Fig. 1

HDACs are required for liver and exocrine but not endocrine pancreas formation. (a) Embryos from Tg(lfabp:RFP; elaA:EGFP) and Tg(fli-1:EGFP) were treated from shield stage with VPA (20 μM) as described in Materials and methods. Liver formation was examined by the appearance of red fluoresce (white arrows) at 3 dpf (A), 4 dpf (C) and 5 dpf (E and F). In VPA-treated embryos, liver was not formed up to 4 dpf as judged by the absence of red fluorescent, while a much smaller liver emerged at 5 dpf (E and F). Blood vessel was monitored by green fluorescence and ISV defect is indicated by thin white arrow (G, H). (b) Liver formation analyzed by liver differentiation marker Ceruloplasmin (cp) expression at various stages of liver formation including 32 hpf (A, B) and 48 hpf (C, D), 3 dpf (E, F), 4 dpf (G, H) and 5 dpf (I, J). Cp expression was absent in liver (white arrow) in VPA-treated embryos up to 3 dpf although its expression in YSL (white arrowhead) was not affected (C, D, E and F). A small number of liver cells start to express Cp at 4 dpf which grew, but never reached the control liver size at 5 dpf (I vs. J). All the images are dorsal views, anterior to the left. (c) Liver and exocrine pancreas formation analyzed in transgenic line and endocrine pancreas formation analyzed by insulin expression. Panels A–D are merged images of z-stack taken by confocal microscope. Liver (in red) is indicated by a thick white arrow and exocrine pancreas (in green) by a thin white arrow. Endocrine pancreas (black arrow, E–J) as indicated by insulin (ins) expression was not affected by VPA treatment from 2 dpf to 5 dpf. All the images are dorsal views, anterior to the left. Scale bar represents 100 μm for all panels except in panel aG, which represent 30 μm and panel cA, which represents 50 μm.