stk3 mutants phenocopy sav1 mutants, but with delayed onset. (A) Schematic depicting wild-type and mutant alleles of zebrafish stk3. stk3 mutant alleles contain a 5 bp deletion (red), causing a frameshift and early stop codon (red box). (B) Western blot of protein lysates from stk3−/−, stk3+/− and stk3+/+ single larvae at 8 dpf. γ-Tubulin staining was used as a loading control. (C-D″) Paraffin histology shows abnormal biliary cells with increased Yap and Taz levels using immunofluorescent and Hematoxylin and Eosin staining in stk3−/− fish at 16 dpf compared with wild-type siblings. Gb, gallbladder; CBD, common bile duct (n=5 wild type; n=5 stk3−/−). Scale bar: 100 μm. (E) TEM example of wild-type canaliculi and hepatocyte-biliary epithelial cell junctions. Canaliculi are outlined with dashed red lines. (F-F′) stk3−/− siblings of E showing examples of abnormal canaliculi, with shorter abnormal shape (F) or no canalicular structure despite tight junctions connecting the hepatocyte to the adjacent biliary epithelial cell (F′). These areas are outlined with dashed red lines and/or labeled with an asterisk for possible canaliculi with abnormal structure. (F″) A dashed yellow line outlines excessive extracellular space filled with electron-dense material. Yellow arrowhead indicates bile salt deposit. Hep, hepatocyte; BEC, biliary epithelial cell; c, canaliculi; TJ, tight junction (n=3 wild type; n=3 stk3−/−). All images are of 16 dpf larvae.
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