Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Mar;14(2):62-8.
doi: 10.1016/j.gep.2013.12.002. Epub 2014 Jan 3.

A novel keratin18 promoter that drives reporter gene expression in the intrahepatic and extrahepatic biliary system allows isolation of cell-type specific transcripts from zebrafish liver

Benjamin J Wilkins  1 , Weilong Gong  2 , Michael Pack  3
Affiliations

A novel keratin18 promoter that drives reporter gene expression in the intrahepatic and extrahepatic biliary system allows isolation of cell-type specific transcripts from zebrafish liver

Benjamin J Wilkins et al. Gene Expr Patterns. 2014 Mar.

Abstract

Heritable and acquired biliary disorders are an important cause of acute and chronic human liver disease. Biliary development and physiology have been studied extensively in rodent models and more recently, zebrafish have been used to uncover pathogenic mechanisms and potential therapies for these conditions. Here we report development of novel transgenic lines labeling the intrahepatic and extrahepatic biliary system of zebrafish larvae that can be used for lineage tracing and isolation of biliary-specific RNAs from mixed populations of liver cells. We show that GFP expression driven by a 4.4 kilobase promoter fragment from the zebrafish keratin18 (krt18) gene allows visualization of all components of the developing biliary system as early as 3 days post-fertilization. In addition, expression of a ribosomal fusion protein (EGFP-Rpl10a) in krt18:TRAP transgenic fish allows for enrichment of translated biliary cell mRNAs via translating ribosome affinity purification (TRAP). Future studies utilizing these reagents will enhance our understanding of the morphologic and molecular processes involved in biliary development and disease.

Keywords: Gallbladder; Gene profiling; Liver; Translation.

PubMed Disclaimer

Figures

Figure 1
Figure 1
krt18 and Tg(krt18:egfp) are expressed in the developing biliary system. (A) Whole-mount in situ hybridization of 5 dpf wild-type larva (left lateral view), showing gallbladder expression of krt18 (arrow). (B) Combined immunostaining and fluorescent in situ hybridization of 5 dpf Tg(tp1bglob:egfp) larva (left lateral view), showing Notch-responsive GFP protein in intrahepatic bile ducts within the liver (l), but not the krt18-expressing gallbladder (g). Red signal anterior to the liver is background staining. (C) Whole-mount right lateral-inferior fluorescent image of 5 dpf wild-type control larva, stained with anti-GFP, showing background autofluorescence in larval tissues. (D) Whole-mount right lateral-inferior fluorescent image of 5 dpf Tg(krt18:egfp) larva stained with anti-GFP, showing EGFP expression in the developing biliary system, skin, and proximal tubule. Abbreviations: l=liver, g=gallbladder, i=intestine, t=trunk, cd=cystic duct, chd=common hepatic duct, cbd=common bile duct, pt=proximal tubule.
Figure 2
Figure 2
krt18-driven reporters allow live detection of the developing biliary system. (A) Fluorescent micrograph of live 6 dpf Tg(krt18:egfp) larva, showing EGFP expression in intrahepatic biliary cells (single arrow) and gallbladder epithelium (double arrow), partly obscured by skin expression. (B–C) Fluorescent micrographs of live 4 dpf (B) and 7 dpf (C) Tg(krt18:TRAP) larvae, showing visualization of EGFP-Rpl10a fusion protein in developing intrahepatic biliary cells (single arrows) and gallbladder epithelium (double arrow). Red dashed line in (B) indicates gallbladder overlying the liver, out of the focal plane.
Figure 3
Figure 3
Tg(krt18:egfp) is expressed at all levels of the developing intrahepatic and extrahepatic biliary system. (A–C) Confocal projections of dissected livers from 3, 4, and 5 dpf Tg(krt18:egfp) fish, indicating biliary proliferation and remodeling (400X magnification). Arrow in panel A indicates rudimentary gallbladder. (D–E) Confocal projections from dissected liver of 5 dpf Tg(krt18:egfp) larva stained with (D) anti-GFP and (E) anti-Mdr to mark biliary cells and hepatocyte canaliculi, respectively. (F) Merged projection from panels D and E, demonstrating colocalization of canaliculi with GFP-positive terminal ductules (D–F: 630X magnification).
Figure 4
Figure 4
Generation of transgenic zebrafish for liver translating ribosome affinity purification (TRAP). (A–C) Confocal projection of dissected liver and gallbladder from a 5 dpf Tg(krt18:TRAP) larva, showing EGFP-Rpl10a fusion protein (A) and biliary marker 2F11 (B), which co-localize (C; EGFP - green; 2F11 - red). EGFP is also visualized in krt18-expressing cells of the esophagus (e) and skin (s). (D) High-magnification confocal scan of liver from the same larva shows localization of TRAP fusion protein to biliary cell cytoplasm and nucleus, with a subset showing nucleolar enrichment (insets). (E–G) Confocal projection of dissected liver and gallbladder from 5 dpf Tg(lfabp:TRAP) larva, showing hepatocyte expression of TRAP fusion protein (E) that is non-overlapping with biliary 2F11 staining (F). (H) High-magnification confocal scan of liver from same larva, showing localization of TRAP fusion protein to the hepatocyte cytoplasm and nucleolus (inset), exclusive of biliary cells stained red with the 2F11 antibody.
Figure 5
Figure 5
Selective mRNA isolation from biliary cells and hepatocytes by translating ribosome affinity purification (TRAP). (A) Schematic of TRAP methodology, demonstrating selective isolation of mRNAs actively translated by EGFP-Rpl10a-containing ribosomes. (B–C) Gel electrophoresis (B) and band densitometry (C) of RT-PCR products from one month-old Tg(krt18:TRAP) and Tg(lfabp:TRAP) fish, demonstrating enhanced cell-type specific recovery of biliary (krt18, cftr) and hepatocyte (lfabp, transferrin) mRNAs, respectively. (D) Quantitation of RT-PCR products from 6–7 dpf Tg(krt18:TRAP) and Tg(lfabp:TRAP) livers and gallbladders, demonstrating enhanced cell-type specific amplification of biliary (krt18, cftr, sox17) and hepatocyte (transferring, lfabp, a2ml) mRNAs, respectively.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Dong PD, Munson CA, Norton W, Crosnier C, Pan X, Gong Z, Neumann CJ, Stainier DY. Fgf10 regulates hepatopancreatic ductal system patterning and differentiation. Nat Genet. 2007;39:397–402. - PubMed
    1. Doyle JP, Dougherty JD, Heiman M, Schmidt EF, Stevens TR, Ma G, Bupp S, Shrestha P, Shah RD, Doughty ML, Gong S, Greengard P, Heintz N. Application of a translational profiling approach for the comparative analysis of CNS cell types. Cell. 2008;135:749–62. - PMC - PubMed
    1. Heiman M, Schaefer A, Gong S, Peterson JD, Day M, Ramsey KE, Suarez-Farinas M, Schwarz C, Stephan DA, Surmeier DJ, Greengard P, Heintz N. A translational profiling approach for the molecular characterization of CNS cell types. Cell. 2008;135:738–48. - PMC - PubMed
    1. Kawakami K. Transgenesis and gene trap methods in zebrafish by using the Tol2 transposable element. Methods Cell Biol. 2004;77:201–22. - PubMed
    1. Korzh S, Pan X, Garcia-Lecea M, Winata CL, Pan X, Wohland T, Korzh V, Gong Z. Requirement of vasculogenesis and blood circulation in late stages of liver growth in zebrafish. BMC Dev Biol. 2008;8:84. - PMC - PubMed

Publication types

LinkOut - more resources