This work was funded by a Wellcome Trust Sir Henry Dale Fellowship to Y. F. 100104/Z/12/Z.
We would like to thank Dr. Carl Tucker and the team of the fish facility at the Queen&#8217;s Medical Research Institute, University of Edinburgh. We like to thank Michael Millar of the immunohistology and imaging facility of the MRC Centre for Reproductive Medicine, University of Edinburgh for support.
We thank Prof. Dr. Matthias Hammerschmidt and Dr. Heiko Loehr of the Institute of Developmental Biology, University of Cologne for providing the p5E-krt4 entry vector. We also would like to thank Dr. Dirk Sieger of the Centre of Neuroregeneration, Univerity of Edinburgh for providing the pDestTol2CG destination vector.

The following experimental procedures were conducted in strict accordance with the Animals (Scientific Procedures) Act 1986.
1. Preparation of Plasmid DNA for Microinjection
<ol>
	<li>Purify the plasmid pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP with a commercial plasmid DNA purification kit according to the manufacturer&#8217;s instructions and dilute the plasmid DNA to a final stock solution concentration of 100 ng/&#956;l.</li>
	<li>Linearize the transposase containing plasmid pT3TS/Tol214 by BamHI restriction digestion, following the manufacturer protocol. This step can be extended overnight. Precipitate the linearized DNA by a phenol/chloroform extraction, following the standard protocol.</li>
	<li>In vitro transcribe the transposase mRNA from the linearized plasmid with a commercial transcription kit for large amounts of capped RNA according to the manufacturer&#8217;s instructions, dilute and aliquot to a final stock solution concentration of 100 ng/&#956;l in nuclease-free water. Ensure quality of the purified RNA with a native 1-2% TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA) agarose gel electrophoresis (80-120 V). Store the RNA at -80 &#176;C.</li>
</ol>
2. Microinjection of Plasmid DNA for Transient Gene Expression
<ol>
	<li>Prepare an agarose injection plate to hold the fertilized eggs during injection. Pour liquid 3% agarose into a 90 mm Petri dish. Let the agarose cool to 40-50 &#176;C and add the wedged-shaped microinjection mold TU-1 on top of the liquid agarose. After hardening at room temperature, let the agarose rest at 4 &#176;C for 30 min before removing the mold. Store microinjection molds at 4 &#176;C and reuse.</li>
	<li>Prepare an aliquot of the injection solution. Perform the following steps on ice. Pipette 1 &#956;l of plasmid DNA (final concentration of 10 ng/&#956;l), 2 &#956;l of transposase mRNA (final concentration 20 ng/&#956;l), 2 &#956;l of 10 mg/&#956;l Rhodamine B isothiocyanate-Dextran, 2 &#956;l of 5x Danieau solution (290 mM NaCl, 3.5 mM KCl, 2 mM MgSO4, 3 mM Ca(NO3)2, 25 mM HEPES, pH 7.6) to a final volume of 10 &#956;l in nuclease-free water.</li>
	<li>Prepare needles from borosilicate glass capillaries of 1 mm diameter containing an inner filament by pulling the capillary in opposite directions with a Micropipette Puller. Use the following program: heat 530, pull 200, velocity 80 and time 150.</li>
	<li>Fill one needle with 2 &#956;l of the injection solution (kept on ice). Avoid bubbles by carefully withdrawing the microloader tip while releasing the solution into the capillary. Carefully break the tip of the needle with a forceps.</li>
	<li>Regulate the drop size with a pneumatic pico pump. Measure the drop diameter/volume (V = 4/3&#960;r3) and adjust the drop size with an ocular micrometer under a stereoscope in dimethylpolysiloxane. We recommend 100 &#956;m, corresponding to 0.5 nl. This assures uniform and reproducible concentrations per each injected embryo. Titrate the injected concentration and adjust for each construct and plasmid preparation.</li>
	<li>Place the zygotes (Figure 1A) obtained from a cross of Tg(UAS:eGFP-HRASG12V)io006 15 with Tg(lysC:dsRed2)nz50Tg 16 into the molds of the injection plate by use of a 3 ml pastette. Meanwhile, keep the tip of the pre prepared injection needle in dimethylpolysiloxane to avoid the solution from drying up and clotting.</li>
	<li>Inject the premeasured drop (100 &#956;m corresponding to 0.5 nl) under a stereoscope through the chorion into the blastodisc17, prior to the first cleavage (Figure 1A). Injection into the one-cell stage increases the potential of a uniform distribution of the plasmid DNA and RNA in the developing cell mass and the possibility for germline transmission. It is notable that the expression of constructs more often tends to be mosaic.</li>
</ol>
3. Conditional Skin Cell Transformation by 4-Hydroxytamoxifen (4-OHT)
<ol>
	<li>Transfer the injected embryos into 0.3x Danieau solution and keep them at 28.5 &#176;C in an incubator. Remove unfertilized eggs from the dish at sphere stage17. The co-injected Rhodamine B isothiocyanate-Dextran can be utilized as an injection-control under a fluorescent stereoscope.</li>
	<li>Screen embryos at 24 hpf (hr post fertilization) for cardiac expression of eGFP. Continue to raise cmlc:eGFP positive zebrafish embryos until 72 hpf. Carefully monitor the water-quality (0.3x Danieau).</li>
	<li>Remove the chorion of those embryos that have not hatched with forceps at 72 hpf. Carefully poke the chorion with 1 forceps and pull it apart by using the other. Select embryos for lysC:dsRed2 expression using a fluorescent stereoscope.</li>
	<li>Add 10 &#956;l of a 10 mM stock of (Z)-4-Hydroxytamoxifen to 20 ml of 0.3x Danieau solution (final concentration of 5 &#956;M). Use the 4-OHT induction-solution for one standard Petri dish (90 mm) on a batch of 40-60 embryos.</li>
	<li>Transfer the embryos into a new Petri dish containing 20 ml of freshly made induction-solution. 4-OHT has to be stored and handled in the dark. The first signs of expression may be detected 2 hr post induction commencement. The induction-step can be extended overnight, keeping in mind that initial host cell: preneoplastic cell interactions can already occur a few hours after treatment.</li>
</ol>
4. Mounting and Live Imaging of Zebrafish Larvae
<ol>
	<li>Prepare a 60 mm Petri dish by introducing a hole with a diameter of 18-20 mm. Use high-vacuum silicone grease to cover and seal the hole on the outer bottom of the Petri dish with a 25 mm cover glass (Figure 1B).</li>
	<li>Prepare 1% low melting point (LMP) agarose. Keep one 1.5 ml tube of LMP agarose at 37 &#176;C in a heating block.</li>
	<li>Preselect Tg(krt4:KalTA4-ERT2;UAS:eGFP-HRASG12V;lysC:dsRed2)&#160;larvae for green fluorescent skin cell and dsRed2 positive innate immune cell expression, under a fluorescent stereoscope. Anesthetize larvae by the addition of 1 ml of 0.4% tricaine18 to the 20 ml induction solution.</li>
	<li>Transfer the preselected larvae with a pastette into the liquid LMP agarose. Let the larvae sink into the LMP agarose and transfer them onto the cover glass (Figure 1B). Orientate the embryos under a stereoscope. The larvae have to be placed directly onto the glass to reduce the working distance (up to 6 larvae).</li>
	<li>Cover the LMP agarose with the tricaine containing induction solution after it has hardened (Figure 1B arrow).</li>
	<li>Image the embedded larvae using an inverted fluorescent, laser-scanning or spinning disc confocal microscope. Use a 40X or 63X objective as objectives with long working distances enable focusing through the entire larvae.</li>
</ol>

