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Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3
Published on: December 20, 2013
Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos
Jelena Hrubik1, Branka Glisic1, Dragana Samardzija1
1University of Novi Sad, Faculty of Sciences, Department of Biology and Ecology, Laboratory for Ecotoxicology, Novi Sad, Serbia.
Abstract:
Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos.
Insights
Xenobiotic-induced Protein Kinase C (PKC) activation in zebrafish embryos caused developmental deformities, apoptosis, and reduced survival. While inhibiting PKC improved survival and reduced apoptosis, it did not prevent deformities or oxidative stress.
Area of Science:
- Developmental Biology
- Toxicology
- Molecular Biology
Background:
- Protein Kinase C (PKC) isoforms play crucial roles in early embryonic development.
- The impact of xenobiotic-induced PKC activation on early embryogenesis remains largely unknown.
- Understanding these effects is vital for assessing developmental risks of environmental toxins.
Purpose of the Study:
- To investigate the consequences of phorbol 12-myristate 13-acetate (PMA)-induced Protein Kinase C (PKC) activation during zebrafish early embryogenesis.
- To determine the effects of PMA on embryonic development, survival rates, apoptosis, and oxidative stress.
- To evaluate the efficacy of PKC inhibition in mitigating PMA-induced adverse effects.
Main Methods:
- Zebrafish embryos were exposed to varying concentrations of phorbol 12-myristate 13-acetate (PMA).
- Developmental endpoints, survival rates, and apoptosis (using acridine orange staining) were assessed.
- Gene expression analysis for caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma), catalase, and superoxide dismutase 2 was performed.
- PKC inhibition was achieved using GF109203X.
Main Results:
- PMA exposure at 200μg/L induced significant developmental abnormalities, including cardiac edema, yolk sac edema, and coagulation.
- PMA exposure led to decreased embryo survival rates and increased apoptosis in the brain region.
- PMA caused upregulation of casp9 and puma mRNA and downregulation of catalase and superoxide dismutase 2 mRNA, indicating apoptosis and oxidative stress.
- PKC inhibition with GF109203X improved survival and reduced apoptosis but did not reverse developmental deformities or oxidative stress.
Conclusions:
- Direct over-activation of Protein Kinase C (PKC) during zebrafish early embryogenesis is linked to apoptosis and reduced survival.
- While PKC inhibition can ameliorate some PMA-induced effects like apoptosis and mortality, it does not fully protect against developmental deformities or oxidative stress.
- These findings highlight the complex role of PKC in embryonic development and its susceptibility to xenobiotic disruption.
