Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans

Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.


Introduction
The elimination of unnecessary or dangerous cells is fundamental to development, tissue homeostasis and disease mitigation in multicellular organisms.The primary mechanism of cell elimination is apoptosis, a form of cell suicide that was originally defined by evolutionarily conserved morphological characteristics that include chromatin condensation, shrinkage of the cytoplasmic volume and membrane blebbing [1] and by biochemical features like phosphatidylserine exposure and DNA fragmentation [2,3].Apoptosis serves as a highly controlled mechanism for the removal and degradation of damaged or unnecessary cells, and blocking apoptosis can lead to catastrophic forms of cell death, such as necrosis, which can cause dangerous inflammatory responses [4].The discovery of the CED-3 caspase as a cell-autonomous executioner of programmed cell death in the nematode Caenorhabditis elegans led to the paradigm that the caspase family of cysteine proteases drives apoptosis through the cleavage of substrate proteins at specific peptide sequences [5,6].Indeed, caspases have evolutionarily conserved roles in apoptosis throughout metazoa [7].
Despite the compelling causal link between caspases and apoptosis, a growing body of evidence indicates that apoptosis can occur in the absence of caspases [4].For example, mouse cells lacking Apaf-1, an activator of the apical caspase Caspase-9, which in turn activates effector caspases, can undergo apoptosis in response to pro-apoptotic stimuli [8].In the presence of caspase inhibitors, TNF (tumor necrosis factor) can induce a form of cell death termed necroptosis, which exhibits characteristics of both necrosis and apoptosis [4,9].The mitochondrial flavoprotein AIF (apoptosis-inducing factor) is thought to promote apoptotic cell death in mammals even in the presence of caspase inhibitors [10].Furthermore, cell death with aspects of apoptotic morphology occurs in non-metazoans, including unicellular eukaryotes and prokaryotes, that lack clear caspase homologs [11,12].Thus, it is possible that apoptosis, as defined morphologically and biochemically, can occur in the absence of caspases.
A standard approach to assaying the caspase-dependence of apoptotic stimuli in tissue cell culture is through the pharmacological inhibition of caspases.However, it is difficult to prove that caspase activity is completely blocked in such experiments, and it is possible for caspase inhibitors to trigger non-apoptotic forms of cell death [13].Studies of caspase-independent apoptosis in metazoans are also complicated by the existence of multiple caspases with potentially redundant functions in promoting cell death.The human genome, for example, encodes at least 10 caspase homologs, seven of which (caspases-2, -3, -6, -7, -8, -9 and -10) have demonstrated roles in apoptosis [14].The genome of Drosophila melanogaster encodes seven caspase homologs (dcp-1, dronc, drice, dredd, decay, damm and strica) [7], several of which are essential for organismal viability.The C. elegans genome encodes three caspase homologs (csp-1, csp-2 and csp-3) in addition to ced-3 [15].Therefore, the use of mutant animals or cell lines deleted for one or two caspases might not eliminate all caspases expressed within a specific cell.Furthermore, since caspases have different substrate specificities [16], the use of a chemical substratecompetitive caspase inhibitor might not completely block all caspase activity.Ideally, experiments that test whether apoptosis can occur in the absence of caspases should be performed using mutant animals or cells that are genetically deleted of all caspase homologs.In this regard, C. elegans is an excellent animal for studies of caspase-independent programmed cell death, because: (1) there are several examples of ced-3-independent programmed cell death in C. elegans [17][18][19]; (2) mutants of ced-3, csp-1, csp-2 and csp-3 are viable [18][19][20][21][22][23]; and, (3) it is relatively easy to generate multiply mutant C. elegans strains.
The ced-3 caspase gene is required for most programmed cell deaths that occur during C. elegans development [5,20].However, a small number of cells die in animals carrying null mutations of ced-3.The male-specific linker cell, which facilitates the connection of the vas deferens to the cloaca and then dies, undergoes a nonapoptotic cell death that bears morphological features (e.g., nuclear membrane crenellation) not seen with other C. elegans programmed cell deaths and that occurs in ced-3 mutants as well as in animals doubly mutant for ced-3 and csp-1, csp-2 or csp-3 [18,20,24].We recently showed that a subset of cells fated to die in the C. elegans embryo are eliminated from ced-3 mutants via a caspase-independent shedding mechanism [19].Interestingly, the shed cells appear apoptotic, exhibiting chromatin condensation, TUNEL-reactive DNA degradation and phosphatidylserine exposure despite the absence of all four caspases.Unlike other apoptotic programmed cell deaths of C. elegans, the shed cells do not undergo phagocytosis by engulfing cells; instead, they are extruded from the developing embryo.By contrast, a small number of apoptotic cell corpses are visible in the heads of ced-3 larvae [17].Like other programmed cell deaths of C. elegans, these ced-3-independent cell corpses have a refractile appearance when viewed with Nomarski optics and are not extruded from the animal, suggesting that a ced-3-independent cell-killing activity contributes to these typical programmed cell deaths.The other caspase homologs, csp-1, csp-2 and csp-3, are obvious candidates for driving this ced-3-independent cell-killing activity.However, it has recently been reported that csp-2 and csp-3 inhibit apoptosis in the germline and soma, respectively [22,23].
To date, the C. elegans caspase homolog csp-1 has no known function in vivo, including in apoptosis.An isoform of CSP-1 can cleave and possibly activate the CED-3 pro-protein in vitro [15].We tested whether csp-1 can promote or inhibit programmed cell death and whether it is regulated by the canonical programmed cell death pathway that activates ced-3.We found that csp-1 encodes a proapoptotic caspase activity that promotes programmed cell death independently of the CED-3 caspase, CED-4 (the Apaf-1 homolog that activates CED-3), and CED-9 (a Bcl-2 family protein that negatively regulates CED-3 activation via inhibition of CED-4).Furthermore, we tested whether csp-1, csp-2 and csp-3 contribute to the ced-3-independent cell-killing activity that generates cell corpses in the heads of ced-3 mutant larvae and found that these apoptotic cell deaths can occur in the complete absence of caspases.Thus, during C. elegans development programmed cell death followed by cell-corpse engulfment is achieved through three redundant pathways: (1) a ced-3-dependent pathway; (2) a csp-1-dependent pathway, which is not regulated by the canonical apoptosis pathway that controls ced-3; and, (3) a caspase-independent pathway.

