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

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 transgenic TetActivator fish lines
   1. Perform microinjection of the TetActivator construct according to standard procedures⁹.
      1. Inject 25-50 pg of the plasmid DNA or up to 250 pg BAC DNA into one-cell-stage embryos together with capped sense RNA coding for tol2 transposase for transposon-mediated transgenesis, or with I-SceI protein for I-SceI-mediated transgenesis.
         NOTE: Considerations on the choice of the transgenesis method can be found in the discussion section.
         NOTE: Inject preferably into the cytoplasm, not the yolk, since efficiency of transgenesis might decrease when the DNA is not delivered into the cytoplasm.
   2. Raise injected G0 embryos to adulthood. Optionally, screen G0 embryos for AmCyan fluorescence and preferentially grow embryos showing the desired expression pattern. This might increase the rate of germ-line transmission, in particular for Tol2-mediated transgenesis.
   3. Assess successful germ-line integration by outcrossing individual G0 fish to wild-type fish and appearance of AmCyan fluorescence in the expected expression pattern in F1 embryos or larvae using a fluorescent stereomicroscope (example is shown in Figure 1D).
   4. Raise fluorescence-positive F1 embryos to adulthood and mate them with wild-type fish to obtain stable F2 transgenic fish.
   5. Raise and characterize at least 3 different sublines derived from different G0 founder fish, since individual lines might differ in expression levels, expression pattern, ability to induce TetResponder lines, leakiness, and their susceptibility to silencing.
      NOTE: Many transgenes that are robustly expressed in embryos are either partially or completely silenced in late-stage larvae or adult fish. Thus, if lines are meant to be used in adult fish, characterize several independent lines for their TetActivator expression and function during adulthood.
      NOTE: We have generated a panel of TetActivator lines for tissue-specific expression of the TetActivator in both embryos and adult fish, which are listed in Table 1. These lines are available from the Weidinger lab upon request.

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 polypeptides^6,10.

