khdrbs1a and khdrbs1b are expressed in BPPCs during liver regeneration. (A) Scheme showing the DMSO or Mtz treatment and analysis stages during liver regeneration. (B) UMAP showing khdrbs1a or khdrbs1b expression (overlaid in red scale) in various liver cell types at 6 dpf and R0 h. BEC, biliary epithelial cell; BPPC, bipotential progenitor cell; Hep, hepatocyte; HSC, hepatic stellate cell; IC, immune cell; VEC, vascular endothelial cell. Black dashed circle indicates BEC/BPPC. (C) WISH images showing the expression patterns of khdrbs1a or khdrbs1b during liver regeneration at 6 dpf, R0 h, R8 h, R24 h, and R48 h. Red arrowheads and white dashed outlines indicate livers. Scale bars: 100 μm. (D) Confocal images showing the expression of khdrbs1a or khdrbs1b in BECs or BPPCs at 6 dpf, R8 h, and R24 h by FISH in Tg(lfabp:Dendra2-NTR; Tp1:GFP) in which hepatocytes and BECs are labeled, respectively. Insets show magnified views of regions of interest. Schematic at the top shows the Mtz treatment and analysis stages. Numbers at the bottom of image panels indicate the proportion of larvae exhibiting the expression shown. Scale bars: 50 μm.

Specific inactivation of khdrbs1 in BECs causes impaired biliary-mediated liver regeneration. (A) Schematic showing CRISPR/dead Cas9-mediated interference (dCas9i) to knock down khdrbs1 in BECs, combined with doxycycline (Dox)-induced Tet-on3G/TRE3G system and constructed in the background of khdrbs1b−/− mutants. (B) Scheme showing the treatment of Dox and Mtz and then analysis at R48 h. Graph shows the percentage of regenerating livers of the control and dCas9i groups at R48 h with or without Dox. Images show examples of the normal, moderate and small categories. ‘Normal’ represents a regenerating liver size comparable to that of wild type. ‘Small’ represents a severe deficiency of liver regeneration compared to wild type. (C) Right-hand images: Epifluorescence images showing the body shape and regenerating livers (white arrowheads). Main panels: Confocal images showing the regenerating livers of control and small regenerated liver in the dCas9i group (dCas9iS) at R48 h. Insets show magnified views of regions of interest. (D) WISH images showing the expressions of gc, cp, and bhmt at R48 h. Red arrowheads and white dashed outlines indicate livers. Scale bar: 100 μm. (E) Confocal images showing the expression of Alcam and Dendra2 at R48 h. Scale bars: 50 μm. (F) Quantification of Alcam+ among Dendra2+ cells in regenerating livers at R48 h. Error bars represent s.e.m. ****P<0.0001 (two-tailed, unpaired t-test; control group, n=7; dCas9iS subgroup, n=8.). Numbers at the bottom of images indicate the proportion of larvae exhibiting the expression shown.

Loss of khdrbs1 impairs the re-differentiation of BPPCs during biliary-mediated liver regeneration. (A) Scheme showing the stage of Mtz treatment and analysis at R48 h. (B) Epifluorescence images showing the body shape and regenerating livers (white arrowheads) of the control and khdrbs1−/− mutant at R48 h. (C) Confocal projection images showing livers of the control and khdrbs1−/− mutant at BT (before treatment), R0 h, R24 h, and R48 h. Scale bars: 100 μm. (D) Quantification of liver area in the control and khdrbs1−/− mutant at BT and R48 h. 5 dpf: control, n=7; khdrbs1−/−, n=6; R48 h: control, n=7; khdrbs1−/−, n=6. (E) WISH images showing expression of the hepatocyte markers hnf4α, ttr, gc, cp, and bhmt in the control and khdrbs1−/− mutant at R48 h. Red arrowheads and white dashed outlines indicate livers. Numbers indicate the proportion of larvae exhibiting the expression/phenotype shown. Scale bar: 100 μm. (F,G) Confocal images and quantification showing the expression of Alcam and Hnf4α or Alcam and Dendra2 at R48 h. Insets show magnified views of regions of interest. F: control, n=8; khdrbs1−/−, n=11; G: control, n=8; khdrbs1−/−, n=6. Error bars represent s.e.m. **P<0.01, ****P<0.0001 (two-tailed, unpaired t-test). Scale bars: 50 μm.

p53 mutation partially rescues liver regeneration in the khdrbs1 mutant. (A) WISH images showing the expression of tp53 and mdm2 in the control and khdrbs1−/− mutant at R8 h. (B) Confocal images and quantification showing the expression of p53 in Anxa4+ cells at R8 h. Insets show magnified views of regions of interest. White arrows point to p53 and Anxa4 co-expressing cells, and magenta arrows point to p53+ and Anxa4 cells. Control, n=9; khdrbs1−/−, n=7. (C) EdU assay (schematic) showing the proliferation of BPPCs after tp53 mutation R24 h. Graph shows quantification of EdU+ among Dendra2 and Anxa4 co-expressing cells. Control, n=7; tp53−/−, n=6; khdrbs1−/−, n=5; khdrbs1−/−;tp53+/−, n=6. (D) Confocal images showing the expression of Alcam and Dendra2 at R48 h. Insets show magnified views of regions of interest. Dashed lines delineate liver region. (E,F) Quantification of Alcam+ cells among Dendra2+ cells (E) or the area of regenerating livers (F) after tp53 mutation at R48 h. Control, n=7; tp53−/−, n=7; khdrbs1−/−, n=6; khdrbs1−/−;tp53+/−, n=7. (G) WISH images showing the expression of hnf4a and bhmt after tp53 mutation at R48 h. Scale bars: 100 μm. In WISH images, red arrowheads and white dashed outlines indicate livers, and numbers indicate the proportion of larvae exhibiting the expression shown. ns, not significant. **P<0.01, ***P<0.001, ****P<0.0001 (two-tailed, unpaired t-test). Error bars represent s.e.m. Scale bars: 100 μm (A); 50 μm (B-D).

Acknowledgments
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