csp-1 promotes the deaths of a subset of somatic cells fated to die
The C. elegans genes csp-1, csp-2 and csp-3 are paralogs of the proapoptotic ced-3 caspase gene [15], which is required for most programmed cell deaths in the worm [5,20].Given the conserved role of caspases in apoptosis, we tested csp-1, csp-2 and csp-3 for roles -both pro-and anti-apoptotic -in programmed cell death.We used mutations of csp-1 (n4967 and n5133) and csp-2 (n4871) that completely remove the genomic sequences encoding their respective predicted caspase active sites (SACRG in the CSP-1 protein, and VCCRG in the CSP-2 protein) and therefore eliminate any potential caspase activity encoded by these genes (ref.[19]; Figure 1A).csp-3 lacks a caspase active site (ref.[15,22]; Figure 1A); we used the csp-3 deletion mutation n4872, which is likely a null allele [19].
Recently, it was reported that mutations in csp-2 and csp-3 cause ectopic cell deaths in the germline and soma, respectively, and hence that csp-2 and csp-3 inhibit apoptosis [22,23].We therefore tested whether csp-1 mutants have ectopic cell deaths indicative of a loss of anti-apoptotic function.Using Nomarski optics and a

Author Summary
Caspases are cysteine proteases that in many cases drive apoptosis, an evolutionarily conserved and highly stereotyped form of cellular suicide with functions in animal development and tissue maintenance.The dysregulation of apoptosis can contribute to diseases as diverse as cancer, autoimmunity, and neurodegeneration.Caspases are often thought to be required for, or even to define, apoptosis.Although there is evidence that apoptosis can occur in the absence of caspase activity, caspase-independence can be difficult to prove, as most animals have multiple caspases.The nematode Caenorhabditis elegans has four caspases, CED-3, CSP-1, CSP-2, and CSP-3.CED-3 has a well-established role in apoptosis, but less is known about the functions of the CSP caspases.In this study, we show that CSP-1 promotes apoptosis in the developing C. elegans embryo and that CSP-1 is regulated differently than its homolog CED-3.Furthermore, we show that apoptosis and the engulfment of dying cells can occur in mutants lacking all four caspases, proving that neither apoptosis nor cell-corpse engulfment require caspase function and that caspase-independent activities can contribute to apoptosis of some cells during animal development.
P mec-3 ::gfp transgene that expresses GFP in the six touch neurons (AVM, two ALM, PVM and two PLM neurons) in addition to the FLP and PVD neurons, we examined csp-1 mutants for missing cells that normally survive.We observed that csp-1(n4967) mutants contained a full complement of touch neurons and pharyngeal cells (Table S1).We also noted that csp-1(n4967) failed to cause ectopic cell deaths in sensitized animals carrying the loss-offunction mutation n2812 in the anti-apoptotic gene ced-9, a homolog of human BCL2 (Table S1; data not shown).These results indicate that csp-1 does not have an obvious anti-apoptotic function in the soma.Consistent with a previous report that csp-2 does not affect somatic cells [23], a mutation in csp-2 did not cause ectopic cell deaths in the somatic cells we examined (Table S1).However, we failed to observe the ectopic cell deaths in csp-3 mutants previously reported [22].Ectopic somatic cell deaths have also been noted in animals with loss-of-function mutations in ced-9 [25] or tat-1 [26,27], which encodes an aminophospholipid translocase required for the asymmetric distribution of phosphatidylserine on the inner leaflet of the plasma membrane.As expected, we found that ced-9 mutant larvae were missing pharyngeal cells and many touch neurons: more than 80% of PLM neurons were not present in ced-9(n2812) larvae (Table S1).However, we failed to detect the previously reported ectopic celldeath defect of tat-1 mutants (ref.[26,27]; Table S1); we used the same deletion alleles for csp-3 and tat-1 and assayed the same cells that had been characterized in the previous studies.
To determine whether the C. elegans caspase homologs csp-1, csp-2 or csp-3 promote programmed cell death in the soma, we examined animals carrying csp deletion mutations for extra cells that failed to undergo programmed cell death in the anterior pharynx.As many as 16 extra cells can be counted in the anterior pharynges of mutants with strong defects in programmed cell death, e.g., ced-3(n3692) (ref.[28]; Table 1).Single mutations in csp-1, csp-2 or csp-3 failed to cause detectable defects in programmed cell death (Table 1; data not shown).However, we observed that mutations in csp-1 (but not csp-2 or csp-3) caused the survival of pharyngeal cells in sensitized strains carrying a weak mutation in the caspase gene ced-3 (Table 1).The partial loss-of-function ced-3 mutations n2427 and n2436 cause slight and intermediate defects in apoptosis, respectively (ref [17]; Table 1; data not shown).The n4967 and n5133 mutations, both of which delete the putative active site of CSP-1 (Figure 1A), enhanced the celldeath defects of ced-3(n2427) and ced-3(n2436) mutants, increasing the number of extra cells in their anterior pharynges on average by 1.4 and 2.4 cells, respectively (Table 1).These results are consistent with our RNAi experiments in which csp-1B dsRNA (which likely inactivated all csp-1 transcripts) was injected into the gonads of rrf-3(pk1426); ced-3(n2436) animals and caused an enhanced cell-death defect in their progeny (Figure 1C); we used the rrf-3 mutation to increase sensitivity to RNAi [29].The cell-death defect conferred by the csp-1(n4967) mutation was rescued by extrachromosomal arrays carrying a 9 kb genomic csp-1 fragment that included the entire csp-1 coding region, 1.5 kb of genomic sequence 59 of the csp-1A translational start codon and 3.5 kb of genomic sequence 39 of the csp-1A/B translational stop codon (Figure 1B; Table S2).These results demonstrate that csp-1 encodes a detectable cell-killing activity that contributes to programmed cell death in C. elegans.Mutation of csp-2 and/or csp-3 neither enhanced nor suppressed the cell-death defects of strains mutant for csp-1 and/or ced-3 (Table 1; Table S3), suggesting that csp-1 and ced-3 are the only C. elegans caspase genes with functions in somatic programmed cell deaths.
The development of the anterior part of the C. elegans pharynx involves 16 programmed cell deaths, all of which appear to be

