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Updated: May 2, 2026
Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3
Published on: December 20, 2013
The role of apoptosis in MCLR-induced developmental toxicity in zebrafish embryos
Cheng Zeng1, Hong Sun2, Ping Xie3
1College of Fisheries, Huazhong Agricultural University, Wuhan 430070, China.
Abstract:
We previously demonstrated that cyanobacteria-derived microcystin-leucine-arginine (MCLR) is able to induce developing toxicity, such as malformation, growth delay and also decreased heart rates in zebrafish embryos. However, the molecular mechanisms by which MCLR induces its toxicity during the development of zebrafish remain largely unknown. Here, we evaluate the role of apoptosis in MCLR-induced developmental toxicity. Zebrafish embryos were exposed to various concentrations of MCLR (0, 0.2, 0.5, 2, and 5.0 mg L(-1)) for 96 h, at which time reactive oxygen species (ROS) was significantly induced in the 2 and 5.0 mg L(-1) MCLR exposure groups. Acridine orange (AO) staining and terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labelling (TUNEL) assay showed that MCLR exposure resulted in cell apoptosis. To test the apoptotic pathway, the expression pattern of several apoptotic-related genes was examined for the level of enzyme activity, gene and protein expression, respectively. The overall results demonstrate that MCLR induced ROS which consequently triggered apoptosis in the heart of developing zebrafish embryos. Our results also indicate that the p53-Bax-Bcl-2 pathway and the caspase-dependent apoptotic pathway play major roles in MCLR-induced apoptosis in the developing embryos.
Insights
Microcystin-leucine-arginine (MCLR) from cyanobacteria causes developmental toxicity in zebrafish embryos. MCLR induces reactive oxygen species (ROS), triggering apoptosis via p53-Bax-Bcl-2 and caspase pathways.
Area of Science:
- Environmental toxicology
- Developmental biology
- Cellular toxicology
Background:
- Cyanobacteria blooms release toxins like microcystin-leucine-arginine (MCLR).
- MCLR is known to cause developmental toxicity in zebrafish embryos, including malformations and growth delays.
- The precise molecular mechanisms underlying MCLR's developmental toxicity are not fully understood.
Purpose of the Study:
- To investigate the role of apoptosis in MCLR-induced developmental toxicity in zebrafish embryos.
- To elucidate the molecular pathways involved in MCLR toxicity.
Main Methods:
- Zebrafish embryos were exposed to varying concentrations of MCLR (0–5.0 mg L⁻¹) for 96 hours.
- Reactive oxygen species (ROS) levels were measured.
- Apoptosis was assessed using Acridine Orange (AO) staining and TUNEL assays.
- Expression of apoptosis-related genes (p53, Bax, Bcl-2) and caspase activity were analyzed.
Main Results:
- MCLR exposure significantly induced ROS at concentrations of 2 and 5.0 mg L⁻¹.
- Apoptosis was confirmed in zebrafish embryos exposed to MCLR.
- MCLR-induced apoptosis in the heart was mediated by ROS.
- The p53-Bax-Bcl-2 pathway and caspase-dependent pathways were identified as key players in MCLR-induced apoptosis.
Conclusions:
- MCLR induces developmental toxicity in zebrafish embryos primarily through ROS-mediated apoptosis.
- Both the p53-Bax-Bcl-2 and caspase-dependent pathways are critical in the apoptotic response to MCLR.
- Understanding these mechanisms is crucial for assessing the risks of cyanobacterial toxins.
