Caspy, a Zebrafish Caspase, Activated by ASC Oligomerization Is Required for Pharyngeal Arch Development*

The pyrin domain was identified recently in multiple proteins that are associated with apoptosis and/or inflammation, but the physiological and molecular function of these proteins remain poorly understood. We have identified Caspy and Caspy2, two zebrafish caspases containing N-terminal pyrin domains. Expression of Caspy and Caspy2 induced apoptosis in mammalian cells that were inhibited by general caspase inhibitors. Biochemical analysis revealed that both Caspy and Caspy2 are active caspases, but they exhibit different substrate specificity. Caspy, but not Caspy2, interacted with the zebrafish orthologue of ASC (zAsc), a pyrin- and caspase recruitment domain-containing protein identified previously in mammals. The pyrin domains of both Caspy and zAsc were required for their interaction. Furthermore, zAsc and Caspy co-localized to the “speck” when co-transfected into mammalian cells. Enforced oligomerization of zAsc, but not simple interaction with zAsc, induced specific proteolytic activation of Caspy and enhanced Caspy-dependent apoptosis. Injection of zebrafish embryos with a morpholino antisense oligonucleotide corresponding tocaspy resulted in an “open mouth” phenotype associated with defective formation of the cartilaginous pharyngeal skeleton. These studies suggest that zAsc mediates the activation of Caspy, a caspase that plays an important role in the morphogenesis of the jaw and gill-bearing arches.

Apoptosis, a morphologically distinguished form of programmed cell death, is critical during development and tissue homeostasis and plays a role in the pathogenesis of a variety of diseases. The morphological features of apoptosis and demise of the cell are because of the cleavage of structural and non-structural proteins by a family of proteases called caspases (for review, see Ref. 1). Upon stimulation by apoptotic stimuli, proximal caspases are activated and cleave downstream effector caspases leading to an amplification of proteolytic activity within the dying cells. Activation of proximal caspases requires upstream regulators that have domains homologous to those present in the N-terminal prodomains of proximal caspases, namely death effector domains (DEDs) 1 and caspase recruitment domains (CARDs). Homophilic interactions between the CARDs or DEDs of upstream regulators and proximal caspases mediate caspase activation (1). For example, homophilic interactions between the DEDs of Fas-associated death domain protein and procaspase-8 and between the CARDs of Apaf-1 and procaspase-9 are essential for caspase activation (2). Activation of proximal caspases is thought to be induced via proximity of their catalytic subunits, a process that is mediated through oligomerization of their upstream regulators. For example, Fas-associated death domain protein and Apaf-1 contain a death domain (DD) and nucleotide-binding domain, respectively, that mediate their oligomerization in a ligand-dependent manner (1).
Bioinformatic and protein structure analyses have revealed that the CARD, DED, and DD share similar structural and functional features (3)(4)(5)(6). These domains are found not only in apoptosis regulators but also in proteins that are associated with inflammation, cell cycle regulation, and cytoskeletal organization (4,7). Thus, these ␣-helix-rich domains mediate homophilic interactions between signaling components of diverse signaling pathways, most notably those involved in apoptosis and inflammation (8 -11).
The pyrin domain (also called DAPIN, PYRIN, and PAAD) was originally found in pyrin, the product of the familial Mediterranean fever-associated gene (12), and in ASC, a component of the "speck," a granular structure induced in the cytosol of certain apoptotic cells (13). Subsequently, pyrin domains were also identified in multiple proteins whose molecular functions are largely unknown (14 -19). Computer modeling has predicted that the pyrin domain is ␣-helix-rich and structurally related to the CARD, DED, and DD. Furthermore, the pyrin domain mediates homophilic interactions between pyrin domain-containing proteins (16,20). However, the functional significance of these pyrin-pyrin homophilic interactions is presently poorly understood.
The vertebrate zebrafish (Danio rerio) is a model organism whose genome encodes the great majority of the components that mediate apoptosis and inflammation in mammals including humans (10,21). Thus, analysis of zebrafish components should provide insight into the mechanisms that mediate apoptosis and inflammation in mammalian systems. Here we report the identification and initial characterization of the zebrafish orthologue of ASC (zAsc) and two zebrafish pyrin-containing caspases, Caspy and Caspy2. We provide evidence that zAsc associates with and activates Caspy through a homophilic pyrin-pyrin domain interaction. In addition, we show that Caspy is required for normal development of the jaw and pharyngeal arches.