<ol>
	<li>Kajita, M., Sugimura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filamin+acts+as+a+key+regulator+in+epithelial+defence+against+transformed+cells.">Filamin acts as a key regulator in epithelial defence against transformed cells.</a> Nature communications. 5, 4428 (2014).</li><li>Feng, Y., Santoriello, C., Mione, M., Hurlstone, A., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+innate+immune+cell+sensing+of+transformed+cells+in+zebrafish+larvae:+parallels+between+tumor+initiation+and+wound+inflammation.">Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation.</a> PLoS Biology. 8 (12), e1000562 (2010).</li><li>Feng, Y., Renshaw, S., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Tumor+Initiation+in+Zebrafish+Larvae+Reveals+a+Trophic+Role+for+Leukocyte-Derived+PGE+2.">Live Imaging of Tumor Initiation in Zebrafish Larvae Reveals a Trophic Role for Leukocyte-Derived PGE 2.</a> Current Biology. 22 (13), 1253-1259 (2012).</li><li>Halpern, M. E., Rhee, J., Goll, M. G., Akitake, C. M., Parsons, M., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gal4/UAS+transgenic+tools+and+their+application+to+zebrafish.">Gal4/UAS transgenic tools and their application to zebrafish.</a> Zebrafish. 5 (2), 97-110 (2008).</li><li>Gong, Z., Ju, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=fluorescent+protein+expression+in+germ-line+transmitted+transgenic+zebrafish+under+a+stratified+epithelial+promoter+from+keratin8.">fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.</a> Developmental Dynamics an Official Publication of the American Association of Anatomists. 223 (2), 204-215 (2002).</li><li>Distel, M., Wullimann, M. F., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Gal4+genetics+for+permanent+gene+expression+mapping+in+zebrafish.">Optimized Gal4 genetics for permanent gene expression mapping in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (32), 13365-13370 (2009).</li><li>Feil, R., Wagner, J., Metzger, D., Chambon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Cre+recombinase+activity+by+mutated+estrogen+receptor+ligand-binding+domains.">Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.</a> Biochemical and Biophysical Research Communications. 237 (3), 752-757 (1997).</li><li>Gerety, S. S., Breau, M. a., Sasai, N., Xu, Q., Briscoe, J., Wilkinson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+transgene+expression+system+for+zebrafish+and+chick.">An inducible transgene expression system for zebrafish and chick.</a> Development. 140 (10), 2235-2243 (2013).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Eisenhoffer, G. T., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Extrusion+from+the+Epidermis+of+Developing+Zebrafish.">Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish.</a> J. Vis. Exp. (52), e2689 (2011).</li><li>Kague, E., Weber, C., Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+Zebrafish+Transgenesis+for+Evaluating+Enhancer+Sequences.">Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences.</a> J. Vis. Exp. (41), e1722 (2010).</li><li>Balciuniene, J., Gene Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+Using+Gal4+in+Zebrafish.">Trapping Using Gal4 in Zebrafish.</a> J. Vis. Exp. (79), e50113 (2013).</li><li>Balciunas, D., Wangensteen, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genetics. 2 (11), e169 (2006).</li><li>Santoriello, C., Gennaro, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kita+driven+expression+of+oncogenic+HRAS+leads+to+early+onset+and+highly+penetrant+melanoma+in+zebrafish.">Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish.</a> PloS One. 5 (12), e15170 (2010).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Developmental Biology. 7, 42 (2007).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics : an Official Publication of the American Association of Anatomists. 203 (3), 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> THE ZEBRAFISH BOOK: A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Le Guellec, D., Morvan-Dubois, G., Sire, J. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+development+in+bony+fish+with+particular+emphasis+on+collagen+deposition+in+the+dermis+of+the+zebrafish+(Danio+rerio).">Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).</a> The International Journal of Developmental Biology. 48 (2-3), 217-231 (2004).</li><li>Kwan, K. M., Fujimoto, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Developmental Dynamics : an Official Publication of the American Association of Anatomists. 236 (11), 3088-3099 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MultiSite+Gateway+Three-+Fragment+Vector+Construction+Kit.">MultiSite Gateway Three- Fragment Vector Construction Kit.</a> Protocols and Product Manuals. 12537-023, (2010).</li><li>Petersen, L. K., Stowers, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gateway+MultiSite+Recombination+Cloning+Toolkit).">A Gateway MultiSite Recombination Cloning Toolkit).</a> PLoS ONE. 6 (9), e10 (2011).</li><li>Magnani, E., Bartling, L., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Gateway+to+MultiSite+Gateway+in+one+recombination+event.">From Gateway to MultiSite Gateway in one recombination event.</a> BMC Molecular Biology. 7, 46 (2006).</li><li>Robertson, D. W., Katzenellenbogen, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+the+(E)+and+(Z)+isomers+of+the+antiestrogen+tamoxifen+and+its+metabolite,+hydroxytamoxifen,+in+tritium-labeled+form.">Synthesis of the (E) and (Z) isomers of the antiestrogen tamoxifen and its metabolite, hydroxytamoxifen, in tritium-labeled form.</a> The Journal of Organic Chemistry. 47 (12), 2387-2393 (1982).</li><li>Murphy, C., Langan-Fahey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+hydroxylated+metabolites+of+tamoxifen+that+control+the+proliferation+of+estrogen-responsive+T47D+breast+cancer+cells+in+vitro.">Structure-function relationships of hydroxylated metabolites of tamoxifen that control the proliferation of estrogen-responsive T47D breast cancer cells in vitro.</a> Molecular. 38 (5), 737-743 (1990).</li><li>Furr, B. J. A., Jordan, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacology+and+clinical+uses+of+tamoxifen.">The pharmacology and clinical uses of tamoxifen.</a> Pharmacology &amp; Therapeutics. 25 (2), 127-205 (1984).</li><li>Akerberg, A. a., Stewart, S., Stankunas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+Temporal+Control+of+Transgene+Expression+in+Zebrafish.">Spatial and Temporal Control of Transgene Expression in Zebrafish.</a> PloS One. 9 (3), e92217 (2014).</li><li>Hans, S., Kaslin, J., Freudenreich, D., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+recombination+in+zebrafish.">Temporally-controlled site-specific recombination in zebrafish.</a> PloS One. 4 (2), e4640 (2009).</li></ol>

