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

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....

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 see^7,8).
   2. Prepare toxin-free plasmid DNA or BAC DNA preparations using commercially available kits.
2. Generation of transg....

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).......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Breeding boxes	Aqua Schwarz	AquaBox 1
Compound fluorescent microscope	e.g., Leica, Zeiss	varies with the manufacturer	to image fluorescent tissue sections
Confocal microscope	e.g., Leica, Zeiss	varies with the manufacturer	to image fluorescent tissue sections
Cryostat	e.g., Leica, Thermo-Scientific	varies with the manufacturer	for cryosectioning
4’, 6- diamidino-2-phenylinodole (Dapi)	Sigma-Aldrich	D9542	use 1/5,000 in PBS for visualization of nuclei
Doxycycline	Sigma-Aldrich	D9891	prepare stocks in 50% EtOH at 50 mg/ml (97 mM) for TetResponder induction
Paraformaldehyde (PFA)	Sigma-Aldrich	P6148	4% (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 slides	Thermo Scientific	1014356190	for collection of tissue sections
Stereo fluorescent microscope	e.g., Leica, Zeiss	varies with the manufacturer	for fluorescence-based genotyping
Thermocycler	e.g., Biorad, Applied Biosystems	varies with the manufacturer	for PCR-based genotyping
Tissue freezing medium (TFM)	Triangel Biomedical Sciences	TFM-C	for embedding of tissue samples
Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)	Sigma-Aldrich	E10521	for anesthesia use at 1 mg/ml in E3 embryo medium