org). (B)
Extrachromosomal arrays carrying a wild-type genomic fragment of the csp-1 locus or a mutant version that expresses only the B or C isoforms can rescue the csp-1(n4967) mutant phenotype.The csp-1 PD-only transgene contains two nonsense mutations that encode early stop codons affecting the B and C mRNA isoforms; the csp-1A-only transgene contains a mutation that changes the B/C start codon to an alanine codon; and the csp-1B/Conly transgene contains two nonsense mutations that encode early stop codons affecting the A isoform.The csp-1 transgenes were injected into csp-1(n4967); ced-3(n2436) animals, and the resulting independent lines were assayed for csp-1 rescuing activity by counting the number of extra undead cells in the anterior pharynx.The transgenes and their constructions are described in detail in Materials and Methods, and the complete data for each transgenic line are provided in Table S2.(C) RNAi knockdown of csp-1 phenocopies the csp-1 mutations.dsRNAs targeting the csp-1 pro-domain or the csp-1B isoform were in vitro transcribed and injected into the gonads of RNAi-sensitive rrf-3(pk1426); ced-3(n2436) adult hermaphrodites.Progeny of the injected adults were assayed for extra undead cells in the anterior pharynx.PD, prodomain.doi:10.1371/journal.pgen.1003341.g001sensitive to ced-3 [17,28,30].To test whether specific pharyngeal programmed cell deaths required csp-1, we used GFP reporters to visualize the survival of cells fated to die, specifically the sister cells of the M4 and NSM neurons.csp-1 was partially required in ced-3(n2427) or ced-3(n2436) sensitized strains for the death of the M4 sister cell (Table S4); by contrast, mutation of csp-1 did not affect the cell deaths of the sister cells of the NSM neurons (data not shown).Likewise, csp-1 did not appear to function in the postembryonic programmed cell deaths of the ventral cord or postdeirid sensilla (Table S4).We conclude that csp-1 promotes cell death in a subset of cells fated to die during C. elegans development.
The csp-1B and/or C isoforms are required for the cellkilling activity of csp-1 The csp-1 locus produces three known mRNA isoforms [15], all of which include the sequence that encodes the presumptive caspase active site (Figure 1A).The csp-1A transcript contains a long prodomain not present in the other transcripts, and it uses an alternative start site that is 3 kb 59 to the start site of the csp-1B and csp-1C isoforms.To determine which isoforms are required for the cell-killing activity of csp-1, we peformed experiments in which the csp-1 rescuing transgene was mutated to express: (1) the A isoform only, (2) the B and C isoforms only, or (3) a truncated version of csp-1A including only the prodomain (PD).Extrachromosomal arrays engineered to express only csp-1-PD or the csp-1A isoform failed to rescue the cell-death defect of csp-1(n4967) mutants (Figure 1B; Table S2).By contrast, a csp-1 transgene lacking the csp-1A translation start codon and predicted to express only the csp-1B and csp-1C transcripts rescued the csp-1(n4967) defect in programmed cell death (Figure 1B; Table S2).Consistent with these results, transgenes expressing the csp-1B cDNA, but not the csp-1A cDNA, under the control of the mec-7 promoter efficiently killed touch neurons (Figure 2A-2B; Table 2; data not shown); we also expresed the csp-1C cDNA under the control of the mec-7 promoter and failed to observe killing of the touch neurons (data Includes animals that were either homozygous for unc-75(+) or unc-75(e950). 3 Homozygous for dpy-20(e1282ts).
For the statistical comparisons between ced-3(n2427) or ced-3(n2436) and double mutants with each csp allele, p values were considered significant if less than 0.01 to correct for multiple comparisons.doi:10.1371/journal.pgen.1003341.t001not shown).Ectopic expression of csp-1B from the ser-2d and flp-15 promoters killed the OLL and I2 neurons, respectively (ref.[31]; N. Bhatla and H.R. Horvitz, unpublished results).However, we noted that tm917, a csp-1 allele that deletes coding regions of only the csp-1A transcript, enhanced significantly (albeit weakly) the celldeath defects of ced-3(n2427) and ced-3(n2436) mutants, increasing the number of extra cells in their anterior pharynges by 0.9 and 1.2 cells, respectively (Table 1).dsRNA targetting the csp-1A prodomain (csp-1-PD) caused a similar slight enhancement of the cell-death defect of ced-3(n2436) mutants (Figure 1C), suggesting that, in addition to the more robust cell-killing activity of the csp-1B transcript, csp-1A might have a weak cell-killing function.

