Establishment of the lineage tracing system specific for the detection of BEC-to-hepatocyte conversion (A) Experimental scheme illustrating the stage of 4OHT treatment to double transgenic line, Tg(tp1:CreERT²; β-actin:loxP-DsRed-loxP-GFP) from 5 dpf to 7 dpf and analysis at 10 dpf. (B) Immunostaining for 2F11 and GFP on livers (2D imaging) showing tp1-CreER labels the 2F11-positive cholangiocytes specifically after 4OHT treatment. Nuclei were stained with DAPI (4',6-diamidino-2-phenylindole) (blue). Quantification of the percentage of GFP⁺ among 2F11⁺ cells in DMSO- (n = 5) and 4OHT (n = 6)-treated livers at 10 dpf. (C) Experimental scheme illustrating the stage of 4OHT treatment to triple transgenic line Tg(tp1:CreERT2; lfabp: loxP-STOP-loxP-DsRed; lfabp:Dendra2-NTR) from 5 dpf to 7 dpf and analysis at 12 dpf. Immunostaining for DsRed and Dendra2 in livers (3D imaging) showing no DsRed positive cells in Dendra2⁺ cells. Nuclei were stained with DAPI (blue). (D) H&E staining images showing normal liver histologies at 7 dpf after DMSO and 4OHT treatment. (E) Whole-mount in situ hybridization images showing the expression of cp and gc in DMSO and 4OHT treatment at 7 dpf. Scale bars: 100 μm. Data are represented as mean ±SEM（Standard Error of Mean）. See also Figure S1.

Tp1-positive BECs contribute to physiological hepatocyte homeostasis in a portion of zebrafish adults (A) Experimental scheme illustrating the stage of 4OHT treatment to triple transgenic line Tg(tp1:CreERT²; lfabp:loxP-STOP-loxP-DsRed; lfabp:Dendra2-NTR) from 5 dpf to 7 dpf and analysis at 1 and 2 years. (B) Live images showing the expression of Dendra2 and DsRed in the adult livers at 1 year. Co-immunostaining for Dendra2 and DsRed in the liver sections after DMSO and 4OHT treatment. (C) Fluorescence activating cell sorter (FACS) analysis showing the ratio of DsRed⁺ among Dendra2⁺ cells in DMSO- and 4OHT-treated livers. Quantification of the percentage of DsRed⁺ among Dendra2⁺ cells in DMSO- (n=3) and 4OHT-treated (no contribution, n=3; low contribution, n=4; high contribution, n=3) livers at 1 year. (D) Live images showing the expression of Dendra2 and DsRed in the adult livers at 2 years. Many clusters of DsRed⁺ cells among livers in 4OHT-treated groups. Co-immunostaining for Dendra2 and DsRed in the liver after DMSO and 4OHT treatment. (E) Fluorescence activating cell sorter (FACS) analysis showing the ratio of DsRed⁺ among Dendra2⁺ cells in DMSO- and 4OHT-treated livers. Quantification of the percentage of DsRed⁺ among Dendra2⁺ cells in DMSO- (n=5) and 4OHT-treated (no contribution, n=4; low contribution, n=5; high contribution, n=3) livers at 2 years. Numbers indicate the proportion of larvae exhibiting the expression shown. Asterisks indicate statistical significance: ∗p<0.05, ∗∗p<0.01, and p value was calculated by Student t tests. Scale bars: 100 μm. Data are represented as mean ±SEM.

Krt18-positive BECs contribute to physiological hepatocyte homeostasis in a portion of zebrafish adults (A) Experimental scheme illustrating the stage of 4OHT treatment to triple transgenic line Tg(krt18:CreERT²; lfabp:loxP-STOP-loxP-DsRed; lfabp:Dendra2-NTR) from 10 dpf to 11 dpf and analysis at 1 year. (B) Live images showing the expression of Dendra2 and DsRed in the adult livers at 1 year. Co-immunostaining for Dendra2 and DsRed in DMSO- and 4OHT-treated livers. (C) Fluorescence activating cell sorter (FACS) analysis shows the ratio of DsRed⁺ among Dendra⁺ cells in DMSO- and 4OHT-treated livers. Quantification of the percentage of DsRed⁺ among Dendra2⁺ cells in DMSO- (n=5) and 4OHT-treated (no contribution, n=5; low contribution, n=8; high contribution, n=3) livers at 1 year. Numbers indicate the proportion of larvae exhibiting the expression shown. Asterisks indicate statistical significance: ∗∗p<0.01, and p value was calculated by Student t tests. Scale bars: 100 μm. Data are represented as mean ±SEM. See also Figure S2.