1. Generation of TetResponder construct
   1. Clone CDS of the gene of interest 3’ of the Tetracycline operator and 5’ of the YPet CDS according to standard procedures. Ensure that the stop codon has been removed from the gene of interest CDS.
   2. Prepare toxin-free plasmid DNA preparations using commercially available kits.
2. Generation of transgenic TetResponder fish lines
   1. Perform microinjection of TetResponder construct according to standard procedures⁹.
      1. Inject 25-50 pg of the plasmid DNA into the cytoplasm of one-cell-stage embryos together with capped sense RNA coding for tol2 transposase for transposon-mediated transgenesis or with I-SceI enzyme for meganuclease-aided transgenesis.
         NOTE: Considerations on the choice of the transgenesis method can be found in the discussion section.
         NOTE: Inject preferably into the cytoplasm, not the yolk, since efficiency of transgenesis might decrease when the DNA is not delivered into the cytoplasm.
   2. Raise injected G0 embryos to adulthood.
   3. Assess successful germ-line integration and inducibility of the transgene by mating individual G0 fish with a previously established TetActivator line followed by Doxycycline treatment of F1 embryos or F1 larvae.
      NOTE: Usually we use a ubiquitin promoter-driven TetActivator line (ubiquitin:irtTAM2(3F)-p2a-AmCyan^ulm2, short ubiquitin:TetA AmCyan³) to screen for functional TetResponder carriers, as this line drives fairly ubiquitous expression in embryos and adult fish and thus is well suited to assess TetResponder inducibility in all tissues of interest. This line is available from the Weidinger lab upon request.
   4. Collect embryos of each clutch in a separate 10 cm Petri dish containing E3 embryo medium (see Materials table) and incubate dishes at 28.5 °C.
   5. Place G0 fish that gave embryos into individual numbered tanks (breeding boxes; see Materials table) until embryos have been screened. This allows for recovery of G0 fish that show germ line transmission of the transgene.
   6. If only half of the embryos are expected to carry the TetActivator transgene (in case a heterozygous TetActivator carrier was crossed to a TetResponder founder fish), select for embryos containing the TetActivator transgene by appearance of AmCyan fluorescence at the appropriate developmental stage.
   7. Induce TetActivator transcriptional activity by treating embryos with Doxycycline (DOX) as described in section 2.3.
   8. Screen F1 embryos for emergence of YPet fluorescence. Prefer lines (G0 fish) that produce homogenous YPet expression, that is embryos in the clutch should display little mosaicism within the expected expression domain and little variation between individual embryos. See arrowheads in Figure 1F and Figure 1G for representative examples.
   9. Mate G0 fish transmitting an inducible TetResponder transgene identified in step 2.2.8 with wild-type fish and raise all F1 embryos to adulthood.
   10. Identify adult F1 transgenic fish either by PCR-based genotyping (see section or by crossing individual F1 fish to a TetActivator line followed by DOX treatment of F2 embryos and emergence of YPet fluorescence (see section 2.5).
   11. Establish and characterize at least 3 different TetResponder sublines derived from different G0 founder fish, since inducibility might differ in individual lines. In particular achievable expression levels and whether the responder can be activated in all cell types might vary. In addition, susceptibility to silencing will differ between lines. Finally, in some cases it might be preferable to select lines that mediate only moderate expression levels of the protein of interest, in particular if leaky expression produced in combination with a TetActivator line in the absence of Doxycycline is expected to cause developmental defects or toxicity.
       NOTE: TetResponder lines that are robustly inducible in embryos may be either partially or completely silenced in late-stage larvae or adult fish. Thus, if lines are meant to be used in adult fish, several independent lines should be characterized for inducibility in adults. If induction of the gene of interest is compatible with further development, it has been proven useful to create double transgenic embryos (TetActivator x TetResponder), to induce GOI expression in embryos and to select and raise only the individuals showing the most robust and least mosaic induction.
   12. To maintain and propagate established TetResponder lines, genotype adult fish as described in section 2.5).
3. Doxycycline treatment of embryos
   1. Prepare 2,000x Doxycycline (DOX) stocks. Solve DOX in 50% EtOH to achieve a concentration of 50 mg/ml (97 mM). Store DOX stocks protected from light at -20 °C.
   2. Decant the majority of E3 medium from embryos and replace it with E3 embryo medium containing DOX at a final concentration of 25 µg/ml DOX (2.5 µl of 2,000x DOX stock per 50 ml E3). Dechorionation is not required.
   3. Return embryos to 28.5 °C for a minimum of 6 hr before assessing expression of the tagged gene of interest by appearance of YPet fluorescence.
   4. Assess leakiness of the TetActivator/TetResponder combination by treating embryos with EtOH solvent only.
      NOTE: In situ hybridization (ISH) or RT-PCR to detect the TetResponder mRNA can be used as more sensitive measures of leakiness than fluorescence of the tagged gene of interest. The extent of leakiness will depend on the particular TetActivator/TetResponder line combination and it may also vary between tissues and developmental stages.
4. Anesthesia of adult zebrafish
   1. Prepare 10 cm diameter Petri dish or small beaker filled with fish system water containing 1 mg/ml Tricaine (Ethyl 3-aminobenzoate methanesulfonate, MS-222).
   2. Transfer fish to container containing anesthetic and wait until it reaches level 4 of anesthesia as indicated by complete loss of equilibrium (fish lies on its side), no movements and no response to touching¹¹.
   3. Carefully transfer fish using tweezers or a spoon onto a glass slide, lid of a plastic petri dish, or agarose-coated petri dish and perform fin amputation or imaging (see section 3.2).
   4. Return fish to a container containing a large volume of fresh fish system water and monitor it. If gill movements do not resume within 3 min, assist by blowing water over the gills using a plastic transfer pipet.
5. PCR-based genotyping of TetResponder fish
   1. Perform partial amputation of the tail fin¹². Place fish into individual, numbered tanks (breeding boxes; see Materials table). This allows for the recovery of transgenic fish following successful genotyping.
   2. Transfer fin tissues to individual wells of a 96-well PCR plate pre-filled with 20 µl DNase-free water for isolation of genomic DNA¹³.
      1. Add 120 µl 1 M NaOH to each well and incubate samples for 20 min at 95°C using a thermocycler.
      2. Chill samples to 4°C, add 14 µl of 1 M Tris-HCl (pH 8.0) to each well, and briefly vortex samples. Centrifuge samples for 5 min at 16,000 x g to pellet cell debris. Store genomic DNA at 4 °C until needed.
   3. Design primers to specifically amplify a fragment of the TetResponder transgene. Use 2 µl of isolated DNA in a standard PCR reaction and analyze PCR product on an agarose gel.

