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¹ Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan
² Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
³ Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan
⁴ Cell-free Science and Technology Research Center, Ehime University, Matsuyama 790-8577, Japan
⁵ Department of Biology, Vertex Pharmaceuticals, Cambridge, MA 02139, USA
⁶ Research Unit for Animal Life Sciences, Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki-Iwama 319-0206, Japan

	Abstract

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Two major apoptotic signaling pathways have been defined in mammals, the extrinsic pathway, initiated by ligation of death receptors, and the intrinsic pathway, triggered by cytochrome c release from mitochondria. Here, we identified and characterized the Xenopus homologs of caspase-10 (xCaspase-10ß), a novel initiator caspase, and Bid (xBid), a BH3-only molecule of the Bcl-2 family involved in both the extrinsic and intrinsic pathways. Exogenous expression of these molecules induced apoptosis of mammalian cells. By biochemical and cytological analyses, we clarified that xCaspase-10ß and xBid exhibit structural and functional similarities to their mammalian orthologues. We also detected xCaspase-10ß and xBid transcripts during embryogenesis by whole-mount in situ hybridization and RT-PCR analysis. Microinjection of mRNA encoding a protease-defect xCaspase-10ß mutant into embryos resulted in irregular development. Enforced expression of active xBid induced cell death in developing embryos. Using transgenic frogs established to allow monitoring of caspase activation in vivo, we confirmed that this form of cell death is caspase-dependent apoptosis. Thus, we demonstrated that the machinery governing the extrinsic and intrinsic apoptotic pathways are already established in Xenopus embryos. Additionally, we propose that the functions of the initiator caspase and BH3-only molecule are evolutionarily conserved in vertebrates, functioning during embryonic development.

	Introduction

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Apoptosis, or programmed cell death, removes unnecessary, superfluous, damaged or harmful cells in multicellular organisms. Apoptosis is important for tissue morphogenesis during development, for the maintenance of homeostasis in adulthood, and for proper immune responses (Raff 1992; Abbas 1996; Jacobson et al. 1997; Vaux & Korsmeyer 1999; Meier et al. 2000). A family of cysteine proteases, known as caspases, is activated to function as executors of apoptosis or regulators of the inflammatory response (Thornberry & Lazebnik 1998). Of the 15 caspases that have been previously identified in mammals, the caspases involved in apoptosis are divided into two categories, the initiator caspases, including caspases-2, -8, -9 and -10, and the effector caspases such as caspases-3, -6 and -7. The activation of caspases represents the convergence of two major signaling cascades, the extrinsic pathway, which is initiated by ligation of cell surface "death receptors," such as Fas (APO-1/CD95), and the intrinsic pathway, which is triggered by cytochrome c release from mitochondria into the cytosol.

Caspase-8 and caspase-10 are involved in the extrinsic signaling pathway (Chen & Wang 2002). Activation of death receptors by ligand binding leads their association with the adaptor molecule FADD (Fas-associated death domain protein, also called MORT1). FADD then recruits caspase-8 (also known as FLICE/MACH1/Mch5) and caspase-10 (FLICE2/Mch4) via interactions between death effector domains (DED) (Boldin et al. 1996; Muzio et al. 1996; Vincenz & Dixit 1997). In the death-inducing signaling complex (DISC), these caspases execute autoprocessing to become active (Kischkel et al. 1995). Activated initiator caspases are released from the complex and cleave downstream effector caspases, leading to cell death (Stennicke et al. 1998). In contrast, the intrinsic signaling pathway is triggered by a wide variety of stimuli, including UV irradiation, steroids, chemotherapeutic drugs and growth factor deprivation. Molecules of the Bcl-2 family are the prime players in this pathway (Gross et al. 1999a). Of the members of the Bcl-2 family, the BH-3-only molecules such as Bid (BH3-interacting domain death agonist) are pro-apoptotic molecules that induce the release of cytochrome c from mitochondria by interacting with other classical Bcl-2 family members (Huang & Strasser 2000). Bid, discovered as a molecule that binds to Bcl-2 and Bax (Wang et al. 1996), is a target of caspase-8 (Li et al. 1998; Luo et al. 1998; Gross et al. 1999b). This cleavage event links the extrinsic and intrinsic apoptotic signaling pathways, allowing the processed form of Bid to translocate to mitochondria to induce intrinsic apoptotic signaling.

In amphibians, degeneration of the tadpole tail during metamorphosis is accompanied by cell death (Kerr et al. 1974; Tata 1994; Nishikawa et al. 1998). Recent reports suggest that apoptosis also occurs during the early development of Xenopus embryos (Hensey & Gautier 1998; Hensey & Gautier 1999; Charrier et al. 1999). Although these apoptotic events occur during different stages of development, several lines of evidence suggest that all apoptotic signaling pathways converge to a common final pathway, requiring a number of evolutionarily conserved genes. Eight caspase types have been identified in Xenopus (Yaoita & Nakajima 1997; Nakajima et al. 2000), each of which exhibits homology to its mammalian counterpart. Expression of the majority of these caspases increases in regressing organs during metamorphosis (Nakajima et al. 2000). Caspase-3 and caspase-9 have been directly implicated in cell death of the tail (Das et al. 2002; Rowe et al. 2005). Caspase-2 works as an executioner of stockpiled oocytes (Nutt et al. 2005). In addition, Bax, a pro-apoptotic molecule of the Bcl-2 family, plays a critical role in the regression of the tail during metamorphosis (Sachs et al. 2004). We previously identified Xenopus FADD as a protein that functions similarly to mammalian FADD, affecting embryonic development following ectopic over-expression (Sakamaki et al. 2004). Thus, the machinery involved in apoptotic signaling appears to be conserved between mammals and Xenopus. Numerous components required for apoptosis, including death ligands and BH-3-only molecules, have not yet been characterized in Xenopus. Further investigation is necessary to elucidate the common mechanisms controlling programmed cell death in vertebrates.

To understand the universal mechanisms that regulate cell death in vertebrates, we examined the apoptosis-inducing molecules in Xenopus that are essential for the extrinsic and intrinsic signaling pathways. Using mammalian cells and Xenopus embryos, we identified and characterized a novel initiator caspase, xCaspase-10ß, and a BH3-only molecule, xBid. Our study clearly demonstrates that these molecules have both structural and functional similarities to their mammalian homologs, confirming the evolutionary conservation in vertebrates of apoptotic signaling mechanisms. In this study, we also present a novel system for the detection of apoptosis associated with caspase activation in vivo.