For in situ hybridization, digoxigenin-labeled antisense RNA probes were synthesized from full-length cDNAs using an in vitro transcription kit (Promega). As a control, sense RNA-labeled probes were synthesized and used for hybridization as above. In situ hybridization and development of whole-mount zebrafish embryos were performed as described (26).
Transfection, Expression, Immunoprecipitation, and Immunodetection of Tagged Proteins-Cells were transfected, and immunoprecipitation assay was performed as described with slight modifications (27). The total amount of transfected plasmid DNA was adjusted with pcDNA3 to always be the same within individual experiments. 1 ϫ 10 6 COS-7 cells were transfected with 2.4 g of expression plasmids using LipofectAMINE-PLUS reagent (Invitrogen) according to the manufacturer's instructions. The supernatant was immunoprecipitated with 20 l of protein G-Sepharose 4B (Amersham Biosciences) conjugated with anti-Myc polyclonal antibody (Santa Cruz Biotechnology, Inc.). Immunoprecipitated proteins were subjected to 16% SDS-polyacrylamide electrophoresis and detected by Western blotting using anti-FLAG monoclonal antibody M2 (Sigma). For cellular localization assay, COS-7 cells were transiently cotransfected with pEGFP-zAsc and pEGFP-Caspy. For coexpression assay, COS-7 cells were transiently cotransfected with pDsRed-zAsc and pEGFP-Caspy. After 24 h, cells were fixed with 70% ethanol. Signals were detected by immunofluorescence microscopy.
Microinjection of Morpholino Oligonucleotides and Phenotypic Analyses-Each morpholino (Gene Tools, LLC) was resuspended in sterile water to a concentration of 1 mM. For injections, the stock solution was diluted to 250 nM in Danieau buffer. A 3-nl volume of the solution was injected into one to four cell-stage embryos at the yolk and cytoplasm interface. The sequences of caspy and control morpholino oligonucleotides were 5Ј-GCCATGTTTAGCTCAGGGCGCTGAC-3Ј and 5Ј-CCTCT-TACCTCAGTTACAATTTATA-3Ј, respectively. For the morphological assay, larvae were euthanized in tricane, fixed in 4% paraformaldehyde, and washed for 3 min in acid-alcohol (0.37% HCl, 70% ethanol).
The fixed larvae were stained for 1 h with 0.1% Alcian blue. After rinsing in ethanol, animals were suspended in 50% glycerol, 0.25% KOH overnight. Stained preparations were clarified with 1% H 2 O 2 and mounted in glycerol-KOH.

Identification of a Novel Class of Caspases with Pyrin Domain-
To identify novel pyrin domain-containing proteins in zebrafish, we screened the zebrafish GenBank TM EST data base for cDNAs encoding amino acid sequences with homology to the pyrin domain (amino acids 1-91) of human ASC by the program TBLASTN. We identified the zebrafish orthologue of human ASC (referred to here as zAsc) and two zebrafish caspases (designated here as Caspy and Caspy2). Sequence analysis of zAsc cDNA revealed that it encodes a protein of 203 amino acid residues composed of a N-terminal pyrin domain linked to a C-terminal CARD as reported for its mammalian orthologues (GenBank TM accession numbers were as follows: human ASC, AB023416; mouse ASC, AB032249; bovine ASC, AB050006; rat ASC, AB053165). Caspy and Caspy2 contained N-terminal pyrin domains and C-terminal caspase catalytic FIG. 2. Genomic mapping of asc, caspy, and caspy2. The chromosomal locations of asc, caspy, and caspy2 were determined as described under "Experimental Procedures." The localization of asc, caspy, and caspy2 is indicated by arrows. The chromosomal localization of human orthologues was determined by NCBI-BLAST programs (www.ncbi.nlm.nih.gov/ BLAST) and are given in parentheses. domains (Fig. 1A). The N-terminal pyrin domain of Caspy was most homologous to that present in zAsc (87% similarity) whereas that of Caspy2 was most homologous to that of human Cryopyrin/PYPAF1 (46% similarity), a pyrin-domain containing Nod-family protein (30,31). Comparison of these zebrafish and human caspases showed that the catalytic domains of Caspy and Caspy2 share highest homology with those of human caspase-1 and caspase-5, respectively (54 and 57% similarity, respectively). Caspy and Caspy2 contained conserved His and Cys amino acid residues, which are essential for proteolytic catalysis (depicted by arrows in Fig. 1A), and also conserved residues, which are essential for interaction with critical core residues of substrates, suggesting that both Caspy and Caspy2 are enzymatically active. Notably, Caspy, like human caspase-1, has His at position 318, corresponding to the residue at position 342, which binds the specific P3 residue of the substrate (Ala of YVAD) in human caspase-1 (32), whereas Caspy2 and human caspase-5, which can not digest peptides carrying Ala residue at P3 (33), have Asn and Asp residues, respectively, at the corresponding positions (arrowhead; see Fig. 1A).