3. Tissue-specific Induction of Transgene Expression in the Adult Regenerating Zebrafish Tail Fin

1. Establishment of TetActivator; TetResponder double transgenic fish
   1. Mate TetActivator carriers with TetResponder carriers.
   2. Select embryos carrying the TetActivator transgene at a developmental stage when the regulatory elements driving the TetActivator cassette are active. Transgenic embryos can easily be identified by appearance of AmCyan fluorescence using a stereo fluorescent microscope.
      NOTE: TetActivator transgene-positive fish may alternatively be identified during adulthood by imaging of anesthetized fish, eg. following amputation of the tail fin and emergence of AmCyan fluorescence in the regenerate.
   3. Raise selected embryos to adulthood.
   4. Identify double transgenic adult fish (fish that carry the TetResponder in addition to the TetActivator) by PCR-based genotyping or (preferably) by their ability to induce expression of the TetResponder in the tissue of interest. A protocol for TetResponder induction and detection in the regenerating tail fin is described in section 2.3 and section 4.
      NOTE: If transient TetResponder expression is compatible with further embryonic or larval development, it is useful to identify double transgenic embryos carrying TetActivator and TetResponder transgenes following DOX treatment during embryonic development as described above (section 2.3, Figure 1H). Double positive embryos will show AmCyan and YPet fluorescence. Raise positive embryos to adulthood.
2. Amputation of zebrafish tail fins and induction of transgene expression in the regenerating fin
   1. Perform partial amputation of the tail fin on anesthetized fish (see section 2.4)¹². Treat fish either immediately with DOX or return them to the fish housing system.
   2. Prepare breeding box (Figure 2A; see Materials table) filled with 1 L of fish system water containing 25 µg/ml DOX (add 500 µl of 2,000x 50 mg/ml stock to 1 L of fish system water).
      ​NOTE: Induction of transgene expression immediately after amputation allows for assessment of effects on early events during regeneration, while induction at 3 days post-amputation (3 dpa) should be used to assess transgene expression or function during regenerative growth.
   3. Transfer up to 10 previously amputated, double transgenic fish to the breeding box and close box with a lid such that fish cannot escape (Figure 2A).
   4. Place breeding boxes into the dark to reduce stress. We usually place the boxes into a 28.5 °C air incubator to ensure constant darkness and temperature.
   5. Treat negative control double transgenic fish with EtOH only (250 µl per 1L fish system water).
      NOTE: Single transgenic or wild-type fish treated with DOX can be used as additional negative control, although we have never observed adverse effects of the DOX doses used here on fin regeneration.
   6. Anesthetize fish as described in section 2.4 and transfer fish using tweezers or a spoon onto a wetted agarose-coated petri dish. Carefully spread the tail fin using tweezers.
   7. Screen fish for appearance of YPet within the regenerate using a fluorescent stereo-microscope (Figure 2B).
      NOTE: Usually, TetResponder-driven fluorescence can be robustly observed following 6 hr of DOX treatment. However, we recommend to characterize the dynamics of TetResponder expression for each line generated.
   8. Return fish to the fish housing system to terminate TetResponder transgene induction or continue treatments.
      NOTE: For treatments >24 hr (long-term treatments) it is essential to exchange water and thoroughly clean the breeding boxes daily.
   9. During long-term treatments, feed fish every 2-3 days by transferring fish into a clean container with fresh fish system water before returning them to breeding boxes after feeding.

