Name: Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration (Video) | JoVE Journal
Uploaded: 2015-06-25T13:00:00.000+00:00
Duration: 10 min 22 s
Description: Learn the TetON system for tissue-specific gene expression in zebrafish tail fin regeneration. This method enables conditional gene manipulation using doxycycline induction and immunofluorescence visualization.

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5 minutes

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 f&#252;r Kardiologie via an Oskar-Lapp-Stipendium and a Klaus-Georg-und-Sigrid-Hengstberger-Forschungsstipendium.

<ol><li>Fang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1%2FStat3+injury+response+required+for+zebrafish+heart+regeneration%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li>
<li>Tryon, R. C., Johnson, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Clonal+analysis+of+kit+ligand+a+functional+expression+reveals+lineage-specific+competence+to+promote+melanocyte+rescue+in+the+mutant+regenerating+caudal+fin%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">Clonal analysis of kit ligand a functional expression reveals lineage-specific competence to promote melanocyte rescue in the mutant regenerating caudal fin.</a> PloS one. 9 (7), e102317(2014).</li>
<li>Wehner, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wnt%2Fbeta-catenin+signaling+defines+organizing+centers+that+orchestrate+growth+and+differentiation+of+the+regenerating+zebrafish+caudal+fin%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Wnt/beta-catenin signaling defines organizing centers that orchestrate growth and differentiation of the regenerating zebrafish caudal fin.</a> Cell Rep. 6 (3), 467-481 (2014).</li>
<li>Huang, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+expression+of+a+myocardium-specific+transgene+in+zebrafish+transgenic+lines%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Conditional expression of a myocardium-specific transgene in zebrafish transgenic lines.</a> Dev Dyn. 233 (4), 1294-1303 (2005).</li>
<li>Knopf, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dually+inducible+TetON+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (46), 19933-19938 (2010).</li>
<li>Provost, E., Rhee, J., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Viral+2A+peptides+allow+expression+of+multiple+proteins+from+a+single+ORF+in+transgenic+zebrafish+embryos%5BTitle%5D)+AND+%22Genesis%22%5BJournal%5D)">Viral 2A peptides allow expression of multiple proteins from a single ORF in transgenic zebrafish embryos.</a> Genesis. 45 (10), 625-629 (2007).</li>
<li>Suster, M. L., Abe, G., Schouw, A., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transposon-mediated+BAC+transgenesis+in+zebrafish%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Transposon-mediated BAC transgenesis in zebrafish.</a> Nat Protoc. 6 (12), 1998-2021 (2011).</li>
<li>Bussmann, J., Schulte-Merker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+BAC+selection+for+tol2-mediated+transgenesis+in+zebrafish%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Rapid BAC selection for tol2-mediated transgenesis in zebrafish.</a> Development. 138 (19), 4327-4332 (2011).</li>
<li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microinjection+of+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> J Vis Exp. (27), (2009).</li>
<li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines%2C+zebrafish+and+mice%5BTitle%5D)+AND+%22PloS+one%22%5BJournal%5D)">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PloS one. 6 (4), e18556(2011).</li>
<li>Matthews, M., Varga, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anesthesia+and+euthanasia+in+zebrafish%5BTitle%5D)+AND+%22ILAR+journal+%2F+National+Research+Council%2C+Institute+of+Laboratory+Animal+Resources%22%5BJournal%5D)">Anesthesia and euthanasia in zebrafish.</a> ILAR journal / National Research Council, Institute of Laboratory Animal Resources. 53 (2), 192-204 (2012).</li>
<li>Hyde, D. R., Godwin, A. R., Thummel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+electroporation+of+morpholinos+into+the+regenerating+adult+zebrafish+tail+fin%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">In vivo electroporation of morpholinos into the regenerating adult zebrafish tail fin.</a> J Vis Exp. (61), (2012).</li>
<li>Meeker, N. D., Hutchinson, S. A., Ho, L., Trede, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+isolation+of+PCR-ready+genomic+DNA+from+zebrafish+tissues%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">Method for isolation of PCR-ready genomic DNA from zebrafish tissues.</a> Biotechniques. 43 (5), 610(2007).</li>
<li>Thermes, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((I-SceI+meganuclease+mediates+highly+efficient+transgenesis+in+fish%5BTitle%5D)+AND+%22Mech+Dev%22%5BJournal%5D)">I-SceI meganuclease mediates highly efficient transgenesis in fish.</a> Mech Dev. 118 (1-2), 91-98 (2002).</li>
<li>Suster, M. L., Kikuta, H., Urasaki, A., Asakawa, K., Kawakami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transgenesis+in+zebrafish+with+the+tol2+transposon+system%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Transgenesis in zebrafish with the tol2 transposon system.</a> Methods Mol Biol. , 56141-56163 (2009).</li>
<li>Grabher, C., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Meganuclease+and+transposon+mediated+transgenesis+in+medaka%5BTitle%5D)+AND+%22Genome+Biol%22%5BJournal%5D)">Meganuclease and transposon mediated transgenesis in medaka.</a> Genome Biol. 8, Suppl 1S10. (2007).</li>
</ol>

