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¹ Department of Animal Development and Physiology, Graduate School of Biostudies, Kyoto University, Kyoto 606-8507, Japan
² Department of Developmental Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan
³ Division of Embryology and Genetics, Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Higashihiroshima 739-8526, Japan
⁴ Department of Zoology, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
⁵ Department of Science for Laboratory Animal Experimentation, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan

	Abstract

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FADD is an adaptor protein that transmits apoptotic signals from death receptors such as Fas to downstream initiator caspases in mammals. We have identified and characterized the Xenopus orthologue of mammalian FADD (xFADD). xFADD contains both a death effector domain (DED) and a death domain (DD) that are structurally homologous to those of mammalian FADD. We observed xFADD binding to Xenopus caspase-8 and caspase-10 as well as to human caspase-8 and Fas through interactions with their homophilic DED and DD domains. When over-expressed, xFADD was also able to induce apoptosis in wild-type mouse embryonic fibroblasts (MEF), but not in caspase-8-deficient MEF cells. In contrast, DED-deficient xFADD (xFADDdn) acted as a dominant-negative mutant and prevented Fas-mediated apoptosis in mammalian cell lines. These results indicate that xFADD transmits apoptotic signals from Fas to caspase-8. Furthermore, we found that transgenic animals expressing xFADD in the developing heart or eye under the control of tissue-specific promoters show abnormal phenotypes. Taken together, these results suggest that xFADD can substitute functionally for its mammalian homologue in death receptor-mediated apoptosis, and we suggest that xFADD functions as a pro-apoptotic adaptor molecule in frogs. Thus, the structural and functional similarities between xFADD and mammalian FADD provide evidence that the apoptotic pathways are evolutionally conserved across vertebrate species.

	Introduction

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Apoptosis, or programmed cell death, is a widespread biological phenomenon in multicellular organisms that is important for tissue morphogenesis during development and the maintenance of cellular homeostasis in adulthood (Raff 1992; Jacobson et al. 1997; Vaux & Korsmeyer 1999). The host immune response employs apoptosis in its surveillance of and protection from pathogens (Abbas 1996). In all of these circumstances, a family of cysteine proteases known as caspases are activated and then work as executors to induce the proteolytic cleavage of many critical proteins leading to cell death (Thornberry & Lazebnik 1998). The activation of caspases is the point of convergence of two major signalling pathways. One pathway is initiated by binding to cell surface ‘death receptors’ including Fas (APO-1/CD95), tumour necrosis factor receptor type 1 (TNFR1), death receptor 3 (DR3), and two receptors for TNF-related apoptosis-inducing ligand (TRAIL-R1 and -R2) (Muzio et al. 1998; Ashkenazi & Dixit 1998). Another apoptotic signalling pathway is triggered by cytochrome c release from the mitochondria into the cytosol (Liu et al. 1996).

FADD (Fas-associated death domain protein, also known as MORT1) is an adaptor molecule that interacts upon apoptotic stimulation with the death receptors including Fas, TNFR1 and TRAIL (Boldin et al. 1995; Chinnaiyan et al. 1995; Zhang & Winoto 1996; Strasser & Newton 1999). FADD is a well-characterized component of the Fas-mediated apoptotic signalling pathway. Following Fas ligation, FADD interacts with Fas through its C-terminal ‘death domain (DD)’ and recruits an initiator caspase, pro-caspase-8 (also known as FLICE/MACH1/Mch5), by the mutual interaction of the ‘death effector domains (DEDs)’ found in FADD and pro-caspase-8 (Boldin et al. 1996; Muzio et al. 1996). FADD thus functions as a connector molecule in a complex with receptor and pro-caspase-8, referred to as the ‘death-inducing signalling complex (DISC)’ (Kischkel et al. 1995). In this complex, pro-caspase-8 undergoes autoprocessing and converts into an active form. Activated caspase-8 is then released from the complex and cleaves the downstream effector caspases or Bid leading to cell death (Hirata et al. 1998; Chou et al. 1999; McDonnell et al. 1999). Thus, FADD is indispensable in Fas-mediated apoptotic signalling. Recently, FADD-deficient mice were generated and are reported to die in utero (Zhang et al. 1998; Yeh et al. 1998). Although the specific physiological roles of FADD in embryonic development are still unknown, this finding suggests that FADD is essential during embryogenesis.

In amphibians, apoptosis plays a pivotal role in tadpole tail degeneration during metamorphosis (Tata 1994; Nishikawa et al. 1998). During this developmental period, apoptosis is also observed in the gills and larval intestine (Nishikawa et al. 1998; Rollins-Smith 1998; Shi & Ishizuya-Oka 2000). Another apoptotic program is initiated at the onset of gastrulation in Xenopus (Hensey & Gautier 1998, 1999; Charrier et al. 1999). Although these apoptotic events occurs at different stages, several lines of evidence suggest that apoptotic signalling pathways converge to a common final pathway, for which a number of evolutionary conserved genes have been identified. An anti-apoptotic molecule, Bcl-XL has been identified, and a pro-apoptotic molecule, Bax, has also been detected in Xenopus (Cruz-Reyes & Tata 1995; Sachs et al. 1997). Furthermore, eight types of caspases exhibiting high homology to their respective mammalian counterparts have been isolated (Yaoita & Nakajima 1997; Nakajima et al. 2000). The expression of most of these caspases is increased in regressing organs during metamorphosis (Nakajima et al. 2000). Thus, the machinery for apoptotic signalling pathways seems to be conserved between amphibians and mammals.