csp-1B encodes a pro-apoptotic caspase
The proteolytic activity of caspases requires an active-site cysteine.Previously, it was shown that the CSP-1B protein can proteolytically process CED-3 in vitro and that this enzymatic activity required the active-site (SACRG) cysteine of CSP-1B, C138 [15].We tested in vivo whether C138 was necessary by assaying the touch neuron-killing activity of mutant P mec-7 ::csp-1B transgenes in which C138 was changed to a serine.We observed that the ectopic cell deaths were entirely dependent on the caspase active site (Table 2).Thus, csp-1B promotes cell death via caspase activity.The cell deaths induced by a P mec-7 ::csp-1B transgene resulted in cell corpses with apoptotic characteristics (Figure 2C-2D).When observed with Nomarski optics, the csp-1B-induced cell deaths exhibited a refractile button-like appearance (Figure 2C) similar to that of developmental programmed cell deaths.Transmission electron micrographs of the cell corpses showed some contraction of the cytoplasmic volume and considerable condensation of the nuclear chromatin (Figure 2D), which are general characteristics of apoptotic cells, including those generated by ced-3 cell-killing transgenes (ref.[32]; data not shown).We conclude that csp-1B encodes a functional caspase that promotes programmed cell deaths with apoptotic morphology.csp-1B cell-killing activity is independent of ced-9 and ced-4 CED-3, like most caspases, is expressed as an inactive zymogen with an inhibitory N-terminal prodomain.Trans-auto-proteolysis  of the CED-3 pro-protein at two aspartate residues removes the pro-domain and yields two subunits that form the active caspase [33].CED-3 auto-activation is dependent on its prodomain and is facilitated by the association of two CED-3 pro-proteins within an octameric complex formed with the Apaf-1 homolog CED-4 [34][35][36].Under normal cellular conditions, CED-4 is sequestered by CED-9 at mitochondria through a direct protein-protein interaction [37][38][39].In response to upstream pro-apoptotic signals and the consequent expression of the BH3-domain-only protein EGL-1, which binds to and inhibits CED-9 [40], CED-4 is released from CED-9 and translocates to the nuclear periphery [37,41], where it facilitates CED-3 activation [38].Thus, the activation of CED-3 is controlled by an apoptosis pathway involving a BH3domain-only protein, a member of the Bcl-2 family of apoptosis regulators, and a homolog of the apoptosome complex protein Apaf-1.The basic elements of this apoptosis pathway are evolutionarily conserved in mammals and are responsible for the activation of caspases in response to cell-intrinsic apoptotic stimuli [7].
We also observed that the expression of a P mec-7 ::csp-1A transgene in ced-3(null) mutant strains failed to cause PLM cell death, even in a ced-9(null) background (Figure S1).These results suggest that the CSP-1A isoform (which contains a long prodomain similar to that of CED-3) does not promote programmed cell death, even in the absence of the anti-apoptotic protein CED-9.A role for csp-1A in cell death cannot be excluded entirely, as it is possible that endogenous CSP-1A requires a cofactor not present in the touch neurons to mediate cell killing.

csp-1 expression in the maternal germline contributes to embryonic programmed cell death
To determine which C. elegans cells express csp-1, we directly visualized endogenous csp-1 transcripts via fluorescence in situ hybridization (FISH) experiments using Cy5-and ALEXAlabelled probes complementary to the csp-1B transcript (i.e., targeted to all csp-1 transcripts) or to the csp-1A prodomain (specific to the csp-1A trancript).To our surprise, csp-1 mRNA was not detectable in the somatic cells of wild-type or egl-1(n1084 n3082) mutant embryos, larvae or adult hermaphrodites (data not shown).By contrast, csp-1 transcripts were present in the germlines of L4stage larval and adult hermaphrodites (Figure 4A-4B).This expression was restricted to the late pachytene stage of meiosis I in both L4 larval gonads (in pachytene nuclei adjacent to differentiating sperm) and adult gonads (in pachytene nuclei adjacent to the bend of the gonad arm) (Figure 4A-4B).Both csp-1A and csp-1B/C transcripts were expressed in the adult pachytene germ cells, as indicated by the presence of FISH foci recognized by the csp-1A prodomain probes and foci recognized primarily by the csp-1B probes and only weakly by the csp-1A probes (Figure 4C).Stochastic and ionizing radiation (IR)-induced germline cell deaths occur during the late pachytene stage of oocyte development in adult gonads [43,44].However, csp-1 (unlike ced-3) was not required for either stochastic or IR-induced germline apoptosis, even in ced-3(n2436) strains sensitized for defects in germ-cell death (Figure 4D).In these experiments, apoptotic germ cells were identified using a transgene that expresses a functional GFP::CED-1 fusion protein that envelopes dying cells engulfed by the gonadal sheath [45,46].We also failed to detect differences in either stochastic or IR-induced germline cell death between csp-1 mutants and wild-type animals in experiments in which apoptotic germ cells were quantified by acridine orange staining or by direct observation of their refractile morphology using Nomarski optics (data not shown).We also noted that the level of csp-1 transcript expression in the germline (as determined by FISH) was not affected by either ionizing radiation or by mutation of egl-1 or ced-3 (data not shown).
Since we detected csp-1 expression in the adult germline but not in somatic cells of the embryo, we tested whether maternally supplied csp-1 transcript was necessary for the zygotic function of csp-1 in programmed cell death.Indeed, in sensitized genetic backgrounds (ced-3(n2427) and ced-3(n2436)), csp-1(+) progeny of csp-1(n4967) hermaphrodites (M2Z+ animals) had more undead pharyngeal cells than the csp-1(+) progeny of csp-1(+) hermaphrodites (M+Z+ animals) or the csp-1(n4967) progeny of csp-1(+) hermaphrodites (M+Z2 animals) (Table 3).Thus, csp-1 expressed in the maternal germline is necessary for the csp-1 pro-apoptotic activity in embryonic programmed cell deaths.Given that we could not detect csp-1 expression in either embryos or larvae, it is therefore not surprising that the postembryonic programmed cell deaths of the ventral cord and postdeirid sensilla were unaffected by mutation of csp-1 (Table S4).

