Method Article

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

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DOI:

10.3791/52756

June 25th, 2015

* These authors contributed equally

In This Article

Summary

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

Abstract

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta–catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Introduction

The zebrafish is a well-established vertebrate model organism to study many aspects of development, physiology, disease, and regeneration. With the growing adoption of zebrafish as a model for post-embryonic biological processes, experimental tools for inducible, tissue-specific manipulation of gene expression or signaling pathways have become increasingly important. Particularly, studies into organ and appendage regeneration in adult zebrafish have suffered from a lack of tools for dissection of the spatio-temporal requirements of signaling pathways during these regenerative processes.

Currently, three different systems have been used to a....

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Protocol

1. Generation of Transgenic TetActivator Fish Lines

  1. Generation of TetActivator construct
    1. Clone regulatory sequences of interest upstream of the TetActivator cassette in vector #1247 using standard techniques. Alternatively, use recombination techniques to generate a BAC, in which the TetActivator cassette (vector #1180) is inserted into the first exon of the target gene, and where the Tol2 inverted repeats are introduced into the BAC backbone (for a detailed recombineering protocol see7,8).
    2. Prepare toxin-free plasmid DNA or BAC DNA preparations using commercially available kits.
  2. Generation of transg....

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Results

To establish a functional TetON system for tissue-specific inducible gene expression, transgenic TetActivator and TetResponder lines need to be generated (Figure 1A). This is accomplished by microinjecting TetActivator (Figure 1B-C) or TetResponder (Figure 1E) constructs into early zebrafish embryos and subsequent germ-line integration. Functional TetActivator constructs can either be generated by cloning of short regulatory sequences (enhancer elements).......

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Discussion

The adult zebrafish has an amazing capacity to successfully regenerate many internal organs and appendages. A thorough understanding of the molecular and cellular mechanisms involved requires tissue-specific analysis of gene functions and signaling pathways. Towards this, the TetON system provides an efficient tool for spatiotemporally controlled gene expression in embryonic and adult zebrafish. The TetON system constructs and methodology described in this manuscript have been successfully used in a recent study of our l.......

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Disclosures

The authors have nothing to disclose.

Acknowledgements

The authors thank Christa Haase, Doris Weber and Brigitte Korte for technical assistance. Work in the Weidinger lab is supported by grants of the Deutsche Forschungsgemeinschaft WE 4223/3-1, WE 4223/4-1 and by the Deutsche Gesellschaft für Kardiologie via an Oskar-Lapp-Stipendium and a Klaus-Georg-und-Sigrid-Hengstberger-Forschungsstipendium.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Breeding boxesAqua SchwarzAquaBox 1
Compound fluorescent microscopee.g., Leica, Zeissvaries with the manufacturerto image fluorescent tissue sections
Confocal microscopee.g., Leica, Zeissvaries with the manufacturerto image fluorescent tissue sections
Cryostate.g., Leica, Thermo-Scientificvaries with the manufacturerfor cryosectioning
4’, 6- diamidino-2-phenylinodole (Dapi)Sigma-AldrichD9542use 1/5,000 in PBS
for visualization of nuclei
DoxycyclineSigma-AldrichD9891prepare stocks in 50% EtOH at 50 mg/ml (97 mM)
for TetResponder induction
Paraformaldehyde (PFA)Sigma-AldrichP61484% (w/v) paraformaldehyde in PBS, pH 7.5
for fixation
1x Phosphat-buffer saline (PBS)1.7 mM KH2PO4, 5.2 mM Na2HPO4, 150 mM NaCl, pH 7.5
1x Phosphat-buffer saline + Tween 20 (PBT)1x PBS with 0.1% Tween 20
Superfrost Ultra Plus adhesion microscope slidesThermo Scientific 1014356190for collection of tissue sections
Stereo fluorescent microscopee.g., Leica, Zeissvaries with the manufacturerfor fluorescence-based genotyping
Thermocyclere.g., Biorad, Applied Biosystemsvaries with the manufacturerfor PCR-based genotyping
Tissue freezing medium (TFM)Triangel Biomedical SciencesTFM-Cfor embedding of tissue samples
Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)Sigma-AldrichE10521for anesthesia
use at 1 mg/ml in E3 embryo medium

References

  1. Fang, Y., et al. Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration. Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).
  2. Tryon, R. C., Johnson, S. L.

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Tags

Tissue Specific Gene ExpressionDoxycycline InductionTransgenic LinesFluorescence MicroscopyTail Fin RegenerationWnt Beta Catenin SignalingConfocal MicroscopyGermline Integration

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