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Takayama E
,
Higo T
,
Kai M
,
Fukasawa M
,
Nakajima K
,
Hara H
,
Tadakuma T
,
Igarashi K
,
Yaoita Y
,
Shiokawa K
.
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We previously demonstrated that overexpression of S-adenosylmethionine decarboxylase (SAMDC) in Xenopus early embryos induces execution of maternal program of apoptosis shortly after midblastula transition, which likely serves as a fail-safe mechanism of early development to eliminate physiologically damaged cells before they entering the gastrula stage. To determine how caspases are involved in this process, we microinjected peptide inhibitors and "dominant-negative forms" of caspase-9 and -1 into Xenopus fertilized eggs, and found that inhibitors of caspase-9, but not caspase-1, completely suppress SAMDC-induced apoptosis. The lysate of SAMDC-overexpressing late blastulae contained activity to cleave in vitro-synthesized [(35)S]procaspase-9, but not [(35)S]procaspase-1, and mRNA for caspase-9, but not caspase-1, occurred abundantly in the unfertilized egg as maternal mRNA. We also found that overexpression of caspase-9 and -1 equally executes the apoptosis, but the apoptosis executed by these mRNAs was only partially rescued by Bcl-2 and rescued embryos did not develop beyond neurula stage. These results indicate that activation of caspase-9 is a key step for execution of the maternally preset program of apoptosis in Xenopus early embryos.
Fig. 1.
Inhibition of execution of post-MBT embryonic apoptosis in SAMDC mRNA-injected Xenopus embryos by peptide inhibitors for caspases. 2000, 200, 20, or 0 pmol/egg of the synthetic peptide inhibitor for caspase-9 (Ac-LEHD-CHO) (A) or caspase-1 (Ac-YVAD-CHO) (B) was co-injected with 100 pg/egg SAMDC mRNA into Xenopus lavies fertilized eggs. Control embryos were injected with distilled water alone. Fifty fertilized eggs from the same batch were used for each experimental group. Percentage of developing embryos was plotted against the ordinate according to the developmental stages. Three independent experiments were done, with essentially the same results.
Fig. 2.
Effects of the injection of the mRNA for “dominant negative type” caspases. One hundred picograms per egg of mRNAs for dn-caspase-9, dn-caspase-1 or β-globin was injected together with 100 pg/egg mRNA for SAMDC into each of the two blastomeres of 2-cell stage Xenopus embryos. Control embryos were injected with distilled water alone.
Fig. 3.
Induction of procaspase-9-cleaving activity in SAMDC mRNA-injected Xenopus embryos. (A) Embryos were injected with 100 pg/egg mRNA for SAMDC (closed circles) or β-globin (open circles) into both of the blastomeres of the two cell stage embryo (200 pg/embryo) and cultured in 1× MBS to protect osmotic embryo lysis throughout the experiment. Fifty embryos were collected and lysed in the homogenizing buffer (cf. Materials and methods) either at stage 6.5 or stage 10.5 to prepare cell-lysates. (B,C) 35S-labelled procaspase-9 (B) and procaspase-1 (C) were incubated with the cell lysates prepared at stage 6.5 or 10.5 (A) from embryos which had been injected with mRNA for SAMDC (+) or β-globin (−). Reaction mixtures were subjected to gel electrophoresis under reducing conditions. Independent experiments were three times repeated, with essentially the same results.
Fig. 4.
Northern blot analysis of caspase mRNAs in Xenopus embryos. Fertilized eggs were injected with SAMDC mRNA (100 pg/egg) or distilled water (uninjected, control embryos) and cultured in 1× Steinberg’s solution throughout the experiment to protect embryos from osmotic shock. RNAs were isolated from embryos, separated on a formaldehyde-containing 1% agarose gel, transferred to a nylon membrane filter, and hybridized with 32P-labelled probes specific for Xenopus caspase-9 or caspase-1. 18S rRNAs were stained with ethidium bromide. The amount of 18S rRNA is practically the same between uninjected and SAMDC mRNA-injected embryos even at stage 12, suggesting the maintenance of undegraded ribosomes in the apoptotic cells kept unruptured until this stage. Note that experiments were not performed in SAMDC mRNA-injected embryos after stage 12 because of the complete embryo death.
Fig. 5.
Induction of apoptosis by injection of mRNA for Xenopus caspase-9 or -1 in Xenopus embryos. Fertilized eggs of the same batch were injected with 100 pg/egg mRNA for caspse-9 (B), caspase-1 (C), or SAMDC (D), and cultured in 1× Steinberg’s solution to protect dissociated cells from rupture due to osmotic shock. White cells without pigmented surface coat are those dissociated due to execution of apoptosis, and these cells appeared first at the area of eggs where mRNAs were injected. Uninjected embryos were those injected with distilled water alone (A). Embryos were filmed at the early gastrula stage.
Fig. 6.
Time-course of the execution of apoptosis in Xenopus embryos injected with mRNAs for Xenopus caspases. Fertilized eggs were injected with 1000, 100, 10, or 0 pg/egg of mRNA for β-globin (A), caspase-9 (B), or caspase-1 (C), and cultured in 1× Steinberg’s solution throughout the experiment. Fifty embryos of the same batch were used for each experimental group.
Fig. 7.
Effects of co-injection of Bcl-2 mRNA on apoptosis executed by the injection of caspase-9 or -1 mRNA. Xenopus fertilized eggs were injected with mRNA for caspase-9 (A) or caspase-1 (B). Fifty fertilized eggs of the same batch were used in each experimental group. Open symbols are for embryos injected with caspase mRNA (100 pg/egg) plus β-globin mRNA (100 pg/egg), and closed symbols are for embryos injected with caspase mRNA (100 pg/egg) plus Bcl-2 mRNA (100 pg/egg). Uninjected embryos were those injected with distilled water alone.