Studying FADD in a non-mammalian species would clarify the generic role of FADD in vertebrates. Xenopus is generally a suitable model organism for the study of many developmental phenomena. Since it would thus also be appropriate for the elucidation of common mechanism(s) of programmed cell death in vertebrates, we sought to identify and characterize an amphibian orthologue of FADD. In the present study, we report the cloning of the Xenopus FADD (xFADD) gene by DNA database searching, as well as its expression and function. Furthermore, we show that the exogenous expression of xFADD in the eye or heart of Xenopus tadpoles affects the organization of those organs. Our study clearly demonstrates that xFADD has structural and functional similarities to its mammalian homologue and suggests that FADD function is evolutionarily conserved among vertebrates.

	Results

Top Abstract Introduction Results Discussion Experimental procedures References

 
Identification of the gene encoding Xenopus FADD

A search for Xenopus DED motif-containing proteins in the GENBANK EST database yielded one cDNA clone (db61b04) that encodes a DED domain similar to those of mammalian FADD and caspase-8. We further sequenced this clone and found one open reading frame encoding 188 amino acids and a death domain (DD) at its carboxyl terminus (Fig. 1A). This sequence clearly possesses an overall structural similarity to the mammalian FADD (40% identity and 62% similarity at amino acid sequence) but not caspase-8 (Fig. 1B). Searching the GENBANK database with the predicted amino acid sequence of the Xenopus orthologue revealed that the avian homologue (GENBANK accession no. XP_421073 [GenBank] ) was most similar, being 46% identical and 67% similar, suggesting the evolutionary conservation of FADD among vertebrates (data not shown). On the other hand, we found significant differences between Xenopus and mammalian FADD sequences. Whereas the human protein possesses a serine at 194 that is involved in cell cycle (Alappat et al. 2003; Hua et al. 2003), the Xenopus orthologue does not, due to a 20-amino acid truncation at its carboxyl terminus. Based on these results, we concluded that this cDNA clone encodes Xenopus FADD (xFADD), which contains the evolutionally conserved DED and DD domains.

View larger version (46K): [in this window] [in a new window]   Figure 1  Primary structure of Xenopus FADD. (A) Nucleotide and predicted amino acid sequences of Xenopus FADD cDNA clone db61b04 are shown. Numbers on the left side of the sequence indicate nucleotide positions, and numbers on the right side indicate amino acid positions. The normal and bold lines under the amino acid sequence indicate the regions of the DED and DD domains, respectively. (B) Amino acid sequences of Xenopus and human FADDs are compared. Identical and similar amino acids between Xenopus and human are indicated by black and shaded boxes, respectively.

It has been shown that over-expression of mammalian FADD induces cell death by activating the apoptotic signalling pathway (Chinnaiyan et al. 1995). Therefore, we examined whether xFADD expressed in mammalian cell lines induces apoptosis. We also examined the ability of Xenopus caspase-8 (xCaspase-8) and caspase-10 (xCaspase-10) to induce apoptosis in mammalian cells. We co-transfected either xFADD, xCaspase-8 or xCaspase-10 cDNA and the EGFP gene into human HeLa-K cells. Cell viability of transfectants was examined by detecting enhanced green fluorescent protein (EGFP)-positive cells under the microscope. Ectopic expression of xFADD resulted in a reduced number of EGFP-positive cells, suggesting that it had induced cell death (Fig. 2B). In the presence of the pan-caspase inhibitor carbobenzoyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), however, the number of EGFP-positive cells was increased and became comparable to that of transfectants expressing EGFP only (Fig. 2A,C). These data suggest that xFADD-induced cell death is in fact apoptotic and is associated with caspase activation. Similarly, Xenopus xCaspase-8 and xCaspase-10 induced cell death in transfected HeLa-K cells (Fig. 2D,F). Cell death induction by xCaspase-8 and xCaspase-10 was also prevented by z-VAD-fmk in these cells (Fig. 2E,G). We further characterized transfectants expressing xFADD, xCaspase-8 or xCaspase-10 with a quantitative flow cytometric method for determining the apoptotic potential of these molecules. As shown in Fig. 2H, over-expression of xFADD, xCaspase-8 and xCaspase-10 in HeLa-K cells increased the cellular population in a sub-G1 fraction, indicating one of apoptotic characteristics (Lamm et al. 1997). In contrast, coexpression of another pan-caspase inhibitor, p35 with xFADD reduced it. Thus, Xenopus xFADD as well as xCaspase-8 and xCaspase-10 was able to induce apoptosis in mammalian cells.

View larger version (63K): [in this window] [in a new window]   Figure 2  Induction of apoptosis in human HeLa-K cells by over-expression of Xenopus FADD (xFADD), caspase-8 (xCaspase-8) and caspase-10 (xCaspase-10). (A–G) Plasmids carrying xFADD (B, C), xCaspase-8 (D, E) or xCaspase-10 (F, G) cDNA, or vector DNA alone (A), were co-transfected with the EGFP-expression vector, pCX-EGFP, into cells. After 24 h culture, transfectants were washed, fixed and photographed using phase contrast (left panels) and fluorescence (right panels) microscopy in the same field. Half of these transfectants were cultured in the presence of 100 µM z-VAD-fmk in medium (C, E, G). (H) The DNA content of transfectants was assessed by flow cytometry. Transfectants carrying control vector DNA (a), xFADD cDNA (b), both xFADD cDNA and the p35 gene (c), xCaspase-8 cDNA (d), or xCaspase-10 cDNA (e) were analysed by flow cytometry for the detection of the DNA content stained with propidium iodide (PI). Percentage indicates the cellular population detected in the sub-G1 fraction of the DNA content.

