Zebrafish as a model for analysis of signaling pathways involved in cell growth, proliferation and development

Abstract

Zebrafish is an emerging and promising model organism to study cancer formation, organogenesis, development, cell signaling, and drug screening applications. Cellular signaling driven by E2F and TOR proteins regulate cell proliferation, growth and development; yet expression of E2F targets and downstream effectors of TOR inhibition have not been studied in zebrafish in detail. In this study, we first demonstrated the conservation of E2F target ortholog expression in zebrafish in response to serum; second our results revealed significant changes in the zebrafish fibroblast cells (ZF4) at the whole transcriptome level upon treatment with rapamycin, an inhibitor of TOR; and third we phenotypically screened zebrafish embryos in vivo when exposed to different doses of rapamycin. Our studies showed that as in mammalian cells, ZF4 cells entered into a quiescent state at G1/S phase in the cell cycle, which was reversed by serum stimulation. We showed that serum response of selected E2F target gene orthologs, namely pcna, mybl2, tyms, mcm7 and ctgf, were conserved between zebrafish and mammals. Using microarray analysis, we demonstrated that rapamycin modulated expression of a large number of genes in ZF4 cells with functions ranging from cell cycle to protein synthesis. Similar to previous findings in mammals, rapamycin treatment downregulated expression of proteasomal subunits in zebrafish. Our findings in zebrafish also implicated a moderate increase in expression of ribosomal subunits; this finding warrants further comparison with mammalian studies. qRT-PCR studies confirmed dkk1b, pah, dcc, cyp26b1 and wif1 as being significantly differentially expressed under rapamycin treatment using a time-course experiment. In zebrafish embryos, in vivo exposure to rapamycin caused a significant dose-dependent developmental delay and in particular prominent reductions in formation of pigments and cartilage, tissues known to be derived from embryonic neural crest cells. Our study implicates a potential role for TOR in the neural crest formation, differentiation or migration in zebrafish. Our study also clearly establish ZF4 cells as a model to further study signaling pathways involved in cell proliferation, growth and development.