|
Figure 1
Spatio temporal expression of fadd during early stages of embryonic development. (A) Total RNA isolated from embryos at stages 1, 8.5/9, 10.25, 11/12, 15/16, 20, 25, and 32 was subjected to RT-PCR analysis (lanes 2–9). The resulting PCR products for fadd and ornithine decarboxylase (odc) were resolved by electrophoresis on a 2.5% agarose gel. (B) Whole-mount in situ hybridization (WISH) of fadd transcripts in embryos was performed at the indicated stage of development using a specific RNA probe. In the magnified images, an arrowhead indicates the distal portion of the forebrain and an arrow shows the thoracic portion including the heart primordium. B, branchial arch; H, heart; O, olfactory placode.
|
|
Figure 2
Effects of enforced expression of wild-type and truncated form of FADD in embryogenesis. (A) A schematic diagram is shown of the full-length and truncated mutant of Xenopus FADD. DED: death effector domain, DD: death domain. (B) An illustration of a four-cell embryo. Microinjection was performed into the adjacent sites, which localize in the vegetal hemisphere near the boundary between the vegetal and animal hemispheres, in two dorsal blastomeres at the four-cell stage and are shown by a red point. (C–E) Morphological and histochemical analyses of the embryos undergoing cell death. Wild-type embryos were uninjected (C) or injected either with 500 pg of wild-type fadd mRNA (D) or with 500 pg of truncated mutant fadd-dd mRNA (E). Embryos developed to stage 18 were stained with acridine orange and photographed on both bright-filed (left panel) and fluorescent (right panel) images. Cell death was observed in more than 95% of embryos injected with fadd mRNA (119 of 124 embryos), but not detected in those injected with fadd-dd mRNA. The arrows indicate the area involved in cell death. (F) Detection of DNA fragmentation in embryos subjected to microinjection with fadd mRNA. Genomic DNAs were isolated from normal embryos or embryos injected with 500 pg of fadd mRNA or fadd-dd mRNA at the four-cell stage that were collected at stages 12, 13.5, or 18, and the same amount of DNA aliquots was applied and resolved by electrophoresis on a 2%-agarose gel. An arrowhead indicates the start position of the electrophoresis. (G) Detection of caspase-3 activation in embryos subjected to microinjection of fadd mRNA. The fadd mRNA-injected embryos were incubated with the caspase-3 detection reagent at stage 18, and green fluorescence was monitored by microscopy. The bright-field (upper panel) and fluorescent (lower panel) images were photographed. The arrows indicate the area undergoing cell death. (H) Fluorescence images of a biosensor, SCAT3, in transgenic embryos injected with fadd mRNA. Cell lysates were prepared from uninjected embryos or embryos injected with 500 pg of fadd mRNA at the four-cell stage that were collected at stages 10, 13, or 18 and then resolved by SDS-PAGE. Fluorescence in the gel was analyzed on a fluorescent image analyzer. As SCAT3 is a fusion consisting of both seCFP and Venus (Nagai et al. 2002) fluorescent proteins, joined by a peptide linker including the caspase-3 recognition sequence, the separation of seCFP and Venus indicates cleavage by active caspase-3 (Takemoto et al. 2003). An arrow indicates full-length SCAT3, while the white and black arrowheads indicate the cleaved Venus and seCFP fragments, respectively.
|
|
Figure 3
Effects of the antisense morpholino oligonucleotide (MO) on heart development in Xenopus embryos. (A) Histological observation of the developing embryos. Normal embryos are collected at stage 46, and their transverse sections were prepared. A representative picture of the thoracic portion including the heart, esophagus, and notochord was shown. ‘V’ indicates a ventricle. Scale bar indicates 100 μm. (B) Histological analyses of microinjected embryos. Five hundred micromolar fadd-MO was injected without or with 50 or 100 pg of MO-resistant fadd mRNA into embryos at the four-cell stage. Embryos were collected at stage 46, and their transverse sections were prepared. Two representative photographs from each group are shown. The arrows indicate the heart and scale bars indicate 100 μm. (C) Statistical analysis. The ventricular size of the heart in each specimen was calculated by measuring its area in serial sections as described in the experimental procedures. Each bar indicates the mean of the size of the ventricle and notochord of five or six animals in each group and error bars represent the standard deviation of the mean.
|
|
Figure 4
Effects of a truncated form, FADDdd, in embryonic development. Injection of fadd-dd mRNA (50 pg) into embryos was performed at the four-cell stage. Images of normal tadpoles (three animals on the right side) and FADDdd-expressing tadpoles (three animals on the left side) were taken at stage 46 under the microscope. Within the range of 66–100% in five experiments, fadd-dd mRNA-injected embryos showed growth retardation.
