskeleton. These results support a model in which CED-2, -5, and -12 act as upstream regulators of CED-10, and all four proteins form a signaling pathway in engulfing cells to regulate the polarized extension of cell surfaces.

4. Engulfment and Degradation of Apoptotic Cells

113

Figure 7 Activation of CED-10 leads to the rearrangement of the actin cytoskeleton, an event required for the extension of pseudopods during the engulfment of cell corpses. (A) Overexpression in Swiss 3T3 cells of active forms of CED-10 or Rac1, the mammalian homolog of ced-10, induces the formation of membrane ruZes (indicated by arrowheads). Nuclei of serum-starved Swiss 3T3 cells were microinjected with construct that express myc-ced10(G12V) or myc-Rac1G12V, a constitutively active form of CED-10 or Rac1. Biotin dextran was also injected as a negative control. Distribution of filamentous actin (F-actin) was examined using phalloidin staining. An arrow indicates an actin bundle, which results from a secondary eVect after the injection of myc-Rac1G12V. (Reproduced from Zhou et al., 2001a.) (B) Current model for the action of the signaling pathway composed of CED-2, CED-5, CED-10, and CED-12. See text for details. The thin double line indicates the plasma membrane of an engulfing cell. The question mark indicates that the transmembrane receptor(s) for the ‘‘eat me’’ signal exposed by apoptotic cells is(are) of unknown identity.

CED-2 and CED-5 are homologs of mammalian CrkII, an SH2 SH3 domain-containing adaptor protein, and Dock180, a CrkII-binding protein, respectively (Fig. 5) (Reddien and Horvitz, 2000; Wu and Horvitz, 1998b). CED-12 has one Drosophila homolog (DCED-12) and three human homologs (ELMO1, ELMO2/HCED-12A, and human BAB14712), all of which contain a pleckstrin homology (PH) domain, a lipid-binding domain, and a proline-rich motif for binding to SH3 domains (Fig. 5) (Gumienny et al., 2001; Wu et al., 2001; Zhou et al., 2001a). Assays performed in vitro or in the yeast two-hybrid protein interaction detection system have shown that CED-12 interacts with CED-5, which interacts with CED-2, and that all three interact simultaneously in vitro (Wu et al., 2001; Zhou et al., 2001a). Thus, CED-2, CED-5, and CED-12 may form a complex in vivo. How might this protein complex activate CED-10? The activity of Rho family GTPases is regulated by a number of proteins, including the guanine

114

Zhou et al.

nucleotide exchange factors (GEFs), which enhance the exchange of bound GDP for GTP and promote the conversion of the Rho family proteins to an active state to regulate downstream signaling events (reviewed in Van Aelst and D’Souza-Schorey, 1997). More than 50 GEFs capable of regulating the Rho family GTPases have been identified, all of which contain a Db1 homology (DH) domain required for the nucleotide exchange activity in tandem with a PH domain, which also makes important contributions to the nucleotide exchange activity (reviewed in Braga, 2002). However, no known C. elegans DH-containing GEF proteins are required for the engulfment of cell corpses and thus are unlikely to act as GEFs for CED-10 during phagocytosis (Lundquist et al., 2001; Wu et al., 2001). Although CED-2, -5, and -12 do not have DH domains, CED-12 does contain a PH domain. Biochemical studies suggest that DOCK180 and ELMO1, the mammalian homologs of CED-5 and CED-12, respectively, could act together as an unconventional bipartite GEF for Rac (Brugnera et al., 2002). DOCK180 interacts with Rac (Kiyokawa et al., 1998; Nolan et al., 1998). The CED-2/-5/-12 complex thus is likely to possess similar GEF activity for CED-10 (Fig. 7B). Not much is known about how the CED-2/-5/-12 protein complex activates CED-10 in response to extracellular cues. CED-10 is membrane bound and is distributed evenly over the plasma membrane of many cells (Lundquist et al., 2001). The recruitment and assembly of the CED-2/-5/-12 complex is likely to be a region-specific event induced by extracellular signals. The PH domain, which binds phopholipids, in particular, phosphatidylinositols (PIs), is proposed to recruit GEFs to membranes (Braga, 2002). CED-12, which contains a PH domain, was detected to be membrane-bound in C. elegans cells (Zhou et al., 2001a). In addition, CED-2 contains an SH2 domain likely to associate with phosphorylated tyrosine residues, possibly in the cytosplasmic domain of an unknown transmembrane receptor (Reddien and Horvitz, 2000). The recruitment of the CED-2/-5/-12 complex to the membrane may therefore occur according to one of two possible models: (1) the binding of the PH domain of CED-12 to particular forms of membrane PIs generated in response to an extracellular signal, or (2) the binding of the SH2 domain of CED-2 to the cytoplasmic domain of an activated transmembrane receptor. It is also possible that both models are correct and the processes act cooperatively, or the complex might be recruited and activated by an unforeseen mechanism. CrkII, Dock180, and ELMO1, the mammalian counterparts of CED-2, -5, and -12, respectively, function cooperatively to promote Rac activation and phagocytosis in assays performed using cultured mammalian cells (Albert et al., 2000; Gumienny et al., 2001). Integrin v 5 was implicated as one of the upstream receptors that links extracellular signals with this Rac-activating complex through the function of p130cas, which interacts with CrkII (Albert et al., 2000). These studies confirmed that the signaling

4. Engulfment and Degradation of Apoptotic Cells

115

pathway led by CED-2, -5, -10, and -12 represents an evolutionarily conserved basic mechanism that regulates cytoskeletal reorganization. However, none of the three integrin subunits identified in C. elegans seem to be involved in engulfment (Gumienny et al., 2001; Wu et al., 2001). The receptor that directly responds to the presence of cell corpses and that activates the CED-2/-5/-12 complex remains to be identified. c. GEX-2 and GEX-3 Are Two Potential New Components of the CED-10Signaling Pathway. Animals homozygous for a ced-10 null allele (n3417) display a maternal-eVect embryonic lethal phenotype (Lundquist et al., 2001). Embryos produced by ced-10(n3417) homozygous mothers arrest development with apparently well-diVerentiated cell types that fail to become properly organized, indicating the existence of multiple defects in morphogenesis and cell migration (Soto et al., 2002). Inactivation of two genes, gex-2 and gex-3 (gut on the exterior), results in embryonic lethality and embryonic developmental defects (e.g., defects in epidermal enclosure) in many ways similar to or more severe than that observed in ced-10(n3417) arrested embryos (Soto et al., 2002). GEX-2 and GEX-3 are homologous to p140/Sra-1 and Hem-2, two mammalian Rac1-interacting proteins, respectively (Soto et al., 2002). Because mammalian p140/Sra-1 can directly interact with filamentous actin, and because the Drosophila homolog of Hem-2 is required for axon pathfinding and correct actin organization, p140/Sra-1 and Hem-2 were proposed to provide a link between the Rac1 GTPase and actin cytoskeleton (Hummel et al., 2000; Kobayashi et al., 1998). Interestingly, arrested embryos generated in gex-2 and gex-3 loss-of-function background contain persistent cell corpses in numbers similar to those found in ced-10(n3417) mutant embryos (Soto et al., 2002). Although it is unknown whether this observation reflects a potential defect in cell corpse engulfment or a secondary consequence of the morphogenesis defects, it is possible that GEX-2 and GEX-3 act in the signaling pathways regulated by CED-10, including cell corpse engulfment (Soto et al., 2002). 3. CDL-1, a Stem-Loop Binding Protein and a Regulator of Core Histone Expression, May Be Involved in the Efficient Engulfment of Cell Corpses In addition to the 10 C. elegans genes that play or may play roles in cell corpse engulfment described previously, cdl-1 (cell death lethal), which encodes a stem-loop binding protein, was found to be required for the progression of programmed cell death and other aspects of embryogenesis in C. elegans (Kodama et al., 2002). In cdl-1 homozygous embryos, which are zygotic lethal, excess cell corpses accumulate, in addition to the defects observed in body elongation, pharyngeal development, and mitotic chromosome condensation (Kodama et al., 2002). A close examination indicates

116

Zhou et al.

that both the appearance and the elimination of cell corpses are delayed and that cell corpses persist much longer in these embryos (Kodama et al., 2002). CDL-1 was shown to bind to the stem-loop structure in the 30 -UTR of C. elegans core histone mRNAs, an event thought to be critical in regulating histone expression (Kodama et al., 2002). This activity and the cdl-1 mutant phenotypes are consistent with a model in which chromosome condensation, global gene expression, or both were perturbed in cdl-1 mutant embryos (Kodama et al., 2002). However, it remains to be determined whether CDL-1 aVects the progression of cell death and the eYciency of cell corpse engulfment by, perhaps, aVecting chromosome condensation during programmed cell death, or by a more indirect action.

D. The Engulfment of Cells That Die by Necrosis Requires the Same Genes That Act to Engulf Apoptotic Cells Necrosis is a form of cell death that results from acute cell injury (Wyllie et al., 1980). Necrotic cells swell and burst open, releasing their cellular contents, rather than shrinking and remaining intact, as is seen in apoptotic death (Wyllie et al., 1980). In C. elegans, dominant mutations in genes encoding several ion channel subunits such as MEC-4 (a subunit of the degenerin Naþ channels) and DEG-3 (acetylcholine receptor Ca2þ channel) induce necrotic death of specific groups of neurons as a result of channel hyperactivity and increased ion influx (Fig. 8) (reviewed in Driscoll and Gerstbrein, 2003). Expression of a constitutively active form of a heterotrimeric G-protein subunit Gs can also induce similar necrotic neuronal death in C. elegans (Driscoll and Gerstbrein, 2003). The induction of necrosis is independent of the functions of ced-3, ced-4, and egl-1 (egg-laying defective), three genes required for programmed cell death in C. elegans, and is not protected by ced-9, which protects against programmed cell death, indicating that the execution of necrosis is mechanistically distinct from that of apoptosis (Chalfie and Wolinsky, 1990; Chung et al., 2000; Ellis and Horvitz, 1986). Like apoptotic cells, necrotic cells in C. elegans are removed through the process of engulfment, and the functions of all seven known engulfment genes are required for this removal (Chung et al., 2000; Hall et al., 1997). A genetic screen was carried out for genes required for the eYcient removal of necrotic cell corpses generated in a mec-4 dominant mutant background, and one allele of ced-12, which was one of the first ced-12 mutant alleles identified, was recovered from this screen (Chung et al., 2000). As observed in the engulfment of apoptotic cells, the seven engulfment genes seem to act in the same two partially redundant pathways for necrotic cell removal: ced-1, -6, -7 in one pathway and ced-2, -5, -10, and -12 in the other (Chung et al., 2000). The requirement for the same engulfment

