Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

Summary

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

Abstract

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

Introduction

The liver is a highly regenerative organ. During liver regeneration, regenerating hepatocytes, the parenchymal cells of the liver, are derived from pre-existing hepatocytes (hepatocyte-driven liver regeneration) or BECs (biliary-driven liver regeneration)^1,2. Liver injury usually elicits the proliferation of pre-existing hepatocytes; however, when hepatocyte proliferation is compromised, BECs can contribute to hepatocytes^2-4. These two modes of liver regeneration are clinically significant. Upon surgical removal of a portion of the human liver (e.g., because of liver tumors or live liver donors), hepatocytes in the remaining liver prolif....

Protocol

Zebrafish were raised and bred according to standard procedure; experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

1. Preparation of Embryos/larvae

1. To conduct timed matings, set up adult male and female Tg(fabp10a:CFP-NTR)^s931 hemizygous or homozygous fishes O/N and place a divider between them. Remove this divider the following morning when mating is desired. Collect embryos by straining the water using a fine plastic mesh strainer.
2. Turn the strainer upside down and rinse the mesh surface with a fine stream of egg water dispensed from a s....

Results

Mtz treatment for 36 hr (A36h, A stands for ablation) from 3.5 to 5 dpf dramatically reduced liver size (Figure 1B). After Mtz washout, considered as the beginning of liver regeneration (R), strong fabp10a:CFP-NTR and fabp10a:dsRed expression reappeared within 30 hr (Figure 1B). The intrahepatic biliary network, as assessed by expression of Alcam that is present in the membrane of BECs²², initially collapsed but was re-established at 54 hr post-washout (R54h).......

Discussion

Severe, but not mild, hepatocyte ablation elicits biliary-driven liver regeneration. Therefore, following Mtz treatment and washout, it is important to examine the liver size of the Mtz-treated larvae, which can be assessed by intrinsic CFP fluorescence from the fabp10a:CFP-NTR transgene. Since a small percentage of the Mtz-treated larvae will display non-ablated (0-5%) or partially ablated (10-20%) livers, it is essential to sort out and only analyze the larvae with a very small liver, which is indicative of se.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
3-amino benzoic acid ethyl ester (Tricaine) powder	Sigma-Aldrich	A5040	Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.
Dimethyl sulfoxide (DMSO)	Sigma-Aldrich	D8418
Metronidazole	Sigma-Aldrich	M1547
Triton X-100	Sigma-Aldrich	T8532
Formaldehyde	Sigma-Aldrich	252549
Calcium sulfate dihydrate	Sigma-Aldrich	C3771
N-Phenylthiourea (PTU)	Sigma-Aldrich	P7629
Mounting media (Vectashield)	Vector Laboratories	H-1000
100 x 15 mm petri dish	Denville Scientific Inc.	M5300
clear nail polish	Fisher Scientific	50949071
fine forceps	Fine Science Tools	11251-20	Dumont #5
glass slide	Fisher Scientific	12-544-1
glass coverslip	Fisher Scientific	12-540-A and 12-542-A	18 x 18 mm
zn-5 (mouse anti-Alcam)	ZIRC	zn-5	1:10 dilution
goat anti-Hnf4a	Santa Cruz	sc-6556 (C-19)	1:50 dilution
rat anti-RFP	Allele Biotechnology	ACT-CM-MRRFP10	1:300 dilution
AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L)	Jackson laboratories	705-605-147	1:500 dilution
AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L)	Invitrogen	A21240	1:500 dilution
Cy3 donkey anti-rat IgG	Jackson laboratories	712-165-150	1:500 dilution
dissecting microscope	Leica Microsystems	S6F
epifluorescence microscope	Leica Microsystems	M205FA
confocal microscope	Carl Zeiss	LSM700
egg water			0.3 g/L of sea salts ‘Instant Ocean’ and 0.5 mM CaSO4 in distilled water.
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10X PBS			25.6 g Na2HPO4·7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.  Bring to 1 L with distilled water.  Adjust pH 7.  Autoclave for 20 min at 121°C.
1X PBSDT			0.2% Triton X-100 and 0.2% DMSO in 1 X PBS.
PEM buffer			30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4.  Bring to 1 L with distilled water.  Adjust pH 7.
Blocking solution			Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT.