The blg mutant exhibits intrahepatic bile duct anomalies. A: Confocal and 3D reconstruction images of the intrahepatic bile ducts in the WT and blg mutant at 5 dpf under the Tg(tp1:Tomato) transgenic line. The dendrite mean diameter colormaps represent bile duct branches (yellow dotted boxes). The blg mutant showed intrahepatic bile duct anomalies with loss of branches. B: Statistical diagram of intrahepatic bile duct diameter in the WT (n = 40) and blg mutant (n = 13) at 5 dpf. Data are expressed as mean ± SEM, Student's t-test. C: Bright-filed micrographs of the WT and blg mutant at 5 dpf, white dotted outlines indicate the liver area. D: ORO staining of the WT and blg mutant at 5 dpf. Black arrows indicate the liver area. E: BODIPY FL C5 fluorescence staining of the WT (18/18) and blg mutant (13/15) at 5 dpf. The blg mutant presents defective bile flow (arrowheads) and a diminished gallbladder (arrows). F: Survival rate of the WT (n = 80) and blg mutant (n = 70). WT, wild-type; ib, intestinal bulb; li, liver; dpf, days post fertilization. Scale bars, 50 μm (A and E). n = number of embryos with indicated phenotype/total analyzed in each class.

The blg phenotypes are caused by ppp1r21 gene mutation. A: Genetic map of the candidate region on chromosome 13 between SSLP markers z818 (0.043 cM) and z9878 (0.235 cM), containing 6 open reading frames. Two hundred and eighty seven mutant embryos were tested, and the numbers in parentheses represent the recombination rate. B: Sequencing results of ppp1r21 gene in the wild-type and blg mutant. The G→A point mutation led to the formation of premature stop codons. Asterisk indicates the blg mutation site. C: Diagram of Ppp1r21 protein domain and the blg mutation site. Asterisk indicates the blg mutation site, which is located in TTKRYEDQ-motif domain. D: Conserved motifs and regions in homologous sequences of Ppp1r21 protein. Asterisk indicates the site of amino acid mutation in blg mutant. E: Confocal images show the bile duct phenotypes of the wild-type, blg mutant, and CRISPR/cas9-induced ppp1r21 mutant. The ppp1r21CRISPR mutant validated that the intrahepatic bile duct anomalies phenotype in blg was caused by the dysfunction of Ppp1r21. F: Confocal 2D images (single optical planes) of the intrahepatic bile ducts in double transgenic lines Tg(hsp70l:ppp1r21-HA-p2a-DsRed; tp1:EGFP). HS represents heat shock induction. Arrows indicate the intrahepatic bile ducts of blg after being rescued. Scale bars, 50 μm (E and F). n = number of embryos with indicated phenotype/total analyzed in each class.

The blg/ppp1r21 mutant develops analogous pathological features of PBC. A: Immunofluorescence staining of PDC-E2 in the WT and blg mutant under Tg(tp1:EGFP) background at 5 dpf. Arrowheads indicate increased PDC-E2 in blg cholangiocytes. B: Immunofluorescence staining showed elevated levels of γH2AX in the blg mutant compared to the WT at 4 dpf. Arrowheads indicate γH2AX positively overlap with blg cholangiocytes. C: Confocal 3D projection and 2D single-optical section images of WT or blg mutant at 5 dpf under Tg(tp1:EGFP, coronin1a:DsRed) background. coronin1a positive cells (red) were gathered around the bile duct (green) in the blg mutant. The dotted outlines indicate the liver area. D: Statistical diagram of the number of the DsRed positive cells in the WT and blg mutant (per group n ≥ 6) from 3–7 dpf under Tg(coronin1a:DsRed) background. Data are expressed as mean ± SEM, Student's t-test. E: Whole-mount in situ hybridization showed that expressions of il1b, tnfa, nfkbiab, and tgfb1a were up-regulated in the liver (arrows) of blg mutant compared to the WT larvae at 5 dpf. Lateral views, anterior left. F: Immunofluorescence staining of Collagen1a in the WT and blg mutant at 5 dpf under Tg(tp1:EGFP) background. Arrowheads indicate increased Collagen1a around the blg cholangiocytes. WT, wild-type; ib, intestinal bulb; li, liver. Scale bars, 50 μm (A–C, F). n = number of embryos with indicated phenotype/total analyzed in each class.

Inhibition of PI3K/AKT/mTOR pathway alleviates blg intrahepatic bile duct anomalies and reduces PDC-E2 content in blg liver. A: Experimental scheme for chemical treatment under Tg(tp1:Tomato) background. B: Confocal 3D images of the intrahepatic bile ducts in the wild-type and blg mutant after LY294002 and Rapamycin treatment at 5 dpf. This treatment could rescue bile duct phenotype of blg mutant. C: Statistical diagram of intrahepatic bile duct diameter in (B) (per group n ≥ 11). Data are expressed as mean ± SEM, Student's t-test. D: Immunofluorescence staining of PDC-E2 in blg mutant with DMSO, LY294002, and Rapamycin treatment 5 dpf under Tg(tp1:EGFP) background. The treatment could reduce PDC-E2 content in mutant bile ducts. Scale bars, 50 μm (B and D). n = number of embryos with indicated phenotype/total analyzed in each class.