BECs act as the major contributor to hepatocyte regeneration after extreme injury to fibrotic liver (A) Experimental scheme illustrating the stage of EtOH and Mtz treatment in transgenic line Tg(lfabp:Dendra2-NTR). (B) Confocal projection images (3D imaging) showing the staining of extracellular matrix protein collagen 1 in the liver region (dashed lines) after DMSO, EtOH, Mtz, and Mtz plus EtOH treatment at R0h (arrows). (C) Confocal projection images (3D imaging) showing the co-immunostaining for Dendra2 and 2F11 in regenerating livers after EtOH and Mtz treatment from R0h to R72h. (D) Single optical images showing the co-immunostaining for Dnedra2 and DsRed in regenerating livers at R24h. Most of the Dendra2⁺ cells are 2F11 positive in Mtz and EtOH treatment (arrows). In EtOH treatment, the 2F11⁺ and Dendra2⁺ cells are not co-stained (arrowhead). Nuclei were stained with DAPI (blue). (E) Experimental scheme illustrating the stage of 4OHT and EtOH treatment to double transgenic line, Tg(tp1:CreERT²; β-actin:loxP-DsRed-loxP-GFP), from 4 dpf to 5 dpf and analysis at 6 dpf. Immunostaining for 2F11 and GFP on livers (2D imaging) showing tp1-CreER labels the 2F11-positive cholangiocytes specifically after 4OHT and EtOH treatment. Nuclei were stained with DAPI (blue). Quantification of the percentages of GFP⁺ among 2F11+ hepatocytes (EtOH, n=5; EtOH+4OHT, n=8). (F) Experimental scheme illustrating the stage of 4OHT, EtOH, and Mtz treatment to triple transgenic line Tg(tp1:CreERT²; lfabp:loxP-STOP-loxP-DsRed; lfabp:Dendra2-NTR) and analysis at R72h. Single optical images showing the co-immunostaining for DsRed and Dendra2 at R72h after 4OHT, EtOH, and Mtz treatment. Quantification of the percentage of the DsRed⁺ among the Dendra2⁺ cells in regenerating livers at R72h (EtOH+4OHT, n=10; EtOH+4OHT+Mtz, n=10). Numbers indicate the proportion of larvae exhibiting the expression shown. Scale bars: 100 μm. Data are represented as mean ±SEM. See also Figures S4 and S5.

BECs fail to contribute to hepatocyte regeneration after PH (A) Live images showing the liver (Green) after PH at 0 hpp. (B) Confocal projection showing (3D imaging) the co-immunostaining for Dendra2 and 2F11 at 1 dpp. (C) Single optical images showing the co-immunostaining for Dendra2 and 2F11 at 1 dpp. The 2F11⁺ (BECs) were Dnedra2^-. Nuclei were stained with DAPI (blue). (D) Experimental scheme illustrating the stage of 4OHT and PH and analysis at 2 wpp in triple transgenic line Tg(tp1:CreERT2; lfabp:loxP-STOP-loxP-DsRed; lfabp:Dendra2-NTR). (E) The confocal projection shows that no DsRed expression in regenerating livers at 2 wpp. (F) The confocal projection (3D imaging) shows that no DsRed expression at 2 wpp in large magnification. Numbers indicate the proportion of larvae exhibiting the expression shown. Scale bars: 100 μm. hpp: hours post-partial hepatectomy; dpp: days post-partial hepatectomy; wpp: weeks post-partial hepatectomy.

BECs contribute to physiological hepatocyte homeostasis and fibrotic and severe liver regeneration (A) A subset of BECs contribute to physiological hepatocyte homeostasis. (B) BEC transdifferentiations are the main cellular sources for fibrotic/severe and severe liver regeneration.