Conservation of zebrafish At3 expression and function. (A-D) In situ hybridization for at3 mRNA demonstrates liver-specific expression in 5 dpf larvae. Larvae were photographed with an Olympus BX-51 upright light microscope using an Olympus DP-70 digital camera. (A-B) Left lateral view at low and high magnification, respectively. (C-D) Dorsal view at low and high magnification, respectively. Arrow indicates the liver. Scale bar, 10 µm (C), 100 µm (D). In situ hybridization with a sense strand probe did not show any signal (not shown). (E) Alignment of the region flanking the AT3 P1 arginine (arrowhead). (F) Western blot analysis (using anti-human thrombin antibody) on recombinant At3 pulled down with human thrombin after expression in HEK 293 cells. Thrombin input only (−), pCDNA3.1 without insert (empty), pCDNA3.1zat3 (At3), and pCDNA3.1zat3R410G (At3^R410G). The intermediate band present at ∼50 kDa is an unknown protein present in the cell lysate and recognized by the antibody, but unrelated to expression of At3. (G) Time to occlusion of larvae (3 dpf) derived from at3^+/− × at3^−/− progeny injected at the 1-cell stage with the zebrafish At3^R410G expressing cDNA transgene regulated by the ubi promoter (at3^−/−, n = 34). Horizontal bars represent the median of time to occlusion.