View larger version (49K): [in this window] [in a new window]   Figure 3  Induction of apoptosis by xFADD in wild-type MEF cells, but not caspase-8-deficient MEF cells. (A) A schematic diagram is shown of the full length and truncated mutant xFADD constructs fused to IRES (Internal Ribosomal Entry Site)-EGFP. (B) xFADD-EGFP was transfected into either wild-type MEF cells (b, c) or caspase-8-deficient MEF cells (f). The truncation mutant xFADDdn-EGFP was introduced into wild-type MEF cells (d), and the control IRES-EGFP gene was transfected into both types of MEF cells (a, e). In caspase-8-deficient MEF cells, mouse caspase-8 cDNA was co-transfected with xFADD-EGFP (g, h) or IRES-EGFP (i). Both wild-type and caspase-8-deficient MEF transfectants were also incubated with 100 µM z-VAD-fmk (c, h). After 24 h culture, cells were washed, fixed and photographed using phase contrast (left panels) and fluorescence (right panels) microscopy in the same field. (C) A summery of the data in (B) is presented. Cell viability was estimated by counting the number of EGFP-positive cells in each experiment. Data present the means and standard deviations from four experiments.

A FADD deletion mutant lacking the N-terminal DED motif functions as a dominant-negative molecule against Fas- and TNFR1-induced apoptosis (Chinnaiyan et al. 1996; Hsu et al. 1996). We examined the possible protective function of the truncated mutant, xFADDdn in human HeLa-K and SK-Hep1 cells. As shown in Fig. 4, xFADDdn acted as a dominant-negative mutant in both cell lines by preventing Fas-mediated apoptotic signalling. In HeLa-K transfectants expressing only EGFP, cells died and exhibited small apoptotic bodies to the same extent as non-transfected cells after treatment for six hours with the agonist anti-Fas antibody CH11 (Fig. 4B). In contrast, xFADDdn-EGFP-expressing cells survived even in the presence of anti-Fas antibody, whereas non-transfected cells were killed (Fig. 4D). Moreover, we examined the susceptibility to Fas-induced apoptosis of SK-Hep1 transfectants expressing xFADDdn-EGFP. These cells exhibited a reduced response to anti-Fas antibody-mediated stimulation of apoptosis, resulting in an increased number of EGFP-positive cells compared to control cells (Fig. 4E). Taken together, these results indicate that xFADDdn expression renders cells resistant to Fas-induced apoptosis.

View larger version (59K): [in this window] [in a new window]   Figure 4  Transfection of xFADD lacking the DED motif, a dominant-negative inhibitor of Fas-mediated apoptosis. (A–E) HeLa-K cells (A–D) and SK-Hep1 cells (E) were transfected with the control vector pIRES-EGFP (A, B and • in E) or the vector carrying mutant xFADD (xFADDdn) cDNA (C, D and in E) fused to the IRES-EGFP gene as shown in Fig. 3A. Transfectants were treated with (B, D) or without (A, C) 500 ng/mL of anti-human Fas antibody CH-11 and 10 µg/mL of CHX for 6 h (A–D) or treated with various concentration of CH11 and 10 µg/mL of CHX for 8 h (E). Cell viability was examined under a microscope (A–D) or calculated by measuring fluorescence in cell lysates (E). Data present the means and standard deviations from four experiments (E).

Mammalian FADD associates with caspase-8 and caspase-10 through a homophilic DED interaction (Boldin et al. 1996; Muzio et al. 1996; Vincenz & Dixit 1997). We investigated whether xFADD interacts with Xenopus xCaspase-8 and xCaspase-10. Flag-tagged xFADD, xFADDdn or mouse FADD were coexpressed with or without either xCaspase-8 or xCaspase-10 in 293T cells, and we examined protein interactions by co-immunoprecipitation. Both xCaspase-8 and xCaspase-10 were detected in immunoprecipitates with xFADD or mouse FADD, but not with DED-deficient xFADDdn (Fig. 5A). These data suggest that xFADD associates with both xCaspase-8 and xCaspase-10 through a homophilic DED interaction, and that mouse FADD is also able to interact with Xenopus caspases. Furthermore, the specificity of the FADD–caspase-8 interaction was confirmed by examining its behaviour together with human caspase-8 in 293T cells (Fig. 5B). In a demonstration of its ability to bind human caspases, xFADD, like its human counterpart, was able to co-immunoprecipitate with human caspase-8. Thus, we demonstrated that FADD and initiator caspases derived from Xenopus interact with mammalian molecules.