Programmed cell deaths occur in animals completely lacking all caspase genes
Most programmed cell deaths in C. elegans require ced-3 [20].However, some cells die in mutants completely lacking ced-3.We previously reported that a subset of cells fated to die can be eliminated from ced-3 mutant embryos via a cell-shedding mechansm [19].In that study, we noted that cell shedding from ced-3 mutants occurs independently of csp-1, csp-2 and csp-3: quadruple mutants lacking all four caspases also generate shed cells, indicating that cell elimination by this mechanism is completely caspase-independent [19].Like most programmed cell deaths, the cells generated by caspase-independent extrusion are apoptotic in appearance.However, unlike caspase-dependent cell corpses, shed cells do not undergo phagocytosis by engulfing cells.
The death of the male linker cell, which also occurs independently of ced-3, is non-apoptotic and requires the heterochronic protein LIN-29, its binding partner MAB-10 [47], and the polyglutamine repeat protein PQN-41 (ref.[18,24]; Table S5).Previously it was shown that this cell death occurs in double-mutant males in which ced-3 and an additional csp gene (csp-1, csp-2 or csp-3) were inactivated [18].We have now examined males lacking all four caspases and observed that the linker cell died in 100% of csp-3;  S5).The csp-3; csp-1; csp-2 ced-3 quadruple mutants were viable and fertile.Thus, both zygotic and maternal caspase contributions were eliminated.Our results therefore confirm that this cell death is indeed completely caspase-independent.
In addition, cell corpses are visible in the heads of larvae carrying null alleles of ced-3 (ref.[17]; Table 4).All programmed cell deaths in the developing heads of wild-type animals occur embryonically and are engulfed and degraded prior to hatching (ref.[30,48]; Table 4).To detect ced-3-independent programmed cell deaths in larval heads, we used mutations (e.g., ced-1(e1735), ced-6(n2095) or ced-7(n1996)) that cause defects in cell-corpse engulfment and result in the persistence of many embryonic cell corpses into larval stages (ref.[49,50]; Table 4).Like most wild-type cell corpses, the ced-3independent cell corpses were refractile in appearance as observed with Nomarski optics and were not extruded from the animal (data not shown).We also observed that larvae mutant for ced-4 or egl-1 contained similar cell corpses, demonstrating that their generation does not require the canonical pro-apoptotic pathway that mediates most programmed cell deaths (Table 4).
We tested whether the small number of cell corpses visible in ced-3 larval heads are generated by the other C. elegans caspase genes and found that all double, triple and quadruple caspase mutants that we examined contained a small number of refractile corpses (Table 4).For example, 39% of csp-3; csp-1; ced-6; csp-2 ced-3 mutant animals contained at least one refractile cell corpse (Table 4), indicating that these programmed cell deaths occur in animals lacking all C. elegans caspases.We observed caspase-independent cell corpses in different regions of the larval head, including positions internal and external to the pharynx, which suggests that multiple cell lineages -at low frequencies -generated caspase-independent cell corpses.Surprisingly, we discovered that engulfment-competent ced-3 and csp-3; csp-1; csp-2 ced-3 mutants also contained refractile cell corpses (Table 4).The number of cell corpses per ced-3 or csp-1; csp-2 ced-3 larva increased until 12 to 24 hours post hatching (see below; data not shown), indicating that at least some of the cell deaths occurred after embryogenesis.Given that all programmed cell deaths in the head normally occur embryonically and that cell corpses are never observed in the heads of wild-type larvae, we concluded that timing of cell deaths in these ced-3 mutants was delayed.Thus, caspase-independent cell corpses can undergo an inefficient programmed cell death with slow kinetics in the absence of CED-3 activity, indicating that these cells likely die via CED-3-mediated apoptosis in wild-type animals.

Caspase-independent cell corpses exhibit apoptotic morphology
Despite the strong causal link between caspase activation and apoptosis, recent studies have demonstrated that many morphological and biochemical changes associated with apoptosis can occur in the absence of caspases [4,19,21].For example, in C. elegans the shed cells of csp-3; csp-1; csp-2 ced-3 quadruple mutants exhibit phosphatidylserine exposure, expression of the pro- apoptotic BH3-only gene egl-1, and chromatin condensation [19].To determine whether these apoptotic attributes are evident in caspase-independent programmed cell deaths that do not involve extrusion of the dying cell from the embryo, we characterized the cell corpses visible in caspase-deleted larvae (Figure 5 and Figure 6).In most of these experiments, we used strains with the wild-type csp-3 allele, because (1) csp-3 lacks a caspase active-site [15]; (2) although previous studies reported that csp-3 has an anti-apoptotic function in somatic cells [22], we were unable to replicate those findings (Table S1); and, (3) the presence or absence of a csp-3 mutation had no effect on the frequency or appearance of caspase-independent corpses (Table 4; Figure 6B; data not shown).Like ced-3-mediated programmed cell deaths in wild-type animals, the caspaseindependent corpses expressed egl-1, the upstream activator of the canonical apoptosis pathway (Figure 5A).Also, these cell corpses displayed phosphatidylserine on their cell surfaces, as indicated by the phosphatidylserine-binding reporter MFG-e8::Venus (Figure 5B), and exhibited many of the morphological hallmarks of apoptosis, including contraction of cytoplasmic volume and, in some but not all cases, condensation of nuclear chromatin (Figure 5C).Additionally, we noted that the caspase-independent cell corpses frequently stained with acridine orange (Figure 6A), suggesting that these corpses are engulfed, internalized and degraded via endosomal pathways, as are canonical programmed cell deaths [30,48,49,51].Indeed, we found that the caspase-independent corpses were recognized by CED-1 (Figure 6B), a receptor expressed on engulfing cells required for the efficient phagocytosis of cell corpses [46,49,50].The recognition of caspase-independent cell corpses by CED-1 appeared to be functionally important, as ced-1; csp-1; csp-2 ced-3 larvae contained more corpses than csp-1; csp-2 ced-3 larvae (Figure 6C).Given that ced-1 and other genes that function in cell-corpse engulfment promote programmed cell death [52,53], it is unlikely that the ced-1(e1735) loss-of-function mutation caused additional cell deaths in the caspase-deleted mutants.Instead, the extra cell corpses in ced-1 mutant larvae likely reflected an engulfment defect, consistent with the comparatively rapid degradation and disappearance of most caspase-independent corpses in ced-1(+) larvae within the 36-hour period after hatching (Figure 6C).We conclude that caspases are not required for programmed cell deaths to be recognized by the engulfment machinery, internalized and degraded.In short, many aspects of apoptosis, including phagocytosis -the ultimate fate of apoptotic cells -can occur without caspases.We conclude that a parallel, caspase-independent pathway contributes to programmed cell death in C. elegans and can execute most cellular changes associated with apoptosis.