|
|
Figure 5
Effect of FADDdd on the Myd88-mediated signaling pathway in embryos. (A) Structure of Myd88 protein. Xenopus Myd88 encodes 283 amino acids and consists of a death domain (DD) and a Toll/IL-1R (TIR) motif at the amino- and carboxyl-terminal halves, respectively. (B) Coimmunoprecipitation and immunoblot analysis on interactions between Xenopus Myd88 and FADD. Human HEK293 cells were transfected with either pCS2 empty vector, pCS2-Flag/xFADD, or pCS2-Flag/xFADDdd in conjunction with pCS2-HA/xMyd88. After 2 days of cultivation, transfectants were harvested and lysed in a lysis buffer. Cell lysates were immunoprecipitated after a 4-h incubation with anti-FLAG M2 affinity gel. After adding a sample buffer, coimmunoprecipitates and aliquots of cell lysates were examined by immunoblot analysis with anti-FLAG, anti-HA, and anti-actin antibodies, respectively. Baculovirus p35 was introduced into all transfected cells to prevent cell death. Abbreviation: MWM, molecular weight marker. (C) The morphology of embryos injected with myd88 mRNA. Uninjected embryo (top) and myd88 mRNA-injected embryos showing the typical phenotype of short trunk (middle) or a curved body shape associated with edema (bottom); embryos were developed to stage 42. The white arrow indicates edema. Scale bar indicates 1 mm. (D) A summary of the morphological analysis. The morphology of embryos, which were injected without or with myd88 mRNA in combination with fadd-dd mRNA in the indicated amounts, was analyzed at stage 42. The numbers of embryos displaying normal (white), short trunk (red), and a curved body shape associated with edema (blue), and other uncharacterized abnormalities (black) were counted under the microscope, and those percentages were calculated. N indicates the total number of embryos examined in three independent experiments.
|
|
Figure 6
Isolation of a FADDdd-interacting molecule in embryos. (A) Silver staining of immunoprecipitates. Cell lysates were prepared from uninjected embryos (lane 1) or embryos injected with fadd-dd mRNA at the four-cell stage (lane 2), which developed to stage 20, and were incubated with an anti-FLAG M2 affinity gel. Immunoprecipitates within the gel were resolved by SDS-PAGE and visualized by silver staining. Peptide bands specifically observed in FADDdd-expressing embryos were indicated by blue arrows and also magnified separately. (B) The amino acid sequence of Xenopus cullin-4 (Cul4). The entire sequence of Cul4 registered in the NCBI database (NCB Accession Number: NP_001090088) is displayed. Amino acids detected by mass spectrophotometry are shown in red. (C) A schematic diagram of the full-length and truncated mutant of Xenopus Cul4. The yellow and green boxes indicate a cullin domain and a neddylation domain, respectively. (D) Coimmunoprecipitation and immunoblot analysis on interactions between FADDdd and Cul4 in embryos. Cell lysates were prepared from either normal embryos, FADDdd-expressing embryos, or both FADDdd- and Cul4-expressing embryos at stage 20 and incubated with anti-FLAG M2 affinity gel. Immunoprecipitates and aliquots of cell lysates were resolved by SDS-PAGE following immunoblotting with antibodies against FLAG-tag, HA-tag, or α-tubulin, respectively. (E) Assessment of the interactions between FADD and Cul4 in mammalian culture cells. HEK293 cells were transfected with either pCS2 empty vector, pCS2-Flag/xFADD, or pCS2-Flag/xFADDdd in conjunction with pCS2-HA/xCul4 or pCS2-HA/xCul4ΔC. After 2 days of cultivation, cell lysates were prepared from those transfectants and incubated with anti-FLAG M2 affinity gel. Immunoprecipitates and aliquots of cell lysates were resolved by SDS-PAGE following immunoblotting with antibodies against FLAG-tag, HA-tag, or actin, respectively. The white arrowheads indicate the truncated xCul4ΔC proteins. The plasmid DNA carrying the baculovirus p35 gene was used to prevent cell death. (F) Palliative effect of FADDdd to Cul4-induced abnormality of embryos. Wild-type embryos uninjected (a) or injected either with 400 pg (b and d) or 800 pg (c and e) of cul4 mRNA without or with 200 pg of fadd-dd mRNA (d and e) into the equatorial area of two dorsal blastomeres at the four-cell stage. The developing embryos were photographed at stage 14 under the microscope. (G) Histological analysis of Cul4-expressing embryos. Embryos uninjected (left panel) or injected with 800 pg of cul4 mRNA (right panel) were harvested at stage 14, and their cross-sections were prepared for interior observation. Scale bars indicate 200 μm.
