Characterization of the tumor suppressor PTEN in zebrafish

Description

The tumor suppressor PTEN is essential in stress-induced senescence and aging in invertebrates. However, because mice with complete PTEN ablation are lethal or develop tumor in the targeted organs, a vertebrate model to study organismal senescence is currently lacking. Zebrafish have two Pten genes, either of which can be homozygously inactivated without impairing development or inducing tumors. Therefore, we established the zebrafish as a versatile model system to examine the role of PTEN in redox-induced senescence in the context of a whole animal. PTENb-deficient embryos displayed p53-dependent hypersensitivity to oxidative stress. Strikingly, we discovered that organismal senescence is activated by certain circulating factor(s) able to trigger senescence in distant cells. We further identified one of these factors as TGF-β1 (Transforming Growth Factor-β1). Activation or silencing of TGF-β1 signaling correlated with the appropriate senescence readout in the whole animal. Importantly, we confirmed that PTEN-deficient adults preserved hypersensitivity to oxidative stress and died significantly earlier than the wild type siblings, suggesting that the embryonic fish model can provide valuable insights into the senescence signaling. Our data demonstrate a novel use of the zebrafish and identified a previously unknown crosstalk between PTEN and TGF-β1 signaling pathways in mediating organismal senescence. RNA interference (RNAi) has been established as a potent gene "knockdown" technology in C. elegans, Drosophila, chicken, and mammalian systems. In this thesis, we demonstrate that both short interfering RNAs (siRNAs) and microRNA-based shRNA system can effectively and specifically inhibit gene function in zebrafish. We first examined the specificity and efficiency of siRNAs by inhibiting the expression of the exogenous genes EGFP and K-RAS V12 , and the endogenous smad5 gene. Next, an siRNA specific for the dominant-negative smad5 dtc24 allele had no effect on wild type fish but rescued the somitabon phenotype in smad5 dtc24 mutant fish, supporting the advantage of siRNAs in allele-specific suppression of target genes. Furthermore, we show that shRNAs display very efficient knockdown of gene expression when the shRNA is embedded in a microRNA context. Our mir-30 mediated shRNA expression system successfully suppressed the expression of EGFP, smad5 and Pten-a genes in zebrafish embryos. Importantly, the mir-30 shRNA system will allow us to achieve stable gene silencing when combined with transgenic fish technology.

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