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

The overall goal of this procedure is to set up a functional TED on system in zebrafish to achieve conditional tissue specific gene expression as demonstrated in the regenerating tail fin in this video. This is accomplished by first generating Tet activator transgenic lines that express a tetracycline inducible transcriptional activator tagged with am cyan in the tissue of interest and Tet responder lines that contain a ype tag gene of interest under control of Tet responsive elements. Next specific combinations of Tet activator Tet responder double transgenic fish are established.

Then tissue specific Tet responder transgene expression is induced through administration of doxycycline. Finally, Tet responder transgene expression is verified by preparing tissue sections here of fin regenerates. Ultimately, immunofluorescence is used to visualize tissue specific Tet responder transgene expression.

The main advantage of this technique over other methods such as heat shock induced gene expression, is that deed on system allows for inducible and reversible gene expression in a tissue specific manner. This method can help to gain a more profound understanding of the molecular signaling networks guiding S fish fin regeneration as it allows for analysis of signaling pathway and gene function in individual tissues. While we use this technology to study zebrafish fin regeneration, it can be instrumental also many other contexts requiring tissue specific manipulation of gene function.

After generating Tet responder fish lines according to the text protocol, assess for germline integration and inducibility of the transgene by mating individual G zero fish with a previously established Tet activator line. Then harvest embryos and raise them to a stage at which Tet responder induction can be assessed. Prepare 2000 x doxycycline stock solution by using 50%ethanol to dissolve docks to arrive at a concentration of 50 milligrams per milliliter.

Store aliquots of docks protected from light at negative 20 degrees Celsius. Next, prepare E three embryo medium containing docks at a final concentration of 25 micrograms per milliliter. Then decant the majority of E three medium from the embryos.

Replace it with dos containing E three medium and return the embryos to 28.5 degrees Celsius for a minimum of six hours. Following the incubation, use a fluorescent stereo microscope to screen embryos for Y pet fluorescence. Establish several stable transgenic Tet responder lines from founders just identified.

Choose lines that induce strongly and display little mosaicism within the expected expression domain as well as little variation between individual embryos. Once TED activator and Tet responder fish lines have been established, make Tet activator carriers with Tet responder carriers to generate double transgenic. Fish select embryos carrying the Tet activator transgene by screening for am cyan fluorescence in a stereo microscope and raise am cyan positive embryos to adulthood.

Identify double transgenic adult fish that carry the Tet responder in addition to the Tet activator by PCR-based genotyping or by their ability to induce expression of the Tet Responder in the tissue of interest to carry out Tail Fin Tation anesthetize Adult zebra fish in a beaker filled with fish water containing 160 micrograms per milliliter of trica. When the fish stops moving, use tweezers to carefully transfer it onto an inverted Petri dish and use a scalpel to perform a partial amputation of the tail fin to treat with docs. Prepare breeding boxes with one liter of fish system water containing 25 micrograms per milliliter of doxycycline.

As a negative control add 250 microliters of ethanol instead of doxycycline to breeding boxes with one liter of fish system water transfer up to 10 double transgenic fish to each breeding box and place the boxes in the dark to reduce stress. After a minimum of six hours, anesthetize and transfer the treated fish to a wet aros coated dish and use tweezers to spread the tail.Fin. Next, under a fluorescence microscope screen the fish for the appearance of Y PET within the regenerating tissue.

Afterwards fish can be used for further treatments or return to the fish housing system to terminate te responder transgene induction following partial fin amputation and docs treatment re amputate fin regenerates at about four to five bony segments proximal to the initial amputation plane and at a time point, depending on your experimental setup as previously described in this video with tweezers and only handling the stump part of the tissue, carefully lay the tissue regenerates flat in a small Petri dish containing 4%weight per volume of PFA in PBS and fix overnight at four degrees Celsius the following day. Use PBS to wash the regenerates twice before transferring them into 0.5 molar E-D-T-A-P-B-S to decalcify the bone matrix at four degrees Celsius overnight. Next, incubate the regenerates in a sucrose PBS series at room temperature for 30 minutes each and finally equal parts of 30%sucrose PBS and tissue freezing medium for an additional 30 minutes.

Transfer the fins to tissue freezing medium and incubate at four degrees Celsius for at least two hours. Then place cryo molds onto a microscope slide and fill them with tissue freezing medium. Transfer the regenerates into the cryo molds and orient the tissue to obtain longitudinal or transverse sections.

Ensure that the regenerate is straight to facilitate sectioning. Together with the microscope slide, transfer the cryo molds onto a metal rack positioned on a bed of dry ice to start the freezing process. When the medium is solid, transfer the cryo molds back to room temperature to allow the medium to thaw at the plastic medium interface.

Then using an instrument such as a pen, push the frozen block out of the cryo mold and store at negative 80 degrees Celsius until sectioning with a cryo microtome. Collect 10 to 14 micrometer sections on adhesion microscope slides and store the slides at negative 20 degrees Celsius until needed. To characterize Tet responder expression on fin sections, treat the sections with 100%MEOH chilled to negative 20 degrees Celsius for 30 minutes.

Carefully remove liquid using a paper towel. Then wash the sections twice with PBT and once with PBS for five minutes each. Incubate the sections for 10 minutes in dappy before using PBS to wash twice for 10 minutes each.

Then use Antifa mounting agent and a cover slip to mount the samples before using confocal or wide field fluorescence microscopy to image the samples having established a functional TET on system in zebrafish. This system can be used to test for gene or signaling pathway function in individual tissues. As shown here, the HER 4.3 promoter driven tet activator induces TED responder expression only in a subset of tissues of the regenerate YFP fluorescence is detected in the proliferative proximal blasa while the distal blasa and the epidermis are devoid of expression.

Expression of the WINT beta-Catenin antagonist acts in one in the HER 4.3 domain containing the proliferative cells of the blasa, surprisingly has no effect on regenerative cell proliferation. This suggests that wind betaine and signaling in the proliferative proximal blastema has no essential role in the regulation of blasts ML cell proliferation. In contrast, using the ubiquitin promoter driven Tet activator line to induce axin one expression in all tissues of the fin regenerate strongly interferes with regenerative cell proliferation.

In addition expression of axon one in the HER 4.3 domain, which does not include osteoblasts, strongly interferes with bone formation in the regenerate. Thus, tissue specific interference with wind signaling using the TE on system reveals unexpected indirect functions of wind signaling in regulation of cell proliferation and bone regeneration. Once master, this technique can be used for inducible targeted gene expression in any tissue of interest.

We have successfully applied this method to probe for tissue specific functions of wind bed nicotine signaling in the regenerating S fish tail fin. Using this method will help to gain a more comprehensive understanding of the molecular determinants regulating tissue regeneration.