	Results

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Identification of the genes for Xenopus caspase-10ß and Bid

In our search for DED-containing proteins in the GENBANK expressed sequence tag (EST) database, we identified two promising cDNA clones (XL070f19 and XL061i12) from the Xenopus tailbud cDNA library. Further sequence analysis revealed that both of these cDNA clones represent a single open reading frame encoding a 512 amino acid protein with a higher degree of homology to Xenopus caspase-10 (84% identity at the amino acid level) than that of caspase-8 (32% identical) (Fig. 1A,B). Alignment of the novel gene with caspase-10 demonstrated the presence of several differing amino acid and one gap. In addition, these molecules exhibit distinct expression profiles during early embryonic development (see below). Therefore, we concluded that this cDNA clone encodes a novel caspase that has diverged evolutionarily from caspase-10 in Xenopus. We refer to this molecule as a second caspase-10 (caspase-10ß).

View larger version (66K): [in this window] [in a new window]   Figure 1  Alignments of xCaspase-10ß and xBid with their homologs. (A) Amino acid sequence comparison of xCaspase-10ß, xCaspase-10, and xCaspase-8. The predicted amino acid sequence of Xenopus caspase-10ß cDNA clone db61b04 was aligned with caspase-10 and caspase-8 from Xenopus. Amino acids that are identical or similar between the three molecules are indicated in black and shaded boxes, respectively. Two DED motifs are underlined and the protease domain is indicated by a box. Asterisks represent the amino acids involved in the formation of an alpha-helical structure. Sharps (#) indicate the amino acids deleted in caspase-10ß, but not caspase-10. The bold arrows underneath the amino acid sequence indicate the primers used for RT-PCR analysis. (B) Comparison of homologies between Xenopus and human initiator caspases. The numbers indicate the percentage identity between the two molecules. (C) Amino acid sequence comparison of Xenopus and human Bid molecules. Amino acids that are identical or similar between Xenopus and human molecules were indicated in black and shaded boxes, respectively. The bold lines over the amino acid sequence indicate the BH3 domain, while the caspase recognition sequence is designated by asterisks.

Characterization of Xenopus caspase-10ß and Bid

In Xenopus, two initiator caspases, caspase-8 (xCaspase-8) and caspase-10 (xCaspase-10), have been identified (Nakajima et al. 2000). These molecules are the functional equivalents of their mammalian homologs, inducing apoptosis in mammalian cells upon exogenous expression (Sakamaki et al. 2004). Therefore, we investigated whether the exogenous expression of Xenopus caspase-10ß (xCaspase-10ß) in mammalian cell lines could induce apoptosis. We constructed egfp-xCasp10ß, a fusion of enhanced green fluorescent protein (EGFP) and the full-length xCaspase-10ß (Fig. 2A), and transfected this gene into human HeLa cells. The cell viability of transfectants was examined by monitoring EGFP-positive cells by microscopy. Ectopic expression of EGFP-xCasp10ß molecules clearly induced cell death in HeLa cells, as evidenced by reduced numbers of EGFP-positive cells and by the appearance of apoptotic bodies compared with control cells expressing EGFP alone (Fig. 2B,C). In the presence of a pan-caspase inhibitor carbobenzoyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), however, an increased number of EGFP-positive cells were detected, exhibiting a normal morphology in a similar manner as the control cells (Fig. 2D). This result suggests an inhibition of apoptotic cell death induced by exogenous xCaspase-10ß. Aggregates termed death-effector filaments (Siegel et al. 1998) were observed in surviving cells expressing EGFP-xCasp10ß in the presence of z-VAD-fmk (Fig. 2E), suggesting that apoptotic induction by xCaspase-10ß requires homophilic oligomerization in a manner similar to the mammalian DED-containing molecules.

View larger version (70K): [in this window] [in a new window]   Figure 2  Over-expression of xCaspase-10ß and xBid induces apoptosis in mammalian cells. (A) Constructs termed EGFP-xCaspase10ß and EGFP-txBid encode fusions of EGFP and either caspase-10ß or truncated xBid. EGFP fusions of full-length wild-type and mutant xBid were named xBid-EGFP and xBid(D52A)-EGFP, respectively. (B–J) HeLa cells were co-transfected with plasmids encoding EGFP alone (B), EGFP-xCasp10ß (C–E), or EGFP-txBid (F–H) with mouse Bcl-XL (H). Transfected cells were cultured in the presence or absence of 100 µM z-VAD-fmk (D,E,G). Mouse embryonic fibroblasts (MEF) deficient in caspase-8 (I) or caspase-9 (J) were also transfected with a plasmid encoding EGFP-txBid. After 24 h of culture, cells were washed and fixed. A single field was then photographed by phase contrast (right panels) and fluorescence (left panels) microscopy. (K and L) HeLa cells transfected with a plasmid encoding control EGFP together in conjunction without (K) or with mBcl-XL (L) were treated with an anti-Fas antibody in the presence of CHX for 24 h. After fixation, cells were photographed by both phase contrast and fluorescence microscopy. The arrowheads in (C) indicate apoptotic bodies, while the dotted line in (E) indicates the edge of the magnified cell.

Translocation of xBid to the mitochondria during apoptosis

In mammals, Bid is processed by activated caspase-8; truncated Bid translocates to the mitochondria (Gross et al. 1999b). To address the localization of the processed form of xBid to mitochondria, we generated a plasmid, pxBid-EGFP, encoding a fusion of full-length xBid and EGFP (Fig. 2A). Following co-transfection of HeLa cells with pDsRed2-Mito, the majority of the transfectants did not die (Fig. 3A). Then, we induced apoptosis in the transfectants by treatment with an agonistic anti-Fas antibody, CH11. The xBid-EGFP fusion clearly translocated to mitochondria in dying cells, as evidenced by the overlap of EGFP fluorescence with that of DsRed, a mitochondrial marker (Fig. 3B). We also generated an xBid mutant, xBid(D52A), in which the LETD sequence, the putative caspase recognition site, is replaced by LETA (Fig. 2A). This mutant failed to translocate to mitochondria, despite the induction of apoptosis in transfectants (Fig. 3C,D). These results demonstrate that the processed form of xBid translocates to mitochondria in a similar manner as observed for mammalian Bid. Immunoblotting analysis confirmed the cleavage of xBid-EGFP, but not the mutant xBid(D52A)-EGFP, after the induction of apoptosis. The processed form of xBid was only detectable in extracts of HEK293Fas cells transfected with the wild-type, but not the mutant, xBid after treatment with an anti-Fas antibody (Fig. 3E). Thus, in a manner similar to the mammalian orthologues, processed xBid translocated to mitochondria upon apoptotic stimulation.