4. Engulfment and Degradation of Apoptotic Cells

117

Figure 8 Nomarski microscope image of a ced-1(e1735);mec-4(e1611dm) larva at L1 stage showing the morphology of cells undergoing necrotic and apoptotic deaths. In this genetic background, a dominant mutation in mec-4 results in the necrotic death of six mechanosensory neurons, three of which are visible and indicated with arrows. These necrotic cells appear swollen and bigger in size compared to apoptotic cells, which appear condensed and highly refractile (indicated by arrowheads). These apoptotic cells are not engulfed at this time (as they would be in wild-type) because of the ced-1 mutation.

genes suggests that the mechanisms behind the recognition and engulfment of necrotic and apoptotic cells are similar. In particular, the requirement for CED-1 and CED-7, two proteins implicated in the recognition of apoptotic cells, suggests that cells dying of apoptosis and cells dying of necrosis may present similar ‘‘eat me’’ signals to induce their engulfment by neighboring cells. Alternatively, CED-1 may possess the ability to recognize multiple types of ‘‘eat me’’ signals. The removal of necrotic cells occurs much more slowly than that of apoptotic cells (Chung et al., 2000). This raises the possibility that the ‘‘eat me’’ signals are actively exposed onto the surface of apoptotic cells in a process triggered by the cell death execution machinery but are only passively released to the surface of necrotic cells as the cells break apart. A comparison of the cell surface properties of apoptotic and necrotic cells may help to identify the ‘‘eat me’’ signal(s). E. The Process of Engulfment Assists in the Execution of Apoptosis As discussed in Section II.B, the notion that engulfing cells are solely responsible for the removal of dying cells but are not involved in the execution of death resulted from the observation that mutations that severely

118

Zhou et al.

impair engulfment do not prevent cells destined to die from undergoing apoptosis. However, recent progress has indicated that engulfing cells play an active role in assisting apoptosis (Hoeppner et al., 2001; Reddien et al., 2001). Genetic and molecular studies in C. elegans have identified the pathway that controls the execution of somatic programmed cell death (reviewed in Metzstein et al., 1998). This pathway is composed of ced-3, the most downstream gene that encodes a caspase (CED-3) crucial for the execution of most if not all cell deaths, and ced-4, ced-9, and egl-1, three genes encoding its positive and negative regulators. According to the current model established through genetic and biochemical studies of these genes (reviewed in Metzstein et al., 1998), in cells destined to die, CED-3 is activated by CED-4, an Apaf-1-like caspase activator. In cells that should normally live, CED-9, a Bcl-2-like survival factor, directly interacts with CED-4 and inhibits CED4’s ability to activate CED-3. CED-4, CED-9, and most likely CED-3 are present in most, if not all, somatic cells. EGL-1, the most upstream protein required for the execution of somatic programmed cell deaths, is a BH3-only protein and is specifically expressed in cells destined to die in which it frees CED-4 from CED-9-mediated interaction and interacts with CED-9 itself (Chen et al., 2000; Conradt and Horvitz, 1998, 1999; Thellmann et al., 2003). Active CED-3 is thought to induce a number of downstream events that together lead to cell death. These events include cell and nuclear shrinkage, nuclear DNA degradation, and the exposure of the ‘‘eat me’’ signal(s) (reviewed in Metzstein et al., 1998). Reddien and co-workers (2001) and Hoeppner and co-workers (2001) each separately observed that in animals carrying homozygous weak partial loss-of-function mutations in the downstream killer genes ced-3 or ced-4, the presence or absence of an intact engulfment system aVects the eYciency of the cell death execution process. For example, animals homozygous for ced3(n2427), a weak loss-of-function allele, have an average of 1.5 extra undead cells in the anterior pharynx of 16 cells that normally die during embryogenesis (Reddien et al., 2001). Although no extra cells were observed in this area in any engulfment mutant alone, animals carrying both ced3(n2427) and mutations in engulfment genes have a greater number of extra cells (4.5–6.2) than in ced-3(n2427) mutants alone (Reddien et al., 2001). This phenomenon is not limited to the anterior pharynx, as the enhancement was also observed in cells that underwent apoptosis postembryonically in the lateral ectoderm and in the posterior ventral nerve cord (Reddien et al., 2001). This enhancement by mutations aVecting engulfment is not limited to mutations of particular engulfment genes; rather, mutations in all seven engulfment genes have a similar eVect, indicating that it is the whole engulfment machinery that plays a role in death execution (Hoeppner et al., 2001; Reddien et al., 2001). Reddien and co-workers (2001) also found that

4. Engulfment and Degradation of Apoptotic Cells

119

expressing CED-1 specifically in engulfing cell types was suYcient to rescue the defects of ced-1 loss-of-function mutants in engulfment-mediated enhancement of cell death execution. Therefore, the death-assisting activity acts from engulfing cells in a cell nonautonomous manner. On the other hand, a deletion of the entire caspase domain of ced-3 results in the presence of 12 extra cells in the anterior pharynx, a phenotype that is not enhanced by any engulfment mutants, suggesting that the death-promoting eVect of the engulfing cells is dependent on some remaining caspase activity (Reddien et al., 2001). In engulfment mutant animals, even when the genes controlling the execution of cell death are wild-type, there is a low penetrance incidence of failure of or delayed cell death. In the posterior ventral cord of ced-6 and ced-7 mutant larvae, Reddien and co-workers (2001) observed that in many cases cells destined to die initially displayed some morphological characteristics of dying cells, then fluctuated between a dying and a living appearance for a period of time, and eventually chose one of the two fates. The living cells that had visibly undergone morphological changes associated with death and then recovered could extend neuronal processes and express a lin-11::gfp reporter, indicating that they had fully recovered and properly diVerentiated (Reddien et al., 2001). Using four-dimensional microscope time course analysis, Hoeppner and co-workers (2001) observed a similar phenomenon in ced-7 mutant embryos. How do engulfing cells promote death and ensure that death is irreversible? Although the molecular mechanism is not clear, several models have been proposed to explain the action of the engulfing cells (reviewed in Conradt, 2002). Cells in which a low-level caspase activity is expressed are not necessarily committed to die. In weak ced-3 mutants, embryonic cell deaths occur but are apparently delayed (Hoeppner et al., 2001; Stanfield and Horvitz, 2000). Moreover, Hoeppner and co-workers (2001) showed that in a weak ced-3 mutant background, some cells that have begun the morphological process associated with programmed cell death have the ability to recover and escape death. Thus, the eVect of low caspase activity is reversible. When the caspase activity within a dying cell is low, an engulfing cell may internalize such a cell and start its degradation before it has time to recover and survive. Alternatively, the neighboring engulfing cells may secret digestive enzymes before a dying cell is fully engulfed, or they may initiate a positive feedback loop through the contact with the dying cells to reinforce the decision of the dying cells to complete apoptosis (reviewed in Conradt, 2002). Consistent with the second and third models, ced-1 and ced-7 have been found to be required for an initial step in the degradation of the nuclear DNA of apoptotic cells, a step that occurs eYciently even when the engulfment of the dying cell is prevented by other engulfment mutations (Wu et al., 2000). These data are consistent with an eVect of the neighboring cell to

120

Zhou et al.

promote an apoptotic process within the unengulfed dying cell (discussed later). The discovery that engulfment assists in the execution of apoptosis is important to further our understanding of the regulation of apoptosis. In mammals, the enhancement of death by phagocytes may ensure an eYcient clearance of apoptotic cells and thus will lead to better prevention of potentially harmful inflammatory and autoimmune responses. Furthermore, macrophages, which are the ‘‘professional phagocytes’’ in mammals, have been shown to promote the apoptosis of certain types of cells, an event important for tissue remodeling. For example, macrophages induce apoptosis in normal vascular endothelial cells in rat eyes during programmed capillary regression (Diez-Roux and Lang, 1997). In vitro, co-culture of interferon-stimulated macrophages with myofibroblast-like mesangial cells was found to induce the apoptosis of the mesangial cells, and this phenomenon has been implicated as a step of the resolution of inflammatory injury in vivo (DuYeld et al., 2000). It remains to be studied whether macrophages in mammals and engulfing cells in C. elegans use similar molecular mechanisms for inducing apoptosis. As discussed in the next section, in C. elegans, there are also several known examples of cell deaths that are solely dependent on engulfment.

F. The Cases of Murder: Programmed Cell Deaths That Are Critically Dependent on Engulfing Cells For a few of the described cases of programmed cell deaths in C. elegans, engulfing cells appear to act as killers, required for the deaths to occur. There are two notable cases in male development where a cell programmed to die must interact with its neighbors in order for its death to occur: the linker cell and the B.a(l/r)apaav equivalence group (Sulston et al., 1980). In the first example, the linker cell leads the extending male gonad from its starting point in the midbody to its final destination in the tail (Kimble and Hirsh, 1979). Once the gonad has reached its target, the linker cell is engulfed by one of two cells, U.lp or U.rp, neighboring to its final position. However, if these cells are ablated or if the linker cell is prevented from coming in contact with them, the linker cell survives (Sulston et al., 1980). In the second case, B.alapaav and B.arapaav are left-right homologs that comprise an equivalence group: one of these two cells fuses to form part of the vas deferens, while the other cell dies and is engulfed by its neighbor, P12.pa. Similar to linker cell death, both B.alapaav and B.arapaav survive when the cell that normally engulfs them is ablated (Sulston et al., 1980). In both of these cases, death occurs through murder instead of suicide (i.e., death required the participation of another cell). ced-1 or ced-2 loss-of-function

4. Engulfment and Degradation of Apoptotic Cells

121

mutations can result in the survival of the linker cell and both B.alapaav and B.arapaav, indicating that engulfment is required for cell death in these two instances (Ellis and Horvitz, 1986; Hedgecock et al., 1983; Reddien et al., 2001). In the case of the linker cell, death can occur even in the absence of ced-3 or ced-4, suggesting that engulfment leads to the activation of a nonclassical killing pathway (Ellis and Horvitz, 1986). In contrast, while the death within the B.alapaav and B.arapaav equivalence group is completely dependent upon engulfment, it also requires the function of ced-3 (Reddien et al., 2001). In this case, the engulfing cell may be able to trigger caspase activation or may be necessary to respond to a sublethal level of caspase activation by promoting cell death execution (Reddien et al., 2001). Another example of cell death that is dependent on engulfment was observed in semidominant mutants of lin-24 and lin-33, two genes required for the specification of vulval cell lineages (Ferguson et al., 1987). In these mutants, up to six of 12 Pn.p cells, which are a group of epithelial precursor cells that should normally live, develop an abnormal morphology and often die. These dying cells undergo morphological changes that resemble necrotic cell deaths (Ferguson et al., 1987). While these deaths appear to be independent of ced-3 and ced-4 activities, they are suppressed by loss-of-function mutations in the engulfment genes ced-2, ced-5, and ced-10, which constitute one of two functional groups required for the engulfment of dying cells, but not by mutations in ced-1, ced-6, or ced-7 (S. C. Kim and H. R. Horvitz, unpublished results). This phenomenon suggests that the deaths of the abnormal Pn.p cells are dependent on the function of CED-2/CED-5/CED-10 and presumably involves aggressive engulfment by neighboring cells.

