id2a knockdown reduces liver size but does not block hepatoblast specification or hepatocyte differentiation. (A) Epifluorescence images revealing a decreased number of Tp1:GFP+ BECs in the liver of id2a MO-injected embryos at 72 hpf compared with controls (squares). Higher magnification images of the square regions are shown in insets. Lateral view, anterior to the left. (B) Confocal projection images revealing fabp10a:dsRed (hepatocytes; red), Tp1:GFP (BECs; green) and Anxa4 (the hepatopancreatic ductal system; blue) expression (Zhang et al., 2014). In id2a MO-injected embryos, liver size was greatly reduced and intrahepatic BECs appeared aggregated, displaying a branching defect. (C–H) id2a MO-injected and uninjected control embryos were processed for WISH with hhex (C), prox1a (D), fabp10a (E), cp (F), sepp1b (G), and cdx1b (H) probes. Overall liver size was greatly reduced in id2a MO-injected embryos as revealed by the hepatoblast markers (hhex and prox1a) and the hepatocyte markers (fabp10a, cp, and sepp1b). However, the expression of these genes was clearly detected in the MO-injected embryos (C–G, arrows), indicating normal hepatoblast specification and hepatocyte differentiation upon id2a knockdown. The induction of the intestinal bulb as assessed by cdx1b expression appeared normal in id2a MO-injected embryos at 36 hpf; however, the intestinal bulb failed to grow at 48 hpf (H, brackets). The percentage of id2a MO-injected embryos exhibiting the representative phenotype shown is indicated in the upper left corner (n = 10–20). The remaining percentage of embryos exhibited an intermediate liver/intestinal bulb phenotype: their liver/intestinal bulb size was still smaller than that of the control embryos. Arrows point to the liver. Scale bar: 250 (A), 20 (B), and 100 (C–H) µm.