View larger version (37K): [in this window] [in a new window]   Figure 3  xBid processing and translocation to the mitochondria following apoptotic stimuli. (A–D) HeLa cells expressing xBid-EGFP (A, B) or xBid(D52A)-EGFP (C, D), stained with DsRed2-Mitotracker, were stimulated without (A, C) or with an anti-Fas antibody and CHX (B, D) for 2 h. Both EGFP and DsRed fluorescence images were examined by confocal microscopy. Scale bars indicate 8 µm (E) HEK293Fas cells untransfected or transfected with xBid-EGFP or mutant xBid(D52A)-EGFP were incubated in the absence or presence of anti-Fas antibody for 6 h. Cell lysates were then analyzed by immunoblotting with an anti-GFP antibody. The sizes of molecular weight standards are shown at the left side.

To examine the protease activity of xCaspase-10ß, we generated and purified recombinant xCaspase-10ß and xCaspase-8 from E. coli. In an in vitro assay using a fluorescent substrate, recombinant xCaspase-10ß exhibited a similar protease activity as recombinant xCaspase-8 (Fig. 4A). The large subunit of xCaspase-10ß alone failed to cleave the substrate. Although the processing activity of caspase-8 for Bid has been recognized, it remains unclear if caspase-10 can process Bid. We next examined this possibility by using recombinant xCaspase-10ß and xBid. We prepared both wild-type and mutant (D52A) xBid by the novel cell-free system (Sawasaki et al. 2002) and mixed them with recombinant xCaspase-10ß in vitro. As shown in Fig. 4B, recombinant xCaspase-10ß directly cleaved wild-type, but not mutant. In addition, xCaspase-10ß protease activity was completely inhibited by the addition of z-VAD-fmk. These results suggest that xCaspase-10ß has a potent protease activity, recognizing and directly cleaving xBid. Recently, human caspase-10 was reported to possess the potency to cleave Bid (Milhas et al. 2005), correlating well with our data.

View larger version (19K): [in this window] [in a new window]   Figure 4  xCaspase-10ß protease activity and the cleavage of xBid in vitro. (A) The protease activities of the following recombinant proteins fused to MBP at the N-terminus: the xCaspase-10 protease domain (xCasp10ß), the large catalytically inactive subunit of xCaspase-10ß (xCasp10ß(p21)), and the xCaspase-8 protease domain (xCasp8) were examined. The fluorescence of AMC released from a synthetic caspase substrate, Ac-IETD-MCA, was measured at the indicated times at 37 °C. The resultant data are presented as arbitrary units (AU). These data are representative of two experiments performed in triplicate. (B) The protease activity of recombinant xCaspase-10ß against xBid was examined. Active xCaspase-10ß (xCasp10ß-His), tagged with a His-tag sequence at both the N- and C-termini of the protease domain, was purified from E. coli. Both the xBid-EGFP and xBid(D52A)-EGFP fusion proteins were synthesized by in vitro translation. After incubation of xBid-EGFP or xBid(D52A)-EGFP with or without active xCasp10ß-His in the presence or absence of z-VAD-fmk, the reaction mixture was resolved by SDS-PAGE and the processing profile analyzed using an image analyzer. FITC-labeled molecular weight markers were applied at left side lane.

We previously demonstrated that Xenopus caspase-10 interacts with the adaptor protein FADD (Sakamaki et al. 2004). In this study, we examined the association of xCaspase-10ß with Xenopus and mouse FADD molecules. By immunoprecipitation assay, xCaspase-10ß as well as mouse caspase-8 was detected in the complex with xFADD or mouse FADD upon co-expression in HEK293T cells, suggesting the interactions between xCaspase-10ß and Xenopus and mouse adaptors (Fig. 5A). We further investigated whether xCaspase-10ß forms homodimeric complexes or heterodimeric complexes with either xCaspase-10 or xCaspase-8 (Fig. 5B). xCaspase-10ß clearly interacted with both xCaspase-10ß and other xCaspase-8 and xCaspase-10 molecules. These data suggest that xCaspase-10ß can interact with other Xenopus initiator caspases via the conformational activity of its DED.

View larger version (44K): [in this window] [in a new window]   Figure 5  xCaspase-10ß interacts with Xenopus FADD, caspase-8 and caspase-10. (A) HEK293T cells were co-transfected with plasmids either EGFP-xCaspase10ß, GFP-mouse caspase-8, Flag-xFADD, Flag-mouse FADD, or control vector. After 24 h of transfection, cells were lyzed. Proteins were then immunoprecipitated from cell lysates with an anti-Flag antibody. Whole cell lysates and immunoprecipitates were analyzed by immunoblotting with anti-Flag and anti-GFP antibodies. (B) HEK293T cells were co-transfected with plasmids either EGFP-xCaspase-10ß, Flag-xCaspase-10ß, HA-xCaspase-8, Myc-xCaspase-10, or control vector. After 24 h of transfection, cells were lyzed. Proteins were immunoprecipitated from cell lysates with an anti-GFP antibody. Whole cell lysates and immunoprecipitates were analyzed by immunoblotting with anti-Flag, anti-HA, anti-Myc, and anti-GFP antibodies. To prevent apoptosis, we introduced the baculovirus p35 gene, a strong caspase inhibitor (Zhou et al. 1998), into all samples.

To investigate the expression patterns of xCaspase-10ß and xBid in Xenopus laevis, we detected their transcripts in a variety of adult and embryonic tissue by reverse transcription-polymerase chain reaction (RT-PCR). In previous studies, we demonstrated that caspase-10 is expressed in adult tissues (Sakamaki et al. 2004) and at very early stages of embryonic development (our unpublished data). To compare the expression patterns of xCaspase-10 and xCaspase-10ß by RT-PCR, we designed new primers capable of distinguishing the PCR products derived from the two genes (see Fig. 1). Using this primer set, we could detect two distinct transcripts, representing xCaspase-10 and xCaspase-10ß, in adult tissues, including the brain, heart, kidney, liver, lung, muscles, and spleen (Fig. 6A). The identities of these transcripts were confirmed by direct sequencing (our unpublished observation). In embryos, xCaspase-10 transcripts were expressed during stages 1-20, while xCaspase-10ß increased beginning at stage 15/16, similar to the pattern seen for xCaspase-8 (Fig. 6B). Thus, the expression patterns of xCaspase-10ß and xCaspase-10 transcripts were distinct during embryogenesis. In contrast, although the expression levels varied, we detected xBid transcripts in all samples of adults and embryos (Fig. 6A,B).