The authors have nothing to disclose.

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...

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&#8211;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&#8217; 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.

<table><tbody><tr><td>Breeding boxes</td><td>Aqua Schwarz</td><td>AquaBox 1</td><td></td></tr><tr><td>Compound fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Confocal microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>to image fluorescent tissue sections</td></tr><tr><td>Cryostat</td><td>e.g., Leica, Thermo-Scientific</td><td>varies with the manufacturer</td><td>for cryosectioning</td></tr><tr><td>4&amp;rsquo;, 6- diamidino-2-phenylinodole (Dapi)</td><td>Sigma-Aldrich</td><td>D9542</td><td>use 1/5,000 in PBS&lt;br/&gt; for visualization of nuclei</td></tr><tr><td>Doxycycline</td><td>Sigma-Aldrich</td><td>D9891</td><td>prepare stocks in 50% EtOH at 50 mg/ml (97 mM)&lt;br/&gt; for TetResponder induction</td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich</td><td>P6148</td><td>4% (w/v) paraformaldehyde in PBS, pH 7.5&lt;br/&gt; for fixation</td></tr><tr><td>1x Phosphat-buffer saline (PBS)</td><td></td><td></td><td>1.7 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, 5.2 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 150 mM NaCl, pH 7.5</td></tr><tr><td>1x Phosphat-buffer saline + Tween 20 (PBT)</td><td></td><td></td><td>1x PBS with 0.1% Tween 20</td></tr><tr><td>Superfrost Ultra Plus adhesion microscope slides</td><td>Thermo Scientific&amp;nbsp;</td><td>1014356190</td><td>for collection of tissue sections</td></tr><tr><td>Stereo fluorescent microscope</td><td>e.g., Leica, Zeiss</td><td>varies with the manufacturer</td><td>for fluorescence-based genotyping</td></tr><tr><td>Thermocycler</td><td>e.g., Biorad, Applied Biosystems</td><td>varies with the manufacturer</td><td>for PCR-based genotyping</td></tr><tr><td>Tissue freezing medium (TFM)</td><td>Triangel Biomedical Sciences</td><td>TFM-C</td><td>for embedding of tissue samples</td></tr><tr><td>Tricaine (L-Ethyl-m-amino-benzoate-methane sulfonate/MS-222)</td><td>Sigma-Aldrich</td><td>E10521</td><td>for anesthesia&lt;br/&gt; use at 1 mg/ml in E3 embryo medium</td></tr></tbody></table>

use of the teton system to study molecular mechanisms of zebrafish regeneration

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

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&#8211;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.

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)...

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 ...

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.

use-teton-system-to-study-molecular-mechanisms-zebrafish

Research

JoVE Journal

Developmental Biology

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein’s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2 days post-injury, and C-bend movement by 3 days post-injury.

Spinal Cord Transection in the Larval Zebrafish

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Learn the TetON system for tissue-specific gene expression in zebrafish tail fin regeneration. This method enables conditional gene manipulation using doxycycline induction and immunofluorescence visualization.

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

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