ZFIN ID: ZDB-FIG-111220-25
Cheung et al., 2012 - Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b. Developmental Biology   361(1):68-78 Full text @ Dev. Biol.
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Anatomical Terms:
Stage Range: Protruding-mouth to Day 5
PHENOTYPE:
Fish:
Observed In:
Stage Range: Protruding-mouth to Day 5

Fig. 4 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.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
cldn15lb AB standard conditions Protruding-mouth gut ISH
Protruding-mouth liver ISH
Protruding-mouth pancreas ISH
cldn15lbfh290/fh290 standard conditions Protruding-mouth gut ISH
Protruding-mouth liver ISH
Protruding-mouth pancreas ISH
Ab1-cldn15lb cldn15lbfh290/fh290; um14Tg standard conditions Day 4 gut epithelium IHC
Ab1-cldn15lb Day 4 hepatic duct epithelial cell IHC
Ab1-cldn15lb um14Tg standard conditions Day 4 gut epithelium IHC
Ab1-cldn15lb Day 4 hepatic duct epithelial cell IHC
DsRed cldn15lbfh290/fh290; gz15Tg standard conditions Day 5 liver IFL
gz15Tg standard conditions Day 5 liver IFL
EGFP cldn15lbfh290/fh290; s942Tg standard conditions Day 4 hepatocyte basal plasma membrane IFL
cldn15lbfh290/fh290; um14Tg standard conditions Day 4 hepatic duct epithelial cell IFL
s942Tg standard conditions Day 4 hepatocyte basal plasma membrane IFL
um14Tg standard conditions Day 4 hepatic duct epithelial cell IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-abcb11 IHC cldn15lbfh290/fh290; s942Tg standard conditions Day 4 bile canaliculus
IHC s942Tg standard conditions Day 4 bile canaliculus
Ab1-pan-Cadherin IHC AB standard conditions Protruding-mouth hepatic duct epithelial cell
IHC Protruding-mouth hepatocyte
IHC cldn15lbfh290/fh290 standard conditions Protruding-mouth hepatic duct epithelial cell
IHC Protruding-mouth hepatocyte
Ab1-tjp1 IHC AB standard conditions Protruding-mouth liver vasculature
IHC cldn15lbfh290/fh290 standard conditions Protruding-mouth liver vasculature
Phenotype Details
Fish Conditions Stage Phenotype
cldn15lbfh290/fh290 standard conditions Protruding-mouth hepatocyte apical-basal polarity, abnormal
cldn15lbfh290/fh290 standard conditions Protruding-mouth hepatocyte spatial pattern, abnormal
cldn15lbfh290/fh290; gz15Tg standard conditions Day 5 liver size, normal
Day 5 whole organism morphology, normal
cldn15lbfh290/fh290; s942Tg standard conditions Day 4 bile canaliculus decreased length, abnormal
Day 4 bile canaliculus increased width, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Biology, 361(1), Cheung, I.D., Bagnat, M., Ma, T.P., Datta, A., Evason, K., Moore, J.C., Lawson, N.D., Mostov, K.E., Moens, C.B., and Stainier, D.Y., Regulation of intrahepatic biliary duct morphogenesis by Claudin 15-like b, 68-78, Copyright (2012) with permission from Elsevier. Full text @ Dev. Biol.