The authors declare that they have no competing financial interests.

During embryogenesis and larval development, the embryonic zebrafish skin is composed of two epithelial layers, the superficial layer called periderm and a layer of basal keratinocytes that are attached to the underlying basement membrane19 (Figure 1C, a). The protocol presented here describes a straightforward method to conditionally induce preneoplastic-cell transformation in the superficial skin layer of zebrafish larvae, and allows for subsequent interaction analysis with innate immune cells by live imaging. The basic methodologies in our protocol follow well-established zebrafish techniques9&#8211;13, and our protocol can be easily adapted and modified.
The krt4 promoter5 used here drives KalTA4-ERT2 expression specifically in the outermost skin layer (Figure 1C, b). However, the modular MultiSite Gateway cloning strategy, used to generate the krt4:KalTA4-ERT2construct (Figure 1D, a), provides the possibility to clone any promoter of interest in front of KalTA4-ERT2 in a single cloning step. An adaptation of the method presented herein is therefore possible for most tissues during embryogenesis and larval stages. The availability of promoter sequences is hereby the limiting factor. Furthermore, almost any gene of interest cloned behind a UAS can be conditionally overexpressed at different developmental stages by use of the spatial and temporally controlled KalTA4-ERT2 expression. Therefore, our protocol can be adapted to perform conditional gene expression in any tissue of interest in zebrafish larvae, in a mosaic manner. One can also optimize the microscope setting in order to live image deeper tissues such as liver or pancreas.
We have described one application using the protocol here, similarly, one can overexpress other inflammation modulators using this protocol to study the regulation of inflammatory responses, as one can easily live image neutrophil and macrophage behavioral changes following the induction and arrest in the expression of a candidate inflammatory modulator. Furthermore, the adapted protocol would also be beneficial for those who want to trace cell movement and behavior change during different stages of tissue morphogenesis and organ formation and, finally live image the interaction of innate immune cell interactions with other specific cell lineages that can be targeted by the inducible KalTA4-ERT2/UAS expression system.
We used the pDestTol2CG vector from the zebrafish Tol2kit20&#8211;23 that contains a cmlc2:eGFP-pA cassette (Figure 1D, a) which allows subsequent selection of embryos by green fluorescent positive hearts as a selection marker20 that simplifies the F0 screening process. A stable insertion of the transposon flanked gene of interest into the genome is facilitated by co-injection of the plasmid DNA together with transposase mRNA. The preparation of plasmid DNA and the transposase mRNA for microinjection follows standard protocols. However, a successful integration of the construct into the genome depends on the Tol2-based transposition14. This highlights the particular attention to be paid to work on ice under sterile conditions to avoid contaminations and degradations by RNases and DNases. Microinjection into one-cell stage embryos is a robust and well-established technique for gain and loss of function studies in zebrafish9,10. Although a well-trained person can easily inject into the yolk of over 1,000 embryos in 1 hr, the injection into the blastodisc (Figure 1A, asterisk) requires more experience and training. Critical during this procedure is the handling of the needle. The needle has to be very thin to penetrate the cell without damage and therefore has to be broken only on its very tip. It is important to regularly control and measure the drop size during the injection to guarantee a uniform injection into each embryo. Carefully handled, the needle can be used to rotate the embryo. This is often needed to find the blastodisc and the optimal penetration angle.