III. The Degradation of Nuclear DNA During Programmed Cell Death in C. elegans Wyllie observed that DNA from thymocytes induced to undergo apoptosis is processed into multimers of 180 base pair subunits (Wyllie, 1980). This nuclear DNA fragmentation is a general phenomenon observed in many types of cells undergoing apoptosis and was demonstrated to be dependent upon caspase activation during cell death (Enari et al., 1996; Liu et al., 1996). Pulse field gel electrophoresis reveals that DNA from apoptotic cells is initially cleaved at the scaVold regions to produce fragments of 50–300 kb, before they are subsequently processed into smaller fragments (Lagarkova et al., 1995; Oberhammer et al., 1993). TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) (Gavrieli et al., 1992) and LM-PCR (linker-mediated-PCR) (Staley et al., 1997), two common assays for cell death, measure the extent of DNA fragmentation by detecting the

122

Zhou et al.

30 -hydroxyl

50 -phosphate

reactive and ends generated in the process, respectively. In this section, we discuss studies investigating nuclear DNA degradation during programmed cell death in C. elegans, and in Section IV, we compare them to what is known in mice and Drosophila.

A. nuc-1 Encodes a DNaseII Homolog Critical for DNA Degradation During Cell Death The first gene in C. elegans identified as playing a role in cell death was nuc-1 (nuclease abnormal). nuc-1 was isolated in a genetic screen seeking to recover mutants with lineage defects in the ventral nerve cord (Sulston, 1976). Sulston (1976) was visualizing cell nuclei by Fuelgen staining (a DNA dye) when he noticed a mutant, nuc-1, in which ingested bacterial DNA was not properly degraded. nuc-1 mutants also retained persistent DNA at positions consistent with the nuclei of cells that had undergone programmed cell death (Hedgecock et al., 1983; Sulston, 1976). An acid endonuclease activity is significantly reduced in protein extracts generated from nuc-1 mutants compared to those from wild-type animals (Hevelone and Hartman, 1988). Although DNA degradation is inhibited in nuc-1 mutants, neither the execution of cell death nor the engulfment of cell corpses appears to be aVected (Hedgecock et al., 1983; Parrish et al., 2001; Sulston, 1976; Wu et al., 2000). To examine the mechanism regulating DNA degradation during apoptosis, Wu and co-workers (2000) developed a TUNEL assay for use on C. elegans embryos. As previously mentioned, TUNEL specifically labels 30 hydroxyl ends present in DNA fragments (Gavrieli et al., 1992) and has been extensively used to label cells undergoing apoptosis. In wild-type C. elegans embryos, during midembryogenesis, at the stage when 14 cell corpses are visible using Nomarski microscopy, an average of only 1.7 nuclei are TUNEL positive (Wu et al., 2000). In contrast, approximately 48 TUNELreactive nuclei can be seen in nuc-1 mutants at the same stage. In addition, the generation of TUNEL-reactive nuclei follows kinetics similar to that of the appearance of cell corpses. NUC-1 must therefore act in an intermediate step for DNA degradation, converting TUNEL-positive ends to TUNEL-negative ends (Wu et al., 2000). Wu and co-workers (2000) cloned nuc-1 and determined that it encodes a DNaseII homolog. DNaseII is an endonuclease that functions at low pH and generates DNA fragments with 50 -hydroxyl and 30 -phosphate groups, which are not substrates for the terminal transferase used in TUNEL assays (Harosh et al., 1991). Thus, the disappearance of a TUNEL-positive signal in wild-type embryos is consistent with the predicted enzymatic properties of NUC-1. Engulfment does not appear to be required for the generation and degradation of TUNEL-reactive ends in cells undergoing programmed cell death.

4. Engulfment and Degradation of Apoptotic Cells

123

Single mutant embryos of the engulfment genes ced-2, -5, -6, and -10 have similar numbers of TUNEL-positive nuclei compared to wild-type embryos, and double mutants between each of these engulfment mutants and nuc-1 produced approximately the same number of TUNEL-staining nuclei as a nuc-1 single mutant (Wu et al., 2000). However, engulfment is required for the completion of DNA degradation, as Feulgen-reactive spots persist in unengulfed cell corpses (Hedgecock et al., 1983). In addition, unengulfed cell corpses are positive for the staining of SYTO-11, another DNA dye, in ced-2, ced-5, ced-6, and ced-10 mutant animals (Wu et al., 2000). The fact that DNA is present in unengulfed cell corpses suggests that the completion of DNA degradation requires a contribution from either the engulfing cell or the engulfment process. In contrast to ced-2, -5, -6, and -10, ced-1 and ced-7 were observed to be required or partially required for the initial step of DNA degradation that results in the generation of TUNEL-reactive ends that are then degraded in a nuc-1-dependent manner. In ced-1;nuc-1, and ced-7;nuc-1 double mutant embryos, an average of only 1.0 and 20 TUNEL-reactive nuclei, respectively, were observed, compared to the 48 TUNEL-positive nuclei observed in nuc-1 embryos (Wu et al., 2000). This requirement for ced-1 and ced-7 might be explained if these two genes, in addition to their functions in promoting engulfment, are required to promote certain aspects of cell death such as nuclear DNA degradation within the dying cell (Wu et al., 2000). As discussed in Section II, CED-1, in particular, functions as a phagocytic receptor, while CED-7 appears to be required for the clustering of CED-1. Wu and co-workers proposed a three-step model for DNA degradation in C. elegans (Fig. 9) (Wu et al., 2000). Initially, intact DNA is cleaved by a CED-1/CED-7-dependent nuclease that produces TUNEL-reactive fragments. These fragments serve as substrates for NUC-1, which processes them into TUNEL-nonreactive fragments. Given that NUC-1 is secreted into the intestinal lumen, it is possible that NUC-1 is produced in the engulfing cell and then is transported to the dying cell (Wu et al., 2000). Alternatively, the observation that NUC-1 functions in unengulfed cell corpses to degrade TUNEL-reactive DNA ends raises the possibility that it acts, at least in part, cell autonomously (Wu et al., 2000). Complete digestion into nucleotides occurs through an unidentified nuclease activity provided in the engulfing cell.

B. cps-6 Regulates Both DNA Degradation and the Timing of Cell Death cps-6 (CED-3 protease suppressor) was identified by Parrish and co-workers (2001) from a genetic screen for suppressors of cell deaths induced by ectopially ced-3 expressed. cps-6 encodes the C. elegans homolog of mitochondrial

124

Zhou et al.

Figure 9 Current model for the process and pathway of nuclear DNA degradation during apoptosis in C. elegans. The process for DNA degradation involves at least three steps. In the first step, chromosomal DNA of dying cells is digested into TUNEL-positive fragments by an unknown endonuclease activity that requires CED-1 and CED-7 functions. In the second step, these fragments are further cleaved into TUNEL-nonreactive fragments. Endonucleases including NUC-1 mediate this step, and they form four partially functional redundant groups as described in the text. In the third step, which also requires the engulfment of the cell corpses, the TUNEL-negative DNA is further degraded into free nucleotides.

endonuclease G. Like NUC-1, CPS-6 is required for the eYcient processing of TUNEL-reactive DNA fragments to a TUNEL-nonreactive form. Inactivation of cps-6 by RNA interference (RNAi) in a nuc-1 mutant background causes the presence of TUNEL-positive nuclei in numbers larger than seen in either nuc-1 mutants or cps-6 (RNAi) animals, indicating that cps-6 and nuc-1 may have partially redundant functions (Parrish et al., 2001). In addition to its role in DNA degradation, cps-6 regulates the timing and execution of cell death, which might explain the finding that inactivating cps-6 could partially suppress ced-3-induced apoptosis (Parrish et al., 2001). cps-6 mutants display a delayed appearance of cell corpses during development similar to that observed in ced-8 mutants (Parrish et al., 2001). ced-8 encodes a transmembrane protein required for the proper kinetics of programmed cell death (Stanfield and Horvitz, 2000). The appearance of cell corpses is further delayed in cps-6;ced-8 double mutants compared to either single mutant. Although a mutation in cps-6 on its own has little eVect in blocking apoptosis, cps-6 loss of function enhances cell survival in both weak and strong mutants of ced-3 and ced-4. These observations support the hypothesis that cps-6 may normally function in promoting cell death (Parrish et al., 2001). Wang and co-workers (2002) have reported that cps-6 functions together with an apoptosis inducing factor (AIF) homolog in C. elegans. AIF is a mitochondrial oxidoreductase that is released into the cytoplasm to induce cell death in response to an apoptotic signal (Joza et al., 2001; LoeZer et al., 2001; Susin et al., 1999). RNAi inactivation of wah-1 (worm AIF homolog) results in an enhancement of ced-3, ced-4, and ced-8 single mutant phenotypes similar to that seen for cps-6 inactivation (Wang et al., 2002). wah-1 promotes DNA degradation: wah-1 (RNAi) embryos contain more TUNEL-reactive nuclei than do embryos treated with control RNAi. In addition, wah-1(RNAi) fails to delay death more than control RNAi in a cps-6;ced-8 double mutant background, and wah-1(RNAi) does not enhance the DNA degradation

4. Engulfment and Degradation of Apoptotic Cells

125

defect seen in cps-6(sm116) animals (Wang et al., 2002). Like cps-6, wah1(RNAi) animals are not deficient in degrading bacterial DNA. Thus, inactivations of cps-6 and wah-1 cause similar phenotypes, and inactivation of both does not cause more severe defects than those seen for either one alone. Finally, recombinant WAH-1 and recombinant CPS-6 display synergetic ability to degrade DNA, a finding that could not be reiterated by sequential addition of CPS-6 and WAH-1 each in the absence of the other (Wang et al., 2002). These results are consistent with a model in which WAH-1 and CPS-6 act together to promote cell killing and degradation of nuclear DNA, a relationship that may be conserved in mammals (Wang et al., 2002).

