Generation of pkd1l1^hsc117 zebrafish mutants. (A) Pkd1l1 is a large membrane-spanning protein with multiple transmembrane domains, with a predicted length of 2153 amino acids (England et al., 2017). The asterix indicates the CRISPR/Cas9 deletion target site. (B) Pairing of nucleotide and amino acid sequences of wild-type and CRISPR/Cas9-derived mutants, revealing mutant F0 zebrafish with a 4 bp deletion, resulting in a premature stop codon. (C,D) F1 embryos were raised, heterozygotes identified and F1 heterozygotes were incrossed to generate pkd1l1^hsc117 mutant embryos. At 5 dpf, wild-type (C) and pkd1l1^hsc117 (D) zebrafish are morphologically indistinguishable. Images are representative of 50 fish (n=50). Scale bars: 1 mm.

pkd1l1 mutant embryos demonstrate laterality defects. (A,B) Whole-mount RNA in-situ hybridization demonstrating lefty 1 and lefty 2 (lefty 1/2) expression in wild-type and pkd1l1 mutant embryos (dorsal view at 19 hpf) (A) and cmlc2 expression in wild-type and pkd1l1 mutant embryos (ventral view at 48 hpf) (B). Scale bars: 0.25 mm. (C) lefty 1/2 expression was only demonstrated on the left side in wild-type embryos (n=45), with expression being dispersed among the left, right and bilateral sides in pkd1l1 mutant embryos (n=48). (D) cmlc2 expression demonstrated normal looping in the majority of wild-type embryos (n=74), but in pkd1l1 mutant embryos (n=36), looping was primarily absent, and a few pkd1l1 mutant embryos showed either normal or reverse looping. (E) Confocal images of the KV of 10-16 somite wild-type (n=5) and pkd1l1 mutant (n=7) embryos demonstrate the presence of motile cilia. Arl-GFP is shown in green and membrane-localized RFP (memRFP) in red. Scale bars: 10 μm. (F) Quantification of the presence of the gallbladder showed no significant differences in gallbladder development in wild-type (n=33) and pkd1l1 mutant (n=39) larvae. (G) Position of the gallbladder as seen from right and left lateral views. Scale bars: 500 µm. (H) pkd1l1 mutations cause laterality defects in gallbladder positioning as demonstrated by an increased frequency of left-sided gallbladders in pkd1l1 mutant (n=156) compared to wild-type (n=166) zebrafish. ****P<0.0001 (Fisher's exact test).

pkd1l1 knockout alters hepatobiliary function. (A) Representative images of PED6 uptake in larvae showing typical normal (top), faint (middle) and absent (bottom) fluorescence (red circles) in gallbladders. Scale bars: 500 µm. (B) Quantification of PED6 uptake showed an increase number of absent gallbladders in pkd1l1 mutant (n=145) compared to wild-type (n=84) larvae (***P<0.001; χ² test), reflecting defects in biliary function.

Pkd1l1 function is necessary for normal bile duct development. (A) Confocal images showing bile ducts (assessed by 2F11 antibody staining) in liver (white outline) in wild type and mutants. GB, gallbladder. (B) Quantification showed reduction of the area of the liver in mutants compared to that in the wild type. (C,D) Whole-mount immunostaining of zebrafish livers (C) showed a decrease number of biliary epithelial cells (BECs) and abnormal intrahepatic biliary network in pkd1l1 mutant larvae compared to those in wild-type larvae (D). The numbers of BECs were measured by counting 2F11 and DAPI double-positive cells and the percentages were calculated by dividing by the total number of DAPI-positive cells from each fish. (E,F) Confocal images (E) showed a reduced length of intrahepatic ducts (white lines) in mutants compared to that in the wild type. The lengths of intrahepatic ducts were calculated by measuring the duct distance between two cell bodies and the average of ten ducts was calculated per image (fish) (F). Graphs show the mean±s.d. Scale bars: 50 µm. ns, not significant; *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA with Tukey post hoc test).

pkd1l1 mutant zebrafish have abnormal liver biochemistry. Whole livers were isolated from 6-week-old zebrafish and homogenized for protein assays. (A) Total liver protein concentration showed no significant differences between wild-type and pkd1l1 mutant livers (n=16). (B,C) ALT (B) and GGT (C) enzyme assays showed a significant increase in enzyme activity in pkd1l1 mutant livers compared to that in wild-type livers (n=15). Graphs show the mean±s.e.m. ns, not significant; ***P<0.001; ****P<0.0001 (unpaired two-tailed Student's t-test).