The concentration of DNA and transposase mRNA used for injection almost certainly influences the expression efficiency. Higher doses can lead to an increased proportion of cells expressing the transgene but with higher concentrations of DNA (&#62;50 ng/&#956;l) and transposase mRNA (&#62;100 ng/&#956;l) also the potential for toxic effects amongst injected embryos does arise. We favor the use of 10 ng/&#956;l DNA and 20 ng/&#956;l transposase mRNA for injection, which corresponds to 50 pg of plasmid DNA and 100 pg of RNA per injected embryo. 100% of embryos injected with these concentrations do develop normally, and between 40-70% show eGFP-HRASG12V expression, after 4-OHT induction, depending on the injection efficiency into the single cell. Amongst positive embryos we normally find approximately 30% that have 1-10 clones, 40% that have 10-50 clones and 30% having more than 50 clones. Due to our research aims, we favor the use of embryos with fewer clones expressing eGFP-HRASG12V. We select embryos with 10-50 clones for our transient experiments. For the generation of transgenic founder fish, we recommend to only select embryos with both the eGFP positive cardiac marker and a large number of eGFP-HRASG12V positive clones in their epidermis.
(Z)-4-Hydroxytamoxifen undergoes a cis-trans (E-Z) interconversion when exposed to light24. It was found that the cis isomer (E) is 100x less anti-estrogenic than its trans counterpart25,26. Exposure to light therefore has to be avoided. The 4-OHT stock solution dissolved in ethanol can be stored at -20 &#176;C in the dark over a long time period. We recommend the use of a box to protect the Petri dishes from light during target gene induction within the incubator and transport. Although the area of imaging is very small and exposure of 4-OHT to UV light is reduced to a minimum, a constant exchange of induction solution every 2 hours while imaging over longer time periods is recommended to maintain maximum expression levels. 4-OHT effectively permeabilizes through the chorion and the outermost skin layers during zebrafish embryogenesis, therefore it can induce gene expression very rapidly. We can readily observe eGFP emission after 2 hr of induction and it has been reported that first signs of target-gene expression can be observed after 1 hour and plateau after 3 hr of treatment8. The differences might be due to the different promoter used in our study. As has been previously reported that 4-OHT treatment is dose dependent8,27, we chose to use the highest reported concentration of 5 &#956;M to achieve robust and reproducible expression levels in our transient approach. This should guarantee that all cells carrying the transgene will induce target gene expression.
It has to be taken into account that the transient approach presented here could lead to varying expression levels due to the possibility of multiple and random genome insertion events. Furthermore, the use of the transposase mRNA in the Tol2 transgenic background of Tg(UAS:eGFP-HRASG12V)io006 can lead to potential transpositions of the UAS transgene that could result in gene silencing or disruption. However, as the method presented here represents a transient approach, the pre-selection of embryos subsequent to injection is mandatory. Many labs have found that UAS sequences tend to be silenced in the transgenic offspring, hence injecting the pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP construct into Tg(UAS:eGFP-HRASG12V)io006 embryos might not be as efficient as injecting a UAS construct into Tg(krt4:KalTA4-ERT2;cmlc2:GFP)&#160;embryos. The use of the stable transgenic line Tg(krt4:KalTA4-ERT2) crossed with Tg(UAS:eGFP-HRASG12V)io006 will allow a precise monitoring of the on/off kinetics of the time-dosage dependent 4-OHT gene activation.
A critical step during the mounting of larvae is the transfer into the LMP agarose and onto the cover glass (Figure 1B). There is only a short time frame for liquid LMP agarose temperature that allows a safe transfer and orientation of the preselected larvae without harming the fish by either heat shock or hardening of the gel. The larvae should be orientated directly onto the cover glass to reduce the working distance for the subsequent microscopic analysis. As this can be a limiting factor during microscopy the choice of appropriate objectives is mandatory. Once mounted, the larva can be maintained from hours to days.
The conditionality of the model system presented herein allows for repeated transformation by 4-OHT activation of the transgene. The expression system depends on the presence of 4-OHT and therefore the KalTA4 controlled expression of a gene of interest is reversible (Figure 1D, b). In contrast to the Cre-ERT2/Lox system, which facilitates an irreversible genomic recombination event28, KalTA4-ERT2/UAS can be activated and deactivated upon addition or removal of 4-OHT and this potential for repeated induction is an advantage of the technique over the Cre-ERT2/Lox system. However, the requirement for constant levels of 4-OHT is a limitation if the experiment has to be prolonged from days to weeks.

