Fig. 7. Ttrap knockdown modulates Nodal-Alk4 signaling. (A) Ttrap knockdown increases activity of ARE-luciferase reporter. ARE-lux plasmid (50 pg) was co-injected with Ttrap^MO (or control^MO) and embryo lysates assayed for luciferase at shield stage. Knockdown results in fivefold greater induction relative to control (4.6±2.5; P0.01; Student's unpaired t-test; eight independent experiments). No significant increase in luciferase was detectable for control pGL3. (B) Ttrap^MO potentiates ARE-lux by sqt or cyc. This experiment was performed as described in A, this time in combination with sqt or cyc mRNA injection (11 pg). The addition of Ttrap^MO induced ARE-lux an additional tenfold relative to induction by either one of the ligands [81.0±15.6 (ARE+sqt+Ttrap^MO) vs 9.0±2.6 (ARE+sqt); 38.0±8.6 (ARE+cyc+Ttrap^MO) vs 3.8±0.8 (are+cyc); P0.0001, One-way analysis of variance (ANOVA)]. y-axis, fold-induction of luciferase. (C-F) bon is visibly upregulated in Ttrap^MO and Smad3b^OE embryos; WISH at 50% epiboly, animal views (asterisks). (G,H) Ttrap^MO-mediated increase in bon expression depends on intact alk4 activity. (G) Ttrap^MO embryo shows strong expression of bon, whereas (H) Ttrap^MO embryo treated with SB431542 no longer expresses bon. Animal views (asterisks). (I-N) Overexpression of Smad3b causes CE and epiboly defects. WISH at 90% epiboly, paraxial mesoderm marker expression in βgal^OE (700 pg) and Smad3b^OE (700 pg) embryos. Dorsal-posterior views, anterior at top. (I,L) papc cells fail to converge near the midline in Smad3b^OE embryos compared with control βgal^OE embryos. Arrowheads indicate blastopore opening, which is wider in Smad3b^OE embryos. (J,M) Distance between myod cells is greater in Smad3b^OE relative to control embryos (double-headed arrows). (K,N) Live observation of β-gal^OE and Smad3b^OE embryos, 90% epiboly. (K) Control β-gal^OE embryo displaying normal epiboly and nearing blastopore closure. (N) Smad3b^OE embryo showing severe delay in epiboly. Red arrowheads, edge of blastoderm margin. Lateral views, anterior at top. Note that the gastrulation defects depicted here are at an earlier timepoint than those shown for Ttrap^MO embryos (see Fig. 2). Importantly however, the same gastrulation defects were also observed for Ttrap^MO embryos at this earlier stage (i.e. 90% epiboly).