Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.
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.
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 achieve conditional, tissue-specific gene expression in regenerating organs of adult zebrafish: the Cre-lox system, mosaic expression of heat-shock inducible transgenes using transposon-mediated somatic transgenesis, and the TetON system1-3. TetON refers to a variant of a tetracycline-controlled transcriptional activation system, where expression is activated in the presence of the antibiotic tetracycline or a derivative, e.g. doxycycline. The Cre-lox system, as it has so far been used in adult fish, relies on a Tamoxifen-controlled Cre recombinase (CreERT2), whose expression is spatially restricted by tissue-specific regulatory elements. Cre-driven removal of a STOP cassette facilitates expression of the gene of interest driven by a promoter that should be active in all cell types1. Transposon-mediated creation of mosaically expressed somatic transgenes provides a system for inducible transgene expression in individual cell lineages. Injection of zebrafish embryos with a Tol2 transposon carrying a gene of interest under transcriptional control of a heat shock promoter results in chimeric individuals typically carrying the transgene only in discrete cell lineages of a regenerating organ2. While both systems allow for conditional tissue-specific gene expression, the Cre-lox system is not reversible, and the strategy using transposon-based clonal labeling suffers from its stochastic nature. Thus, we and others have recently adapted transgenic TetON systems for use in zebrafish, which facilitate temporally and spatially-controlled gene expression that is in addition tunable and reversible3-5.
The TetON system used here comprises a transgenic driver line (TetActivator) in which a Doxycycline (DOX)-inducible transcriptional activator (improved reverse tetracycline transactivator, irtTA, short TetA) is under control of tissue-specific genomic regulatory sequences. Secondly, it requires a transgenic responder line (TetResponder) that harbors a gene of interest under transcriptional control of the Tetracycline operator (Tet response element; TetRE) (Figure 1A). Thus, the use of specific combinations of TetActivator and TetResponder lines allows for conditional tissue-specific manipulation of gene expression.
We have recently utilized the TetON system to probe for tissue-specific functions of Wnt/beta–catenin signaling in the adult regenerating zebrafish tail fin3. In the protocol outlined here we describe a work flow for set-up and use of the TetON system in zebrafish, in particular for studies of fin regeneration. This includes detailed instructions on how to generate stable transgenic TetActivator and TetResponder lines and a protocol for transgene induction in embryos and adult zebrafish. Furthermore, we describe techniques for verification of tissue-specific gene expression in the fin regenerate, including a protocol for the preparation of cryosections of adult zebrafish fins. Additionally, we discuss considerations for the design of the TetActivator transgene, the choice of a transgenesis method, and detection of TetResponder expression. Hence, the overall goal of this protocol is to serve as a blueprint for setting-up a functional TetON system in zebrafish to achieve conditional tissue-specific gene expression, which can be applied to any tissue of interest.
We have created a TetActivator vector allowing for I-SceI or Tol2-mediated generation of stable TetActivator lines using short genomic regulatory sequences (enhancer fragments; Weidinger lab plasmid database no. 1247; Figure 1B). This construct contains a TetActivator cassette consisting of the M2 mutant variant of the reverse Tet repressor domain fused with the Herpes simplex virus VP16 transactivation domain-derivative 3F [irtTAM2(3F)]. Expression of the TetActivator (TetA) can be easily monitored since it is co-expressed with the fluorophore AmCyan from the same open reading frame; a p2a peptide mediates ribosomal skipping, which should result in production of TetA and AmCyan as separate proteins at a 1:1 ratio5,6. The construct also contains a poylinker 5’ of the TetActivator cassette to facilitate insertion of genomic regulatory sequences of interest using conventional cloning methods.
Additionally, we have created a construct consisting of the above described TetActivator cassette plus a Kanamycin selection cassette (Weidinger lab plasmid database no. 1180; Figure 1C), which can be recombined into a bacterial artificial chromosome (BAC) containing a large genomic region (usually into the start codon of a gene whose expression pattern is to be mimicked by the transgene). Both constructs are available from the Weidinger lab upon request.