그렇지 않으면 정상 상피 시트 내에서 발암 세포 자손의 전면에 자라 강하게의 미세 환경과의 상호 작용에 따라 달라집니다. 숙주 조직과의 상호 작용은 종양 전 셀이 상기 부푼 암으로 발전하는 클론 틈새를 확립 할 수 있는지의 주요 결정 요인이 될 가능성이 높다. 개발의 중요성에도 불구하고, 암의 진행이 초기 단계는 생체 내에서, 가장 일반적으로 사용되는 모델 시스템에 액세스 할 수 없습니다. 제브라 피쉬, 다니오 레 리오 (rerio)은, 때문에 개발 전반에 걸쳐 투명성, 수용성 약물의 유전자 조작에 대한 가능성과 접근성의 라이브 영상 검사에 대한 잘 확립 된 모델 생물이다. 상피 세포를 변환하는 과발현, 인간의 종양 유전자 HRAS G12V을 애벌레 제브라 피쉬 모델을 사용하여 최근의 연구는 숙주 세포가 특정 H 2 O 2 (으)로에서 신호를 유도 것을 보여 주었다선천성 면역 세포의 모집. 흥미롭게도, 모집 면역 세포, 호중구 및 대식 세포는 종양 전 세포 증식을 지원 2,3- 영양 역할을하는 것으로 하였다. 종양의 개시 및 진행의​​ 초기 사건뿐만 아니라 염증 반응의 기여를 이해하기 위하여, 하나는 이후 초기 시점에서 이러한 이벤트 이미지 할 수 있어야한다. 그러므로 시간적 및 조직 특이 적으로 세포 변환을 제어 할 필요가있다. 우리는 성공적으로 조건부 피부 특이 적 프로모터 각질 4 (krt4)의 제어하에 인간 종양 유전자 HRAS G12V을 표현하는 초파리 4에서 적응 된 GAL4 / UAS 시스템을 5 활용합니다. GAL4 전사 활성 즉, KalTA4 (6)의 수정 된 버전은, 상류 활성화 서열의 제어 (UAS하에 HRAS G12V의 발현을 가능하게). 조건부 전사 활성의 발현을 유도하기 위하여, 특별히 KalTA4 -4- Hydroxytamoxifen 결합 인간 에스트로겐 수용체 α의 돌연변이 리간드 결합 도메인 (ER의 T2) (7)에 융합 된 (4-OHT) 1. 4- OHT의 부재에서 ER-T2는 열 충격 단백질에 의해 세포질 바인딩된다. 4-OHT에 관심 8의 유전자를 제어 UAS의 KalTA4-ER T2의 핵 전위 이후 활성화 있도록, 단백질이 해리 열 충격 바인딩 (그림 1C을, B). 이러한 발현 시스템은 4 OHT의 존재에 의존 같이, 관심 유전자의 발현 조절 KalTA4 가역적이다. 비가역 재조합 이벤트 리드 치운다 ER의 T2 / 된 LOX 시스템과는 달리, KalTA4 ER-T2 의한 전사 활성화의 유도는 또한 4-OHT 제거함으로써 가역적이다. 다음 프로토콜여보세요<html> 제브라 피쉬 애벌레 피부 세포의 조건 변화와 형광 단백질 발현에 의한 선천성 면역 세포와의 상호 작용의 후속 모니터링 WS. 13 -이 프로토콜에서 사용되는 기본적인 방법은 제브라 피쉬 커뮤니티 (9) 내에서 일반적으로 사용되는 기술과 유사하다. 세대와 함께 유전자 변형 구조의 미세 주입으로 유전자 변형 라인의 조합 적 사용은 모자이크 방식으로 단일 세포를 변환하는 일시적인 접근 할 수 있습니다. 배아와 유충 지브라 피쉬 피부의 산소 투과도는 일 시간이다 고해상도 현미경으로 시간 경과 라이브 영상 검사를위한 가능성을 제기한다. 또한, 수용성 약물 접근성 미래 기전 연구 및 암 발생의 초기 단계의 이해로 이어질 수 생체 내에서 세포의 생물학적 이벤트의 감시를 허용 할 것이다. _content는 &quot;&gt;이 프로토콜은 모자이크 방식으로, 제브라 피쉬 유충에서 관심있는 조직 조건부 유전자 발현을 수행하도록 할 수있다. 하나는 또한 피부 이외의 화상 깊은 조직을 살 수 있도록 설정 현미경을 최적화 할 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="1104">
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			<td height="21" style="height:21px;width:316px;">Name of Material/Equipment</td>
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			<td style="width:124px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
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			<td height="21" style="height:21px;">(Z)-4-Hydroxytamoxifen&#160;</td>
			<td>Sigma Aldrich</td>
			<td>H7904</td>
			<td>dilute in ethanol and store in the dark at -20&#176;C</td>
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			<td height="21" style="height:21px;">20x Objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>20x/0,5 Ph2 &#8734;/0,17</td>
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		<tr height="21">
			<td height="21" style="height:21px;">40x Objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>40x/1,2W Korr &#8734;/0,14-0,18</td>
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		<tr height="21">
			<td height="21" style="height:21px;">63x Objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>63x/1.4 Oil Ph3 &#8734;/0,17</td>
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		<tr height="21">
			<td height="21" style="height:21px;">BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 510 META</td>
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			<td height="21" style="height:21px;">Cover glass</td>
			<td>VWR</td>
			<td>631-0171</td>
			<td>Diameter 25mm</td>
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			<td height="21" style="height:21px;">Dimethylpolysiloxane</td>
			<td>Sigma Aldrich</td>
			<td>DMPSV</td>
			<td></td>
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			<td height="21" style="height:21px;">Dow Corning high-vacuum silicone grease&#160;</td>
			<td>Sigma Aldrich</td>
			<td>MKBL4135V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming/Brown P-97 Micropipette Puller</td>
			<td>Sutter Instruments Co.</td>
			<td>P-97</td>
			<td>Program: heat 530, pull 200, velocity 80 and time 150</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent stereoscope&#160;</td>
			<td>Leica</td>
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			<td>M205 FA</td>
