|
Fig. 5
Reduced ESCO2 function is associated with mTOR inhibition, p53 activation, and dramatic developmental phenotypes in zebrafish.
Total levels of S6, S6K1, and tubulin serve as loading controls. (A). Embryos were microinjected with 4 ng ESCO2-5mismatched morpholino (ESCO2-5mis) or ESCO2-ATG morpholino (ESCO2-MO) and photographed at 24 hours post fertilization (h.p.f.) and 3 days post fertilization (d.p.f.). Scale bar = 200 µm. (B). Embryos were microinjected with 1, 2, or 4 ng ESCO2-5mis or ESCO2-MO and photographed at 3 d.p.f. Scale bar = 200 μm. (C). Embryos were microinjected with 4 ng ESCO2-5mis or 1, 2, or 4 ng ESCO2-MO to test the effect of dosage on phophorylation of S6 by Western blot at 2 d.p.f. (D). ESCO2 morphant embryos (2 ng) show inhibition of the TOR pathway and accompanying activation of p53 at 24 h.p.f.. (E). ESCO2-transgenic mutant zebrafish embryos show gross developmental abnormalities compared with WT embryos at 24 h.p.f. and 3 d.p.f.. Bar = 200 μm. (F). ESCO2 mutant embryos show reduced S6 phosphorylation and upregulation of p53, Mdm2, and p27 by Western blot analysis.