C. Multiple Pathways Are Involved in DNA Degradation in C. elegans A paper by Parrish and Xue (2003) describes the identification of several cell death-related nucleases (crn genes) via a functional genomics approach. Seventy-seven predicted genes were selected for inactivation based on the predicted nuclease activities of their protein products. Animals treated with RNAi against each of these 77 candidates were screened for alterations in the pattern of TUNEL staining. Nine nucleases were identified, including two products of previously characterized genes nuc-1 and cps-6. The seven remaining nucleases were named crn-1 through crn-6 and cyp-13 (a cyclophilin E homolog); each of the identified genes has a mammalian homolog. Parrish and Xue (2003) proposed that these proteins normally perform housekeeping functions but when needed are recruited to execute cell death. The 10 genes that have been identified to function in DNA degradation (Table II) have been classified into four epistasis groups: (1) nuc-1; (2) wah-1, cps-6, crn-1, crn-4, crn-5, cyp-13; (3) crn-2, crn-3; and (4) crn-6 (Fig. 9) (Parrish and Xue, 2003; Parrish et al., 2001; Wang et al., 2002). Interestingly, animals mutant for cps-6 and inactivated for a gene in the crn-2 pathway may display a synthetic engulfment phenotype. Cell corpses persist 55% longer in cps-6(sml16);crn-2(RNAi) animals compared to corpses in the wild-type, cps-6(sm116) mutants, or crn-2(RNAi) animals. This observation hints of a possible link between DNA degradation, possibly as a checkpoint for the successful progression of apoptosis, and cell corpse engulfment (Parrish and Xue, 2003). Alternatively, it has been observed that cells fated to die in which the execution process has been impaired often persist longer than do dying cells in the wild-type, suggesting that the persistent cell corpses of cps-6(sm116);crn-2(RNAi) animals may result from a defect in the execution of death (Hoeppner et al., 2001; Stanfield and Horvitz, 2000). In addition, proteins encoded by members of the cps-6 pathway can interact in vitro, indicating that they may form a DNA degradation complex, named the degradeosome (Parrish and Xue, 2003). A more extensive

126

Zhou et al.

Table II Genes Whose Functions Are Involved in the Degradation of Nuclear DNA During Programmed Cell Death in C. elegans Identity of Mammalian Homologs

Gene Name cps-6

Endonuclease G

cm-1

Flap endonuclease I

cm-2 cm-3

cm-4

TatD 100 kDa polymyositis/ scleroderma autoantigen (PM/Scl-100) 30 to 50 exonuclease

cm-5

Rrp46

cm-6 cyp-13 nuc-1 wah-1

DNase II Cyclophilin E DNase II Apoptosis inducing factor (AIF)

Function of the Mammalian Homologs

Reference

Caspase-independent cell death DNA replication, damage repair No known function Ribonuclease component of the exosome

Parrish et al., 2001

tRNA processing, DNA replication Ribonuclease component of the exosome Lysomomal nuclease RNA splicing Lysosomal nuclease Caspase-independent cell death

Parrish and Xue, 2003

Parrish and Xue, 2003 Parrish and Xue, 2003 Parrish and Xue, 2003

Parrish and Xue, 2003 Parrish and Xue, 2003 Parrish and Xue, 2003 Wu et al., 2000 Wang et al., 2002

characterization of crn-1 function supports the model that the distinct components of the degradeosome provide diVerent enzymatic activities that cooperate in degrading DNA (Parrish and Xue, 2003; Parrish et al., 2003). Thus, the emerging picture of apoptotic DNA degradation appears to be far more complex than initially anticipated, with multiple pathways contributing to the digestion of apoptotic DNA and linking DNA degradation with the execution of cell death and, possibly as a consequence, the engulfment of dying cells (Fig. 9) (Parrish and Xue, 2003).

IV. Study of Engulfment and DNA Degradation in Drosophila A. Hemocytes Act as Engulfing Cells In Drosophila, the vast majority of apoptotic cells is cleared by hemocytes (blood cell), although engulfment by nonprofessional phagocytes (epidermal cells in the eye and glial cells in the central nervous system [CNS]) has also been reported (Sonnenfeld and Jacobs, 1995; WolV and Ready, 1991). Using an antibody against the extracellular matrix (ECM) protein peroxidasin as a hemocyte-specific marker, Tepass and co-workers (1994) traced

4. Engulfment and Degradation of Apoptotic Cells

127

the embryonic origin of hemocytes and studied how cell death aVects the hemocyte-to-macrophage transition during development. They found that hemocytes arise from about 700 progenitors generated in the procephalic mesoderm. These cells migrate from their birthplace to populate the entire embryo, and their number remains constant during embryonic development. Earlier in development, hemocytes are typically small and round mesodermal cells. Coincident with their migration and the initiation of cell death in the embryo, they develop an extensive endoplasmic reticulum network and filipodia that extend from the cell body (Tepass et al., 1994). At this point, hemocytes near the brain and nerve cord become phagocytic. These phagocytic cells contain dense inclusions composed of apoptotic cells that have been engulfed (Tepass et al., 1994). Hemocytes containing ingested cells (or cell fragments) are defined as macrophages (Tepass et al., 1994). At later stages of development, approximately 90% of the peroxidasin-positive hemocytes have become phagocytic, with only the hemocytes in the gut remaining nonphagocytic, possibly due to the absence of cell death there (Tepass et al., 1994). Interestingly, complete conversion of hemocytes to macrophages was observed in embryos in which ectopic cell death had been genetically induced. This suggests that a cell death-dependent signal is suYcient to induce the hemocyte-to-macrophage transition and that peroxidasin-expressing cells define a homogenous population capable of undergoing this conversion (Tepass et al., 1994). On the other hand, programmed cell death is unaVected in embryos from which the hemocyte linage has been ablated (Tepass et al., 1994).

B. croquemort Encodes a Phagocytic Receptor in Drosophila Phagocytosis has not been studied as extensively in Drosophila as in C. elegans. The functions of myoblast city (mbc) and Rac1, homologs of the worm engulfment genes ced-5 and ced-10, respectively, have been characterized in flies. Although MBC and RAC1 have been demonstrated to regulate pseudopod extension and cytoskeletal reorganization (Erickson et al., 1997; Kiyokawa et al., 1998; Nolan et al., 1998)—roles consistent with those proposed for their C. elegans homologs—they have not been reported to function in the engulfment of apoptotic cells. However, Croquemort (French for pallbearer), a transmembrane receptor, has been demonstrated to be required for the engulfment of apoptotic cells (Franc et al., 1996, 1999). croquemort (crq) encodes a single-pass transmembrane protein that bears homology to human CD36, a scavenger receptor expressed in macrophages, and can mediate recognition of apoptotic cells (Franc et al., 1996). Expression of crq in COS7 cells promotes recognition and internalization of

128

Zhou et al.

apoptotic murine thymocytes by COS7 cells. Moreover, binding of apoptotic thymocytes is specifically inhibited by pre-incubation of transfected cells with antisera against the extracellular domain of Crq (Franc et al., 1996). To examine the in vivo function of crq, Franc and co-workers (1999) examined embryos homozygous for deficiencies that span the crq locus. Embryos homozygous for these deficiencies display a 20-fold decrease in the ability of hemocytes to engulf apoptotic cells compared to that seen in wild-type hemocytes, and this defect can be rescued with ubiquitous expression of crq (Franc et al., 1999). There appears to be a specific role for crq in clearing apoptotic cells, as other macrophage functions including phagocytosis of bacteria, endocytosis of acetylated low-density lipoprotein (AcLDL) particles, and production of ECM components are unaVected in crq-deficient embryos (Franc et al., 1999). Strong crq expression starts coincident with the initial wave of embryonic cell death and is detected in macrophages that contain ingested TUNELreactive apoptotic bodies (Franc et al., 1996). Cell death appears to promote crq expression, as a 74% decrease in crq levels was observed in H99 embryos, which contain a small deficiency that deletes the genes grim, reaper, and hid required for embryonic cell death (Franc et al., 1999; White et al., 1994). Moreover, increases in crq expression and phagocytic activity were detected in wild-type embryos and a hemocyte-derived cell line l(2)mbn induced to undergo ectopic apoptosis (Franc et al., 1999). crq expression thus seems to depend on the presence of apoptotic cells. The studies of crq raise multiple questions. First, the ligand for Crq has yet to be identified. In addition to chemically modified LDL, the homologous mammalian scavenger receptor CD36 was reported to bind other polyanionic ligands (Krieger and Herz, 1994). It is tempting to speculate that apoptotic cells present such a signal—perhaps phosphatidylserine—that mediates their recognition by Crq. Second, components of the signaling pathway downstream of crq need to be identified in order to understand how the phagocytic machinery is activated. In addition, loss-of-function alleles that specifically aVect crq have not been reported. The ability of hemocytes to phagocytose apoptotic cells was not totally abolished in crq-deficient embryos, indicating that other receptors may mediate recognition of dying cells (Franc et al., 1999). Other receptors known to be expressed by Drosophila macrophages, such as the scavenger receptor dSR-C1 (Pearson et al., 1995), Malvolio (Rodrigues et al., 1995), and the phosphatidylserine receptor (Fadok et al., 2000), may perform redundant functions (Fig. 10). Given the severe reduction in engulfment in crq-deficient embryos, Crq probably functions as the major phagocytic receptor in flies (Franc et al., 1999).

4. Engulfment and Degradation of Apoptotic Cells

129

Figure 10 Model of Croquemort function as a major phagocytic receptor that mediates the engulfment of apoptotic cells in Drosophila. Recognition of apoptotic cells leads to multiple events, including cytoskeletal rearrangement, that are required for the engulfment of apoptotic cells. The recognition and/or engulfment of apoptotic cells by engulfing cells also promotes Crq expression and diVerentiation of hemocytes into macrophages.

C. Engulfment of Apoptotic Cells Is Required for Proper Patterning of the CNS Many nerve cells die during Drosophila development, and these apoptotic cells in the CNS are continuously cleared by phagocytes (Sonnenfeld and Jacobs, 1995). In a 2003 paper, Sears and co-workers suggested that the engulfment of apoptotic cells is required for normal CNS morphogenesis in Drosophila by studying mutants in PDGF- and VEGF-receptor related (Pvr), a receptor tyrosine kinase. One significant feature of Pvr loss-of-function mutants is that hemocytes fail to migrate, instead remaining at their birthplace in the head mesoderm (Sears et al., 2003). Additionally, although CNS architecture was largely normal in Pvr mutants, the precise ladder-like structure of the axonal scaVold was disrupted (Sears et al., 2003). The nerve cord in Drosophila consists of a set of two longitudinal axon tracts that run parallel to the ventral midline. In Pvr mutants, these two longitudinal axon tracts appear to be compressed much closer to each other. There was also an abnormal accumulation of glia in the midline, indicating that Pvr function is required for correct positioning of these cells. CNS axonal pathfinding appears to be normal, with no inappropriate crossing at the midline (Sears et al., 2003).