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			<td height="21" style="height:21px;">Kwik-Fill Brosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Microinjection mold TU-1</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader tip</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Axiovert 200</td>
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			<td height="21" style="height:21px;">mMESSAGE mMACHINE Kit</td>
			<td>Ambion</td>
			<td>AM1348</td>
			<td></td>
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			<td height="21" style="height:21px;">Nuclease-free water</td>
			<td>Invitrogen</td>
			<td>AM9937</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Pneumatic pico pump</td>
			<td>World Precision Instruments&#160;</td>
			<td>PV820&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pneumatic pico pump</td>
			<td>Warner Instruments</td>
			<td>PLI-90A</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP</td>
			<td></td>
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			<td>Dr. Thomas Ramezani/Yi Feng Lab/The University of Edinburgh</td>
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			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit</td>
			<td>Quiagen</td>
			<td>27106</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Rhodamine B isothiocyanate-Dextran</td>
			<td>Sigma Aldrich</td>
			<td>R8881</td>
			<td>10mg/&#956;l in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscope</td>
			<td>Leica</td>
			<td></td>
			<td>M80</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tg(lysC:dsRed2)nz50Tg&#160;</td>
			<td></td>
			<td></td>
			<td>BMC developmental biology 7, 42, doi:10.1186/1471-213X-7-42 (2007)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tg(UAS:eGFP-HRASG12V)io006</td>
			<td></td>
			<td></td>
			<td>PloS one 5 (12), e15170, doi:10.1371/journal.pone.0015170 (2010)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Tricaine/MS-222</td>
			<td>Sigma Aldrich</td>
			<td>A5040</td>
			<td></td>
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			<td height="21" style="height:21px;">UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>16500-100</td>
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		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Low Melting Point Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>16520-050</td>
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			<td height="21" style="height:21px;">UltraPure Phenol:Chloroform:Isoamyl Alcohol</td>
			<td>Invitrogen</td>
			<td>15593-031</td>
			<td>25:24:1, v/v</td>
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live imaging of innate immune and preneoplastic cell interactions using an inducible gal4/uas expression system in larval zebrafish skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Zygotes of a cross between Tg(UAS:eGFP-HRASG12V)io006 and Tg(lysC:dsRed2)nz50Tg were collected 10-15 min post fertilization (Figure 1A) to obtain 1 cell-stage embryos. After injection the embryos were raised until 3 dpf, before induction with 4-OHT and mounting for live imaging (Figure 1B). 2-6 hr post induction the mounted embryos were placed either under an inverted fluorescent microscope (Figure 2A) or an inverted confocal microscope (Figure 2B-C) and imaged for 2-3 hr with 1.5 min intervals. Preneoplastic superficial skin cells can be identified by their more rounded morphology compared to normal keratinocytes, as well as the green fluorescent emission of the membrane localized eGFP-HRASG12V. Areas of imaging were selected for co-localization of preneoplastic skin cells and innate immune cells (Figure 2A arrowhead). Due to the fast migration process of the neutrophils the time-lapse intervals were chosen to be between 1-1.5 min with Z stack step sizes of 0.7-1.7 &#956;m to obtain maximum intensity projections (Figure 2, Video 1). There is a wide range of behaviors that can be observed while live imaging: neutrophils migrate next to and under preneoplastic cells (Figure 2C, Video 1), form direct contacts to cells (Figure 2B and C), remove remnants of preneoplastic cells that undergo apoptosis (Video 1), or actively engulf parts of preneoplastic cells (not shown). To capture the full spectrum of interactions, it is advisable to follow their behaviors by time-lapse live imaging.
