Zebrafish as a model for analysis of signaling pathways involved in cell growth, proliferation and development
Author
Sucularlı, Ceren
Advisor
Konu, Özlen
Date
2011Publisher
Bilkent University
Language
English
Type
ThesisItem Usage Stats
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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.