View larger version (61K): [in this window] [in a new window]   Figure 6  Expression profiles of xCaspase-10ß and xBid in adult tissues and during early embryogenesis. (A, B) Total RNAs isolated from the brain, heart, kidney, liver, lung, muscles, and spleen of adult frogs (A) and from embryos at stages 1, 8.5/9, 10.25, 11/12, 15/16, 20, 25, and 32 (B) were subjected to RT-PCR analysis of xCaspase-10ß, xCaspase-10, xCaspase-8, and xBid transcript expression. The resulting PCR products were resolved by 2.5% agarose gel electrophoresis. As an internal control, EF1 (A) and ornithine decarboxylase (ODC) (B) transcripts were examined. Molecular weight markers (M.W.M.) were applied in lane 1 of both panels.

Functional analysis of xCaspase-10ß in embryonic development

To determine if xCaspase-10ß is critical to embryogenesis, we examined the phenotype of embryos injected with an mRNA encoding a protease-deficient form of xCaspase-10ß. This molecule is expected to act as a dominant-negative mutant by interacting with and sequestering endogenous xCaspase-10ß. Indeed, HeLa cells expressing a protease-deficient xCaspase-10ß, xCaspase-10ß(C384S) exhibited resistance to Fas-mediated apoptotic stimulus compared with control cells, resulting in an increased number of EGFP-positive cells even in the presence of anti-Fas antibody (Fig. 7A). This result suggests that an xCaspase-10ß(C384S) molecule is able to function as a dominant-negative molecule for initiator caspases. Following injection of the mRNA into four-cell-stage blastocysts, we analyzed the morphology of embryos after developing to stage 45. Injection into the equatorial areas of the two dorsal blastomeres resulted in embryos with shortened trunks in comparison with controls (Fig. 7B,a,c). We also observed edema in a number of embryos (Fig. 7B,b,c; shown by arrows). In contrast, abnormal embryos associated with scrape of the abdomen were observed after microinjection of the mRNA into the ventral area (Fig. 7B,d,e; shown by arrowheads). These phenotypes occurred in dose-dependent manner, but were not observed in embryos subjected to microinjection of control mRNA (our unpublished observation). These data (summarized in Fig. 7C) suggested that irregular development of embryos occurs at high efficiency following ectopic expression of protease-deficient xCaspase-10ß. These results suggest that xCaspase-10ß is involved in the early development of Xenopus embryos.

View larger version (42K): [in this window] [in a new window]   Figure 7  A protease-deficient xCaspase-10ß causes the irregular development of Xenopus embryos. (A) Anti-apoptotic assay of an xCaspase-10ß mutant. HeLa cells were transfected with pCSII-xCaspase-10ß(C384S) (panels c and d) or control vector (panels a and b) together with pEGFP-C1, cultured for 24 h and treated with (lower panels) or without (upper panels) 200 ng/mL of anti-Fas antibody CH11 and 5 µg/mL of CHX for 8 h. Viable cells were detected as EGFP-positive cells under the fluorescent microscope. (B) Morphological analysis of embryos expressing an xCaspase-10ß mutant. Wild-type embryos were injected without (a) or with 20 pg (panels b and d) or 200 pg (panels c and e) mRNA encoding a protease-deficient form of xCaspase-10ß, xCaspase-10ß(C384S), at the equatorial area of two dorsal (panels b and c) or two ventral (panels d and e) blastomeres at the four-cell-stage. Images of the developing embryos were acquired at stage 45. The arrows and arrowheads indicate the edema and the abdominal constriction of the dorsally and ventrally injected embryos, respectively. Scale bars indicate 2 mm (C). A summary of the data in (B) is presented. The numbers of embryos displaying short trunk shape, edema, or abdominal constriction were counted under the microscope. Data represent the percentages calculated from duplicate experiments. Abbreviations: D, dorsal; V, ventral.

To investigate if xBid could induce apoptosis in Xenopus embryos, we enforce expression of a truncated xBid (txBid) by microinjection of mRNA into 4-cell-stage blastocysts. We detected cell death in a region of the embryo at stage 8 (Fig. 8A). Embryos showed a hollow inside due to the loss of some cells. The area involved in the induced cell death is likely derived from the blastomere into which the txBid mRNA was injected. To confirm the pro-apoptotic activity of truncated xBid in embryos, we examined the activation of caspase-3 by txBid in embryos derived from a transgenic frog. We have established a novel transgenic Xenopus line expressing a fluorescent indicator, SCAT3, throughout the animal (Supplementary Fig. S2B). SCAT3 is a fusion of super enhanced cyan fluorescent protein (seCFP) and Venus (a variant of yellow fluorescent protein YFP) joined by a peptide linker (Supplementary Fig. S2A). This substrate is specifically cleaved at the DEVD recognition sequence within the linker by activated caspase-3 (Takemoto et al. 2003). The cleaved form of SCAT3 in embryos treated with a txBid mRNA injection during development could clearly be detected (Fig. 8B). The ratio of cleaved to intact SCAT3 significantly increased in embryos at stage 10 over the levels seen in embryos at stage 7. In contrast, no cleaved products of SCAT3 could be detected in healthy control embryos during stages 5–10. These results suggest that enforced expression of active xBid induces cell death in embryos in a caspase-3-dependent manner.

View larger version (70K): [in this window] [in a new window]   Figure 8  Enforced expression of truncated xBid induces apoptosis during development. (A) Morphological analysis of the embryos occurring cell death. Wild-type embryos were injected with 50 pg (middle panel) and 200 pg (lower panel) of truncated xBid (txBid) mRNAs into the equatorial area of two dorsal blastomeres at 4-cell-stage and photographs were taken of developing embryos at stage 8. The arrows indicate the area involved in cell death. (B) Fluorescence images of SCAT3 in transgenic embryos were acquired after injection of txBid mRNA. Cell extracts were prepared from uninjected embryos or embryos injected at the 4-cell stage with 50 pg of txBid mRNA isolated, at stages 5, 7 or 10. Cell extracts derived from whole embryos were resolved by SDS-PAGE. Fluorescence was analyzed on a fluorescent image analyzer (upper panel), followed by staining of the gels with Coomassie Brilliant Blue (lower panel). FITC-labeled molecular weight markers are visible in the right lane. An arrow indicates full-length SCAT3, while the white and black arrowheads indicate the cleaved Venus and seCFP fragments, respectively. The numbers on the fluorescent bands indicate the ratios of intact to processed SCAT3 proteins calculated by fluorescence measure using an image analyzer.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