View larger version (38K): [in this window] [in a new window]   Figure 5  xFADD interaction with Xenopus and mammalian initiator caspases. (A) 293T cells were transfected with p35 in conjunction with empty vector (lane 1) or plasmids encoding Flag-xFADD (lanes 2–4), Flag-xFADDdn (lanes 5, 6), Flag-mouse FADD (lanes 7, 8), HA-xCaspase-8 (lanes 3, 5 and 7), or Myc-xCaspase-10 (lanes 4, 6 and 8). Twenty-four hours after transfection, cells were lysed and immunoprecipitated with an anti-Flag antibody. Immunoprecipitates and whole cell lysates were analysed by immunoblotting with anti-Flag, anti-HA and anti-Myc antibodies for the detection of xFADD, mouse FADD, xCaspase-8 and xCaspase-10, respectively. Asterisk indicates the presence of a nonspecific anti-Flag immuno-reactive molecule in the cell lysates. (B) 293T cells were transfected with p35 in conjunction with empty vector (lane 1) or plasmids encoding Flag-xFADD (lanes 2 and 4), Flag-human FADD (lanes 3 and 5), or Myc-human caspase-8 (lanes 4 and 5). Fourteen hours after transfection, cells were lysed and immunoprecipitated with an anti-Flag antibody. Immunoprecipitates and whole cell lysates were analysed by immunoblotting with anti-Myc antibody for the detection of human caspase-8. Baculovirus p35 was introduced into all cells to prevent cell death. Abbreviation, Ig: immunoglobulin.

View larger version (32K): [in this window] [in a new window]   Figure 6  Recruitment of xFADD into the DISC complex after Fas-ligation. 293Fas cells were transfected with p35 (all lanes) together with empty vector (lanes 1 and 2) or plasmids encoding Flag-xFADD (lanes 3 and 4). Twenty-four hours after transfection, cells were stimulated with (lanes 2 and 4) or without (lanes 1 and 3) 500 ng/mL of anti-Fas antibody for 40 min, lysed and immunoprecipitated with an anti-Fas antibody. Immunoprecipitates and whole cell lysates were analysed by immunoblotting with anti-Flag and anti-human Fas antibodies for the detection of xFADD and Fas, respectively.

To investigate the tissue distribution of xFADD in Xenopus, we examined xFADD transcripts in various adult tissues by RT-PCR. As shown in Fig. 7, xFADD transcripts were detected in the brain, heart, liver, lung, muscle and spleen, overlapping substantially with expression pattern seen in mice (O’Reilly et al. 2004). We also detected both xCaspase-8 and xCaspase-10 transcripts in these adult tissues with the exception of xCaspase-8 in muscle. Thus, these data suggest that the adaptor FADD and initiator caspases-8/10, which are essential components of the DISC complex, are expressed in adult frogs.

View larger version (64K): [in this window] [in a new window]   Figure 7  Tissue distribution of xFADD and xCaspases-8/10 transcripts in adult frogs. Total RNAs isolated from the brain, heart, kidney, liver, lung, muscle and spleen of adult frogs were subjected to RT-PCR analysis for the detection of xFADD, xCaspase-8 and xCaspase-10 transcripts, and the resulting PCR products were resolved by a 2.5% agarose-gel electrophoresis (lanes 1–7). As an internal control, EF1 transcripts were also examined. Negative control (no first-strand cDNA added) and molecular-weight markers (M.W.M.) are shown in lanes 8 and 9, respectively.

To examine the in vivo function of xFADD, we generated transgenic animals that exogenously expressed xFADD and examined their developmental phenotypes. We generated a transgene CA-GFP3 consisting of the chicken crystallin A (CA) promoter, GFP3 (a variant of GFP) cDNA and SV40 polyA sequence (Fig. 8A), and investigated the tissue-specific expression of GFP3 under the control of the CA promoter in transgenic animals. As shown in Fig. 8B, we detected GFP3 expression in the eye of transgenic tadpoles, suggesting that the chicken CA promoter can drive the tissue-specific transgenic expression even in amphibians. To direct eye-specific expression of xFADD, we introduced a transgene, CA-xFADD/EGFP, in which the GFP3 cDNA was substituted with the xFADD-IRES-EGFP construct, as shown in Fig. 3A (Fig. 8A), by injecting with sperm nuclei into Xenopus eggs. We observed several tadpoles at stage 45 with small or pinhead-shaped eyes (Fig. 8C). This phenotype was observed at a high frequency in transgenic animals carrying the CA-xFADD/EGFP transgene, but not with other transgenes (data not shown). We confirmed by PCR analysis that these animals with eye-defect indeed carry the transgene (Fig. 8D). Thus, exogenous expression of xFADD in the eye affected its development.