Discussion
Our experiments revealed unexpected complexities in the execution of apoptosis in C. elegans.While the CED-3 caspase is clearly the primary effector of programmed cell death, we demonstrated the existence of additional caspase-dependent and caspase-independent contributions to developmental apoptosis.Specifically, we observed that maternally-expressed caspase gene csp-1 (but not csp-2 or csp-3) promotes the deaths of a subset of cells programmed to die during C. elegans embryogenesis (Figure 1 and Figure 4; Table 1 and Table 3).Furthermore, ectopic expression of the csp-1B isoform of csp-1 is sufficient to cell-autonomously kill cells that normally survive.These ectopic apoptotic cell deaths require the active site cysteine (C138) of CSP-1B, indicating that a caspase-like proteolytic function is responsible for its cell-killing activity (Table 2).The C. elegans genome therefore expresses at least two pro-apoptotic caspases, CED-3 and CSP-1B, to mediate programmed cell deaths.Nevertheless, the additional caspase activity conferred by csp-1 cannot account for ced-3-independent programmed cell deaths that have been observed in C. elegans.For example, the non-apoptotic death of the male linker cell and the extrusion of shed cells were already known to be caspaseindependent [18,19].Here we demonstrate that cells in caspasedeleted animals can undergo an apoptosis-like programmed cell death followed by engulfment, indicating that the complete apoptotic program can occur in the absence of caspases.Thus, in addition to CED-3 and CSP-1B, there are caspase-independent cell-killing activities that contribute to programmed cell deaths.

CSP-1B is regulated by a mechanism distinct from that of CED-3
The caspases CED-3 and CSP-1B appear to be regulated differently.The auto-activation of CED-3 is facilitated by the Apaf-1 homolog CED-4 in a protein-protein interaction that requires the CED-3 prodomain [34][35][36].In the absence of a proapoptotic signal, CED-9 sequesters CED-4 [37], thereby preventing its association with the inactive CED-3 proprotein.The CSP-1B proprotein lacks a long prodomain, suggesting that it is not activated through an association with the CED-4 octamer in cells undergoing apoptosis.Consistent with this expectation, we observed that the cell-killing activity of csp-1B transgenes, unlike that of ced-3 transgenes, was not negatively regulated by ced-9 (Figure 3).Furthermore, based on our genetic experiments (Figure 3) and the in vitro studies of Shaham [15], it does not appear that CSP-1B is activated by CED-3.We therefore propose that CSP-1B is regulated by a mechanism different from the canonical programmed cell death pathway that activates CED-3 and that CSP-1B likely promotes cell killing in parallel to CED-3 (Figure 3E).
There are no known or candidate regulators of csp-1.It is possible that csp-1 is controlled entirely at the transcriptional level and that csp-1 contributes a minor, sub-lethal pro-apoptotic activity to all cells within the C. elegans embryo.Indeed, only using sensitized backgrounds with partial defects in programmed cell death did we detect the pro-apoptotic function of csp-1.Nevertheless, we expect that it will be possible to identify regulators and effectors of csp-1 through genetic screens for mutants that modify the cell-killing activity of csp-1B transgenes.

Do the csp genes have non-apoptotic functions?
Given the minor contribution of csp-1 to programmed cell death and the lack of a detectable role of csp-2 or csp-3 in apoptosis (Table 1; Table S1; data not shown), it is tempting to speculate that the csp genes have non-apoptotic functions in C. elegans.In C. elegans, ced-3 functions in axon regeneration following laser axotomy [54].In mammalian and Drosophila neurons, caspases have functions in dendritic pruning, axon guidance and the synaptic changes underlying long-term depression [14].Caspase function is also required for the maturation of Drosophila sperm [55].Interestingly, we observed robust expression of csp-1 in the germlines of L4 and adult hermaphrodites, specifically in the late pachytene nuclei (Figure 4).We also observed temporally and spatially restricted csp-2 and csp-3 mRNA expression in the late pachytene nuclei of the L4 larval germline (data not shown), suggesting that the csp genes might have functions in germ cell development.However, mutant hermaphrodites and males carrying all tested combinations of csp-1, csp-2 and csp-3, including the triple csp mutant were viable, fertile and failed to exhibit obvious brood-size defects that would suggest abnormalities in sperm or oocyte differentiation (data not shown).