1. Generation of Transgenic TetActivator Fish Lines
2. Generation of Transgenic TetResponder Fish Lines
NOTE: We have generated a TetResponder construct allowing for I-SceI or Tol2-mediated generation of stable TetResponder lines, which is available from the Weidinger lab upon request (Weidinger lab plasmid database no. 1444; Figure 1E). This construct contains a Tetracycline operator, followed by a polylinker region facilitating the insertion of coding sequences (CDS) of a gene of interest and the YFP-derivative YPet coding sequence. Thus, the construct is designed for TetA-mediated expression of a C-terminal fusion of the protein of interest with YPet. If expression of a tagged fusion protein has to be avoided, a p2a or t2a peptide can be introduced with the gene of interest CDS, which facilitates co-expression of the protein of interest and YPet as separate polypeptides6,10.
3. Tissue-specific Induction of Transgene Expression in the Adult Regenerating Zebrafish Tail Fin
4. Characterization of TetResponder Expression in Fin Regenerates
NOTE: We usually verify tissue-specific gene expression in the regenerating tail fin using fluorescent imaging of cryosections at 3 dpa. At this time point the different tissue compartments of a regenerate have formed and can be clearly identified on tissue-sections, and cryosectioning is simpler than at later stages of regeneration. Figure 2C depicts the tissue domains that can be distinguished in the regenerate and lists a few molecular markers for these domains. The following protocol describes the preparation of longitudinal or transverse cryosections for direct imaging or immunostaining.
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) upstream of the TetActivator cassette (Figure 1B), or by recombining the TetActivator cassette into a BAC containing regulatory elements of the gene of interest (Figure 1C). Germline integration of the TetActivator construct can be assessed by outcrossing individual injected G0 fish and appearance of AmCyan fluorescence in F1 embryos in the expected expression pattern (Figure 1D). Germline integration and functionality of the TetResponder transgene can be assessed by crossing individual G0 injected fish to established TetActivator fish (here ubiquitin:TetA AmCyan), followed by treatment with DOX and appearance of YFP/YPet fluorescence (Figure 1F). Fish showing strong and homogenous YFP/YPet expression should be preferred to establish a stable TetResponder line (Figure 1G). Having established stable TetActivator and TetResponder fish lines, specific TetActivator/TetResponder combinations can be generated by crossing. Carriers of TetActiator and TetResponder transgenes can be identified during embryogenesis following DOX treatment end emergence of YFP/Ypet at the developmental stage the TetActivator is active (Figure 1H). Alternatively, double transgenic fish can be identified following DOX treatment and TetResponder transgene induction in the regenerating tail fin (Figure 2A–B).
We routinely use this system to achieve inducible, tissue-specific gene expression in the regenerating zebrafish tail fin. We verify tissue-specific TetResponder transgene expression by fluorescence imaging of cryosections of 3 dpa fin regenerates, since the different tissue compartments of a regenerate can be clearly identified on tissue-sections at this time-point (Figure 2C). Longitudinal or transverse fin regenerate sections are obtained by cryosectioning after cryoprotecting tissue and embedding fin regenerate appropriately (Figure 2D). Following sectioning, TetResponder transgene expression (Ypet/YFP fluorescence) can be imaged using fluorescence or confocal microscopy (Figure 2E). Note that the her4.3 promoter-driven TetActivator induces TetResponder expression in the proximal medial blastema, while the distal blastema (arrowhead) and the epidermis (arrow) are devoid of expression.
