Fig. 1.

Inducible gene expression in the sea urchin embryo using the Tet-On system. (A) Schematic of the transactivator and responder constructs used to induce GFP expression in PMCs (see Materials and Methods). (B) GFP expression in the PMCs of transgenic embryos exposed to 5 μg/ml Dox overnight, beginning at the early blastula or late gastrula stage (see Fig. S1A). Top: GFP fluorescence in live embryos. Bottom: GFP fluorescence overlaid onto differential interference contrast (DIC) images. (C) Dose-dependent induction of GFP expression by Dox. Embryos were treated with increasing concentrations of Dox (0, 2, 5, 10, 20, 40, 80 and 160 μg/ml) and relative GFP protein levels were quantified by ELISA (see Fig. S1B). There was a moderate but statistically significant decrease in relative GFP protein levels (1.28-fold) when comparing embryos treated with 5 and 20 μg/ml Dox (see Table S1). (D) Plot showing the time-dependent inducibility of GFP expression following Dox treatment. GFP expression was induced with 5 μg/ml Dox at the late gastrula stage and relative GFP protein levels were quantified by ELISA (see Fig. S1C,D). There was a statistically significant increase in relative GFP protein levels (7.6-fold) at 4 h compared with the first hour of Dox exposure (see Table S1). Error bars represent s.d. from three independently repeated experiments. Scale bars: 50 μm.