Fig. 2. Assays of Shroom2 activity. (A-C) Shroom2 activity was measured by blind-scoring of pigment accumulation in injected embryos. Panels A-C show representative embryos displaying normal, mild and severe phenotypes for the scoring shown in D-F. (D) Shroom2 (576-882) blocked the ability of ectopic Shroom2 to induce apical pigment accumulation in a dose-dependent manner. Shroom2 (0.4 ng) was injected alone or in combination with 0.8 ng or 1.6 ng of Shroom2(576-882) in one blastomeres at the four-cell stage. (E) Shroom2 does not function through the Rap1B GTPase. Shroom2 (0.75 ng) was injected alone or in combination with 2 ng of Rap1B-N17 or RapGAP into one blastomere at the four-cell stage. (F) Shroom2 acts upstream of Rab27a as co-expression of either dominant-negative Rab27a-L130P or Rab27a-A152P inhibited Shroom2 activity. Shroom2 (0.4 ng) was injected alone or in combination with 1.2 ng of Rab27a-L130P or Rab27a-A152P into one blastomere at the four-cell stage.