Both caspase-8 and caspase-10 have been identified in humans, while only caspase-8 has been identified in rodents (Varfolomeev et al. 1998; Grenet et al. 1999). In amphibians, functional homologs of both caspase-8 and caspase-10 have been identified (Nakajima et al. 2000; Sakamaki et al. 2004). In this study, we identified yet another functional initiator caspase, xCaspase-10ß. By RT-PCR analysis, we found that xCaspase-10ß and xCaspase-10 exhibit distinct expression patterns in the early stages of embryogenesis (Fig. 6B). xCaspase-10ß increased markedly beginning in the neurula stage, while the expression of xCaspase-10 was maternally derived in eggs and decreased during the neurula stage. In Xenopus laevis, two forms of Smad4 (Smad and Smadß) have been identified (Masuyama et al. 1999). The exchange of Smadß for Smad expression during embryogenesis is similar to that observed between xCaspase-10 and xCaspase-10ß. xCaspase-10 and xCaspase-10ß are likely transcribed from two distinct genes. Physical map analysis of the caspase-8 and caspase-10 genes in several species revealed that the genomes of chicken (Gallus gallus) and West African clawed frog (Xenopus tropicalis) include an additional caspase-8/10-like gene between these two genes (our unpublished observation). xCaspase-10ß and xCaspase-10 may correspond to the caspase-10 and caspase-8/10-like genes observed in these animals, respectively. It is also possible that these are paralogues in tetraploid animals. Further genomic analysis will be necessary to clarify the conservation and diversification of initiator caspases throughout evolution. Importantly, the expression pattern of xCaspase-10ß coincided with that of xCaspase-8, but not xCaspase-10, suggesting synchronized expression in embryos. Ectopic expression of a protease-deficient xCaspase-10ß mutant molecule in developing embryos disrupted organogenesis by inhibiting the function of the endogenous xCaspase-10ß (Fig. 7). In these abnormal phenotypes, edema is thought to be caused by defect in the blood vessels (Bergers & Song 2005), indirectly suggesting the involvement of caspase-10ß in the development of the cardiovascular system. Caspase-8-deficient mouse embryos display malformations of cardiogenesis and angiogenesis (Varfolomeev et al. 1998; Sakamaki et al. 2002). Thus, our study suggests the involvement of caspase-10/caspase-10ß in the early embryonic development of animals. In humans, caspase-10 is unable to substitute for caspase-8 function in death receptor-mediated apoptotic signaling (Sprick et al. 2002). Defects in caspase-10 cause autoimmune disease (Wang et al. 1999), suggesting that caspase-8 is unable to compensate for this deficiency. In Xenopus, however, the possibility remains that the xCaspase-10ß mutant influenced the activity of xCaspase-8 and xCaspase-10 in embryos through homophilic DED interactions. Consequently, defining the distinct and complimentary roles during development of amphibian xCaspase-10ß, xCaspase-10, and xCaspase-8 will be important in understanding the physiological roles of these initiator caspases in vertebrates.

The previous report showed that mouse truncated Bid is also able to induce apoptosis in a caspase-independent manner and Bcl-XL sequesters killing activity of this molecule (Cheng et al. 2001). In this study, however, we obtained contradicting results using xBid (Fig. 2). Mammalian Bid can interact with Bax and Bcl-XL, as well as with lipids, such as cardiolipin. These additional interactions confer a supplementary role in the induction of cell death (Esposti 2002). It is likely that xBid either cannot interact or has only weak interactions with Bcl-XL and lipids, but strongly promotes caspase-9-dependent intrinsic apoptotic signaling in mammalian cells. Although the genes encoding the pro-apoptotic molecules Bak and Bok and the anti-apoptotic protein Bcl-XL, all members of the Bcl-2 family, have been identified within the genome of the ascidian, Ciona (Dehal et al. 2002), we could not find a homolog of bid, as reported by Bates (2004). This evidence supports the possibility that Bid first appeared evolutionarily in vertebrates. A database search identified Bid from several species of fish and other vertebrates, with conservation of both the BH-3 domain and the initiator caspase recognition sequence in all animals examined (Supplementary Fig. S3). Phosphorylation of mouse Bid Ser61 and Ser64 by casein kinase attenuates caspase-8-mediated cleavage (Desagher et al. 2001). These amino acid residues are unlikely to be conserved in a subset of vertebrate species including Xenopus. Thus, the existence of Bid from fish to mammals suggests a common physiological role in vertebrates associating with diversified regulations in some animals such as rodents.

Fluorescence analysis of fusions of xCaspase-10ß and xBid with EGFP enabled us to observe the subcellular localization of these molecules upon apoptotic stimulation. Death-effector filament formation occurs in cells undergoing apoptosis (Siegel et al. 1998); following induction of apoptosis with an anti-Fas antibody, we also observed the generation of death-effector filaments by EGFP-xCaspase10ß in those cells surviving due to inhibition of cell death by z-VAD-fmk treatment (Fig. 2E). This result suggests that xCaspase-10ß oligomerizes upon encountering death-receptor-mediated signals. Furthermore, we observed the translocation of an xBid-EGFP fusion into mitochondria by fluorescence microscopy. A previous study reported that, in mammalian cells, fusions of human Bid with YFP and CFP at the N- and C-termini is cleaved upon Fas-mediated apoptotic signaling, measured by the loss of fluorescent resonance energy transfer (FRET) (Onuki et al. 2002). Thus, the fluorescent labeling of apoptotic molecules involved in the extrinsic pathway permits the monitoring of the molecular dynamics associated apoptosis. In this study, we established a novel transgenic frog line that is useful for monitoring the activation of effector caspases in vivo. This transgenic line expresses the SCAT3 hybrid molecule, a fusion of CFP and Venus joined by a peptide linker, in the whole bodies of adults (Supplementary Fig. S2B) and in embryos at various developmental stages (our unpublished observation). This system enables caspase activation to be monitored in vivo throughout an animal's lifetime. In mammalian cell lines, as SCAT3 is specifically cleaved by activated caspase-3 and results in the loss of FRET, it enabled to detect cascapse-3 activation by measuring the dynamics of fluorescence (Takemoto et al. 2003). We demonstrated that enforced apoptosis induced SCAT3 cleavage, which was associated with cell death during development. We also examined the dynamics of FRET in the degenerating tail of transgenic animals. Detection of a ratio change in FRET intensity suggested cleavage of SCAT3 by caspase-3 (our unpublished observation). As the activation of caspase-3 in the degenerating tail has already been established (Das et al. 2002), our transgenic line may serve as a method to image caspase activation by monitoring changes in FRET in living animals. This system proves to be an extremely useful methodology for the biomonitoring of apoptosis in living organisms.

