Fig. 2

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.