View larger version (40K): [in this window] [in a new window]   Figure 8  Effects of xFADD in Xenopus larvae. (A) A schematic structure of the constructed transgenes is shown. The transgene, CA-GFP3, is composed of the fluorescence reporter gene GFP3 and the SV40 polyA signal sequence under the control of the chicken crystallin A (CA) promoter. A second transgene, CA-xFADD/EGFP, is composed of the CA promoter, the xFADD-EGFP construct as shown in Fig. 3A, and the SV40 polyA sequence. A third transgene, XMLC2-xFADD, is composed of xFADD cDNA and SV40 polyA sequence under the control of the Xenopus myosin light chain 2 (XMLC2) promoter. Primers (P1-P5) specific to each of the transgenes were used to genotype animals by PCR. (B) Transgenic tadpoles were generated that expressed GFP3 in the eye. A transgenic tadpole carrying the CA-GFP3 transgene at stage 45 was imaged using both bright-field (left panel) and fluorescence (right panel) dissecting microscope. (C, D) Morphological and genomic analyses of transgenic tadpoles reveal xFADD expression in the eye. Xenopus embryos were injected with the CA-xFADD/EGFP transgene and allowed to develop to stage 45. Animals displaying irregular phenotypes of the developing eye were collected and observed under the microscope (C). Two tadpoles (lower and middle) exhibit pinhead-shaped and small left eyes. Transgenic animals were genotyped by PCR-amplifying genomic DNA using two sets of primers-P1 and P2, for the detection of the transgene and P6 and P7, for the detection of the genomic -crystallin gene- followed by 1.5% agarose-gel electrophoresis (D). Plasmid DNA carrying CA-xFADD/EGFP was used as a control. Molecular weight markers (M.W.M.) were applied in lane 1. (E–G) Genomic and histological analyses were performed on transgenic animals expressing xFADD in the heart. Xenopus embryos were co-injected with the transgenes XMLC2-xFADD and CA-GFP3. At stage 46/47, six animals expressing GFP3 in the eye were collected as Tg#1-Tg#6, and their anterior and posterior halves were used for histological analysis and for preparation of genomic DNA, respectively. Animals were genotyped by PCR amplification of genomic DNA using two sets of primers-P2 and P3, for detection of the XMLC2-xFADD transgene and P4 and P5, for the detection of GFP3 cDNA- followed by 1.5% agarose-gel electrophoresis (E). Plasmid DNA carrying XMLC2-xFADD or CA-GFP3 was used as control (lane 8) and molecular weight markers (M.W.M.) were applied in lane 1. Transverse sections containing the heart were prepared and triply stained with azocarmin B, aniline blue and orange G (F). All upper panels show photographs of animals carrying the XMLC2-xFADD transgene, and the lower left panel shows a photograph of an animal not carrying the XMLC2-xFADD transgene, and the lower three right panels are normal tadpoles. Scale bar indicates 100 µm. Abbreviation, v: ventricle. The ventricular volume in each animal was calculated by measuring its area in the serial sections (G). Each bar indicates the ratio of the ventricular volume of each sample to the mean of the ventricular volume of control animals.

	Discussion

Top Abstract Introduction Results Discussion Experimental procedures References

 
The present study provides a unique and direct observation of the function of the Xenopus FADD orthologue. We demonstrated that Xenopus FADD (xFADD), identified from an EST database, has a similar structure and function to mammalian FADD. xFADD interacted not only with Xenopus caspase-8 and caspase-10 but also with mammalian caspase-8 and Fas. Ectopically expressed xFADD induced apoptosis in mammalian cells but not in caspase-8-deficient cells. Moreover, a dominant-negative form of xFADD (xFADDdn) prevented Fas-induced apoptosis. Thus, xFADD was able to transmit apoptotic signals through Fas to caspase-8. In addition, we verified that the exogenous expression of xFADD in Xenopus embryos affects tissue organization during development, suggesting an important role of xFADD during embryonic development in amphibians. Based on these results, we conclude that xFADD is an amphibian counterpart of the mammalian adaptor molecule, FADD. Thus, we provide the first direct evidence of the conserved function of a pro-apoptotic adaptor molecule across vertebrate species.

Caspase-8 and caspase-10 are key molecules for the apoptotic pathway mediated through death receptors and FADD. In humans, it has been reported that both caspase-8 and caspase-10 are recruited and activated to the DISC complex in a FADD-dependent manner but also that caspase-10 can not substitute for caspase-8 (Sprick et al. 2002). Based on this evidence, it has been proposed that the functions of caspase-8 and caspase-10 might differ from each other. As Xenopus caspase-8 (xCaspase-8) and caspase-10 (xCaspase-10) show high amino acid sequence similarity to their mammalian counterparts, it was hypothesized but not shown that these molecules are functionally similar to their orthologues (Nakajima et al. 2000). In this study, we showed that both xCaspase-8 and xCaspase-10 are equally adept at inducing apoptosis in mammalian cells and can interact not only with xFADD but also with mouse FADD, suggesting no differences in their function. Therefore, it is likely that in amphibian, these caspases are simult-aneously recruited to and activated within the DISC complex by xFADD and that they act cooperatively.

Thus far, members of the FADD family have been identified in mammals and insects. dFADD has been identified as a Drosophila homologue of FADD; it physically interacts with and activates DREDD, a Drosophila caspase-8 counterpart (Hu & Yang 2000). However, it has been reported that dFADD does not interact with mammalian caspase-8 or any death receptors in mammalian cells. Furthermore, genomic analysis suggests that there are no death receptor orthologues in Drosophila (Aravind et al. 2001). Recently, it has been shown that dFADD acts together with DREDD in immune defense against bacterial infection (Leulier et al. 2002; Naitza et al. 2002). The ability, demonstrated here, of Xenopus xFADD to activate mammalian caspases, and of the dominant-negative mutant xFADDdn to inhibit Fas-induced apoptosis in mammalian cells, demonstrates clearly that FADD's function is conserved between amphibians and mammals. The functional differences between Drosophila and Xenopus FADD homologues suggest that the molecular machinery underlying apoptosis increased in complexity during evolution, and that the function of FADD orthologues diverges significantly between vertebrates and invertebrates.

In mammals, the extrinsic apoptotic signalling pathway is well defined. Most of the components required for this pathway have now been identified in the frog. In addition, it has been reported that a Fas-like molecule is expressed on splenocytes in Xenopus and shares structural and functional homology with human Fas (Mangurian et al. 1998). More recently, two types of DD-containing receptors have been identified (Tamura et al. 2004). In Xenopus, we have also identified Bid, which is a target molecule recognized by caspase-8 that links apoptotic signals from the extrinsic pathway to the intrinsic pathway in mammals, and detected the translocation of the cleaved Bid to the mitochondria during Fas-induced apoptosis (our unpublished data). In the current study, we verified that xFADD acts as an adaptor molecule, promoting apoptosis even in mammalian cells. Consequently, we propose that xFADD is essential for the death receptor-mediated apoptotic signalling in the frog.