Oligomerization via interactions of DEDs is essential for the activation of initiator caspases. In this study, we observed the interactions of xCaspase-10ß with both xCaspase-10 and xCaspase-8 via homophilic interactions of their DEDs (Fig. 5). Enforced dimerization of caspase-8 and caspase-10 by addition of an FKBP-binding domain fails to activate these proteases (Chen et al. 2002). As we have not yet examined the catalytic activity of complexes of initiator caspases in Xenopus, it is difficult to confirm the physiological roles of heterologous complex of xCaspase-10ß, xCaspase-10, and xCaspase-8. It was recently reported that the DED-containing protein cFLIP interacts with and modulates caspase-8 activation (Micheau et al. 2002). The resultant heterodimer can cleave distinct substrates, such as RIP, implicating the existence of interactions between xCaspase-10ß, xCaspase-10, and xCaspase-8 with differential activities and substrate specificities in vivo.

The Xenopus system is a powerful tool for the dissection of both the basic mechanisms and the complexities of apoptosis, especially those occurring during development. Our studies clarified that the components required for the extrinsic apoptotic signaling pathway, FADD and Bid, affect embryonic development. FADD and Bid, however, exhibit differential killing activities, despite similar expression of their transcripts at early stages of development. Exogenous expression of truncated xBid in embryos induced apoptosis at stage 7 by activating the intrinsic apoptotic pathway (Fig. 8). Over-expression of FADD, however, induced cell death at later stages (approximately stage 18) (our unpublished observation). As xCaspase-10ß and xCaspase-8, but not xCaspase-10, were transcribed in stages 15-20 (Fig. 6B), these molecules may be required for the activity of FADD in vivo. The expression of both xCaspase-10 (and/or xCaspase-10ß) and FADD is up-regulated in the degenerating tail during metamorphosis (Rowe et al. 2005). The mitochondria-dependent intrinsic apoptotic pathway, which is triggered by Bid, is involved in the removal of abnormally developing embryos during the early gastrula transition (stage 10) (Van Stry et al. 2004). In Xenopus, caspase-2 functions in the clearance of metabolically inactivated oocytes/eggs (Nutt et al. 2005). As Bid is essential for caspase-2-induced apoptosis (Gao et al. 2005), Bid may function as a downstream activator in this event. Our study also supports a role for Bid in these processes. Thus, the apoptosis-inducing machinery is already established early in embryogenesis. Consequently, the extrinsic and intrinsic apoptotic pathways may play distinct physiological roles during development.

	Experimental procedures

Top Abstract Introduction Results Discussion Experimental procedures References

 
Animals, embryos and cell lines

Adult wild-type Xenopus laevis were purchased from Hamamatsu Seibutsukyozai Co. (Shizuoka, Japan). In vitro fertilization of Xenopus eggs was performed as described (Suzuki et al. 1994). Fertilized embryos were dejellied in 3% cysteine hydrochloride, washed several times in water, and used for mRNA microinjection, whole-mount in situ hybridization, and RT-PCR analysis. Developing embryo stages were determined according to the scheme described by Nieuwkoop & Faber (1967). Mouse embryonic fibroblasts (MEF cells) isolated from the wild-type, caspase-8-deficient (Sakamaki et al. 2002), and caspase-9-deficient (Kuida et al. 1998) embryos, human cervical carcinoma HeLa-S3 (HeLa) cells, and two variants of human embryonal kidney 293 (HEK293) cells, HEK293Fas and HEK293T cells, that express high levels of Fas and large T antigen, respectively, were cultured in Dulbecco's Modified Eagle's medium supplemented with 10% fetal calf serum.

Database searches and DNA sequencing

To identify Xenopus homologs of DED-containing molecules and the BH3-only molecule Bid in the GENBANK database, we utilized BLAST software (Altschul et al. 1990). Two Xenopus EST clones (GENBANK accession nos. BJ063173 and BJ0590094) were identified using an amino acid sequence corresponding to the DED of human FADD as a probe. A single EST clone (GENBANK accession no. AW641666) was homologous to human Bid. The nucleotide sequences of both strands of these clones were confirmed by DyeDeoxyterminator Cycle sequencing (Applied Biosystems Inc., Foster City, CA, USA) using automated DNA sequencers (PRISM^TM 310, Applied Biosystems Inc. and LI-COR 4000, LI-COR Biosciences, Lincoln, NE, USA). The complete sequences of these Xenopus EST clones have been deposited as the GENBANK accession nos. AY519262 and AY518731, respectively. We compared the aligned amino acid sequences of the Xenopus and human molecules using CLUSTAL W software (Higgins & Sharp 1988).

Sequence alignment of Bid

To align the vertebrate Bid sequences, we cited the amino acid sequences previously published for human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), and chicken (Gallus gallus) Bid (GENBANK accession nos. NP_001187 [GenBank] , NP_031570 [GenBank] , NP_073175 [GenBank] , and NP_989883 [GenBank] , respectively). We also predicted the Bid amino acid sequences of macaque monkey (Macaca mulatta) (GENBANK accession nos. CB554325 and CB554668), cows (Bos taurus) (GENBANK accession no. CK831238), pigs (Sus scrofa) (GENBANK accession no. CK460098), salamanders (Math Ambystoma mexicanum) (GENBANK accession no. CN036937), frogs (Xenopus tropicalis) (GENBANK accession no. AL774229), and five fish species, including Danio rerio (GENBANK accession nos. CO359216 and AL924614), Ictalurus punctatus (GENBANK accession no. CB937949), Oryzias latipes (GENBANK accession no. BJ707272), Oncorhynchus mykiss (GENBANK accession no. CA350756), and Salmo salar (GENBANK accession no. CB513070) from EST databases. To compare the aligned amino acid sequences, we utilized CLUSTAL W software (Higgins & Sharp 1988).

Construction and transfection of expression vectors

To express a fusion protein of Xenopus caspase-10ß (xCaspase-10ß) and EGFP in mammalian cell lines, the DNA fragment containing the PCR-amplified caspase-10ß cDNA was inserted into pEGFP-C1 (BD-Clontech, Palo Alto, CA, USA). To express Flag-tagged xCaspase-10ß, caspase-10ß cDNA was cloned into pME18S or pCAGGS (Niwa et al. 1991; Sakamaki et al. 1992) with the Flag-tag DNA sequence. A protease-deficient xCaspase-10ß mutant, xCaspase-10ß(C384S), was generated by replacing the cysteine residue (amino acid 384) in the QACQG active site of the protease region with serine by exchange of the PCR-amplified DNA fragment containing the mutant sequence. The resulting mutant xCaspase-10ß(C384S) cDNA was then inserted into the pCSII vector. To express a fusion of the truncated form of Xenopus Bid (xBid) with EGFP, we cloned the BglII-SalI DNA fragment of Xenopus bid cDNA into pEGFP-C1. This truncated form of xBid (txBid) was also inserted into the pCSII vector. To monitor mitochondrial translocation and processing of xBid, we generated a plasmid construct, pxBid-EGFP, by inserting the Xenopus bid-coding region into pEGFP-N1 (BD-Clontech). A mutant form of xBid, xBid(D52A), was generated by replacing the putative site (IETD) recognized by initiator caspases, with the sequence IETA and subcloned into pEGFP-N1 to create pxBid(D52A)-EGFP. The GFP-mCasp8 DNA fragment, encoding a mouse caspase-8 cDNA (Sakamaki et al. 1998) fusion with the gfp gene, the Flag-tagged mouse Fadd cDNA (Hsu et al. 1996), the kind gift of Dr D. Goeddel (Amgen), and the mouse bcl-xL cDNA were cloned into pME18S. The pCX-p35 plasmid, carrying the baculovirus p35 gene, has been described (Sakamaki et al. 2004).

