Effects of rapamycin on the early development of zebrafish (Danio rerio)

Abstract

Rapamycin, isolated from the soil bacteria Streptomyces hygroscopicus as an antibiotic, previously was shown to have negative effects on the immune system, cancer development, and cell cycle using different model organisms. Specific effects of rapamycin on the TOR protein activity also was determined by these previous studies. In general, TOR is a kinase that play an important role in the transmission of the signals from growth factors and amino acids to proteins involved in translation processes. In spite of the increasing popularity of zebrafish (Danio rerio) as a model organism in the developmental biological studies, no previous study about the effects of rapamycin on zebrafish exists in the literature. In the present thesis, zebrafish TOR (DrTOR) gene structure was first characterized in silico and then compared in terms of its homology with the mammalian TORs at the cDNA and protein levels. Expression of DrTOR was shown at different embryonic/larval stages and in different tissue samples. Furthermore, different doses of rapamycin were given to the embryos during the early developmental time (2, 3, 4, 5 dpf). Based on the morphometric analyses, rapamycin concentrations greater than 1 µM caused a significant reduction in the larval size. Additionally, 20 µM rapamycin significantly and completely abolished any larval growth supposed to take place during this time period. In the 20 µM rapamycin experiment, also a retardation in the pigment cell (melanocyte) and cranial cartilage development was observed. Expression profiles of the mitfA and DrTOR were analyzed by using the real-time RTPCR. While DrTOR expression was upregulated (3.5 fold) by 20 µM rapamycin, mitfA expression was downregulated (% 40). Finally, a siRNA designed against the TOR gene was tested for its inhibitory activity when given to embryos in a solution.