In adult frogs, although expression levels varied, xFADD expression was detected in all tissues examined. Similarly, the downstream molecules xCaspase-8 and xCaspase-10 were also identified in the same tissues except for muscle. All three molecules were consistently detected in the heart, lung and spleen, making it likely that they are involved in physiological and pathological cell death in these adult tissues. However, previous studies in mice have suggested an additional immunological role for the FADD/caspase-8 pathway in T-cell proliferation and homeostasis (Zhang et al. 1998; Kabra et al. 2001; Salmena et al. 2003). Moreover, It has been shown that phosphorylation of a serine residue in the C-terminal region of mammalian FADD is essential for T-cell proliferation (Hua et al. 2003). To further clarify the nonapoptotic and immunological roles of FADD, xFADD expression should be examined in Xenopus T-cells.

We have established a system of regulating tissue-specific transgenic expression in frogs. Here, we expressed xFADD in the developing heart and eye and detected abnormalities during development in transgenic animals. As shown in panel f of Fig. 3B, if downstream initiator caspase was defective, over-expression of xFADD would not be effective in transfectants and would fail to induce apoptosis. Therefore, the components required for FADD-mediated apoptotic signalling appear to be present and functional in the tissues examined in the Xenopus larvae. It has been reported that caspases are involved in organelle loss during lens development in the eye (Ishizaki et al. 1998; Foley et al. 2004). Therefore, it was thought that over-expression of xFADD triggers apoptosis in the eye, especially in the lens, even in the absence of developmental programs. Similarly, heart-specific expression of xFADD resulted in irregular heart formation, as evidenced by the development of an undersized ventricle. In mouse, FADD plays an important role of cardiac development (Yeh et al. 1998), a role shown clearly here to be conserved in amphibians. Detailed examination of these abnormalities will be pursued in the future.

Our results clearly demonstrate that the function of FADD is evolutionarily conserved between mammals and amphibians. Although the function of apoptotic molecules has been analysed in rodents by establishing gene-deficient and transgenic mice, Xenopus will provide an excellent model system for understanding apoptosis, especially as it occurs during development. Our detection of xFADD, caspase-8 and caspase-10 transcripts during early stages of development (our unpublished data) permits detailed analysis of the role of these molecules not only in cell death but also in other physiological phenomena previously identified in mammals such as cell proliferation, the cell cycle and differentiation.

	Experimental procedures

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Animals, cell lines and reagents

Adult Xenopus laevis were purchased from Hamamatsu Seibutsukyozai Co. (Shizuoka, Japan) and maintained in water at 22 °C. Mouse embryonic fibroblasts (MEF cells) isolated from both wild-type and caspase-8-deficient embryos (Sakamaki et al. 2002), human cervical carcinoma HeLa-K cells, human liver adenocarcinoma SK-Hep1 cells, human embryonic kidney 293 cells and their sublines 293T and 293Fas, which highly express large T antigen or Fas, respectively, were cultured in Dulbecco's Modified Eagle's medium with 10% foetal calf serum. Monoclonal anti-Flag (M2) and anti-HA (3F10) antibodies were purchased from Sigma Chemicals Co. (St. Louis, MO, USA) and Roche Diagnostics (Mannheim, Germany), respectively. Anti-Myc antibody (9E10) was isolated from a hybridoma purchased from ATCC (Manassas, VA, USA). Anti-human Fas antibodies CH-11 and HFE7A were prepared as previously described (Yonehara et al. 1989; Ichikawa et al. 2000) and 3D5 was purchased from Alexis Co. (San Diego, CA, USA). Horseradish peroxidase-conjugated anti-mouse IgG antibody, Protein-G and the peptide caspase inhibitor carbobenzoyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk) were purchased from Cell Signalling Technology Inc. (Beverly, MA, USA), Amersham Biosciences (Arlington Heights, IL, USA) and Kamiya Biomedical Co. (Seattle, WA, USA), respectively.

Database search and DNA sequencing

We used the BLAST algorithm (Altschul et al. 1990) to search for the Xenopus homologue of mammalian FADD in the GENBANK database. A Xenopus EST clone (db61b04, GENBANK accession no. BE188907) was identified using the DED domain of human FADD as an amino acid search sequence. The nucleotide sequence of this clone was confirmed on both strands using TaqDyeDeoxyterminator Cycle sequencing (Applied Biosystems Inc., Foster City, CA, USA) on automated DNA sequencers (PRISM^TM 310, Applied Biosystems Inc. and LI-COR 4000, LI-COR Biosciences, Lincoln, NE, USA).