Transfection of the constructed plasmid DNAs into cells was performed using LipofectAMINE PLUS Reagent (Invitrogen Corp., Carlsbad, CA, USA), according to the manufacturer's protocol.

Construction of the transgene and generation of transgenic frogs

To construct a plasmid carrying the CAG-SCAT3 transgene (Takemoto et al. 2003), we inserted the DNA fragment encoding a hybrid protein composed of seCFP and Venus joined by a linker peptide followed by the rabbit ß-globin polyadenylation (polyA) signal sequence into the pCAGGS expression vector (Niwa et al. 1991). SCAT3 is a fluorescent indicator allowing the detection of cell death. This substrate is cleaved by activated caspase-3 in mammalian cells. To generate transgenic frogs expressing SCAT3, we utilized the established method (Kroll & Amaya 1996; Amaya & Kroll 1999), with the modifications detailed previously (Sakamaki et al. 2004). We established a transgenic line expressing SCAT3 throughout the whole body of adult animals and confirmed that SCAT3 is also expressed in eggs (our unpublished data). In this study, we used F1 generation females from the established transgenic frog lines for experiments.

Pro-apoptotic activity assays of xCaspase10ß and xBid in cell cultures

Cells were transfected either with plasmids carrying EGFP-xCaspase10ß or EGFP-txBid in the absence or presence of 100 µM carbobenzoyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) (Kamiya Biomedical Co., Seattle, WA, USA). A subset of cells was transfected with txBid and a plasmid encoding mouse Bcl-XL. Twenty-four hours after transfection, cells were fixed in PBS containing 3.7% formaldehyde. The number of EGFP-positive cells was assessed in photographs taken under the microscope (DMIRE2, Leica Microsystems, Wetzlar, Germany). MEF cells isolated from caspase-8-deficient and caspase-9-deficient mice were transfected with EGFP-txBid, cultured for 24 h, and examined by microscopy. To examine the ability of a protease-deficient xCaspase-10ß mutant, xCaspase-10ß(C384S) as a dominant-negative molecule, HeLa cells were co-transfected with pCSII-xCaspase-10ß(C384S) and pEGFP-C1. After a 24 h culture period, cells were treated for an additional 8 h with 200 ng/mL of an agonistic anti-human Fas antibody, CH11 (MBL, Nagoya, Japan) in the presence of 5 µg/mL cycloheximide (CHX). Similarly, to examine the anti-apoptotic activity of Bcl-XL in HeLa cells, cells were co-transfected with Bcl-XL and pEGFP-C1. After a 24 h culture period, cells were treated with 200 ng/mL of CH11 and 5 µg/mL of CHX for 24 h.

Cytological analysis by confocal laser scanning microscopy

After co-transfection of HeLa cells with pDesRed2-Mito (BD-Clontech) with either pxBid-EGFP or pxBid(D52A)-EGFP, apoptosis was induced with 200 ng/mL of an anti-Fas antibody and 5 µg/mL of CHX. The subcellular localization of xBid was examined by confocal laser scanning microscopy (TCS SP2, Leica Microsystems).

Synthesis of active xCaspase-10ß recombinant protein and protease activity assay in vitro

To generate recombinant xCasp10ß and xCasp8, DNA sequences encoding the protease domains were amplified by PCR. The resulting fragments were then cloned into the pMAL-c2E plasmid (New England Biolabs, Beverly, MA, USA). As a negative control, we also generated a construct encoding xCasp10ß(p21), which only contained the large, catalytically inactive subunit. After transformation of E. coli Rosetta (DE3) (Novagen, Madison, WI, USA) with the appropriate constructs, maltose-binding protein (MBP)-tagged recombinants were produced following induction of protein expression with IPTG. Recombinant proteins were purified with amylose resin (New England Biolabs), according to the manufacturer's instructions. To detect protease activity, assays examined cleavage of an acetyl-Ile-Glu-Thr-Asp--(4-ethylcoumaryl-7-amide) (Ac-IETD-MCA) substrate (50 µM) (Peptide Institute Inc., Osaka, Japan) in the presence of 1 µg recombinant enzyme in 200 µL of protease assay buffer (50 mM PIPES (pH 7.2), 10% sucrose, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, and 10 mM dithiothreitol) at 37 °C. The production of 7-amino-4-methylcoumarin (AMC) from the substrate was measured at the indicated times in a plate reader (ARVO-SX, PerkinElmer Life Science-Wallac Oy, Turku, Finland) using excitation and emission wavelengths of 355 nm and 460 nm, respectively. All values were assessed in triplicate. To insure for any effect of the MBP modification, we also produced a recombinant xCasp10ß-His, containing only the protease domain fused to His-tags at both termini, in the transformed DE3 cells. Recombinant proteins were purified using nickel-NTA column chromatography according to the manufacturer's recommendations (Qiagen, Valencia, CA, USA).

To analyze the processing activity of xCasp10ß protein against xBid in vitro, we synthesized both wild-type and mutant forms of xBid fused to EGFP in a cell-free system as described (Sawasaki et al. 2002). Briefly, xBid-EGFP or xBid(D52A)-EGFP were transcribed from the SP6 promoter of the pEU expression vector; the resulting transcripts were translated in vitro within wheat embryo extracts. An in vitro cleavage assay mixed active xCasp10ß-His with xBid-EGFP or xBid(D52A)-EGFP in conjunction without or with 200 µM z-VAD-fmk in the protease assay buffer at 37 °C for 3 h. Following resolution by SDS-PAGE, the processed products were detected with an imaging analyzer (LAS-3000, FUJIFILM, Tokyo, Japan). For estimation of the molecular size of products, FITC-labeled molecular weight markers (SP-0130, APRO Life Science Institute, Tokushima, Japan) were used.

