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

Abstract

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.

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/C-only 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.

Figure 2. csp-1B overexpression induces ectopic cell deaths.

(A) Fluorescence image of a transgenic nIs290[P_mec-3::gfp] larva expressing GFP from the mec-3 promoter in the touch neurons (AVM, ALMs, PVM and PLMs, yellow arrows). mec-3 is also expressed in the FLP and PVD neurons (white arrowheads). (B) Fluorescence image of a transgenic nIs309[P_mec-7::csp-1B, P_mec-3::gfp] larva expressing CSP-1B from the mec-7 promoter (which is expressed in the AVM, ALM, PVM and PLM neurons) and GFP from the mec-3 promoter. Note the absence of touch neurons. (C) Nomarski differential interference contrast (DIC) image of a refractile PLM cell corpse (arrow) in a ced-1(e1735); ced-4(n1162); nIs309 L1 larva. (D) Transmission electron microscopic image of the cell corpse in (C). “n”, nucleus of the cell corpse; scale bar, 0.5 microns.

Figure 3. csp-1B cell-killing activity is not regulated by the canonical programmed cell death pathway.

(A–D) The percentages of PLM cells that survive in strains carrying P_mec-7::ced-3, P_mec-7::csp-1B or P_mec-7::ced-4 transgenes. (A) ced-9 protects against ced-3- but not csp-1B-cell-killing transgenes. (B) The cell-killing activity of csp-1B transgenes is mostly independent of ced-3 and ced-4. The cell-killing activities of (C) ced-3 and (D) ced-4 transgenes do not require csp-1. PLM survival was scored based on the presence of GFP expressed from the mec-3 promoter. Asterisks indicate p<0.05 in a Fisher's exact test. All strains in (A) contained ced-3(n3692). (E) A model depicting the genetic pathways regulating the caspase genes csp-1 and ced-3 (see text).

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.

Figure 5. The cell corpses of caspase-deleted mutants are cytologically and morphologically apoptotic.

(A) Nomarski DIC and fluorescence images of a cell corpse within the head of a ced-1(e1735); csp-1(n4967); csp-2(n4871) ced-3(n3692) L1 larva carrying the integrated transgene nIs342[P_egl-1::gfp], a transcriptional reporter that expresses GFP under the control of the BH3 domain-only encoding gene egl-1. (B) Nomarski DIC and fluorescence images of a cell corpse within the head of a ced-1(e1735); csp-1(n4967); csp-2(n4871) ced-3(n3692) L1 larva carrying the extrachromosomal array nEx1646[P_dyn-1::mfg-e8::Venus], a fusion protein that binds to cell-surface exposed phosphatidylserine. (C) Representative transmission electron micrographs of cell corpses from ced-1(e1735); csp-1(n4967); csp-2(n4871) ced-3(n3692) larvae 24 hrs post hatching. “n”, nuclei of the cell corpses; scale bars, 0.5 microns. Note the difference in chromatin condensation between the two cell corpses.

Figure 6. Caspase-independent cell corpses are engulfed and degraded.

(A) Nomarski DIC and fluorescence images of a cell corpse from a ced-1(e1735); csp-1(n4967); csp-2(n4871) ced-3(n3692) L1 larva carrying the integrated transgene nIs400[P_ced-1::ced-1ΔC::gfp], which expresses a non-rescuing CED-1ΔC::GFP fusion protein. CED-1 is a transmembrane receptor that is expressed on engulfing cells, binds to apoptotic cell corpses, and is required for phagocytosis . (B) Nomarski DIC and fluorescence images of a cell corpse from a csp-3(n4872); csp-1(n4967); csp-2(n4871) ced-3(n3692) L1 larva stained with acridine orange (AO), which fluoresces in engulfed cell corpses undergoing degradation in endosomal compartments. (C) The fraction of csp-1(n4967); csp-2(n4871) ced-3(n3692) and ced-1(e1735); csp-1(n4967); csp-2(n4871) ced-3(n3692) with 0, 1, 2 or >2 cell corpses at different time points post hatching. Asterisks indicate p<0.05 in a Mann-Whitney test comparing the two genotypes at a given time point.