Shiokawa K , Aso M , Kondo T , Uchiyama H , Kuroyanagi S , Takai J , Takahashi S , Kajitani M , Kaito C , Sekimizu K , Takayama E , Igarashi K , Hara H .

	Figure 1. Induction of apoptosis by injection of Xenopus SAMDC mRNA. (A) A control embryo injected with distilled water. (B) A fertilized egg injected with Xenopus SAMDC mRNA (100 pg), and cultured in a slightly hypertonic 1 X Steinberg’s solution in order to protect dissociated cells from osmotic shock. White cells are dissociated cells. These cells appear in the region where mRNA was injected. Embryos were filmed at early gastrula stage. From Takayama et al. (2004).
	Figure 2. Apoptosis induced in a half portion of an embryo. Only one blastomere of a 2-celled embryo was injected with a mixture of SAMDC mRNA (1 ng/egg) and GFP mRNA (100 pg/egg), and embryos were filmed at early blastula (A, B) and early gastrula (C, D) stage using the visible light (A, C) and UV light (B, D). In B, cells expressing GFP, hence containing SAMDC mRNA, cleaved normally, but after MBT one half portion of the embryo which expressed GFP (D) underwent cell dissociation (C). (Kuroyanagi S, Shiokawa K, unpublished).
	Figure 3. Cell dissociation induced by microinjection of 5-aza-2′-deoxycytidine (4 pmole/egg). Embryos were cultured in 1 X Steinberg’s solution and filmed at late blastula stage. From Kaito et al. (2001).
	Figure 4. Apoptosis-like reaction in colchicine-treated Xenopus embryos. Xenopus fertilized eggs were cultured in 1 X Steinberg’s solution containing 1 mM colchicine. Embryos were filmed at early blastula stage. (Suwa M, Shiokawa K, unpublished).
	Figure 5. Rescue of SAMDC mRNA-injected Xenopus embryos by Bcl-2. When Xenopus fertilized eggs were injected with SAMDC mRNA (1 ng/egg) alone, embryos were dissociated shortly after MBT (A), and remained dissociated in 1 X Steinberg’s solution (B) even after 12 hrs (at which time control embryos reached stage 22 tailbud embryos). When fertilized eggs were co-injected with SAMDC mRNA (1 ng/egg) and Bcl-2 mRNA (2 ng/egg), cell dissociation did not take place at late blastula stage (C), and embryos developed to the tailbud stage (stage 22) (D). The extent of the rescue by Bcl-2 from SAMDC-induced apoptosis varied depending on the dosage-combination of SAMDC and Bcl-2 mRNAs (E). From Kai et al. (2000).
	Figure 6. Northern blot analysis of caspase mRNAs in Xenopus embryos. Fertilized eggs were injected with either SAMDC mRNA (100 pg/egg) or distilled water, and RNAs were isolated from embryos, and subjected to northern blot analysis. From Takayama et al. (2004).
	Figure 7. Northern blot and RT-PCR analyses for caspase-8 and -9 mRNAs in p53 mRNA- or SAMDC mRNA-injected embryos. Fertilized eggs were injected (+) or not (−) injected with p53 or SAMDC mRNA (1 ng each/embryo), and cultured in 1 X Steinberg’s solution. RNAs were isolated from embryos at stage 6.5 (late cleavage stage) or stage 8.5 (midblastula stage). Upper white panels: RNAs separated on a 1% agarose gel containing formaldehyde were transferred to a nylon membrane, and hybridized with 32P-labelled specific probes for Xenopus caspase-8 and 9. Middle black panel: 28S and 18S rRNAs stained with ethidium bromide on 1% agarose gel. This profile is before blotting. Lower black panels: RNAs were subjected to RT-PCR. The signal obtained for caspase-8 and caspase-9 mRNA was 396 bp and 539bp, respectively. Caspase-8 mRNA expression was induced only in SAMDC mRNA-injected cleavage stage and midblastula stage embryos, whereas caspase-9 mRNA occurred as a maternally-provided RNA. From Shiokawa et al. (2005).
	Figure 8. A model which shows sequence of events in activation of caspase system in SAMDC mRNA-overexpressed and p53 mRNA-overexpressed embryos, with special reference to the recruitment of mRNAs.
	Figure 9. GFP-tracing of SAMDC mRNA-injected cells at the tadpole stage. Embryos were injected with processing-defective (A) or wild-type (B) SAMDC mRNA together with GFP mRNA into one animal side blastomere at the 8-cell stage, and GFP luminescence was examined at the tadpole stage (32 hrs post-fertilization). Note that the embryo in B is shorter in body length than that in A due to apoptotic loss of a certain amount of cell mass at MBT within the blastocel. Both embryos were too large to be taken in a photograph, so embryos were taken in two photographs and tadpoles were constructed by combining the two photographs together. The bar is 5 mm. From Kai et al. (2003).
	Figure 10. Effects on development of SAMDC mRNA injection into an animal side blastomere of either future dorsal or ventral side of the 16-cell stage embryo. Embryos were injected with SAMDC mRNA (130 pg) into one animal side blastomere of either future dorsal (B) or ventral (C) side at 16-cell stage as schematically shown in the diagram in the left, and cultured until the tailbud stage. (A) A control uninjected embryo. In B, the cement gland is missing (arrowheads), and furthermore, head part is absent (acephaly) (lower arrowhead). In C, posterior and ventral structures such as trunk and tail are poorly developed (arrowheads), or tail is curved upwards (lower arrowhead). From Kai et al. (2003).
	Figure 11. A model which shows how early development proceeds. This model suggests possible occurrence of apoptotic check point which functions as a surveillance or “fail-safe” mechanism in Xenopus early embryonic development. Fertilized eggs cleave rapidly until the early blastula stage. At MBT, the “first developmental checkpoint” comes when G1 phase first appears. We assume that this check mechanism determines cell-autonomously if the cell continues or stops development to be eliminated by execution of the maternally-inherited program of apoptosis. However, even when apoptosis was executed, embryos follow two different courses. If the number of apoptotic cells is large, the whole embryo stops development and dies. However, if the number of apoptotic cells is small, they are confined within the blastocoel and the embryo itself continues on development. Cells to be eliminated are segregated into the blastocoel. If such cells came out to the perivitteline space, the whole embryo will be dissolved due to osmotic shock (eggs are laid in the water), and the maternally-inherited program of apoptosis will not serve as a fail-safe mechanism.
	Figure 12. Three characteristic profiles of RNA synthetic patterns as studied by gel electrophoresis of radioactively-labeled RNA at each stage. Dotted, shaded and black areas are for the product of RNA polymerase II, III, and I, respectively, which are heterogeneous mRNA-like RNA, tRNA, and rRNA, respectively. Distance of migration and amount of radioactivity are in arbitrary units. From Shiokawa et al. (1941).

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