<img alt="Figure 1" src="/files/ftp_upload/52107/52107fig1highres.jpg" width="700px" /> 
Figure 1: Schematic for conditional skin cell transformation in zebrafish larvae. (A) An image of a zygote of Tg(UAS:eGFP-HRASG12V;lysC:dsRed2) zebrafish at 10 min post fertilization (mpf). The fertilized egg is surrounded by the chorion (arrowhead). The site for injection is the blastodisc (asterisk), formed by cytoplasmic streams from the yolk (arrow) that defines the animal pole of the embryo. (B) Mounting of zebrafish larvae. 60 mm Petri dish with an 18-20 mm hole (arrowhead) that is sealed by a 25 mm cover glass. The zebrafish larvae are immobilized and mounted onto the cover glass by 1% LMP agarose. 0.3x Danieau solution (arrow) containing 5 &#956;M 4-OHT is used to cover the LMP agarose. (C) 4-OHT induced transformations of superficial skin cells. (a) Schematic cross sectional profile of larval skin. The embryonic and larval skin is composed of two epithelial cell layers, a superficial cell layer and a basal cell layer of keratinocytes that are connected to the underlying basement membrane (BM). (b) Transient expression system to induce a preneoplastic cell clone amongst superficial skin cells. KalTA4-ERT2 is exclusively expressed in the outermost skin layer driven by the krt4 promoter (yellow). Mosaic expression of KalTA4-ERT2 activates UAS controlled (blue) eGFP-HRASG12V expression in superficial cells, leading to a clone of preneoplastic cells (green). (D) Schematic of the transient expression system. (a) Tol2 cassette of the pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP expression vector used transiently. (b) Conditional induction of the transcriptional activator expression. KalTA4 is fused to the mutant ligand-binding domain of the human estrogen receptor &#945; (ERT2) which binds specifically 4-Hydroxytamoxifen (4-OHT). In the absence of 4-OHT the ERT2 is bound in the cytoplasm by heat-shock proteins (Hsp90). Upon 4-OHT binding Hsp90 dissociates, allowing a nuclear translocation of KalTA4-ERT2 and subsequent activation of the UAS controlled eGFP-HRASG12V expression. Scale bar A = 500 &#956;m. <a href="/files/ftp_upload/52107/52107fig1highres.jpg" target="_blank">Please click here to view a larger version of the figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52107/52107fig2highres.jpg" width="700px" /> 
Figure 2: Live imaging of preneoplastic and innate immune cell interaction. (A-C) Images of a 4 dpf Tg(UAS:eGFP-HRASG12V;lysC:dsRed2) embryo, which has been injected with pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP and transposase mRNA at one cell stage. Images were taken after 6 hours of 4-OHT induction. (A) Lateral view of the tailfin region at 6 hours post treatment, showing patches of eGFP-HRASG12V expressing skin cells (arrow), and lysC:dsRed2+ neutrophils (arrowhead). (B-C) Representative images taken from a confocal time-lapse movie, showing neutrophil (red) interactions with eGFP-HRASG12V expressing skin cells (green). (B) A preneoplastic cell forms a short filopodial extension with the contacting neutrophil (arrowhead). (C) Neutrophils (red) is in close contact to a clone of preneoplastic skin cells (green). Scale bar in A = 100 &#956;m; B/C = 50 &#956;m. <a href="/files/ftp_upload/52107/52107fig2highres.jpg" target="_blank">Please click here to view a larger version of the figure.</a>
<a href="https://www.jove.com/files/ftp_upload/52107/Video_1.mov" target="_blank">Video 1</a>: Time-lapse live imaging of preneoplastic and innate immune cell interaction. Time-lapse of a 4 dpf Tg(UAS:eGFP-HRASG12V;lysC:dsRed2) embryo, injected with pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP and transposase mRNA at one cell stage. Confocal time-lapse movie from 6-9 hours after 4-OHT induction showing lysC:dsRed2+ neutrophil (red) interactions with eGFP-HRASG12V expressing skin cells (green). Neutrophils migrate next to and under preneoplastic cells and remove remnants of a preneoplastic cell that undergoes apoptosis. Time-lapse intervals of 1.5 min over 3 hr with Z stack step sizes of 1.3 &#956;m.

