Yap but not Taz drives sav1−/− phenotypes. (A-A‴) Whole-mount GFP staining of ctgfa:d2GFP wild-type and sav1−/− embryos. Gb, gallbladder. Numbers indicate the ratio of fish that displayed the phenotype/total fish analyzed. Scale bar: 100 μm. (B,B′) Images of ctgfa:d2GFP+ wild-type and sav1−/− larvae; dashed yellow line outlines the liver. Scale bar: 100 μm. (C) Mean pixel intensity of ctgfa:d2GFP reporter in livers as outlined in B: n=16 wild-type; n=9 sav1−/−. Data are mean±s.d. Unpaired t-test with equal s.d. resulted in P<0.0001. (D-E″) Immunofluorescent staining of paraffin wax-embedded sections for Yap and Taz in sav1−/− and wild-type siblings, and colocalization with the biliary marker annexin A4. Gb, gallbladder; IBD, intrahepatic bile duct. Scale bar: 100 μm. (F-F‴) Whole-mount staining for annexin A4, EdU and Taz in wild-type larvae. Gb, gallbladder; IBD, intrahepatic bile duct. (G-G‴) Whole-mount analysis of sav1−/− larvae using the markers described in F (n=6). (H-H‴) Whole-mount analysis of sav1−/−;yap−/− larvae using the markers described in F (n=8). Scale bar: 100 μm. (I-K‴) Repeated whole-mount analysis for the markers as described in F-H (although n=5 for sav1−/−;taz−/− embryos). Scale bar: 100 μm. (L) Toluidine Blue stained sections of liver and gallbladder in wild-type, sav1−/− and sav1−/−;yap−/− larvae at 10 dpf (n=9). Gb, gallbladder; CBD, common bile duct. n=7 wild type; n=5 sav1−/−; n=4 sav1−/−;yap−/−. Scale bars: 100 μm. (M) Ratiometric analysis at 8 dpf of BODIPY FL C5 gallbladder uptake in larvae from a sav1+/−;yap+/− incross. Analyses performed at 8 dpf unless otherwise indicated.
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