tomm22 knockdown reduces liver size. (A) Fluorescence images showing hepatic fabp10a:GFP expression (arrows) in tomm22 MO-injected larvae. Note very weak fabp10a:GFP expression until 6 days postfertilization (dpf) but its strong expression at 8 dpf. (B–E) Whole-mount in situ hybridization images showing the expression of prox1a, foxa3, fabp10a, sepp1b, and cp in the MO-injected embryos. Numbers indicate the proportion of larvae exhibiting the representative expression shown. Arrows point to the liver bud or the liver; arrowheads point to the dorsal pancreas. Scale bars: 100 μm.

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Prim-25 to Days 7-13
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage Range: Prim-25 to Day 6

Cell death and proliferation in tomm22 MO-injected larvae. (A) Confocal projection images showing Prox1 expression (gray) and terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) (green) in the liver bud (dashed lines) at 36 h postfertilization (hpf). Arrow points to TUNEL+ cells. (B) Confocal projection images showing TUNEL (green) and the expression of fabp10a:CFP-NTR (blue) and Tp1:H2B-mCherry (red) in the liver at 4 dpf. Arrow points to TUNEL+ hepatocytes. (C) Confocal projection images showing Prox1 expression (green) and 5-ethynyl-2′-deoxyuridine (EdU) labeling (gray) in the liver (dashed lines) at 45 hpf. Arrows point to EdU/Prox1 double-positive cells. (D) Graph showing the percentage of EdU+ cells among Prox1+ cells in the liver at 45 hpf. There was no significant difference in the proliferation rate between the control and tomm22 MO-injected larvae. Red marks indicate the embryos shown in (C); n indicates the number of larvae examined. Scale bars: 20 μm; error bars: ±SEM.

EXPRESSION / LABELING:
Genes:
Fish:
Knockdown Reagent:
Anatomical Terms:
Stage: Day 4
PHENOTYPE:
Fish:
Knockdown Reagent:
Observed In:
Stage Range: Prim-25 to Day 4

Biliary epithelial cells (BECs) give rise to hepatocytes in tomm22 MO-injected larvae. (A) Confocal single-optical section images showing Tp1:H2B-mCherry (red) and Hnf4a (gray) expression in the liver. Arrows point to H2B-mCherry/Hnf4a double-positive cells. (B) A graph showing the percentage of H2B-mCherry+ cells among Hnf4a+ cells in the livers of the tomm22 MO-injected larvae. Red dots indicate the larvae shown in (A). (C) Confocal projection images showing the hepatic expression of ubb:mCherry (red, Cre-labeled cells) and Alcam (gray, BECs) at 9 dpf. 4-OHT was treated from 48 to 84 hpf. (D) Graph showing the percentage of ubb:mCherry+ hepatocytes, which were derived from BECs. fabp10a:CFP-NTR expression was used to define hepatocytes. Red marks indicate the larvae shown in (C). (E) Graph showing the percentage of mCherry+ cells among Alcam+ BECs at 9 dpf, indicating Cre-mediated labeling efficiency. n indicates the number of larvae examined. Scale bars: 20 μm; error bars: ±SEM.

Surviving hepatocytes become hybrid hepatocytes. (A) Confocal single-optical section images showing the expression of Hnf4a (green), Tp1:H2B-mCherry (red), and Bhmt (gray) in the liver. Arrows point to hepatocytes that express Tp1:H2B-mCherry; arrowheads point to BECs negative for Hnf4a and Bhmt. (B) Confocal projection images showing Tp1:H2B-mCherry (red) and Anxa4 (gray) expression in the liver. Arrows point to H2B-mCherry/Anxa4 double-positive cells. (C) Confocal single-optical section images showing the hepatic expression of ubb:mCherry (red, Cre-labeled cells) and Hnf4a (gray) in tomm22 MO-injected larvae. Arrows point to mCherry/Hnf4a double-positive cells. (D) Graph showing the percentage of ubb:mCherry+ cells among Hnf4a+ cells, which were derived from BECs. Red dots indicate the larvae shown in (C); n indicates the number of larvae examined. Scale bars: 20 μm; error bars: ±SEM.