130

Zhou et al.

Several lines of evidence suggest that Pvr function is required in hemocytes for proper development of the CNS. Inducing the expression of a dominant negative form of Pvr(DN-Pvr) in the hemocyte linage results in migration defects comparable to those in Pvr-mutants. In addition, the CNS phenotypes observed in Pvr-mutants were recapitulated by hemocyte-specific expression of DN-Pvr, suggesting that hemocyte function is required for proper CNS patterning (Sears et al., 2003). To test this hypothesis, the hemocyte lineage was genetically ablated using a mutation in serpent (srp), a gene that encodes a GATA transcription factor required for hemocyte development (Rehorn et al., 1996). Axonal tract and glial abnormalities indistinguishable from Pvr mutants were observed in srpneo45 mutant embryos (Sears et al., 2003). Embryos in which crq expression had been knocked down using RNAi have compressed longitudinal axon tracts and glial positioning defects that are very similar to those found in Pvr and srp mutants (Sears et al., 2003). Given these observations, Sears et al. (2003) proposed that clearance of apoptotic cells plays a critical role in patterning the Drosophila CNS. Defects in phagocytosis may result in the accumulation of apoptotic cells in the CNS, which could constitute a physical barrier that disrupts glial and axonal positioning in mutants lacking hemocytes. However, the possibility that Pvr and crq could function in CNS regulation independent of phagocytosis cannot be ruled out.

D. Caspase-Independent Engulfment In order to follow the engulfment of apoptotic cells during development, Mergliano and Minden (2003) developed an in vivo assay in Drosophila that employs resorufin- -galactoside-polyethylene glycol1,900 (VGAL). VGAL is a fluorogenic substrate that is activated after being internalized into macrophages by lysosomal -galactosidase. VGAL is injected into the common cytoplasm of early Drosophila embryos so that it can be taken up by all cells. In wild-type embryos, the pattern of engulfment detected using the VGAL assay closely mirrors that of cell death as observed by acridine orange staining (Mergliano and Minden, 2003). Cell death-deficient H99 embryos have very few acridine orange-positive nuclei indicative of apoptotic cells and develop only a few macrophages (White et al., 1994). Observations made in H99 embryos earlier in development using the VGAL assay agree with the results generated by acridine orange staining. However, later in development, striped VGAL-positive spots in the epidermis were observed in H99 embryos in a pattern that resembles that seen in wild-type embryos, indicating the engulfment of these cells (Mergliano and Minden, 2003). Likewise, ubiquitous expression of the pan-caspase inhibitor Baculovirus

4. Engulfment and Degradation of Apoptotic Cells

131

p35 protein in wild-type embryos does not aVect the production of these VGAL-positive stripes in the epidermis, despite the fact that it eYciently blocks apoptosis, suggesting that a form of cell engulfment proceeds even in the absence of caspase activity (Mergliano and Minden, 2003). This observation suggests a possibility that living cells are being engulfed or that certain cells undergo programmed cell death in a caspase-independent manner. Because dying epidermal cells are usually engulfed by neighboring cells instead of hemocytes, to test if the inhibition of cell death aVects engulfment by hemocytes, p35 was expressed in a patch of neurogenic region, in which dying cells are known to be engulfed by hemocytes (Mergliano and Minden, 2003). Neural precursors deficient for caspase activity and presumably remaining alive were nonetheless recognized and engulfed by hemocytes, again suggesting that certain signals can trigger phagocytosis in the absence of caspase activity in cells otherwise fated to die of caspase-dependent apoptosis (Mergliano and Minden, 2003). In C. elegans, blocking phagocytosis can weakly promote cell survival, an event that can be more strikingly seen as an enhancement of mild defects in cell death (Hoeppner et al., 2001; Reddien et al., 2001). This raises an interesting possibility that the observed engulfment of caspase-deficient cells in Drosophila could reflect an analogous mechanism for promoting cell death in cells that have begun the process of apoptosis. Alternatively, the engulfed cells may die of a very diVerent mechanism or may be engulfed alive (Mergliano and Minden, 2003).

E. DNA Degradation in Drosophila Proceeds in Two Steps 1. Cell-Autonomous DNA Degradation The major cell-autonomous mechanism by which the DNA of apoptotic cells is degraded was initially discovered through elegant biochemical studies performed in mammalian systems. Caspase-activated DNase (CAD) was purified from mouse T-cell lymphoma as the protein responsible for the nuclease activity in caspase-3-treated cytosol (Enari et al., 1998). CAD is normally kept inactive by binding to its inhibitor, ICAD (inhibitor of CAD), and is only de-repressed by active caspases. Caspase activation results in the cleavage of ICAD and the release of functional CAD (Enari et al., 1998; Sakahira et al., 1998). Protein refolding experiments suggest that in addition to inhibiting CAD activity, ICAD functions as a specific chaperone that is required for the proper folding of CAD (Sakahira et al., 2000). CAD and ICAD were also independently identified and characterized as DFF40/ CPAN and DFF45, respectively (Halenbeck et al., 1998; Liu et al., 1997).

132

Zhou et al.

Homologs of CAD and ICAD have been identified in Drosophila (Mukae et al., 2000; Yokoyama et al., 2000), but there are no identifiable homologs of CAD and ICAD in the C. elegans genome. In contrast to mouse and human CAD (mCAD and hCAD, respectively), which are not known to require protease processing, Drosophila CAD (dCAD) is cleaved by eVector caspases into large (p32) and small (p20) subunits during activation (Yokoyama et al., 2000). Activated dCAD behaves as a 100-kDa tetramer of (p32)2(p20)2 in a gel filtration column, while mCAD and hCAD do not seem to display any subunit structure. Additionally, dCAD lacks a C-terminal nuclear translocation signal found in mCAD and hCAD. While dCAD, like its mammalian homolog, has nuclease activity in vitro, it fails to degrade DNA in intact nuclei in standard fragmentation assays (Yokoyama et al., 2000). dICAD is a Drosophila protein approximately 17% identical in sequence to mouse and human ICAD (Yokoyama et al., 2000). Despite this low sequence resemblance, dICAD possesses the biochemical properties of a Drosophila ICAD homolog: it copurifies with the pro-form of dCAD, it inhibits dCAD activity, and it is required to produce functional dCAD (Fig. 11) (Yokoyama et al., 2000). To assess the contribution of dCAD to DNA degradation in apoptotic cells, Mukae and co-workers inactivated the dICAD gene by P element mutagenesis (Mukae et al., 2002). Because ICAD is essential for the production of functional CAD, dICAD-null flies are likewise inactive for CAD. dICAD-null flies are fertile and appear to have no discernable phenotype. However, DNA laddering was detected in wildtype but not ICAD-nulls, indicating that CAD is the major nuclease responsible for the degradation of DNA of apoptotic cells during fly oogenesis and embryogenesis (Fig. 11) (Mukae et al., 2002).

2. Cell Nonautonomous DNA Degradation McIlroy and co-workers generated transgenic mice that ubiquitously express a caspase-resistant form of ICAD (Sdm-ICAD), which should therefore prevent the caspase-induced activation of CAD (McIlroy et al., 2000). No DNA fragmentation was observed when thymocytes from transgenic mice were induced to undergo apoptosis in vitro. However, subsequent analyses in vivo demonstrated that lysosomal DNaseII from phagocytes provides an auxiliary mechanism by which DNA from apoptotic cells that have been engulfed is degraded (McIlroy et al., 2000). McIlroy and co-workers (2000) proposed a two-step model whereby DNA degradation proceeds initially in a cell-autonomous manner through the action of CAD and then proceeds further in a non-cell-autonomous manner through the action of lysosomal DNaseII in engulfing cells.

4. Engulfment and Degradation of Apoptotic Cells

133

Figure 11 The model for the mechanism that activates dCAD in apoptotic cells. dICAD functions as both a molecular chaperone and an inhibitor of dCAD. dICAD is required for correct folding of the newly translated dCAD protein. However, dCAD activity is inhibited upon its binding with dICAD. During apoptosis, an activated caspase in the dying cell cleaves dICAD and releases dCAD, which is further processed by the caspase into two fragments, p32 and p20. These fragments form an active tetramer (p32)2(p20)2, which degrades nuclear DNA into LM-PCR-detectable fragments.

Mukae and co-workers (2002) report a Drosophila model that is deficient for apoptotic DNA degradation. As discussed previously, CAD is the major nuclease responsible for the fragmentation of nuclear DNA detected by LMPCR (Mukae et al., 2002). Oogenesis proceeds in egg chambers housed in the Drosophila ovary. At a late stage of oogenesis, nurse cells transfer their cytoplasm to provide maternal determinants to the oocyte. The nuclei of nurse cells are subsequently degraded in a caspase-dependent manner (Cavaliere et al., 1998; McCall and Steller, 1998). However, despite the lack of DNA fragmentation in dICAD-null flies, no observable DNA accumulation was detected in egg chambers (Mukae et al., 2002). This could be explained if a mode of non-cell-autonomous DNA degradation similar to that seen in mammals operates in flies to promote the degradation of DNA in the absence of cell-autonomous CAD activity. In the same report, Mukae and co-workers described the cloning and characterization of Dnase-1lo, a loss-of-function mutation in Drosophila DNaseII (Mukae et al., 2002). DNaseII loss-of-function mutations do not prevent the generation of DNA laddering in apoptotic cells, indicating that DNaseII is not responsible for the cell-autonomous DNA degradation occurring in apoptotic cells. No DNA laddering in dying nurse cells was observed in flies deficient for both dICAD and dDNaseII. Instead, acridine orange-reactive vesicles accumulate in ovaries of Dnase-1lo single mutants and dICAD, Dnase-1lo double mutants, indicating that DNA from the dying nurse cells is not degraded. Thus, at least in nurse cells, dCAD and dDNaseII define two independent mechanisms for DNA degradation (Fig. 12) (Mukae

134

Zhou et al.