Construction of expression vectors

To express Xenopus FADD (xFADD) in mammalian cell lines, xFADD cDNA was fused with the Flag-tag fragment and inserted into the mammalian expression vector pME18S (Sakamaki et al. 1992) or into the pIRES-EGFP (BD-Clontech, Palo Alto, CA, USA). To generate DED-deficient xFADD (xFADDdn), DNA sequence corresponding to the DED domain was deleted out by digestion with NcoI and HincII and the plasmid was religated. This mutant was also fused with the Flag-tag and inserted into the pIRES (Internal Ribosomal Entry Site)-EGFP. Furthermore, Xenopus caspase-8 (xCaspase-8) and caspase-10 (xCaspase-10) cDNAs (Nakajima et al. 2000) were tagged with HA (haemagglutinin) and Myc at the 5'-end, respectively, and human caspase-8 and FADD cDNAs (Hirata et al. 1998; Shimada et al. 2002) were also tagged with Myc and Flag, respectively. The resulting fusion constructs were inserted into pME18S or the expression vector pCAGGS (Niwa et al. 1991) which was a gift from Dr J. Miyazaki (Osaka University). Flag-tagged mouse FADD (Hsu et al. 1996) was a gift from Dr D. Goeddel (Tularik Inc.). To prevent cell death, the p35 gene derived from baculovirus (Sugimoto et al. 1994) (a gift of Dr A. Sugimoto, RIKEN) was also inserted into pCAGGS. The EGFP-expression vector, pCX-EGFP (Okabe et al. 1997) was a gift from Dr M. Okabe (Osaka University).

Transfection of cells

For transient assays, cells were transfected with the various plasmid DNAs using the LipofectAMINE PLUS Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions.

Apoptosis assays

Twenty-four hours after transfection with xFADD, xCaspase-8 or xCaspase-10, together with pCX-EGFP in the presence or absence of 100 µM z-VAD-fmk, HeLa-K cells were fixed in PBS containing 3.7% formaldehyde, and the number of EGFP-positive cells was examined. Positive cells were photographed under a microscope (DMIRE2, Leica Microsystems, Wetzlar, Germany). Similarly, wild-type and caspase-8-deficient MEF cells were transfected with xFADD or xFADDdn alone or together with mouse caspase-8 (Sakamaki et al. 1998) in the presence or absence of z-VAD-fmk, and then fixed cells were examined for the EGFP expression under the microscope. In xFADDdn-expressing HeLa-K transfectants, 500 ng/mL of anti-human Fas antibody CH11 and 10 µg/mL cycloheximide (CHX) were added to the medium 24 h after transfection, and the cells were incubated for an additional six hours. Cells were then fixed and photographed under the microscope. In SK-Hep1 transfectants transiently expressing xFADDdn, cells were treated with various concentrations of CH11 for 8 h in the presence of 10 µg/mL CHX, washed with PBS twice and lysed in passive lysis buffer (Promega, Madison, WI, USA). Viability of transfectants was estimated by measuring EGFP fluorescence in cell lysates with a multilabel counter (ARVO^TM, PerkinElmer Life Science-Wallac Oy, Turku, Finland).

Propidium iodide (PI) staining and flow cytometric analysis

Twenty-four hours after transfection with xFADD, xCaspase-8 or xCaspase-10 with or without the p35 gene, HeLa-K cells were fixed in PBS containing 3.7% formaldehyde at 4 °C for 1 h, washed twice with cold PBS and postfixed in 70% ethanol at –20 °C for 1 h as described (Lamm et al. 1997). Following fixation, cells were washed with PBS, treated with RNase A (50 µg/mL) in PBS at 37 °C for 30 min and stained with 50 µg/mL of PI in PBS for 30 min. The DNA content of cells was then analysed by flow cytometry (XL^TM, Beckman-Coulter, Miami, FL, USA).

Immunoprecipitation and immunoblotting

To analyse interactions between xFADD and other molecules, 293T cells were transiently transfected with expression vectors carrying baculovirus p35 in conjunction with Flag-xFADD, Flag-xFADDdn or Flag-human FADD with or without HA-xCaspase-8, Myc-xCaspase-10 or Myc-human caspase-8, and suspended in lysis buffer (50 mM Tris-HCl, pH 7.5, 20 mM MgCl₂, 0.5% Nonidet P-40, 150 mM NaCl) containing a cocktails of protease inhibitors (Nacali tesque, Kyoto, Japan). Whole cell lysates were immunoprecipitated with anti-Flag antibody and Protein-G. Immunoprecipitates were resolved by SDS-PAGE and analysed by immunoblotting with anti-HA, anti-Myc or anti-Flag antibody as appropriate for the expressed tag. For examination of DISC (death-inducing signalling complex) formation, 293Fas cells were transfected with either control vector or Flag-xFADD, stimulated with 500 ng/mL of anti-Fas antibody (CH11) for 40 min after 48 h culture, and suspended in lysis buffer. Whole cell lysates were immunoprecipitated with another anti-Fas antibody (HFE7A) and with Protein-G. Immunoprecipitates were then resolved by SDS-PAGE and analysed by immunoblotting with anti-Flag or anti-human Fas (3D5) antibody. Immunoblot analyses were performed as previously described (Sakamaki et al. 1993).