csp-1B as a tool for the genetic ablation of cells
Genetically encoded cell-killing activities provide an efficient and convenient method for determining cellular function through cell ablation.Killer genes such as ced-3 have been used under the control of various promoters to ablate specific cells [32,45,56,57].However, the potent cell-killing activity of ced-3 transgenes can cause organismic inviability, particularly if the promoter expression is not exclusive to a small number of cells (see below).csp-1B overexpression using the mec-7 and flp-15 promoters efficiently killed the touch and I2 neurons, respectively (Figure 2; Table 2; N. Bhatla and H.R. Horvitz, personal communication).The mec-7 and flp-15 promoters are relatively strong, as they also robustly induced gfp expression in these cells, such that the neural processes were visible with a dissecting microscope equipped with fluorescence optics.By contrast, the odr-1 promoter did not produce detectable GFP expression in the neurites of the AWB, AWC and I1 neurons, and csp-1B under the control of the odr-1 promoter failed to kill these cells even when injected at plasmid concentrations as high as 100 ng/ml (N.Bhatla and H.R. Horvitz, unpublished results).Thus, high levels of csp-1B expression might be required to kill most cells, making the use of csp-1B as a cellablation tool appropriate in situations in which the promoter sequence strongly drives expression in targeted cells and/or weakly promotes expression in additional cells not intended to be targets.For example, the P mec-7 ::csp-1B constructs, which were injected at a concentration of 15 ng/ml, produced csp-1B expression outside of the touch neurons that was detectable by fluorescence in situ hybridization.However, this level of csp-1B expression was sublethal and did not induce cell death or other cellular defects outside of the touch neurons (data not shown).By contrast, P mec- 7 ::ced-3 constructs were toxic to the animals when injected at concentrations above 1 ng/ml, suggesting that cells are very sensitive to ectopic ced-3 and that using ced-3 as a cell ablation tool is potentially problematic when promoter expression is not restricted to a small number of targeted cells.
What is the role of ced-3-independent cell-killing activities that have minor contributions to programmed cell death?
Although the csp-1 gene contributes a cell-killing activity to normal programmed cell deaths (Table 1), csp-1 and the other csp genes are not responsible for the ced-3-independent programmed cell deaths present in the heads of ced-3 larvae (Table 4).These deaths, like those of the male linker cell (ref.[18]; Table S5) and the embryonic shed cells [19], are caspase-independent -a surprising result in light of our observations that these cell corpses are morphologically apoptotic (Figure 5) and are engulfed (albeit with slower kinetics) like normal programmed cell deaths (Figure 6).Thus, the complete apoptotic program including cellcorpse internalization can occur in the absence of caspases in C. elegans, suggesting that the cellular changes accompanying apoptosis do not require proteolysis by the caspase family of proteases.Moreover, it is clear that apoptotic programmed cell deaths are achieved through the integration of independent cellkilling activities from CED-3, CSP-1B and an unknown caspaseindependent source.
Given the minor cell-killing effects of the CSP-1B and the caspase-independent pathways, why might cell-killing activities in addition to that of CED-3 have evolved?It is possible that different cells, even within the set of C. elegans cells fated to die, are differentially sensitive to pro-apoptotic signals and that additional caspase and caspase-independent pathways ensure efficient and complete cell death under diverse environmental and developmental conditions.Interestingly, the postembryonic programmed cell deaths of the ventral cord are more sensitive to weak ced-3 mutations than are the embryonic programmed cell deaths in the presumptive anterior pharynx: ced-3 mutations that have weak effects in the anterior pharnyx typically have stronger effects in the ventral cord (ref.[17]; data not shown).We observed a complementary function for csp-1, which promotes apoptosis in the anterior pharynx (Table 1) but not in the ventral cord (Table S4).
In summary, multiple pro-apoptotic caspases function in programmed cell death in C. elegans, Drosophila and vertebrates.Furthermore, as we and others have shown, there are additional caspase-independent contributions to programmed cell deaths in C. elegans.We identified C. elegans caspase-independent cell deaths that are essentially identical to wild-type programmed cell deaths based on their apoptotic appearance and their recognition and internalization by engulfing cells.We expect that caspaseindependent pro-apoptotic activities are present in other metazoans and that their identification will be of major importance to our understanding of cell death in the contexts of development and disease.

Strains
amplicon was digested with BglII and XhoI and then ligated into pL4440.The RNAi plasmid pL4440::csp-1B (pDD061) was constructed using PCR to amplify the csp-1B cDNA with the primers 59gcgagatctatgccgagaacggacgccaag and 59-cgcctcgagttacatcgaccttgaaaagtgcc, which incorporate the restriction sites BglII and XhoI, respectively.The resulting csp-1B amplicon was digested with BglII and XhoI and then ligated into pL4440.

Fluorescence in situ hybridization
The fixation of embryos and larval and adult animals, the conjugation of Cy5 or ALEXA594 fluorescent probes to in situ oligo probes, and the hybridization of oligos to fixed samples were performed as described previously [62].All images were acquired using an inverted Nikon TE-2000 compound microscope equipped for fluorescence microscopy (Prior Scientific).Images were acquired with a PIXIS camera (Princeton Instruments) controlled by MetaMorph software (Molecular Devices) and modified for publication with ImageJ software (NIH).The ''total csp-1'' set of probes included 32 distinct 20-nucleotide sequences complementary to csp-1B (Biosearch Technologies, Inc).This set of oligos was conjugated to the fluorophore Cy5 (GE Healthcare) and hybridized to all three csp-1 mRNA isoforms (csp-1A, csp-1B and csp-1C).The ''csp-1A'' set of probes included 32 distinct 20nucleotide sequences complementary to the region of csp-1A that encodes the prodomain.This set of oligos was conjugated to the fluorophore ALEXA594 (Invitrogen) and hybridized specifically to the csp-1A mRNA isoform.Probe sequences are listed in Table S6.

Cell-death assays and microscopy
The numbers of undead cells that failed to undergo programmed cell death in the anterior pharynges and postdeirid sensilla of L3 larvae were determined by direct observation using Nomarski optics as described previously [28].Persistent cell corpses in larval heads also were quantified by direct observation using Nomarski optics; for this assay, larvae were staged by the time of hatching.For other cell-death assays, the ventral cord cells of young adults, the M4 neuron and its undead sister cell of L3 larvae, the touch neurons of L4 larvae, and the germ cell corpses of adult hermaphrodite gonads were identified using previously described GFP reporter transgenes [45,52,59].For experiments involving ionizing radiation, L4 larvae were exposed to gamma irradiation from a Co-60 source.All strains were analyzed using a Zeiss Axioskop II compound microscope equipped for fluorescence microscopy.Images were acquired with an ORCA camera (Hammamatsu) controlled by OpenLab software (Perkin Elmer) and modified for publication using ImageJ (NIH).

Transmission electron microscopy
L1-stage larvae were fixed, stained and sectioned for transmission electron microscopy as described previously [43].Stained sections were imaged with a JEM-1200EX II microscope (JEOL) using an AMT XR41 CCD camera.

