Hepatocyte polarization and bile canaliculi development are affected in cldn15lbfh290 mutant livers. a, Schematic of the TILLING mutation. The nonsense mutation is situated at the end of exon1a making this mutation specific to cldn15lb_tv1. The mutation is situated in the sequence encoding the extracellular region immediately before the second transmembrane domain. b–c, Brightfield pictures of wildtype (b) and mutant (c) larvae at 5 dpf. The overall body morphology and liver size, as assessed by Tg(fabp10:RFP)gz12 expression, appear unaffected. d–e, In situ hybridization analysis of wildtype (d) and mutant (e) larvae at 80 hpf with a probe that recognizes both transcript variants. The mutant larvae exhibit decreased expression of cldn15lb in the liver and pancreas while gut expression appears unaffected. f–g, 150 μm transverse sections through the liver at 100 hpf. Green: Tg(Tp1bglob:eGFP); red: Cldn15lb antibody. Wildtype larvae (f) express Cldn15lb in BECs (arrows). Cldn15lb expression in mutant larvae (g) is severely reduced (arrowhead). h–i, Whole-mount analysis of 80 hpf larvae stained for pan-cadherin (green) and ZO-1(mouse) (red). Anti-pan-cadherin outlines hepatocytes at lower intensity while it marks BECs at higher intensity. Anti-mouse ZO-1 outlines the intrahepatic vasculature. h′–i′, Red: vasculature; Yellowish-green: biliary network. Hepatocytes are color-coded by their cell shape and location relative to the biliary and vascular networks. Hepatocytes that are sandwiched between the networks and columnar in shape are labeled in light blue. These cells appear to be fully polarized. Hepatocytes that are either columnar but not sandwiched between the two networks or are cuboidal and between the networks are labeled in light purple. Hepatocytes that are not polarized are non-uniformly shaped and not lined by the networks. These cells are labeled in dark purple. Most hepatocytes in mutant larvae do not appear to be polarized and are found in rosettes (* in i′) while a higher number of hepatocytes in wildtype larvae are polarized. Gallbladder (gb) j–k, Whole-mount analysis of 100 hpf Tg(fabp10:rasGFP)s942 larvae stained for BSEP (red) which marks canaliculi. Canaliculi in mutant livers are shorter and wider than those in wildtype livers. Insets highlight the boxed areas (red channel only). l, Percentage of hepatocytes that are polarized (light blue), semi-polarized (light purple), and non-polarized (dark purple). Wildtype livers have significantly more polarized cells while having almost three times fewer non-polarized cells (* p = 0.0018, ** p = 0.0012). Error bars represent SEM. All scale bars are 10 μm.
|