|
|
Figure 7
Assessment of the transcriptional activity of NF-κB in Cul4-expressing mammalian cells and Xenopus embryos. (A) A schematic diagram of the reporter plasmid constructs generated for the detection of NF-κB activity. The plasmid pTAL-mCherry consists of a basal TATA-like (TAL) promoter of the Herpes simplex virus thymidine kinase gene and the coding region of mCherry cDNA. The plasmid pNFκB-mCherry contains four contiguous NF-κB enhancer elements (4κBs) at the upstream of the TAL promoter in pTAL-mCherry. (B) Cytological analysis of NF-κB transcriptional activity induced by Cul4 in mammalian cells. Plasmid DNA mixtures: empty pCS2 vector and pNFκB-mCherry, pCS2-HA/xCul4 and pNFκB-mCherry, pCS2-xMyd88 and pNFκB-mCherry, and pCS2-HA/xCul4 and pTAL-mCherry were transiently cotransfected with pCAG-Venus into HeLa cells. After culturing for 48 h, NF-κB activity was detected by monitoring the intensity of the red fluorescence of mCherry proteins in Venus-positive cells by fluorescent microscopy. Numbers in parentheses indicate the number of mCherry-positive cells and Venus-positive cells. Scale bars indicate 50 μm. (C) Enzymatic analysis of NF-κB transcriptional activity induced by Cul4. Plasmid DNA mixtures: empty pCS2 vector and pNFκB-Luc, pCS2-HA/xCul4 and pNFκB-Luc, pCS2-xMyd88 and pNFκB-Luc, and pCS2-HA/xCul4 and pTAL-Luc were transiently cotransfected with pRL-TK into HeLa cells. After culturing for 24 h, NF-κB activity was examined by measuring the enzyme activity of dual luciferases produced in transfected cells using a luminometer. Data are presented as the means and standard deviations of samples counted from four experiments. The statistically significant difference between the two groups was evaluated by Student's t-test. (D) Fluorescent analysis of the transgenic animals. Plasmid DNA mixtures: empty pCS2 vector and pNFκB-mCherry, pCS2-HA/xCul4 and pNFκB-mCherry, and pCS2-HA/xCul4 and pTAL-mCherry were microinjected with pCAG-Venus into four-cell embryos. After developing to stage 42, images of three transgenic tadpoles carrying plasmid constructs from each group were captured as both bright-field (left panels) and yellow fluorescence of Venus (right panels) under the microscope. Representative photographs from two independent experiments were shown. Seven to eight Venus-positive tadpoles in each group were further analyzed their red fluorescence. Scale bars indicate 1 mm. (E) Assessment of the transcriptional activity of NF-κB in Cul4-expressing animals. The transcription activation by NF-κB was detected by monitoring the intensity of the red fluorescence of mCherry in Venus-positive tadpoles by fluorescent microscopy. Magnified images of three transgenic tadpoles from each group were captured to indicate the fluorescence of Venus (left panels) and mCherry (right panels). Scale bars indicate 100 μm.
|
|
Figure 8
Detection of the non-apoptotic effect of FADD in Xenopus embryos. (A) The morphology of embryos coinjected with fadd and p35 mRNAs. The fadd mRNA-injected or both fadd and p35 mRNAs-injected embryos at 4-cell stage were developed to stage 46. The typical phenotypes of fadd mRNA-injected embryos showing caudal defects (middle) or short trunk (right) were photographed by microscopy. Scale bar indicates 1 mm. (B) A summary of the morphological analysis. The morphology of embryos, which were injected without or with p35 mRNA in combination with fadd mRNA in the indicated amounts, was analyzed at the late tailbud stages. The numbers of embryos displaying normal (white), short trunk (red), caudal defects (blue), and other uncharacterized abnormalities (black) were counted under the microscope, and those percentages were calculated. N indicates the total number of embryos examined in three independent experiments. (C) Histological analysis of the heart in FADD-expressing embryos. Embryos showing short trunk in morphology (A) with uninjected embryos were dissected for histological observation of the heart. The arrows indicate the heart and the arrowheads point to ventricular trabeculae. Scale bars indicate 50 μm. (D) Statistical analysis. The ventricular size of the heart in each specimen was calculated by measuring its area in the serial sections as described in the experimental procedures. Data are presented as the means and standard deviations of six animals in two groups. Significant differences between the two groups were evaluated by the t-test.
|
|
Figure 9
Schematic representation of the biological functions via FADD in the Xenopus embryo. When FADD was excessively expressed in embryos, it induced cell death by activating the intrinsic apoptotic machinery including caspase-3. By contrast, the down-regulation of FADD caused irregular development of the heart. Expression of a truncated mutant, FADDdd, which has lost pro-apoptotic activity, perturbed the expression level of the NF-κB-responsive genes, resulting in growth retardation in the embryos. In addition, FADDdd interacts with Cul4. Each of these three experiments indicated a different phenotype of embryos from the other two. Given these results, we concluded that the DED motif of FADD is required for apoptosis in embryos, whereas DD is crucial for the regulation of NF-κB activity. Moreover, Xenopus FADD as well as murine FADD is involved in heart development.
|
|
Figure S1 Detection of caspase-3 activation in embryos subjected to microinjection with fadd mRNA.
|
|
Figure S2 The target specificity of fadd-MO for reporter plasmids in mammalian cells.
|
|
Figure S3 Comparison of Xenopus Tlrs5 and Tank with their human homologs.
|
|
Figure S4 Identification of the NF-κB binding sites on the tlrs5 and tank genes.
|
|
Figure S5 Whole-mount in situ hybridization of cul4 transcripts in embryos.
|
|
Figure S6 Histological analysis of Cul4-expressing embryos.
|
|
Figure S7 Assessment of inhibitive effect of FADDdd on NF-κB activation by Cul4.
|
|
Figure S8 Acridine orange staining of the embryos.
|
