copeb is required for achieving proper liver size but is dispensable for hepatic patterning. (a–b) fabp10 WISH reveals that the small liver in 4 dpf copeb morphants extends across the midline. Two representative morphants are shown. L indicates the left side of the embryo, where the liver bud forms. (c) The number of embryos with a liver size that was less than 50% of normal was scored in 3 clutches. The total n = 75 for morphants and for controls at 3 dpf n = 71 for each at 4 dpf. p < 0.01 when comparing morphants to controls for both time points. (d) mRNA expression of genes implicated in hepatocyte function were determined by qPCR in control embryos compared to copeb morphants at 4 dpf. Expression levels were normalized to control embryos, the experiments were repeated in 3 independent clutches using 30 embryos per sample. Errors bars show standard deviation. All genes are significantly down-regulated (p < 0.05). (e–h) WISH analysis of gata6 (e, f) and prox1 (g, h) in control embryos (e, g) and copeb morphants (f, h) at 48 hpf. All control embryos had the same staining pattern. The percent of copeb morphants with staining that is indistinguishable from control embryos, as shown in the representative image, is indicated in the lower right corner of f and g (gata6: n = 41, prox1: n = 70). (i–l) Serial sections of control embryos and copeb morphants on 3 and 4 dpf were stained with H&E and the largest section through the liver (outlined) was imaged, illustrating the reduced size of the liver. Arrows point to sinusoids containing nucleated endothelial cells. Hepatocyte morphology is the same in 4 dpf control and copeb morphants (inset; scale bar = 40 μM).
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