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First published online May 30, 2008
Journal of Experimental Biology 211, 1874-1881 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012690
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Extensive apoptosis and abnormal morphogenesis in pro-caspase-3 transgenic zebrafish during development

Michiaki Yamashita*, Nanami Mizusawa, Misako Hojo and Takeshi Yabu

National Research Institute of Fisheries Science, 2-12-4 Fukuura, Yokohama 236-8648, Japan


Figure 1
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  		Fig. 1. The plasmid construct. GFP, green fluorescent protein.

 

Figure 2
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  		Fig. 2. Expression of the transgene product, His-tagged pro-caspase-3, in the transgenic embryos. The embryos were stained with anti-His-tag antibody and Cy3-fluoro-labelled anti-mouse IgG secondary antibody, and were observed by fluorescence microscopy. (A) GFP control. (B) Transgenic embryo expressing pro-caspase-3 (casp-1).

 

Figure 3
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  		Fig. 3. Abnormal morphology of the pro-caspase-3 transgenic zebrafish. Arrows indicate abnormal morphology of the eye (e), heart (h), notochord (n) and yolk sac (y) of transgenic fish lines casp-1 (B,C) and casp-2 (D), at 48 h.p.f. (hours post-fertilization). An embryo expressing GFP was used as a control (A).

 

Figure 4
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  		Fig. 4. Abnormal morphology of the heart of the pro-caspase-3 transgenic zebrafish. (A) wild-type embryo. (B,C) casp-1 and casp-2, transgenic embryos expressing pro-caspase-3. Arrows indicate the ventricles of embryos at 48 h.p.f. The heart cavity of the transgenic embryos was enlarged (casp-1 line). The ventricle formed an elongated structure and was reduced in size.

 

Figure 5
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  		Fig. 5. Cross-sections of the eye of (A) wild-type and (B) pro-caspase-3 transgenic zebrafish (casp-1 line) at 48 h.p.f. le, lens; ipl, inner plexiform layer; inl, inner nuclear layer; pcl, photoreceptor cell layer; pe, pigmented epithelium.

 

Figure 6
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  		Fig. 6. Transverse sections of the notochord in (A) wild-type and (B) pro-caspase-3 transgenic zebrafish (casp-1 line) at 48 h.p.f. Arrowheads indicate abnormal (irregular) formation of the notochord in the transgenic embryo.

 

Figure 7
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  		Fig. 7. Caspase activity in the transgenic zebrafish. Acetyl-DEVD-{alpha}-methylcoumaryl-7-amide (Ac-DEVD-MCA)-hydrolysing activity was measured in the embryos of casp-1 and casp-2 lines at 3, 6, 9 and 24 h.p.f. Pooled samples, each containing 10 embryos, were used for the assay. Each value is the mean ± s.d. of three independent experiments. Asterisks denote significant differences between wild-type and transgenic embryos (P<0.05, ANOVA).

 

Figure 8
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  		Fig. 8. Detection of apoptotic cells by fluorescent TUNEL staining of transgenic embryos. wild-type, GFP control embryos at 12 h.p.f. (A) and 24 h.p.f. (C). casp-1, transgenic embryos expressing pro-caspase-3 at 12 h.p.f. (B) and 24 h.p.f. (D).

 

Figure 9
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  		Fig. 9. Immunohistochemical detection of TUNEL-positive cells in the eye of a transgenic embryo expressing pro-caspase-3 (casp-1 line) at 36 h.p.f. Arrowheads indicate TUNEL-positive cells. (A,C) GFP-expressing embryo used as a control. (B,D) Transgenic embryo expressing pro-caspase-3.

 

Figure 10
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  		Fig. 10. Survival of transgenic embryos following UV irradiation. The casp-1 line (filled circles, N=90) and wild-type (open circles, N=90) at 12 h.p.f. were UV irradiated at 0 mJ cm–2 (A), 2 mJ cm–2 (B) or 5 mJ cm–2 (C), maintained at 28.5°C, and monitored daily for survival. The data were analysed using Kaplan–Meier survival analysis, and an asterisk indicates a statistically significant difference (P<0.05). Each value is the mean ± s.e.m.

 

Figure 11
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  		Fig. 11. Repression of caspase-3 expression in the caspase-3-morpholino antisense oligonucleotide (MO)-injected embryos. The active form of caspase-3 was detected by immunohistochemistry with anti-active caspase-3 antibody. (A) control, embryos injected with MO (1 ng) at 9 h.p.f. (B) caspMO, embryos injected with caspase-3-MO (1 ng) at 9 h.p.f. (C) Caspase-3 activity against Ac-DEVD-MCA was compared between the control MO-injected and caspase-3-MO-injected embryos at 9 h.p.f. Each value indicates the mean ± s.d. of three independent experiments. An asterisk denotes a statistically significant difference between the control MO-injected and the caspase-MO-injected embryos (P<0.05, Student's t-test). The activity of the caspase-3-MO-injected embryos was significantly lower than that of the controls.

 

Figure 12
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  		Fig. 12. Enhanced stress tolerance in the caspase-3-MO-injected embryos following UV irradiation. Survival ratio was compared between the control MO-injected and the caspase-MO-injected embryos. MO was introduced by microinjection into one-cell stage embryos. Open triangles, control embryo injected with control MO (N=90); filled triangles, wild-type embryo injected with caspase-3-MO (N=90). The data were analysed using the Kaplan–Meier survival analysis, and an asterisk indicates a statistically significant difference (P<0.05). Each value indicates the mean ± s.e.m.

 

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