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Shiokawa K, Takayama E, Higo T, Kuroyanagi S, Kaito C, Hara H, Kajitani M, Sekimizu K, Tadakuma T, Miura K, Igarashi K, Yaoita Y.
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Overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus fertilized eggs activates caspase-9 and executes maternal program of apoptosis shortly after midblastula transition (MBT). We find that overexpression of caspase-8 and p53, like that of SAMDC, induces apoptosis in Xenopus late blastulae. The apoptosis induced by p53 was abolished by injection of mRNA for xdm-2, a negative regulator of p53, and by injection of a peptide inhibitor or a dominant-negative type mutant of caspase-9, but not caspase-8. The apoptosis induced by SAMDC was not abolished by injection of xdm-2 mRNA, but was abolished by injection of a peptide inhibitor or a dominant-negative type mutant mRNA of both caspase-9 and caspase-8. Unlike caspase-9 mRNA, caspase-8 mRNA did not occur as a maternal mRNA rather induced to be expressed during cleavage stage (pre-MBT stage) by overexpression of SAMDC but not p53. Furthermore, while activities to process procaspase-8 and procaspase-9 appeared in SAMDC-overexpressed apoptotic embryos, the activity to process procaspase-8 did not appear in p53-overexpressed apoptotic embryos. We conclude there are at least two pathways in the execution of the maternal program of apoptosis in Xenopus embryos; one being through do novo expression of caspase-8 gene during cleavage stage, and the other without involvement of caspase-8.
Fig. 1.
Appearance of embryos injected with various mRNAs. (A) Uninjected (mRNA was not injected, but only distilled water was injected; same for all the “uninjected†in the following figures). (B) Injected with SAMDC mRNA. (C) Injected with caspase-8 mRNA. (D) Injected with p53 mRNA. (E) Injected with SAMDC mRNA. (F) Injected with β-globin mRNA plus β-globin mRNA. mRNAs were injected at 1000 pg/egg (A–D) at 1-cell stage or injected into only one blastomere at 2 cell stage (E,F). Embryos were cultured in 1× Steinberg’s solution. (E) No cell dissociation took place. (F) Only dissociated cells carry blue color.
Fig. 2.
Effects of microinjection of mRNAs for caspase-8, p53, SAMDC, and β-globin. mRNAs were injected at different doses (10, 100, and 1000 pg/egg) into both of the blastmeres of 2 celled embryos and development of the injected embryos was examined by culturing them in 1× Steinberg’s solution. For each dose, 50 embryos of the same batch were used. Each set of experiment was repeated twice with similar results. (A) Caspase-8 mRNA, (B) p53 mRNA, (C) SAMDC mRNA, and (D) β-globin mRNA as a reference. (E) Tests for “DNA-ladder†formation. Fifty fertilized eggs were injected with indicated mRNA (all at 100 pg/embryo) and cultured in 1× Steinberg’s solution. DNAs extracted from injected embryos at stage 12 were electrophoresed on a 1.5 % agarose gel, and stained with ethidium bromide. (F) A rescue experiment of apoptosis by coinjection of Xenopus Bcl-2 mRNA. Bcl-2 mRNA (500 pg/embryo) was injected together with mRNA (100 pg/embryo) for caspase-8 or p53 into both of blastomeres of 2-cell stage embryos. Each experimental group consisted of 50 embryos. β-Globin mRNA (500 pg/embryo) was injected as a control.
Fig. 3.
Effect of coinjection of Xdm-2 mRNA on the effect of the injection of p53 or SAMDC mRNA. Fertilized eggs were coinjected with 500 pg/egg of Xdm-2 mRNA (triangle) and 100 pg/egg of either p53 mRNA (A) or SAMDC mRNA (B). mRNA for β-globin (circle) was also coinjected into p53 or SAMDC mRNA as a control. Fifty embryos of the same batch were used for each experimental group. Experiments were repeated twice with similar results.
Fig. 4.
Effects of coinjection of peptide inhibitors and dominant-negative type mutants of caspase-9 and caspase-1 into p53 or SAMDC mRNA-injected embryos. Fertilized eggs were coinjected with 1000 pmol of peptide inhibitor for caspase-9 together with the mRNA for p53 (A) or SAMDC (B) both at 100 pg/egg. Fertilized eggs were also coinjected with 1000 pg/egg for mRNA of dominant-negative type mutant of caspase-9 (dncaspase-9) or caspase-1 (dncaspase-1) together with mRNA for p53 (C) or SAMDC (D). Fifty embryos of the same batch were used for each dose in each set of experiments. Each set of experiments was repeated twice with similar results.
Fig. 5.
Effects of coinjection of peptide inhibitors for caspase-8 and caspase-1, and dominant-negative type mutants of caspase-8 and caspase-1 into p53 or SAMDC mRNA-injected embryos. Fertilized eggs were coinjected with 1000 pmol of peptide inhibitor for caspase-8 or caspase-1 together with mRNA for p53 (A) or SAMDC (B) both at 100 pg/egg. Fertilized eggs were coinjected with 1000 pg/egg of mRNA for dominant-negative type mutant of caspase-8 (dncaspase-8) or caspase-1 (dncaspase-1) together with mRNA for p53 (C) or SAMDC (D). Fifty embryos of the same batch were used for each group. Each set of experiments was repeated twice with similar results.
Fig. 6.
Appearance of the activity to cleave pro-caspases in p53- and SAMDC-overexpressed Xenopus apoptotic embryos. [35S]Methionine-labelled enzymatically defective pro-caspases were prepared and incubated with lysates prepared from embryos injected with mRNA (1000 pg/embryo) (+) for p53 or SAMDC. Lysates were also prepared from embryos injected with β-globin mRNA alone (1000 pg/embryo) (−) as control experiments. Reaction products were analyzed on SDS–PAGE under the reducing condition and autoradiographed. (A) p53-overexpressed embryos. (B) SAMDC-overexpressed embryos. Molecular size markers, locations of pro-caspase (open arrowheads), and cleaved products (closed arrowheads) are shown.
Fig. 7.
Northern blot and RT-PCR analyses for caspase-8 and -9 mRNAs in p53- or SAMDC-overexpressed embryos. Fertilized eggs were injected (+), or not (−) injected, with p53 or SAMDC mRNA (1000 pg/embryo) and cultured in 1× Steinberg’s solution throughout the experiment. RNAs were isolated from embryos at different stages. (Upper panel) RNAs were separated on a 1% agarose gel containing formaldehyde, transferred to a nylon membrane, and hybridized with 32P-labelled specific probes for Xenopus caspase-8 and -9. (Lower panel) RNAs were also subjected to RT-PCR as described in details under Materials and methods. The signal obtained for caspase-8 mRNA was 396 bp, and that for caspase-9 mRNA was 539 bp. 28S and 18S rRNAs were stained with ethidium bromide. Three independent experiments were done, with essentially the same results.
