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. Author manuscript; available in PMC: 2015 Nov 1.
Published in final edited form as: Hepatology. 2014 Sep 10;60(5):1753–1766. doi: 10.1002/hep.27285

Fig. 2.

Fig. 2

A population of cells, responsive to Notch signaling, maintains their capacity to regenerate as hepatocytes in adult zebrafish. Liver morphology (white dashed lines) of 12-month-old [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] adults treated with dimethyl sulfoxide (DMSO) (A), MTZ (B) or 1% EtOH followed by MTZ (C). (D–F”) Confocal images of Tg(fabp10a:CFP-NTR), Tg(Tp1:mCherry), and type I collagen expression in vibratome sections of adult zebrafish livers. (D–D”) In the DMSO-treated controls, expression of Tg(fabp10a:CFP-NTR) did not overlap with that of Tg(Tp1:mCherry) (D”, inset) with almost undetectable type I collagen deposition (D’). (E–E”) Hepatocytes were ablated by 10mM MTZ treatment for 8 hours and allowed to regenerate for 3 days. Numerous cells co-expressing hepatocyte-specific CFP and NRC-specific nuclear mCherry were observed throughout the MTZ-treated regenerating livers (E”, inset, white arrows) with a modest increase in type I collagen deposition (E’), while bright red NRCs remained CFP negative (E”, inset, yellow arrowhead). (F–F”) The fish were pretreated with 1% EtOH for 72 hours followed by 10mM MTZ treatment for 8 hours and allowed to regenerate for 3 days. Significantly elevated type I collagen deposition was observed in the EtOH/MTZ-treated regenerating livers (F’), while a population of cells co-expressing hepatocyte-specific CFP and NRC-specific nuclear mCherry was appreciated (F”, inset, white arrows). The bright red NRCs remained CFP negative (F”, inset, yellow arrowheads). (G) qRT-PCR analysis showed upregulation of collagen 1a1a and collagen 1a2 mRNA in the EtOH/MTZ-treated regenerating livers. n=3 dissected adult fish liver per condition in three experiments. Asterisks indicate statistical significance: **p<0.01 and ***p<0.001. (H) Representative western blot showed upregulation of type I collagen protein in the EtOH/MTZ-treated regenerating livers. Numbers show relative expression of type I collagen protein after normalization against β-actin protein. n=3 dissected adult fish liver per condition in three experiments. A–C, bright-field images. D–D”, E–E”, and F–F”, confocal single-plane images. For each experiment, after capturing each bright-field image, the liver from the same fish was dissected and processed for section immunostaining to get confocal images (n=6 fish per condition in three experiments). Scale bars: A–C, 2mm; D–D”, E–E”, and F–F”, 20µm. EtOH, ethanol; SD, standard deviation.