Figure 12 Degradation of nuclear DNA of apoptotic cells is a multistep process in Drosophila. In wild-type animals, caspase-activated dCAD promotes the processing of nuclear DNA within apoptotic cells into fragments that can be detected by LM-PCR. DNaseII activity, likely provided by the engulfing cell, eventually degrades these DNA fragments into free nucleotides. In dCAD/ animals, however, DNaseII activity is suYcient to bypass the requirement for dCAD and provides an auxilliary mechanism of degrading the nuclear DNA of apoptotic cells.

et al., 2002). These observations are in agreement with the two-step model proposed in mice (McIlroy et al., 2000; Mukae et al., 2002). Comparisons made between the mechanism of apoptotic DNA degradation in C. elegans and Drosophila/mammalian systems led to the following notions. In both organisms, initial processing of DNA occurs through the action of a nuclease activity that functions in the dying cell: the CED-1, CED-7-dependent nuclease in worms and CAD in flies/mammals. In C. elegans, NUC-1, a DNaseII homolog, is required in the dying cell to process DNA intermediates to ensure the eYcient execution of downstream steps. In contrast, DNaseII activity has been demonstrated to function in engulfing cells in flies and mammals to direct the complete degradation of nuclear DNA from apoptotic cells, while in C. elegans, yet-unidentified nucleases accomplish this step. 3. Undegraded DNA Can Trigger an Immune Response Mukae and co-workers examined whether accumulation of undegraded DNA could activate innate immunity in flies (Mukae et al., 2002). dICAD deficiency had little or no impact on the expression of antibacterial and antifungal peptides compared to the expression seen in the wild-type. In

4. Engulfment and Degradation of Apoptotic Cells

135

lo

contrast, the Dnase-1 mutation, which resulted in the accumulation of chromosomal DNA from apoptotic cells, resulted in the constitutive expression of antibacterial peptides diptericin and attacin but had no eVect on the expression of the antifungal peptide drosomycin (Mukae et al., 2002). The expression level of antibacterial peptides is enhanced in double mutant flies deficient for both dICAD and dDNAse II (Mukae et al., 2002). The specific upregulation of antibacterial peptides indicates that chromosomal DNA left undigested during apoptosis leads to the induction of innate immune response. Interestingly, mice deficient for both CAD and DNaseII suVer from a block in T cell development and display a strong upregulation of interferon- (Kawane et al., 2003). Kawane and co-workers (2003) hypothesized that DNA from apoptotic cells that remain undigested can activate innate immunity, leading to defects in thymic development.

V. Concluding Remarks Studies in C. elegans and Drosophila have contributed much to our understanding of how apoptotic cells are recognized, cleared, and degraded. Genetic analyses have made it possible to identify key components of the engulfment and degradation machinery, which include receptor molecules, downstream signaling components, regulators of polarized cell surface extension, and digestive enzymes. Accumulative evidence has shown that multiple emerging signaling pathways regulate the events of engulfment and degradation. Our future goal is to delineate these pathways by identifying and characterizing the functions of missing components. For example, it remains to be confirmed whether PS acts as one of the ‘‘eat me’’ signals to promote the recognition of apoptotic cells by engulfing cells. Whether there are any cell surface molecules that act as potential ‘‘eat me’’ signals other than PS remains unknown. One field of study that will associate with the identity of the ‘‘eat me’’ signal(s) is to identify the mechanisms that lead to the generation, presentation, and recognition of such signal(s) for the initiation of engulfment. The success of engulfment and degradation relies on the proper coordination of multiple cellular and subcellular events, including cytoskeletal reorganization, membrane remodeling, membrane fusion, the initiation of lysosome-phagosome fusion, and much more. A major question to answer is what molecules link the phagocytic receptor(s) to the downstream molecules responsible for each of these events. Current studies in C. elegans have revealed that CED-10, a member of the Rho-family small GTPases, acts to control cytoskeletal reorganization. However, this is only the tip of the iceberg. Identifying additional downstream components of the CED-1 and the CED-10 pathways using molecular genetic methods will lead to better understanding of what takes place inside the engulfing cells.

136

Zhou et al.

Furthermore, in C. elegans, two parallel, partially redundant signaling pathways (represented by ced-7, -1, -6 and ced-2, -5, -10, -12, respectively) act together to control cell corpse engulfment. It is important to know how these two pathways cooperate with each other and at what point they converge. Last but not least, the mechanism that leads to the degradation of apoptotic cells inside engulfing cells has so far been under-studied, and the only known protein acting in this process is DNaseII. However, this is an important process in mammals, as it is associated with antigen presentation, resolution of inflammatory responses, and development. Genetic screens specifically seeking mutants in which apoptotic cells are engulfed but not degraded, in both worms and flies, should help unravel the mechanism. While much still remains to be discovered about how these processes occur, past and current studies of the mechanisms behind the engulfment and degradation of apoptotic cells provide us a basic framework to be filled in. Given that many components of the engulfment machinery appear to be conserved during evolution, these invertebrate systems prove to be invaluable in vivo models and starting points for studying their mammalian counterparts. Although engulfment and subsequent degradation of apoptotic cells have traditionally been considered as downstream events to cell death, there is an emerging consensus that there is some cross-talk among these pathways. Unraveling the intricate relationship that exists among the execution of apoptosis, engulfment, and degradation will provide us, at least in the cellular sense, an understanding of the diVerence between life and death.

Acknowledgments We apologize to many authors whose papers are not cited here due to page limit. We thank Hillel Schwartz, Andreas Bergmann, Xiaohong Leng, and Kavita Oommen for critical reading of this manuscript. Z. Z. is supported by the National Institutes of Health (GM067848), the Cancer Research Institute, and the March of Dimes Birth Defects Foundation.

References Adachi, H., Tsujimoto, M., Arai, H., and Inoue, K. (1997). Expression cloning of a novel scavenger receptor from human endothelial cells. J. Biol. Chem. 272, 31217–31220. Albert, M. L., Kim, J. I., and Birge, R. B. (2000). v 5 integrin recruits the CrkIIDock180-Rac1 complex for phagocytosis of apoptotic cells. Nat. Cell Biol. 2, 899–905. Arur, S., Uche, U. E., Rezaul, K., Fong, M., Scranton, V., Cowan, A. E., Mohler, W., and Han, D. K. (2003). Annexin I is an endogenous ligand that mediates apoptotic cell engulfment. Dev. Cell 4, 587–598.

4. Engulfment and Degradation of Apoptotic Cells

137

Basson, M., and Horvitz, H. R. (1996). The Caenorhabditis elegans gene sem-4 controls neuronal and mesodermal cell development and encodes a zinc finger protein. Genes Dev. 10, 1953–1965. Botto, M., Dell’Agnola, C., Bygrave, A. E., Thompson, E. M., Cook, H. T., Petry, F., Loos, M., Pandolfi, P. P., and Walport, M. J. (1998). Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat. Genet. 19, 56–59. Braga, V. M. (2002). GEF without a Db1 domain? Nat. Cell Biol. 4, E188–190. Brugnera, E., Haney, L., Grimsley, C., Lu, M., Walk, S. F., Tosello-Trampont, A. C., Macara, I. G., Madhani, H., Fink, G. R., and Ravichandran, K. S. (2002). Unconventional Rac-GEF activity is mediated through the Dock180-ELMO complex. Nat. Cell Biol. 4, 574–582. Callebaut, I., Mignotte, V., Souchet, M., and Mornon, J. P. (2003). EMI domains are widespread and reveal the probable orthologs of the Caenorhabditis elegans CED-1 protein. Biochem. Biophys. Res. Commun. 300, 619–623. Campbell, I. D., and Bork, P. (1993). Epidermal growth factor-like modules. Curr. Opin. Struct. Biol. 3, 385–392. Cavaliere, V., Taddei, C., and Gargiulo, G. (1998). Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster. Dev. Genes Evol. 208, 106–112. Chalfie, M., and Wolinsky, E. (1990). The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans. Nature 345, 410–416. Chen, F., Hersh, B. M., Conradt, B., Zhou, Z., Riemer, D., Gruenbaum, Y., and Horvitz, H. R. (2000). Translocation of C. elegans CED-4 to nuclear membranes during programmed cell death. Science 287, 1485–1489. Chung, S., Gumienny, T. L., Hengartner, M. O., and Driscoll, M. (2000). A common set of engulfment genes mediates removal of both apoptotic and necrotic cell corpses in C. elegans. Nat. Cell Biol. 2, 931–937. Conradt, B. (2002). With a little help from your friends: Cells don’t die alone. Nat. Cell Biol. 4, E139–143. Conradt, B., and Horvitz, H. R. (1998). The C. elegans protein EGL-1 is required for programmed cell death and interacts with the Bcl-2-like protein CED-9. Cell 93, 519–529. Conradt, B., and Horvitz, H. R. (1999). The TRA-1A sex determination protein of C. elegans regulates sexually dimorphic cell deaths by repressing the egl-1 cell death activator gene. Cell 98, 317–327. Diez-Roux, G., and Lang, R. A. (1997). Macrophages induce apoptosis in normal cells in vivo. Development 124, 3633–3638. Driscoll, M., and Gerstbrein, B. (2003). Dying for a cause: Invertebrate genetics takes on human neurodegeneration. Nat. Rev. Genet. 4, 181–194. DuYeld, J. S., Erwig, L. P., Wei, X., Liew, F. Y., Rees, A. J., and Savill, J. S. (2000). Activated macrophages direct apoptosis and suppress mitosis of mesangial cells. J. Immunol. 164, 2110–2119. Ellis, H. M., and Horvitz, H. R. (1986). Genetic control of programmed cell death in the nematode C. elegans. Cell 44, 817–829. Ellis, R. E., Jacobson, D. M., and Horvitz, H. R. (1991a). Genes required for the engulfment of cell corpses during programmed cell death in Caenorhabditis elegans. Genetics 129, 79–94. Ellis, R. E., Yuan, J. Y., and Horvitz, H. R. (1991b). Mechanisms and functions of cell death. Annu. Rev. Cell Biol. 7, 663–698. Enari, M., Sakahira, H., Yokoyama, H., Okawa, K., Iwamatsu, A., and Nagata, S. (1998). A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD. Nature 391, 43–50. Enari, M., Talanian, R. V., Wong, W. W., and Nagata, S. (1996). Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Nature 380, 723–726.

138

Zhou et al.

