Abstract
The zebrafish has many advantages as a vertebrate model organism and has been extensively used in the studies of development. Its potential as a model in which to study tumour suppressor and oncogene function is now being realized. Whilst in situ hybridization of mRNA has been well developed in this species to study gene expression, antibody probes are in short supply. We have, therefore, generated a panel of anti-zebrafish p53 monoclonal antibodies and used these to study the p53 response in zebrafish embryos. By immunohistochemistry, we show that the exposure of zebrafish embryos to p53-activating agents such as R-roscovitine and γ-irradiation results in the accumulation of p53 protein in the gut epithelium, liver and pancreas. A combination of R-roscovitine and γ-irradiation results in massive p53 induction, not only in the pharyngeal arches, gut region and liver but also in brain tissues. Induction of apoptosis and expression of p53 response genes are seen in regions that correspond to sites of p53 protein accumulation. In contrast, although zebrafish tp53M214K mutant embryos showed a similar accumulation of p53 protein, a complete lack of a downstream p53-dependent response was observed. In this system the p53 gene is identified as a p53-responsive gene itself. Our results demonstrate that zebrafish p53 protein can readily be induced in embryos and detected using these new antibody tools, which will increase the usefulness of zebrafish as a model in compound-based screening for novel drugs in cancer research.
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Acknowledgements
We thank Dr Hwang Le-Ann from the Monoclonal Antibody Unit at the Institute of Molecular and Cell Biology for assistance in screening the hybridoma clones. The pCS-myc-Zp53 plasmid was a generous gift from Dr Jacques Piette (Centre de Recherche en Cancérologie, France). The zebrafish tp53M214K mutant line was kindly provided by Professor A Thomas Look (Dana-Farber Cancer Institute, USA). Zebrafish embryos were processed and sectioned by the Biopolis Shared Facility Histology unit. Professor David Lane is a Gibb Fellow of Cancer Research UK. This work was supported by the BioMedical Research Council of Singapore.
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Lee, KC., Goh, W., Xu, M. et al. Detection of the p53 response in zebrafish embryos using new monoclonal antibodies. Oncogene 27, 629–640 (2008). https://doi.org/10.1038/sj.onc.1210695
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DOI: https://doi.org/10.1038/sj.onc.1210695
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