Figure 1: Generation of transgenic TetActivator and TetResponder lines. (A) Cartoon showing strategy for tissue-specific inducible gene expression using the TetON system. (B) Transgenic TetActivator construct (Weidinger lab plasmid database no. 1247). A polylinker 5’ of the TetActivator irtTAM2(3F) facilitates cloning of regulatory sequences of interest. The AmCyan coding sequence is separated from the TetActivator via a p2a ‘self-cleaving’ peptide. The cassette also contains a SV40 polyadenylation signal, is flanked by Tol2 transposon inverted repeats and contains a single I-SceI meganuclease recognition site. (C) TetActivator cassette for BAC recombineering (Weidinger lab plasmid database no. 1180). The cassette includes the Kanamycin gene for selection, which is driven by a gb3 prokaryotic promoter. FRT sites flanking the Kanamycin resistance gene allow for its Flp-recombinase mediated removal following successful integration into the BAC. (D) Identification of G0 founder fish transmitting the her4.3:TetA AmCyan construct through their germ-line. Carriers are identified via emergence of AmCyan fluorescence in some F1 embryos (arrowheads). (E) Transgenic TetResponder construct (Weidinger lab plasmid database no. 1444). This constructs consists of a polylinker and YPet CDS under control of optimized Tet response elements (TetRE-tight, Clontech) and terminated by the SV40 polyadenylation signal. It is flanked by Tol2 transposon inverted repeats and contains a single I-SceI meganuclease recognition site. (F) Identification of G0 founder fish transmitting a functional TetResponder TetRE:Axin1-YFP construct through their germline. Carriers are identified by crossing with an established TetActivator line (here ubiquitin:TetA AmCyan) and induction of YFP fluorescence following DOX treatment for 6 hr (arrowheads). (G) Close up ofembryos shown in (F) after a total of 12 hr of DOX treatment. Note fairly ubiquitous induction of YFP fluorescence. (H) Identification of double transgenic embryos carrying TetActivator (her4.3:TetA AmCyan) and TetResponder (TetRE:Axin1-YFP) transgenes (arrowheads). Embryos were treated with DOX for 6 hr. (A–H) Scale bar: 500 µm Please click here to view a larger version of this figure.
Figure 2: Use of the TetON system for tissue-specific gene expression in the regenerating zebrafish tail fin. (A) Breeding boxes used for DOX treatment of adult zebrafish. (B) Induction of TetRE:Axin1-YFP TetResponder transgene by the her4.3:TetA AmCyan TetActivator in the regenerating tail fin following 12 hr of DOX treatment. Note the absence of YFP fluorescence in the EtOH-treated control fish. (C) Cartoon depicting some of the tissue compartments found within a fin regenerate at 3 dpa in a longitudinal section view. (D) Sample preparation for cryosectioning. Fin regenerates are placed in tissue-freezing medium (TFM)-filled cryomolds, oriented appropriately to obtain either longitudinal or transverse sections of the regenerate, and transferred to a metal rack sitting on top of dry-ice until TFM has solidified. Sectioning plane is indicated in red. Abbreviations: dist.: distal, prox.: proximal, vent.: ventral, dors.: dorsal. (E) Confocal image of YFP fluorescence in a longitudinal section of a fin ray regenerate derived from fins shown in (B). Note that the her4.3:TetA AmCyan TetActivator line drives YFP induction specifically in the proximal-medial blastema. YFP fluorescence is not detected in a variety of compartments, including the wound epidermis (arrow), and the distal-most domain of the mesenchyme (arrowhead) (B-C) Scale bar: 500 µm; (E) Scale bar: 100 µm. Please click here to view a larger version of this figure.
TetActivator line | Reference | Regulatory elements used | Primarily expressed in |
7xTCF-Xla.Siam:irtTAM2(3F)-p2a-AmCyan | Wehner &Weidinger, unpublished | 7xTCF-Xla.Siam Wnt reporter 1 | Wnt-responsive tissues |
myl7: irtTAM2(3F)-p2a-mCherry | Haase & Weidinger, unpublished | myl7 (cmlc2) 2 | mature cardiomyocytes |
her4.3: irtTAM2(3F)-p2a-AmCyanulm6 | 3 | her4.3 4 | central nervous system |
keratin4: irtTAM2(3F)-p2a-AmCyanulm5 | 3 | keratin4 5 | epidermis, in the adult fin excluding the basal layer |
keratin18: irtTAM2(3F)-p2a-AmCyanulm4 | 3 | keratin18 6 | epidermis, in the adult fin exclusively in the basal layer |
sp7: irtTAM2(3F)-p2a-AmCyanulm3 | 3 | sp7/osx 7 | (committed) osteoblasts |
ubiquitin:irtTAM2(3F)-p2a-AmCyanulm2 | 3 | ubiquitin 8 | (fairly) ubiquitous |
Table 1: Available transgenic TetActivator lines. This table lists transgenic TetActivator lines for tissue-specific expression of the TetActivator in both embryonic and adult fish, which are available from the Weidinger lab upon request.