Immunoblotting and immunoprecipitation

To examine xBid cleavage following apoptotic stimulation, xBid and the xBid(D52A) mutant were transiently transfected into HEK293Fas cells. Two days later, transfectants were incubated in the presence or absence of 200 ng/mL anti-Fas antibody for 6 h. Samples were then suspended in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1% TritonX-100, and 0.5% sodium deoxycholate. Cell extracts were resolved by SDS-PAGE and examined by immunoblotting with an anti-GFP antibody (IE4) (MBL) as described (Sakamaki et al. 2004).

To analyze the interactions between xCaspase-10ß and Xenopus and human apoptotic signaling molecules, HEK293T cells were transiently transfected with an expression vector encoding either EGFP-xCaspase10ß or GFP-mouse caspase-8 without or with Flag-xFADD or Flag-mouse FADD. After 24 h of cultivation, transfectants were suspended in lysis buffer; proteins were immunoprecipitated from cell lysates with an anti-Flag antibody (M2) (Sigma Chemicals Co., St. Louis, MO, USA) and Protein-A Sepharose (Amersham Biosciences, Arlington Heights, IL, USA). Immunoprecipitates were analyzed by SDS-PAGE and immunoblotting with anti-Flag and anti-GFP antibodies. To examine the formation of homodimeric complexes of xCaspase10ß or heterodimeric complexes of xCaspase-10ß with xCaspase-10 or xCaspase-8, we co-transfected a plasmid encoding EGFP-xCaspase10ß into HEK293T cells without or with plasmids encoding Flag-xCaspase10ß, Myc-xCaspase-10, or HA-xCaspase-8 (Sakamaki et al. 2004). After 24 h cultivation period, transfectants were suspended in lysis buffer. Proteins were immunoprecipitated from cell lysates with an anti-GFP antibody. Immunoprecipitates were then analyzed by SDS-PAGE and immunoblotting with anti-GFP, anti-Flag, anti-Myc (9E10) (ATCC, Manassas, VA, USA), and anti-HA (ab-hatag) (InvivoGen, San Diego, CA, USA) antibodies. Bound antibody was visualized with Mouse IgG TrueBlot^TM (eBioscience, San Diego, CA, USA) or horseradish peroxidase-conjugated anti-mouse IgG antibody (Cell Signaling Technology Inc., Beverly, MA, USA). For preventing apoptosis of transfectants, the p35 gene derived from baculovirus, which is well known as a strong inhibitor of caspases (Zhou et al. 1998), was introduced into all examined samples with an expression vector (Sakamaki et al. 2004).

RT-PCR analysis

Total RNAs were isolated from several tissues of adult animals and from embryos at various stages with ISOGEN reagent (Nippongene, Toyama, Japan), according to the manufacturer's instructions. First strand cDNAs were synthesized from each RNA sample (1 µg) using random hexameric primers on Ready-To-Go RT-PCR beads (Pharmacia), according to the manufacturer's protocol. These cDNAs were amplified with several sets of primers to detect both xCaspase-10ß and xCaspase-10 (forward: 5'-CAAGACCTTTCTGGATGTGTTGTG-3', reverse: 5'-TTTGCTTGAAACCTGGATGGAG-3'), xCaspase-8 (forward: 5'-CGCTTCTATACTGGAAATATTCTT-3', reverse: 5'-TTGTACTGAAATCTTCTCAAAT-3'), and xBid (forward: 5'-GGAAACGTCCAATTAAGATCT-3', reverse: 5'-CCTCTGTCTGGCGAGACGCTC-3'). As positive controls in embryos and adult tissues, we examined transcription of EF1 and ornithine decarboxylase using two sets of primers (forward: 5'-CAGATTGGTGCTGGATATGC-3', reverse: 5'-ACTGCCTTGATGACTCCTAG-3') and (forward: 5'-CAGCTAGCTGTGGTGTGG-3', reverse: 5'-CAACATGGAAACTCACACC-3'), respectively. PCR was performed as previously described (Sakamaki et al. 2004). Amplified PCR products were analyzed by 2.5% agarose gel electrophoresis.

Microinjection of xCaspase-10ß(C384S) and txBid mRNAs

Capped mRNAs encoding the protease-deficient xCaspase-10ß(C384S) mutant or the truncated xBid were synthesized from pCSII plasmid construct templates using an mMESSAGE mMACHINE SP6 kit (Ambion, Austin, TX, USA). mRNAs were purified using a Sephadex G-50 column (Amersham Biosciences) as described (Yamamoto et al. 2001). Synthetic mRNAs were microinjected at either 20, 50 or 200 pg into the equatorial areas of two dorsal or two ventral blastomeres at the four-cell-stage. The injected embryos were cultured in 3% Ficoll and 0.1x Steinberg's solution (Yamamoto et al. 2001). Photographs were acquired at the appropriate stages of development.

Fluorescent analyses of cell extracts of transgenic animals

After mRNA microinjection, SCAT3-expressing embryos developed to the indicated stages and were lyzed in lysis buffer in the presence of a protease inhibitor cocktail (Nacalai tesque, Kyoto). Whole cell lysates were then obtained by centrifugation. After resolution of the extracts by SDS-PAGE without heat denaturation, the fluorescence was analyzed using an imaging analyzer (Typhoon 9410, Amersham Biosciences). Then, gels were stained with 0.2% Coomassie Brilliant Blue-R250, 40% methanol, and 10% acetic acid and destained in a mixture of 40% methanol and 10% acetic acid.

Whole-mount in situ hybridization

Embryos were fixed in 4% paraformaldehyde in 0.1 M potassium phosphate buffer (pH 7.8), washed with PBS. Digoxigenin-labeled RNA probes were prepared according to the manufacturer's recommendations (Roche Diagnostics, Mannheim, Germany). Whole-mount in situ hybridization of various stages of embryos was performed as previously described (Harland 1991). Hybridized RNAs were detected with alkaline-phosphatase-conjugated anti-DIG antibody (Roche) and developed using BM purple (Roche).

	Acknowledgements

 
The authors are grateful to Drs D. Goeddel (Amgen), Y. Tsujimoto (Osaka University) and M. Miura (University of Tokyo) for providing mouse FADD and Bcl-XL cDNAs and a SCAT3 construct, and to Drs T.S. Yamamoto (National Institute for Basic Biology) and Y. Satou and S. Yonehara (Kyoto University) for their technical assistance and advice and experimental support. A part of this work was supported by a Grant-in-Aid for Creative Scientific Research to N.M and K.S. from the Japan Society for the Promotion of Science (13GS0008).

	Footnotes

 
Communicated by: Shigekazu Nagata

^* Correspondence: E-mail: ksakamak{at}lif.kyoto-u.ac.jp

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