Genomic Mapping of asc, caspy, and caspy2-Genomic mapping experiments using the heat shock doubled haploid meiotic mapping panel revealed that caspy and caspy2 are located on linkage groups 16 and 1, respectively (Fig. 2). Human CASP1 and CASP5, which are homologues of caspy and caspy2, are located as tandem genes on human chromosome 11. We did not find any conclusive evidence supporting conserved syntenies between the zebrafish and human regions containing the zebrafish and human caspase genes (Fig. 2). Notably, caspy was adjacent to asc on linkage group 16 (z06s003336 at www. ensembl.org/Danio_rerio/blastview), whereas the human orthologue of asc is located on chromosome 16. Sequence analysis also showed that the pyrin domain of Caspy2 exhibited significant homology not only to pyrin domains but also to the CARDs of Xenopus caspase-1 (49% similarity) and bovine caspase-13 (45% similarity, previously known as human caspase-13) (Fig. 1B). Thus, both the pyrin domain and CARD are predicted to be ␣-helix rich and to share a similar organization in their predicted secondary structure (Fig. 1B).
zAsc, Caspy, and Caspy2 mRNA Exhibit a Similar Pattern of Expression in the Zebrafish Embryo-We performed in situ hybridization to assess the expression of zAsc, Caspy, and Caspy2 mRNA in zebrafish embryos. At embryonic development of 48 and 72 h post-fertilization, zAsc (Fig. 3, A-C), Caspy (Fig. 3, E-G), and Caspy2 (Fig. 3, I-K) mRNA exhibited a similar labeling pattern with expression being detected primar-ily in epidermis (arrows), mouth (single arrowhead), and pharyngeal arches (white arrowheads) shown in Fig. 3. Significantly, the expression of asc and caspy2 appeared stronger than that observed for caspy. Similar results were observed in embryos at 36 h post-fertilization. 2 The expression pattern of asc (Fig. 3, A and B) is consistent with that of human ASC, which was reported to be expressed in epithelial cells including squamous epithelium of skin and mouth (34).
Caspy and Caspy2 Induce Apoptosis in Mammalian Cells-To begin to assess the molecular function of Caspy and Caspy2, we constructed expression plasmids of tagged and non-tagged Caspy and Caspy2 and transiently transfected the plasmids into 293T cells, respectively. A significant percentage of Caspy-and Caspy2-transfected cells displayed morphological features of adherent cells undergoing apoptosis such as rounding, membrane blebbing, and detachment from the dish (Fig. 4A). At 24 h post-transfection, about 20 -40 and 95% of the cells transfected with the highest dose of both tagged and non-tagged Caspy and Caspy2 constructs, respectively, displayed morphological features of apoptosis compared with less than 1% of the cells transfected with control plasmid (Fig. 4A).