EXPRESSION / LABELING:
Gene:
Antibodies:
Fish:
Condition:
Knockdown Reagent:
Anatomical Terms:
Stage Range: Day 4 to Days 7-13
PHENOTYPE:
Fish:
Condition:
Knockdown Reagent:
Observed In:
Stage Range: Day 4 to Days 7-13

Suppression of Wnt/β-catenin signaling represses hepatocyte proliferation and the formation of hybrid hepatocytes in tomm22 MO-injected larvae. (A) Confocal single-optical section images showing WRE:d2GFP (green) and Tp1:H2B-mCherry (red) expression in the liver at 6 dpf. Arrows point to d2GFP/H2B-mCherry double-positive cells; arrowheads point to H2B-mCherry single-positive cells. Quantification of the percentage of d2GFP+ cells among H2B-mCherry+ cells is shown. (B) Scheme illustrating the period of XAV939 treatment for (C)–(F). (C) Confocal single-optical section images showing the hepatic expression of Tp1:H2B-mCherry (red) and Hnf4a (gray) at 7 dpf. Arrows point to H2B-mCherry/Hnf4a double-positive cells; arrowheads point to H2B-mCherry/Hnf4a+ cells. Quantification of the percentage of H2B-mCherry cells among Hnf4a+ cells is shown. (D) Confocal single-optical images showing the hepatic expression of fabp10a:mAGFP-gmnn (green), Tp1:H2B-mCherry (red), and Hnf4a (gray) at 7 dpf. Arrows point to mAGFP-gmnn/H2B-mCherry/Hnf4a triple-positive cells; arrowheads point to mAGFP-gmnn/Hnf4a double-positive cells. (E) Graph showing the percentage of mAGFP-gmnn+ cells among Hnf4a+ cells. Red marks indicate the larvae shown in (D). (F) Graph showing the percentage of mCherry+ or mCherry proliferating hepatocytes. Red marks indicate the larvae shown in (D). n indicates the number of larvae examined. Scale bars: 20 μm; error bars: ±SEM.

Macrophage ablation impairs the formation of hybrid hepatocytes in tomm22 MO-injected larvae. (A) Confocal single-optical section images showing the expression of mpeg1:Dendra2 (green, macrophages) and fabp10a:CFP-NTR (blue, hepatocytes) in the liver at 5 dpf. Arrows point to macrophages. (B) Confocal single-optical section images showing the expression of mpeg1:Dendra2 (green), Tp1:H2B-mCherry (red), and fabp10a:CFP-NTR (blue) in the liver at 7 dpf. Arrows point to macrophages in the control liver; arrowheads point to a macrophage engulfing a hepatocyte in tomm22 MO-injected larvae. (C) Fluorescence images showing macrophage NTR-mCherry expression before and after metronidazole (Mtz) treatment. Mtz (10 mM) was treated from 4 to 7 dpf. Note the great reduction in the number of NTR-mCherry+ macrophages in the Mtz-treated larvae at 7 dpf compared with the DMSO-treated controls. Lateral views, dorsal up, anterior to the left. Scale bar: 250 μm. (D) Scheme illustrating the period of Mtz treatment and harvest stage for macrophage ablation for (E–I). (E) Confocal single-optical section images showing the expression of Tp1:H2B-mCherry (red) and Hnf4a (gray) in the liver at 7 dpf. Arrows point to H2B-mCherry/Hnf4a double-positive cells; arrowheads point to Hnf4a single-positive cells. (F) Graph showing the percentage of H2B-mCherry cells among Hnf4a+ cells. Red marks indicate the larvae shown in (E). (G) Confocal single-optical section images showing the expression of fabp10a:mAGFP-gmnn (green), Tp1:H2B-mCherry (red), and Hnf4a (gray) in the liver at 7 dpf. Arrows point to mAGFP-gmnn/H2B-mCherry/Hnf4a triple-positive cells; arrowheads point to mAGFP-gmnn+/H2B-mCherry/Hnf4a+ cells. (H) Graph showing the percentage of fabp10a:mAGFP-gmnn+ cells among Hnf4a+ cells. Red marks indicate the larvae shown in (G). (I) Graph showing the percentage of H2B-mCherry+ or H2B-mCherry proliferating Hnf4a+ cells. Red marks indicate the larvae shown in (G). n indicates the number of larvae examined. (J) Confocal single-optical section images showing the expression of WRE:d2GFP (green, Wnt activity) and Tp1:H2B-mCherry (red) in the liver at 6 dpf. Anti-GFP antibody was used to reveal WRE:d2GFP expression. Note the complete absence of hepatic d2GFP expression in the XAV939-treated, tomm22 MO-injected larvae, whereas its faint expression in macrophage-ablated, tomm22 MO-injected larvae. Scale bars: 20 μm, except (C); error bars: ±SEM.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Gene Expr.