Erickson, M. R., Galletta, B. J., and Abmayr, S. M. (1997). Drosophila myoblast city encodes a conserved protein that is essential for myoblast fusion, dorsal closure, and cytoskeletal organization. J. Cell Biol. 138, 589–603. Fadok, V. A., Bratton, D. L., Rose, D. M., Pearson, A., Ezekewitz, R. A., and Henson, P. M. (2000). A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405, 85–90. Ferguson, E. L., Sternberg, P. W., and Horvitz, H. R. (1987). A genetic pathway for the specification of the vulval cell lineages of Caenorhabditis elegans. Nature 326, 259–267. Franc, N. C., Dimarcq, J. L., Lagueux, M., HoVmann, J., and Ezekowitz, R. A. (1996). Croquemort, a novel Drosophila hemocyte/macrophage receptor that recognizes apoptotic cells. Immunity 4, 431–443. Franc, N. C., Heitzler, P., Ezekowitz, R. A., and White, K. (1999). Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila. Science 284, 1991–1994. Gavrieli, Y., Sherman, Y., and Ben-Sasson, S. A. (1992). Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J. Cell Biol. 119, 493–501. Gitai, Z., Yu, T. W., Lundquist, E. A., Tessier-Lavigne, M., and Bargmann, C. I. (2003). The netrin receptor UNC-40/DCC stimulates axon attraction and outgrowth through enabled and, in parallel, Rac and UNC-115/AbLIM. Neuron 37, 53–65. Golstein, P., Aubry, L., and Levraud, J. P. (2003). Cell-death alternative model organisms: Why and which? Nat. Rev. Mol. Cell Biol. 4, 798–807. Gumienny, T. L., Brugnera, E., Tosello-Trampont, A. C., Kinchen, J. M., Haney, L. B., Nishiwaki, K., Walk, S. F., Nemergut, M. E., Macara, I. G., Francis, R., Schedl, T., Qin, Y., Van Aelst, L., Hengartner, M. O., and Ravichandran, K. S. (2001). CED-12/ELMO, a novel member of the CrkII/Dock180/Rac pathway, is required for phagocytosis and cell migration. Cell 107, 27–41. Gumienny, T. L., Lambie, E., Hartwieg, E., Horvitz, H. R., and Hengartner, M. O. (1999). Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline. Development 126, 1011–1022. Halenbeck, R., MacDonald, H., Roulston, A., Chen, T. T., Conroy, L., and Williams, L. T. (1998). CPAN, a human nuclease regulated by the caspase-sensitive inhibitor DFF45. Curr. Biol. 8, 537–540. Hall, A. (1998). Rho GTPases and the actin cytoskeleton. Science 279, 509–514. Hall, A., and Nobes, C. D. (2000). Rho GTPases: Molecular switches that control the organization and dynamics of the actin cytoskeleton. Philos. Trans. R. Soc. Lond. B Biol. Sci. 355, 965–970. Hall, D. H., Gu, G., Garcia-Anoveros, J., Gong, L., Chalfie, M., and Driscoll, M. (1997). Neuropathology of degenerative cell death in Caenorhabditis elegans. J. Neurosci. 17, 1033–1045. Hamon, Y., Broccardo, C., Chambenoit, O., Luciani, M. F., Toti, F., Chaslin, S., Freyssinet, J. M., Devaux, P. F., McNeish, J., Marguet, D., and Chimini, G. (2000). ABC1 promotes engulfment of apoptotic cells and transbilayer redistribution of phosphatidylserine. Nat. Cell Biol. 2, 399–406. Hanayama, R., Tanaka, M., Miwa, K., Shinohara, A., Iwamatsu, A., and Nagata, S. (2002). Identification of a factor that links apoptotic cells to phagocytes. Nature 417, 182–187. Harosh, I., Binninger, D. M., Harris, P. V., Mezzina, M., and Boyd, J. B. (1991). Mechanism of action of deoxyribonuclease II from human lymphoblasts. Eur. J. Biochem. 202, 479–484. Hedgecock, E. M., Sulston, J. E., and Thomson, J. N. (1983). Mutations aVecting programmed cell deaths in the nematode Caenorhabditis elegans. Science 220, 1277–1279. Henson, P. M., Bratton, D. L., and Fadok, V. A. (2001). Apoptotic cell removal. Curr. Biol. 11, R795–805.

4. Engulfment and Degradation of Apoptotic Cells

139

Hevelone, J., and Hartman, P. S. (1988). An endonuclease from Caenorhabditis elegans: Partial purification and characterization. Biochem. Genet. 26, 447–461. Hoeppner, D. J., Hengartner, M. O., and Schnabel, R. (2001). Engulfment genes cooperate with ced-3 to promote cell death in Caenorhabditis elegans. Nature 412, 202–206. Horvitz, H. R. (2003). Worms, life, and death (Nobel lecture). Chembiochem. 4, 697–711. Hubbard, E. J., and Greenstein, D. (2000). The Caenorhabditis elegans gonad: A test tube for cell and developmental biology. Dev. Dyn. 218, 2–22. Hummel, T., Leifker, K., and Klambt, C. (2000). The Drosophila HEM-2/NAP1 homolog KETTE controls axonal pathfinding and cytoskeletal organization. Genes Dev. 14, 863–873. Jacobson, M. D., Weil, M., and RaV, M. C. (1997). Programmed cell death in animal development. Cell 88, 347–354. Joza, N., Susin, S. A., Daugas, E., Stanford, W. L., Cho, S. K., Li, C. Y., Sasaki, T., Elia, A. J., Cheng, H. Y., Ravagnan, L., et al. (2001). Essential role of the mitochondrial apoptosisinducing factor in programmed cell death. Nature 410, 549–554. Kawane, K., Fukuyama, H., Yoshida, H., Nagase, H., Ohsawa, Y., Uchiyama, Y., Okada, K., Iida, T., and Nagata, S. (2003). Impaired thymic development in mouse embryos deficient in apoptotic DNA degradation. Nat. Immunol. 4, 138–144. Kimble, J., and Hirsh, D. (1979). The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditis elegans. Dev. Biol. 70, 396–417. Kishore, R. S., and Sundaram, M. V. (2002). ced-10 Rac and mig-2 function redundantly and act with unc-73 Trio to control the orientation of vulval cell divisions and migrations in Caenorhabditis elegans. Dev. Biol. 241, 339–348. Kiyokawa, E., Hashimoto, Y., Kobayashi, S., Sugimura, H., Kurata, T., and Matsuda, M. (1998). Activation of Rac1 by a Crk SH3-binding protein, DOCK180. Genes Dev. 12, 3331–3336. Klein, I., Sarkadi, B., and Varadi, A. (1999). An inventory of the human ABC proteins. Biochim. Biophys. Acta 1461, 237–262. Kobayashi, K., Kuroda, S., Fukata, M., Nakamura, T., Nagase, T., Nomura, N., Matsuura, Y., Yoshida-Kubomura, N., Iwamatsu, A., and Kaibuchi, K. (1998). p140Sra-1 (specifically Rac1-associated protein) is a novel specific target for Rac1 small GTPase. J. Biol. Chem. 273, 291–295. Kodama, Y., Rothman, J. H., Sugimoto, A., and Yamamoto, M. (2002). The stem-loop binding protein CDL-1 is required for chromosome condensation, progression of cell death and morphogenesis in Caenorhabditis elegans. Development 129, 187–196. Krieger, M., and Herz, J. (1994). Structures and functions of multiligand lipoprotein receptors: Macrophage scavenger receptors and LDL receptor-related protein (LRP). Annu. Rev. Biochem. 63, 601–637. Kwiatkowska, K., and Sobota, A. (1999). Signaling pathways in phagocytosis. Bioessays 21, 422–431. Lagarkova, M. A., Iarovaia, O. V., and Razin, S. V. (1995). Large-scale fragmentation of mammalian DNA in the course of apoptosis proceeds via excision of chromosomal DNA loops and their oligomers. J. Biol. Chem. 270, 20239–20241. Liu, Q. A., and Hengartner, M. O. (1998). Candidate adaptor protein CED-6 promotes the engulfment of apoptotic cells in C. elegans. Cell 93, 961–972. Liu, Q. A., and Hengartner, M. O. (1999). Human CED-6 encodes a functional homologue of the Caenorhabditis elegans engulfment protein CED-6. Curr. Biol. 9, 1347–1350. Liu, X., Kim, C. N., Yang, J., Jemmerson, R., and Wang, X. (1996). Induction of apoptotic program in cell-free extracts: Requirement for dATP and cytochrome c. Cell 86, 147–157.

140

Zhou et al.

Liu, X., Zou, H., Slaughter, C., and Wang, X. (1997). DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89, 175–184. LoeZer, M., Daugas, E., Susin, S. A., Zamzami, N., Metivier, D., Nieminen, A. L., Brothers, G., Penninger, J. M., and Kroemer, G. (2001). Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. FASEB J. 15, 758–767. Lundquist, E. A., Reddien, P. W., Hartwieg, E., Horvitz, H. R., and Bargmann, C. I. (2001). Three C. elegans Rac proteins and several alternative Rac regulators control axon guidance, cell migration and apoptotic cell phagocytosis. Development 128, 4475–4488. McCall, K., and Steller, H. (1998). Requirement for DCP-1 caspase during Drosophila oogenesis. Science 279, 230–234. McIlroy, D., Tanaka, M., Sakahira, H., Fukuyama, H., Suzuki, M., Yamamura, K., Ohsawa, Y., Uchiyama, Y., and Nagata, S. (2000). An auxiliary mode of apoptotic DNA fragmentation provided by phagocytes. Genes Dev. 14, 549–558. Mergliano, J., and Minden, J. S. (2003). Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos. Development 130, 5779–5789. Metzstein, M. M., Stanfield, G. M., and Horvitz, H. R. (1998). Genetics of programmed cell death in C. elegans: Past, present and future. Trends Genet. 14, 410–416. Mevorach, D., Mascarenhas, J. O., Gershov, D., and Elkon, K. B. (1998). Complementdependent clearance of apoptotic cells by human macrophages. J. Exp. Med. 188, 2313–2320. Mukae, N., Yokoyama, H., Yokokura, T., Sakoyama, Y., and Nagata, S. (2002). Activation of the innate immunity in Drosophila by endogenous chromosomal DNA that escaped apoptotic degradation. Genes Dev. 16, 2662–2671. Mukae, N., Yokoyama, H., Yokokura, T., Sakoyama, Y., Sakahira, H., and Nagata, S. (2000). Identification and developmental expression of inhibitor of caspase-activated DNase (ICAD) in Drosophila melanogaster. J. Biol. Chem. 275, 21402–21408. Nagase, T., Nakayama, M., Nakajima, D., Kikuno, R., and Ohara, O. (2001). Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res. 8, 85–95. Nera, M. S., Vanderbeek, G., Johnson, R. O., Ruben, L. N., and Clothier, R. H. (2000). Phosphatidylserine expression on apoptotic lymphocytes of Xenopus laevis, the South African clawed toad, as a signal for macrophage recognition. Dev. Comp. Immunol. 24, 641–652. Nishiwaki, K. (1999). Mutations aVecting symmetrical migration of distal tip cells in Caenorhabditis elegans. Genetics 152, 985–997. Nolan, K. M., Barrett, K., Lu, Y., Hu, K. Q., Vincent, S., and Settleman, J. (1998). Myoblast city, the Drosophila homolog of DOCK180/CED-5, is required in a Rac signaling pathway utilized for multiple developmental processes. Genes Dev. 12, 3337–3342. Oberhammer, F., Wilson, J. W., Dive, C., Morris, I. D., Hickman, J. A., Wakeling, A. E., Walker, P. R., and Sikorska, M. (1993). Apoptotic death in epithelial cells: Cleavage of DNA to 300 and/or 50 kb fragments prior to or in the absence of internucleosomal fragmentation. EMBO J. 12, 3679–3684. Parrish, J., Li, L., Klotz, K., Ledwich, D., Wang, X., and Xue, D. (2001). Mitochondrial endonuclease G is important for apoptosis in C. elegans. Nature 412, 90–94. Parrish, J. Z., and Xue, D. (2003). Functional genomic analysis of apoptotic DNA degradation in C. elegans. Mol. Cell 11, 987–996. Parrish, J. Z., Yang, C., Shen, B., and Xue, D. (2003). CRN-1, a Caenorhabditis elegans FEN-1 homologue, cooperates with CPS-6/EndoG to promote apoptotic DNA degradation. EMBO J. 22, 3451–3460.