To determine whether the proapoptotic activity of Caspy and Caspy2 is because of the their enzymatic activities, the ability of these proteins to induce apoptosis was tested in the presence of the caspase inhibitors, p35, CrmA, and zVAD-fmk. Expression of baculoviral p35 attenuated apoptosis induced by both Caspy and Caspy2 whereas incubation with zVAD-fmk greatly inhibited apoptosis (Fig. 4B). Interestingly, CrmA, an inhibitor of human caspase-1 and certain caspases (35), inhibited apoptosis induced by Caspy but not by Caspy2 (Fig. 4B). These data were also similar compared with the results using nontagged Caspy and non-tagged Caspy2 (Fig. 4B). These results suggest that apoptosis induced by Caspy and Caspy2 is dependent on caspase activity and that the proapoptotic activity of Caspy and Caspy2 can be dissociated based on the inhibition with CrmA.
Caspy and Caspy2 Are Active Caspases with Different Substrate Specificity-To test more directly whether Caspy and Caspy2 are active enzymes, a panel of fluorogenic peptide substrates of mammalian caspases were incubated with Caspy or Caspy2 immunoprecipitated from extracts of cells transfected with plasmids producing the relevant caspases. Both caspases cleaved several caspase peptide substrates, demonstrating that Caspy and Caspy2 are active caspases (Fig. 4, C and D). Nota-2 W. Zhou and J. Y. Kuwada, unpublished data. bly, Caspy preferentially cleaved AcYVAD-AMC, a caspase-1 substrate, whereas Caspy2 was more active on AcWEHD-AFC, a preferred substrate of caspase-5, and it did not cleave AcY-VAD-AMC (Fig. 4, C and D). These results suggest that Caspy and Caspy2 are enzymatically active caspases that exhibit different substrate specificity.
Zebrafish ASC Interacts with Caspy but Not with Caspy2-Because Caspy and Caspy2 contain a pyrin domain highly homologous to that of zAsc, the ability of both caspases to interact with zAsc was tested using a co-immunoprecipitation assay. zAsc was co-immunoprecipitated with Caspy but not with Caspy2 (Fig. 5). Mutational analysis revealed that a zAsc mutant containing the pyrin domain but lacking the CARD was co-immunoprecipitated with Caspy (Fig. 5, FLAG-zAsc-PD), suggesting that the pyrin domain of zAsc mediates the binding to Caspy. Furthermore, a zAsc mutant-lacking pyrin domain did not co-immunoprecipitate with Caspy (Fig. 5, FLAG-zAsc-⌬PD), indicating the pyrin domain of zAsc is necessary and sufficient for the interaction with Caspy. In addition, a Caspy mutant lacking the pyrin domain (Fig. 5, Myc-Caspy-⌬PD) failed to interact with a zAsc mutant containing a pyrin domain (Fig. 5, FLAG-zAsc-PD) suggesting that zAsc and Caspy associate through a homophilic pyrin-pyrin domain interaction. To further assess the association between zAsc and Caspy, we constructed plasmids to express GFP-Caspy, GFP-zAsc, and DsRed-zAsc fusion proteins to facilitate the analysis of their subcellular localization. Expression of GFP-Caspy and GFP-  zAsc induced the formation of specks (Fig. 6B, GFP-Caspy and GFP-zAsc, respectively), and double labeling analysis of the same cells revealed that both GFP-Caspy (Fig. 6A, Green) and DsRed-zAsc (Fig. 6A, Red) co-localized to the same speck visualized in yellow (Fig. 6A, Merge). These observation demon-strates that zAsc interacts with Caspy in the same speck.