Supporting Information
Figure S1 Transgenes that ectopically express csp-1A in the touch neurons lack cell-killing activity in both the presence and absence of the apoptosis regulator CED-9.The percentages of PLM cells that survive in strains carrying P mec-7 ::csp-1A transgenes.All strains contained the ced-3(n3692) mutation, which suppresses ced-9(n2812) inviability.n.s., p.0.05 in a Fisher's exact test.(PDF) Table S1 The deletion of csp-1, csp-2 or csp-3 does not cause the deaths of cells that normally survive.(A).The touch neurons survive in csp mutants.The survival of AVM, ALML/R, PVM and PLML/R was scored using the transcriptional reporters P mec-3 ::gfp (nIs290) a or P mec-4 ::gfp (bzIs8) b .n, animals scored.(B).Mutants carrying csp deletions have the same number of pharyngeal cells as wild-type animals.The following pharyngeal cells were scored: the neurons I1, I2, I3, MC, MI, M3, M4 and NSM; the epithelial cells e1, e2, and e3; and, the muscle cells m1 and m2.In total, 34 cells were scored per pharynx.n, animals scored; SD, standard deviation.
(DOC) Table S2 The defect in programmed cell death of csp-1(n4967) animals is rescued by transgenes that contain the endogenous csp-1 promoter and coding regions.Mutations that alter the start of the B and C splicing isoforms of csp-1 disrupt the rescuing activity of the csp-1 transgene.The transgenes are described in detail in the legend of Figure 1 and in Materials and Methods.A Student's ttest was used to compare the csp-1(n4967); ced-3(n2436) strains with csp-1 transgenes to the csp-1(n4967); ced-3(n2436) parental strain.p values were considered significant if less than 0.01 to correct for multiple comparisons.(DOC) Table S3 The deletion of csp-2 or csp-3 does not modify the defects in programmed cell death of csp-1 and ced-3 mutants.The average number of extra, undead cells in the pharynx was determined for each genotype.n, number of animals scored; SD, standard deviation.For the statistical comparisons between ced-3(n2427) or ced-3(n2436) and double mutants with each csp allele, p values were considered significant if less than 0.02 to correct for multiple comparisons.(DOC) Table S4 csp-1 promotes the programmed cell death of (A) the M4 sister cell but not those of (B) the VC-like cells in the ventral cord or of (C) the V5.praap cell in the postdeirid sensillum.The survival of the M4 sister cell was scored using the integrated transgene nIs177[P ceh-28 ::gfp].The number of extra VC-like cells was determined using the integrated transgene nIs106[P lin-11 ::gfp].The survival of V5Rpaapp was determined via direct observation using Nomarski optics.(DOC) Table S5 The male linker cell dies in animals lacking all four caspases.(DOC) Table S6 Sequences of DNA probes used for fluoresence in situ hybridization (FISH) experiments.The csp-1A oligos hybridize to the region of csp-1A that encodes the prodomain and are therefore specific to the csp-1A isoform.The ''total'' csp-1 oligos hybridize to a region present in all known csp-1 mRNA isoforms.(DOCX)

Figure 1 .
Figure 1.The B and/or C isoforms of csp-1 promote programmed cell death.(A) Representations of the intron-exon organization of the three known csp-1 mRNA isoforms (A, B and C).Red bars indicate the csp-1 deletion alleles used in this study; arrowheads indicate the SACRG sequence that encodes the caspase active-site.The graphic was generated using the Intron-Exon Graphic Maker (N.Bhatla; www.wormweb.org).(B) Extrachromosomal arrays carrying a wild-type genomic fragment of the csp-1 locus or a mutant version that expresses only the B or C isoforms can rescue the csp-1(n4967) mutant phenotype.The csp-1 PD-only transgene contains two nonsense mutations that encode early stop codons affecting the B and C mRNA isoforms; the csp-1A-only transgene contains a mutation that changes the B/C start codon to an alanine codon; and the csp-1B/Conly transgene contains two nonsense mutations that encode early stop codons affecting the A isoform.The csp-1 transgenes were injected into csp-1(n4967); ced-3(n2436) animals, and the resulting independent lines were assayed for csp-1 rescuing activity by counting the number of extra undead cells in the anterior pharynx.The transgenes and their constructions are described in detail in Materials and Methods, and the complete data for each transgenic line are provided in Table S2.(C) RNAi knockdown of csp-1 phenocopies the csp-1 mutations.dsRNAs targeting the csp-1 pro-domain or the csp-1B isoform were in vitro transcribed and injected into the gonads of RNAi-sensitive rrf-3(pk1426); ced-3(n2436) adult hermaphrodites.Progeny of the injected adults were assayed for extra undead cells in the anterior pharynx.PD, prodomain.doi:10.1371/journal.pgen.1003341.g001

Figure 4 .
Figure 4. csp-1 is expressed in late pachytene cells of the L4 and adult hermaphrodite germline.Fluorescence in situ hybridization images of gonad arms of (A) an L4 hermaphrodite and (B) an adult hermaphrodite hybridized with Cy5-labelled probes complementary to csp-1B.The Cy5-labelled probes are visible as green puncta; the gonads are outlined in white.A schematic representation is shown above each micrograph.(C) Fluorescence in situ hybridization images of an adult hermaphrodite gonad hybridized with ALEXA594-labelled probes (red puncta) complementary to the region of csp-1A that encodes the prodomain (csp-1A) and Cy5-labelled csp-1B probes (green puncta) that hybridize to all csp-1 transcript isoforms (total csp-1).White arrowheads indicate csp-1A-specific puncta; orange arrows indicate csp-1B-specific puncta, which are recognized strongly by the total csp-1 probes but only weakly by the csp-1A-specific probes.(D) The number of CED-1::GFP-positive apoptotic cells in the gonads of caspase mutants exposed to 0 Gy and 120 Gy of ionizing radiation at the L4 larval stage.The strains were scored at 24 hrs and 48 hrs post L4-stage.Error bars indicate standard deviations.doi:10.1371/journal.pgen.1003341.g004

Table 1 .
The caspase homolog csp-1 promotes programmed cell death in the C. elegans anterior pharynx.The deletion of csp-1, csp-2 or csp-3 alone does not cause a defect in programmed cell death.Deletion of csp-1, but not csp-2 or csp-3, enhances the defects in programmed cell death caused by partial loss-of-function alleles of ced-3 and ced-4.

Table 2 .
Ectopic expression of csp-1B from the mec-7 promoter can kill touch neurons, and this killing activity requires the conserved cysteine in the putative caspase active site.

Table 3 .
csp-1 is maternally required for programmed cell deaths that occur embryonically in the presumptive anterior pharynx.

Table 4 .
Cell deaths occur in the absence of all C. elegans caspase genes.
The number of refractile cell corpses per head was counted in L1 larvae within one hour of hatching.doi:10.1371/journal.pgen.1003341.t004