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Updated: Jul 4, 2026
Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3
Published on: December 20, 2013
Extensive apoptosis and abnormal morphogenesis in pro-caspase-3 transgenic zebrafish during development
Michiaki Yamashita1, Nanami Mizusawa, Misako Hojo
1National Research Institute of Fisheries Science, 2-12-4 Fukuura, Yokohama 236-8648, Japan. mic@affrc.go.jp
Abstract:
The pro-apoptotic caspase-3 gene has been shown to have key functions in the execution of apoptosis (programmed cell death) in vertebrate cells. However, the central role of caspase-3 in morphogenesis during development remains unclear. In this study, transgenic zebrafish that overexpress full-length pro-caspase-3 were generated to determine the effects of caspase genes on vertebrate morphogenesis and stress tolerance. The enhanced expression of the full-length pro-caspase-3 cDNA induced extremely high levels of caspase activity and extensive apoptosis in the transgenic embryos, and 33-46% of F2 embyos in the transgenic lines exhibited some form of morphological abnormality. Pro-caspase-3 transgenic zebrafish exhibited abnormal morphogenesis in the eyes, notochord, heart and yolk sac, suggesting that enhanced processing of pro-caspase-3 triggers significant apoptotic responses in the specific target tissues that are undergoing morphogenesis during development. The transgenic fish had reduced eye size and showed degeneration of the retina, including the photoreceptor cell layers, whereas pigmentation and lens formation were not affected. In addition, heart failure due to a weakened heartbeat and reduced circulation was noted in the pro-caspase-3 transgenic embryos. The transgenic embryos were markedly sensitive to stress conditions, such as UV irradiation at 2 or 5 mJ cm(-2). On the other hand, caspase-3 deficiency through injection of antisense morpholino oligo into embryos repressed apoptosis and enhanced stress tolerance after UV irradiation. Therefore, the caspase-3-mediated pro-apoptotic signalling pathway and its activation play critical roles in the induction of apoptosis and stress tolerance during zebrafish embryogenesis.
Insights
Overexpressing caspase-3 in zebrafish embryos caused developmental abnormalities and increased apoptosis. Caspase-3 deficiency, however, enhanced stress tolerance, highlighting its role in development and stress response.
Area of Science:
- Developmental Biology
- Molecular Biology
- Genetics
Background:
- Caspase-3 is crucial for apoptosis (programmed cell death) in vertebrates.
- Its specific role in embryonic development and morphogenesis is not fully understood.
Purpose of the Study:
- To investigate the function of caspase-3 in vertebrate development and stress tolerance using transgenic zebrafish.
- To determine the impact of enhanced caspase-3 activity on embryonic morphogenesis.
Main Methods:
- Generation of transgenic zebrafish overexpressing full-length pro-caspase-3.
- Analysis of morphological abnormalities and apoptosis levels in transgenic embryos.
- Assessment of stress tolerance (UV irradiation) in transgenic and morpholino-injected zebrafish.
Main Results:
- Overexpression of pro-caspase-3 led to high caspase activity, extensive apoptosis, and significant morphological defects in zebrafish embryos (eyes, notochord, heart, yolk sac).
- Transgenic fish showed reduced eye size, retinal degeneration, heart failure, and increased sensitivity to UV stress.
- Caspase-3 deficiency via morpholino injection repressed apoptosis and improved stress tolerance.
Conclusions:
- Enhanced pro-caspase-3 processing significantly impacts zebrafish embryogenesis, affecting morphogenesis and stress tolerance.
- The caspase-3-mediated apoptotic pathway is critical for regulating apoptosis and stress response during zebrafish development.
- Caspase-3 plays a vital role in normal embryonic development and stress adaptation.