Enforced Oligomerization of zAsc Induces Caspy-dependent Apoptosis-To determine the functional relevance of the interaction between Caspy and zAsc, we first tested whether coexpression of zAsc enhances apoptosis induced by Caspy and  lanes 1 and 2), and 1 g of pcDNA3 (lanes 1 and 2)  Caspy2. In these experiments we expressed low amounts of plasmid so that we could assess whether zAsc could enhance tagged and non-tagged Caspy-and Caspy2-induced apoptosis. Under these experimental conditions, expression of zAsc did not augment apoptosis induced by either Caspy or Caspy2 (Fig.  7, A and B). This result suggested that the interaction between Caspy and zAsc was not sufficient to induce caspase activation. Enforced oligomerization of the CARD of Apaf-1 and that of the DD of Fas, which are protein modules structurally related to the pyrin domain, is known to induce the activation of target caspases (36,37). Therefore, we hypothesized that zAsc might similarly promote Caspy activation through zAsc oligomerization. To test this hypothesis, we constructed a plasmid to express a zAsc-Fpk3 fusion protein that can be oligomerized with the ligand AP1510 (14). Co-expression of zAsc-Fpk3, like that of zAsc, did not enhance apoptosis induced by Caspy in the absence of AP1510 (Fig. 7, A and B). In contrast, greater than 50% of the cells underwent apoptosis when both zAsc-Fpk3 and Caspy were co-expressed in the presence of AP1510 (Fig. 7, A  and B). In control experiments, co-expression of zAsc-Fpk3 did not enhance the ability of Caspy2 or caspase-9 to induce apoptosis in the presence of AP1510 (Fig. 7B). In reciprocal experiments, co-expression of Apaf-1, a regulator of caspase-9, enhanced apoptosis induced by caspase-9, but it did not augment Caspy-or Caspy2-mediated apoptosis (Fig. 7B). These results suggest that oligomerization of zAsc is required for enhancement of Caspy-dependent apoptosis. Oligomerization of zAsc is presumably induced through an interaction between the Cterminal CARD of zAsc and a putative upstream regulator that needs to be identified. Because Caspy2 did not interact with zAsc, we suggest that Caspy2, which possesses caspase-5 like enzymatic activity, mediates apoptosis and/or inflammation in a zAsc-independent manner.
Enforced Oligomerization of zAsc Induces Proteolytic Processing of Caspy-To examine whether the activation of Caspy is enhanced by zAsc, we determined whether oligomerization of zAsc induces the processing of Caspy. Analysis of cell extracts by immunoblotting revealed that a processed form of Caspy was immunodetected in extracts from cells co-expressing zAsc-Fpk3 and Caspy in the presence but not in the absence of dimerizer AP1510 (Fig. 7C). When Caspy was immunoprecipitated with antibody, processing of Caspy was observed in extracts from cells co-expressing Caspy and zAsc even in the absence of the dimerizer (Fig. 7C). A possible explanation for the latter finding is that Caspy activation can be induced through antibody-mediated aggregation of zAsc-Caspy complexes in vitro resulting from the immunoprecipitation procedure. Significantly, zAsc was still required for processing of Caspy in vitro, suggesting that the interaction of zAsc with Caspy is important for caspase processing and activation.
Morpholino Antisense Oligonucleotide-based Knocked-down of Caspy Affects Development of the Jaw and Posterior Pharyngeal Arches-To assess whether Caspy was required for normal development of zebrafish, we designed a morpholino antisense oligonucleotide to interfere with caspy translation. Microinjection of the blocking antisense oligonucleotide into one-to fourcell stage embryos resulted in an open mouth phenotype as observed by simple morphological examination of larvae at 6 days post-injection. This phenotype was observed at least 4 days post-injection. To assess the phenotype in more detail, we injected caspy and control morpholino oligonucleotides into early cleavage embryos and stained the head cartilage of the resulting larvae with Alcian blue. The analysis revealed that 12/19 larvae injected with the caspy antisense morpholino exhibited several abnormalities in the development of the cartilaginous pharyngeal skeleton. The abnormal phenotype in-cluded deformed and thinned Meckel's and palatoquadrate cartilages, which form the jaw, deformed ceratohyal cartilages, which are pointing posteriorly, and disorganized branchial cartilages (Fig. 8). The larvae were still alive approximately 1 week post-fertilization but then started to die as yolk disappeared. Of the seven remaining larvae derived from early embryos injected with the caspy antisense oligonucleotide, 5/19 showed a similar but less marked phenotype, whereas 2/19 larvae appeared normal. The phenotype is consistent with the expression of caspy in the pharyngeal arches (Fig. 3). No other abnormalities were identified in any other part of the body including the brain, eyes, or pectoral fins of the larvae injected with the caspy antisense oligonucleotide. The latter is consistent with the observation that caspy is not expressed in the brain, eyes, or pectoral fins during embryonic development (Fig. 3). None of the embryos injected with control morpholino oligonucleotide showed any alteration in the cartilaginous structures of the jaw or gills. DISCUSSION In this report we provide evidence that the zebrafish ASC mediates the activation of Caspy, a pyrin domain-containing caspase. Although mammalian orthologues of zebrafish Caspy have not been yet identified, ASC is highly conserved in vertebrates. It is possible that mammals have Caspy-like molecules with typical pyrin domains, but they need to be identified. Because the CARD is highly related to the pyrin domain, it is also possible that one of the mammalian CARD-containing caspases is the Caspy orthologue. Caspy exhibits the highest homology to human caspase-1 and preferentially cleaved caspase-1 substrates whereas Caspy2 showed the highest homology to human caspase-5 and preference for caspase-5-like substrates. Apoptosis induced by Caspy was blocked by CrmA, a caspase inhibitor that inhibits human caspase-1 at low concentrations (35) (Fig. 4B). Both caspase-1 and caspase-5 belong to the same subfamily of CARD-containing caspases (33), but their activator(s) are unknown or are poorly understood. Thus, it will be important to test for interactions between these known CARD-containing caspases and mammalian ASC.
