Retinoic acid signaling enhances embryonic liver development. (A–F) In situ hybridization for lfabp at 72 hpf reveals differences in liver size after chemical exposure at 18 hpf. ATRA treatment leads to greater lfabp expression, whereas DEAB treatment leads to reduced lfabp expression. First row, lateral view. Second row, dorsal view. (G–I) Fluorescence microscopy of lfabp:GFP reporter fish at 72 hpf confirms in situ hybridization results in (A–F). (J–L) Fluorescence microscopy of cyp26a1:eYFP reporter fish at 72 hpf demonstrates changes in RA signaling activity in chemically treated embryos. Arrowheads highlight the liver. (M–O) BrdU whole mount immunostaining of 24 hpf embryos demonstrates that ATRA-treated embryos show higher levels of proliferation in the liver primordium (black rectangle) compared to DEAB and DMSO-treated controls. (M2–O2) Enlarged images of (M–O) illustrating individual proliferating cells in the liver primordium. (P) Quantification of the number of BrdU+ cells/field (black rectangle in O). (Q) FACS quantification of percent GFP+ cells in chemically treated lfabp:GFP embryos reveals that ATRA-treated embryos contain more hepatocytes and DEAB-treated embryos contain fewer hepatocytes than DMSO-treated controls. (R) qPCR analysis reveals differences in the relative expression of the hepatocyte marker transferrin in ATRA and DEAB-treated embryos at 72 hpf compared to DMSO-treated controls (where control expression levels were normalized to 1.0). Scale bars: 100 ¼m.
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