Genetic interaction among man1a2, arf6 and EGFR signaling in intrahepatic biliary network formation. (A,C) Epifluorescence images of larvae treated with PED6 and its quantification. Suboptimal doses of man1a2 (1.5 ng) and arf6 (0.5 ng) MOs were singly or in combination injected into embryos at the one-cell stage (A). Embryos were first injected with the suboptimal dose of man1a2 MO (1.5 ng) and from 60 hpf treated with a suboptimal dose of AG1478 (1 μM) (C). Arrows point to the gallbladder. Graphs show the percentage of larvae in each group. Numbers in the graph indicate the number of larvae in each group. (B,D) Confocal images of the liver showing the intrahepatic biliary network, as revealed by BODIPY-FL C5 feeding (green, biliary ductal network) and Tp1:H2B-mCherry (red, BEC nuclei) expression. Single injection of suboptimal doses of either man1a2 or arf6 MO did not impair proper intrahepatic biliary network formation, whereas their co-injection resulted in defective biliary network formation (B). The biliary network formation was not impaired in larvae only treated with AG1478, whereas it was impaired in AG1478-treated larvae with man1a2 partially knockdown (D). Numbers in the upper right corner are the fraction of larvae exhibiting the representative phenotype shown. Dotted lines outline the liver. Scale bars: 50 μm.
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