Identification of Stat3 phosphorylation as a possible mediator of Gitrl effects during embryogenesis. (A) Total protein lysates were generated from embryos injected with 10 ng of control1 (C) or zGL1 (MO) and harvested at 6, 8, 11 and 19 hpf. The lysates were analysed by western blotting using antibodies specific for phosphorylated (P-Stat3) and total (Stat3) Stat3 content. Embryos (AB*) treated with (B-F) DMSO or (G-K) 5 μM of stattic at 2-4 hpf were compared for phenotypic overlap with the gitrl morphant phenotype shown in figure 3. Stattic-treated embryos displayed incomplete blastoderm margin advancement (arrowhead in (G)) compared to controls (arrowhead in (B)), and indistinct somite boundary formation (H-K) similar to that seen in gitrl morphants. (L) TL embryos were more sensitive than AB* embryos to Stat3 inhibition. (M) TL embryos treated with a low dose of zGL1 (2.5 ng) were additionally treated with either DMSO or 2 mM of stattic at 2 hpf. Embryos that received low-dose MO plus DMSO were viable, but showed incomplete blastoderm margin advancement at 75 % epiboly, while embryos treated with low-dose zGL1 in addition to treatment with stattic showed a lethal phenotype.
|