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 laboratory3. The following additional issues should be considered when establishing the TetON system:
TetActivator transgene design considerations
We have previously shown that a single-inducible TetActivator consisting of the M2 mutant variant of the reverse Tet repressor domain fused with the Herpes simplex virus VP16 transactivation domain-derivative 3F [irtTAM2(3F), in short TetA-M2] confers efficient induction, but can show low, albeit measurable leakiness, that is background inducing activity in the absence of Doxycycline (DOX)5. Therefore, we have described dually inducible systems using fusions with a glucocorticoid receptor (TetA-GBD) or a domain of the Ecdysone receptor (TetA-EcR), which eliminate leakiness5. Thus, if the system will be used to express toxic or very potent proteins (e.g., oncogenes) the use of a dually inducible variant should be considered. Alternatively, during establishment of TetActivator and TetResponder lines a range of lines producing different expression levels can be selected and weaker inducers chosen in case leakiness is an issue with strongly inducing lines. TetActivator constructs containing the TetA-GBD or TetA-EcR variant are available from the Weidinger lab upon request.
Transgenesis methods for generation of transgenic zebrafish lines
Generation of stable transgenic zebrafish lines is accomplished by microinjection of DNA constructs into the early embryo and subsequent germ-line insertion of the injected DNA. Two methods have been routinely used to achieve efficient germ-line transformation in zebrafish: i) enhancement of plasmid integration efficiency using I-SceI meganuclease14, and ii) Tol2 transposon-mediated transgenesis15. The first relies on co-injection of plasmid DNA together with I-SceI meganuclease protein, which results in linearization of the plasmid and thereby is thought to increase the efficiency of plasmid integration into the host genome16. The latter requires co-injection of tol2 transposase RNA together with DNA containing Tol2 transposable elements for transposase-mediated integration into the genome. The Tol2-mediated transgenesis strategy is generally thought to be more efficient and can be used to integrate large DNA constructs, e.g., bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs), however frequently results in multiple single-copy insertions at several genomic loci, which can make establishment of a stable transgenic line showing uniform expression levels and Mendelian inheritance of the transgene time-consuming. In contrast, the I-SceI meganuclease technique is usually less efficient and not recommended for BAC transgenesis, but yields single-copy or tandem array transgene integration at a single genomic locus. We have used both transgenesis methods to successfully create functional TetActivator or TetResponder lines.
TetResponder transgene expression analysis
To detect tissue-specific TetResponder transgene expression in adult fins we usually use fluorescent imaging of cryosections. In our experience, fluorescence of fluorescent proteins expressed by the transgene is preserved throughout the fixation and cryosectioning procedure and thus can be directly imaged. Additional methods for detection of TetResponder expression, which have been described elsewhere, are: i) immunostaining on sections, preferably against the fluorescent protein tag, here YPet, using an anti-GFP antibody, ii) fluorescent or chromogenic ISH on sections, or iii) whole-mount ISH followed by sectioning.
The authors have nothing to disclose.
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.
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 1/5000 in PBS for visualization of nuclei |
Doxycycline | Sigma-Aldrich | D9891 | prepare stocks in 50% EtOH at 50 mg/ml (97 mM) for TetResponder induction |
E3 embryo medium | 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2· 2 H2O, 0.33 mM MgSO4·7 H2O, 0.2 ‰ (w/v) methylene blue, pH 6.5 for embryo/larvae husbandry |
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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 |