zAsc, like its mammalian orthologues, is composed of an N-terminal pyrin domain linked to a C-terminal CARD. Recent studies of the three-dimensional structure of several apoptosis regulators have suggested that the stoichiometry of the homophilic interaction between the CARD and related domains is one by one (38,39). Therefore, it is unlikely that the pyrin domain of zAsc binds to other pyrin or CARD-containing factors in the Caspy/zAsc signaling complex. Enforced oligomerization of zAsc enhanced Caspy-induced apoptosis. These findings suggest that the C-terminal CARD of zAsc and that of its mammalian orthologues might be important for oligomerization. In this model, zAsc acts as an adaptor molecule linking a putative upstream factor to caspases such as Caspy. Potential upstream factors include Defcap/NAC/CARD7/NALP1 and Ipaf-1/ CARD12/CLAN, Cryopyrin/PYPAF1, CARD-, or pyrin domaincontaining proteins with structural homology to a family of Apaf-1-like molecules that include Nod1 and Nod2 (9, 15-16, 30 -31, 40 -45). Such a factor may interact via its CARD with Caspy, an event that may lead to zAsc oligomerization and association with Caspy, leading to caspase activation. Alternatively, self-oligomerization of zAsc might be mediated via its CARD, which might be induced by upstream molecules such as protein kinases or other factors. Consistent with the latter, it has been shown that a mutant form of human ASC carrying the CARD alone can self-associate (20). Further studies are required to understand the mechanism by which caspases are activated by zAsc and its mammalian orthologues.
Analysis of animals in which caspy activity was knocked down by morpholino antisense oligonucleotide revealed abnormal development of the pharyngeal skeleton containing the jaw and branchial arches. The pharyngeal skeleton in vertebrates derives from migrating neural crest cells, and its development involves a complex program of interactions among neural crest cells, mesodermal mesenchyme, and surrounding epithelia (46). The abnormal phenotype involving the anterior and posterior arch cartilages indicates that Caspy is required for proper development of neural crest derivatives. It is possible that Caspy is involved in the execution of a program of neural crest cell death required for proper development and assembly of the cartilages. For example, cell death may be required to release the ceratohyal from ventral attachment to the basibranchials allowing it to assume its normal anterior projection. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) analysis did not reveal any differences in the level of apoptosis between caspy knocked-down and control morphants. 2 However, we cannot rule out a subtle defect in apoptosis that was not revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis. Another possibility is that Caspy is involved in processing a zebrafish homologue of interleukin-1/interleukin-18 and/or another factor that is required for normal development of pharyngeal arch cartilages. For example, mutant mice defi-cient in the endothelin-1 converting enzyme, which processes the endothelin-1 precursor to biologically active endothelin-1, exhibit defects in branchial arch-derived craniofacial tissues (47). Furthermore, endothelin-1 is essential for development of the ventral aspect of zebrafish pharyngeal arches (48). Genetic screens in the zebrafish have revealed a large number of loci required for pharyngeal arch development (49,50). Some of the mutant fish exhibit phenotypes that are similar but not identical to that observed in caspy morpholino knocked-down animals. Thus, it is possible that Caspy is a component of a signaling pathway that includes some of the genes identified in the genetic screens. Additional studies are needed to understand the precise role of Caspy in the genetic program involved in the development of the jaw and branchial arch cartilages.