Watch this Scientific Journal Video about Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin at JoVE.com

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

애벌레 Zebrafish의 피부에 유도 GAL4 / UAS 식 시스템을 사용하여 선천성 면역과 종양 전 세포 상호 작용의 라이브 영상

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

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11.9K 조회수.  The University of Edinburgh. Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

비디오: Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

수질 흉선 상피 세포의 증식, 분화 및 무차별 유전자 발현을 지원하는 3D의 Organotypic 공동 문화 모델

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

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Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

살아있는 Zebrafish의 배아에서 세포 이하 구조를 이미징

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Photoconvertible PSmOrange 시스템을 사용하여 GFP 유전자 변형 Zebrafish의 세포를 추적

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

분석<em&gt; 생체</em&gt; 셀 마이그레이션 Zebrafish의 배아에서 세포 이식 및 시간 경과 이미징을 사용하여

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

생체 외에서 라이브 셀 이미징의 접합 자 양극 화와 애기 thaliana 패터 닝 태아 난 재배

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Mouse Secondary Palate Fusion의 라이브 이미징

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

조사 Trypanosoma cruzi 운동 비보 를 동물 모델로 서 애벌레 Zebrafish의 설립

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Live Cell Imaging of Chromosome Segregation During Mitosis

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled by multiple mechanisms that include the Spindle Assembly Checkpoint (SAC). The SAC is part of a complex feedback system that is responsible for prevention of a cell progress through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. Chromosomal lagging and abnormal chromosome segregation is an indicator of dysfunctional cell cycle control checkpoints and can be used to measure the genomic stability of dividing cells. Deregulation of the SAC can result in the transformation of a normal cell into a malignant cell through the accumulation of errors during chromosomal segregation. Implementation of the SAC and the formation of the kinetochore complex are tightly regulated by interactions between kinases and phosphatase such as Protein Phosphatase 2A (PP2A). This protocol describes live cell imaging of lagging chromosomes in mouse embryonic fibroblasts isolated from mice that had a knockout of the PP2A-B56&#947; regulatory subunit. This method overcomes the shortcomings of other cell cycle control imaging techniques such as flow cytometry or immunocytochemistry that only provide a snapshot of a cell cytokinesis status, instead of a dynamic spatiotemporal visualization of chromosomes during mitosis.

This protocol describes an easy and convenient method to label and visualize live chromosomes in mitotic cells using Histone2B-GFP BacMam 2.0 label and a spinning disc confocal microscopy system.

유사 분열 동안 염색체 분리의 라이브 셀 이미징

Chromosomes must be reliably and uniformly segregated into daughter cells during mitotic cell division. Fidelity of chromosomal segregation is controlled ...

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

스폰지 팁 전극 A 소설 원추형을 사용 하 여 애벌레 Zebrafish에 기록 하는 Electroretinogram

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For ...