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.

1. Cryosectioning of adult tail fins
   1. Treat fish whose tail fins were amputated from 2 dpa until 3 dpa with DOX as described in section 3.2
   2. Anesthetize fish as described in section 2.4
   3. Use a spoon or tweezers to gently transfer fish onto a lid of a plastic Petri dish.
   4. Re-amputate the fin regenerate at about 4-5 bony segments proximal to the initial amputation plane using a scalpel and return the fish to fresh fish system water.
   5. Carefully transfer regenerates to a small petri dish (e.g., 35 mm) containing 4% (w/v) paraformaldehyde (PFA) in 1x Phosphate-buffered saline (PBS; see Materials table) using fine tweezers. Do not touch the regenerate but only the stump part of the tissue.
   6. Place tissue flat onto the petri dish bottom and fix O/N at 4 °C.
   7. Wash regenerates twice in 1x PBS containing 0.1% Tween (PBT) to remove PFA and incubate regenerates o/n at 4 °C in 0.5 M EDTA-PBS (pH 7.5) to decalcify bone matrix.
   8. Incubate regenerates at RT in 10% sucrose-PBS, 20% sucrose-PBS, 30% sucrose-PBS and 30% sucrose-PBS:tissue freezing medium at a 1:1 ratio (TFM; see Materials table) for 30 min each. Subsequently, incubate fins at 4 °C for >2 hr in tissue freezing medium.
   9. Place cryomolds onto a microscope slide and fill them with tissue freezing medium. Avoid introducing air bubbles.
   10. Transfer regenerates into cryomolds and orient tissue appropriately to obtain longitudinal or transverse sections of fin regenerates (Figure 2D). Ensure that regenerate is straight, which facilitates sectioning.
   11. Transfer cryomolds together with microscope slide onto a metal rack positioned on a bed of dry ice to start the freezing process (Figure 2D).
   12. Once the tissue freezing medium has become solid, place cryomold onto bench and let sit at RT for a few minutes such that the tissue freezing medium thaws at the plastic-medium interface.
   13. Use a pen or similar to push the frozen block out of the cryomold. Store cryoblocks in a box at -80 °C until tissue sectioning.
   14. Prepare 10-14 µm thick cryosections using a Cryo-microtome and collect sections on adhesion microscope slides (see Materials table). Store slides at -20 °C until needed.
2. Characterization of TetResponder expression on fin sections based on YPet fluorescence
   1. Treat fin sections for 30 min with 100% MeOH chilled to -20 °C to improve adherence to the microscope slide followed by two washes at RT in PBT and 1 wash in PBS, 5 min each.
   2. Visualize nuclei by incubating sections for 10 min in 4’, 6- diamidino-2-phenylinodole (Dapi) in PBS (1:5,000) followed by two washes 10 min each in PBS.
   3. Mount sections in 75% Glycerol-PBS or commercially available antifade mounts, and cover with a cover slide.
   4. Image YPet and AmCyan fluorescence under a confocal or wide-field fluorescent microscope (Figure 1E).
      NOTE: To facilitate unambiguous identification of cell types expressing YPet, immunofluorescence or immunohistochemistry with antibodies against cell-type specific antigens together with anti-GFP antibodies (which react with YPet, but not AmCyan) can be performed. Alternatively, to facilitate detection of weak YPet expression, RNA in situ hybridization against YPet RNA can be performed either on whole-mount fins or paraffin sections of fins.