4. Engulfment and Degradation of Apoptotic Cells

141

Pawson, T., and Scott, J. D. (1997). Signaling through scaVold, anchoring, and adaptor proteins. Science 278, 2075–2080. Pearson, A., Lux, A., and Krieger, M. (1995). Expression cloning of dSR-CI, a class C macrophage-specific scavenger receptor from Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 92, 4056–4060. Platt, N., da Silva, R., and Gordon, S. (1998). Recognizing death: The phagocytosis of apoptotic cells. Trends Cell Biol. 8, 365–372. Reddien, P. W., Cameron, S., and Horvitz, H. R. (2001). Phagocytosis promotes programmed cell death in C. elegans. Nature 412, 198–202. Reddien, P. W., and Horvitz, H. R. (2000). CED-2/CrkII and CED-10/Rac control phagocytosis and cell migration in Caenorhabditis elegans. Nat. Cell Biol. 2, 131–136. Rehorn, K. P., Thelen, H., Michelson, A. M., and Reuter, R. (1996). A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila. Development 122, 4023–4031. Ridley, A. J., and Hall, A. (1992). The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70, 389–399. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A. (1992). The small GTP-binding protein Rac regulates growth factor-induced membrane ruZing. Cell 70, 401–410. Robertson, A. M. G., and Thomson, J. N. (1982). Morphology of programmed cell death in the ventral nerve cord of Caenorhabditis elegans larvae. J. Embryol. Exp. Morph. 67, 89–100. Rodrigues, V., Cheah, P. Y., Ray, K., and Chia, W. (1995). malvolio, the Drosophila homologue of mouse NRAMP-1 (Bcg), is expressed in macrophages and in the nervous system and is required for normal taste behaviour. EMBO J. 14, 3007–3020. Sakahira, H., Enari, M., and Nagata, S. (1998). Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391, 96–99. Sakahira, H., Iwamatsu, A., and Nagata, S. (2000). Specific chaperone-like activity of inhibitor of caspase-activated DNase for caspase-activated DNase. J. Biol. Chem. 275, 8091–8096. Savill, J., and Fadok, V. (2000). Corpse clearance defines the meaning of cell death. Nature 407, 784–788. Savill, J. S., Fadok, V., Henson, P., and Haslett, C. (1993). Phagocyte recognition of cells undergoing apoptosis. Immunol. Today 14, 131–136. Savill, J., Hogg, N., Ren, Y., and Haslett, C. (1992). Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J. Clin. Invest. 90, 1513–1522. Schlegel, R. A., and Williamson, P. (2001). Phosphatidylserine, a death knell. Cell Death DiVer. 8, 551–563. Sears, H. C., Kennedy, C. J., and Garrity, P. A. (2003). Macrophage-mediated corpse engulfment is required for normal Drosophila CNS morphogenesis. Development 130, 3557–3565. Shaham, S., and Horvitz, H. R. (1996). Developing Caenorhabditis elegans neurons may contain both cell-death protective and killer activities. Genes Dev. 10, 578–591. Smits, E., Van Criekinge, W., Plaetinck, G., and Bogaert, T. (1999). The human homologue of Caenorhabditis elegans CED-6 specifically promotes phagocytosis of apoptotic cells. Curr. Biol. 9, 1351–1354. Song, Z., and Steller, H. (1999). Death by design: Mechanism and control of apoptosis. Trends Cell Biol. 9, M49–52. Songyang, Z., and Cantley, L. C. (1995). Recognition and specificity in protein tyrosine kinasemediated signalling. Trends Biochem. Sci. 20, 470–475.

142

Zhou et al.

Sonnenfeld, M. J., and Jacobs, J. R. (1995). Macrophages and glia participate in the removal of apoptotic neurons from the Drosophila embryonic nervous system. J. Comp. Neurol. 359, 644–652. Soto, M. C., Qadota, H., Kasuya, K., Inoue, M., Tsuboi, D., Mello, C. C., and Kaibuchi, K. (2002). The GEX-2 and GEX-3 proteins are required for tissue morphogenesis and cell migrations in C. elegans. Genes Dev. 16, 620–632. Staley, K., Blaschke, A., and Chun, J. (1997). Apoptotic DNA fragmentation is detected by a semi-quantitative ligation-mediated PCR of blunt DNA ends. Cell Death DiVer. 4, 66–75. Stanfield, G. M., and Horvitz, H. R. (2000). The ced-8 gene controls the timing of programmed cell deaths in C. elegans. Mol. Cell 5, 423–433. Su, H. P., Brugnera, E., Van Criekinge, W., Smits, E., Hengartner, M., Bogaert, T., and Ravichandran, K. S. (2000). Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells. J. Biol. Chem. 275, 9542–9549. Su, H. P., Nakada-Tsukui, K., Tosello-Trampont, A. C., Li, Y., Bu, G., Henson, P. M., and Ravichandran, K. S. (2002). Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP). J. Biol. Chem. 277, 11772–11779. Sulston, J. E. (1976). Post-embryonic development in the ventral cord of Caenorhabditis elegans. Philos. Trans. R. Soc. Lond. B Biol. Sci. 275, 287–297. Sulston, J. E., Albertson, D. G., and Thomson, J. N. (1980). The Caenorhabitis elegans male: Postembryonic development of nongonadal structures. Dev. Biol. 78, 542–576. Sulston, J. E., and Horvitz, H. R. (1977). Post-embryonic cell lineages of the nematode, Caenorhabditis elegans. Dev. Biol. 56, 110–156. Sulston, J. E., Schierenberg, E., White, J. G., and Thomson, N. (1983). The embryonic cell lineage of the nematode Caenorhabditis elegans. Dev. Biol. 100, 64–119. Susin, S. A., Lorenzo, H. K., Zamzami, N., Marzo, I., Snow, B. E., Brothers, G. M., Mangion, J., Jacotot, E., Costantini, P., LoeZer, M., et al. (1999). Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441–446. Tepass, U., Fessler, L. I., Aziz, A., and Hartenstein, V. (1994). Embryonic origin of hemocytes and their relationship to cell death in Drosophila. Development 120, 1829–1837. Thellmann, M., Hatzold, J., and Conradt, B. (2003). 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 130, 4057–4071. Van Aelst, L., and D’Souza-Schorey, C. (1997). Rho GTPases and signaling networks. Genes Dev. 11, 2295–2322. van den Eijnde, S. M., Boshart, L., Baehrecke, E. H., De Zeeuw, C. I., Reutelingsperger, C. P. M., and Vermeij-Keers, C. (1998). Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved. Apoptosis 3, 9–16. Wang, X., Yang, C., Chai, J., Shi, Y., and Xue, D. (2002). Mechanisms of AIF-mediated apoptotic DNA degradation in Caenorhabditis elegans. Science 298, 1587–1592. White, K., Grether, M. E., Abrams, J. M., Young, L., Farrell, K., and Steller, H. (1994). Genetic control of programmed cell death in Drosophila. Science 264, 677–683. WolV, T., and Ready, D. F. (1991). Cell death in normal and rough eye mutants of Drosophila. Development 113, 825–839. Wu, Y. C., Cheng, T. W., Lee, M. C., and Weng, N. Y. (2002). Distinct Rac activation pathways control Caenorhabditis elegans cell migration and axon outgrowth. Dev. Biol. 250, 145–155. Wu, Y., and Horvitz, H. R. (1998a). The C. elegans cell corpse engulfment gene ced-7 encodes a protein similar to ABC transporters. Cell 93, 951–960.

4. Engulfment and Degradation of Apoptotic Cells

143

Wu, Y., and Horvitz, H. R. (1998b). C. elegans phagocytosis and cell-migration protein CED-5 is similar to human DOCK180. Nature 392, 501–504. Wu, Y. C., Stanfield, G. M., and Horvitz, H. R. (2000). NUC-1, a Caenorhabditis elegans DNase II homolog, functions in an intermediate step of DNA degradation during apoptosis. Genes Dev. 14, 536–548. Wu, Y. C., Tsai, M. C., Cheng, L. C., Chou, C. J., and Weng, N. Y. (2001). C. elegans CED-12 acts in the conserved CrkII/DOCK180/Rac pathway to control cell migration and cell corpse engulfment. Dev. Cell 1, 491–502. Wyllie, A. H. (1980). Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556. Wyllie, A. H., Kerr, J. F., and Currie, A. R. (1980). Cell death: The significance of apoptosis. Int. Rev. Cytol. 68, 251–306. Yokoyama, H., Mukae, N., Sakahira, H., Okawa, K., Iwamatsu, A., and Nagata, S. (2000). A novel activation mechanism of caspase-activated DNase from Drosophila melanogaster. J. Biol. Chem. 275, 12978–12986. Yuan, J. Y., and Horvitz, H. R. (1990). The Caenorhabditis elegans genes ced-3 and ced-4 act cell autonomously to cause programmed cell death. Dev. Biol. 138, 33–41. Zhou, Z., Caron, E., Hartwieg, E., Hall, A., and Horvitz, H. R. (2001a). The C. elegans PH domain protein CED-12 regulates cytoskeletal reorganization via a Rho/Rac GTPase signaling pathway. Dev. Cell 1, 477–489. Zhou, Z., Hartwieg, E., and Horvitz, H. R. (2001b). CED-1 is a transmembrane receptor that mediates cell corpse engulfment in C. elegans. Cell 104, 43–56.