PCR and reverse transcription (RT)-PCR analyses

For genotyping, PCR amplification was performed on genomic DNA isolated from transgene-injected animals or the tails of transgenic animals, using following primer sets: forward primer P1 (5'-ACGTTGCCTTCGTCGTGAGATCAT-3' from the CA promoter) and reverse primer P2 (5'-GGAGCAGGTTCTCCATGTTGGCT-3' from xFADD cDNA) for the detection of the CA-xFADD/EGFP transgene, forward primer P3 (5'-GCCTGGAGCTATTTTAGCTATGCC-3' from the XMLC2 promoter) and reverse primer P2 for the detection of the XMLC2-xFADD transgene, and forward primer P4 (5'-CTGTTCCATGGCCAACACTTGTCA-3' from GFP3 cDNA) and reverse primer P5 (5'-AGGACCATGTGGTCTCTCTTTTCG-3' from GFP3 cDNA) for the detection of GFP3 cDNA. Similarly, the following primers for the detection of the Xenopus-crystallin gene were used as internal controls: forward primer P6 (5'-GGATGAACTGGGAATGCTGTCTAG-3' from the promoter region) and reverse primer P7 (5'-TGGTCAGTGACCATTGAAGCTGGA-3' from the first intron) (Smolich et al. 1993). PCR was carried out as follows: initial denaturation at 95 °C for 3 min following by 40 cycles of 94 °C for 30 s, 58 °C for 45 s, and 72 °C for 90 s, and a final extension at 72 °C for 5 min. Amplified PCR products were analysed by 1% agarose-gel electrophoresis.

Total RNA was isolated from a variety of tissues from adult animals using ISOGEN (Nippongene, Toyama, Japan) according to the manufacturer's instructions. One µg of total RNA was used for first-strand cDNA synthesis with random hexameric primers on Ready-To-Go RT-PCR beads (Amersham Biosciences), according to the manufacturer's instructions. First-strand cDNA was amplified using the following primers: primers (forward: 5'-ACCGAAGTCGGCAGCCTGAAGTT-3' and reverse: 5'-GGAGCAGGTTCTCCATGTTGGCT-3' for the detection of xFADD transcripts; forward: 5'-CGCTTCTATACTGGAAATATTCTT-3' and reverse: 5'-TTGTACTGAAATCTTCTCAAAT-3' for xCaspase-8 transcripts; forward: 5'-GCAACAGTTCCTGTAGAA-3' and reverse: 5'-TATGGACTGTTTTCTTGA-3' for xCaspase-10 transcripts; and forward: 5'-CAGATTGGTGCTGGATATGC-3' and reverse: 5'-ACTGCCTTGATGACTCCTAG-3' for EF1 transcripts, as a positive control. PCR was carried out as follows: initial denaturation at 94 °C for 5 min followed by 35 cycles of 96 °C for 15 s, 55 °C for 15 s, and 72 °C for 30 s. Amplified PCR products were analysed by 2.5% agarose-gel electrophoresis.

Construction of the transgene and generation of transgenic frogs

Beginning with pBlusescript-KS (Stratagene, La Jolla, CA, USA), we constructed a plasmid carrying a transgene, XMLC2-xFADD, comprising the Xenopus myosin light chain 2 (XMLC2) promoter (Latinkic et al. 2004), xFADD cDNA and the SV40 polyA sequence. We also created another construct, CA-xFADD/EGFP, consisting of the chicken crystalline A (CA) promoter (Ogino & Yasuda 1998), which was a gift from Dr K. Yasuda (Nara Institute of Science and Technology), the xFADD-EGFP construct as described above, and the SV40 polyA sequence. To easily find transgenic animals expressing XMLC2-xFADD, we generated a construct, CA-GFP3, consisting of the CA promoter, GFP3 (a variant of GFP) cDNA, and the SV40 polyA sequence that expresses green fluorescent proteins in the eye.

To generate transgenic frogs, we followed a previously established method (Kroll & Amaya 1996; Amaya & Kroll 1999), without the use of restriction enzymes. Animals injected with the transgene developed to appropriate stages. Developmental stages of animals were monitored according to Nieuwkoop and Faber (Nieuwkoop & Faber 1967). Some of these animals were used for histological analysis and others were used for observation under a dissecting microscope (MZFLIII, Leica Mycrosystems, Wetzlar) and for genotyping of their genomic DNA.

Histological analysis and size estimation of the ventricle of transgenic animals

For histological analysis, transgenic animals were fixed in PBS containing 1% glutaraldehyde overnight, washed twice in PBS and dehydrated. Samples were then embedded in paraffin wax (Paraplast Plus, Sigma Chemicals) and serially cut into 10-µm sections. All transverse sections were stained with 0.1% azocarmin B (Chroma, Muenster, Germany) for 20 s and a mixture of 0.5% aniline blue (Merck, Darmstadt, Germany) and 1% orange G (Chroma) for 7 min as previously described (Goto et al. 2001), and the resulting images were captured using a CCD camera (Coolpix990, Nikon, Tokyo).

Both the length and width of the cardiac ventricle were measured for each section, and the volume of the ventricle was estimated.

	Acknowledgements

 
The authors greatly thank Drs J. Miyazaki and M. Okabe (Osaka University, Suita), A. Sugimoto (RIKEN, Kobe), D. Goeddel (Tularik Inc., South San Francisco), E. Amaya (Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge), K. Yasuda (Nara Institute of Science and Technology, Ikoma), and T. Mohun (National Institute for Medical Research, London) for their respective gifts of pCAGGS and pCAGGS-EGFP plasmids, p35 genomic DNA, mouse FADD cDNA, GFP3 cDNA, the chicken crystalline CA promoter and the Xenopus MLC2 promoter. The GENBANK accession number for xFADD is AY521029. This work was supported by a Grant-in-Aid for Creative Scientific Research to K.S. from the Japan Society for the Promotion of Science (13GS0008).

	Footnotes

 
Communicated by: Shigekazu Nagata

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

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Received: 24 August 2004
Accepted: 14 September 2004

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