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>

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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.

The overgrowth of a preneoplastic cell progeny within an otherwise normal epithelial sheet strongly depends on an interaction with its microenvironment. The interaction with its host tissue is likely to be the major determinant of whether a preneoplastic cell is able to establish a clonal niche to further develop into a full blown cancer. Despite its importance in development, this initial phase of cancer progression is inaccessible in most commonly used model systems, in vivo.
The zebrafish, Danio rerio, is a well-established model organism for live imaging studies because of its transparency throughout development, the possibility for genetic manipulation, and accessibility for water-soluble drugs. Recent studies using a larval zebrafish model, overexpressing the human oncogene HRASG12V to transform epithelial cells, showed that host cell derived signals, in particular H2O2 lead to the recruitment of innate immune cells. Interestingly, the recruited immune cells, neutrophils and macrophages, were shown to have a trophic role in supporting preneoplastic cell proliferation2,3.
In order to understand the early events of tumor initiation and progression as well as the contribution of an inflammatory response, one needs to be able to image these events from the earliest time point onwards. Therefore it is necessary to control cell transformations in a temporal and tissue specific manner. We utilize the Gal4/UAS system that has been successfully adapted from Drosophila4 to conditionally express the human oncogene HRASG12V under the control of the skin specific promoter keratin 4 (krt4)5. The modified version of the transcriptional activator Gal4, namely KalTA46, enables expression of HRASG12V under the control of the Upstream Activating Sequence (UAS). To conditionally induce the transcriptional activator expression, KalTA4 has been fused to the mutant ligand-binding domain of the human estrogen receptor &#945; (ERT2)7 which binds specifically 4-Hydroxytamoxifen (4-OHT)1. In the absence of 4-OHT the ERT2 is bound in the cytoplasm by heat-shock proteins. Upon 4-OHT binding the heat-shock proteins dissociate, allowing a nuclear translocation of KalTA4-ERT2 and subsequent activation of the UAS controlled gene of interest8 (Figure 1C, b). As this expression system depends on the presence of 4-OHT, the KalTA4 controlled expression of a gene of interest is reversible. In contrast to the Cre-ERT2/Lox system, which leads to an irreversible recombination event, the induction of transcriptional activation by KalTA4-ERT2 is reversible by addition or removal of 4-OHT.
The following protocol allows a conditional transformation of skin cells in zebrafish larvae and a subsequent monitoring of the interaction with innate immune cells by fluorescent protein expression. The basic methodologies employed in this protocol are similar to some commonly used techniques within the zebrafish community9&#8211;13.
The generation and combinatory use of transgenic lines together with the microinjection of transgenic constructs enables a transient approach to only transform single cells in a mosaic manner. The oxygen permeability of the embryonic and larval zebrafish skin raises the possibility for time-lapse live imaging studies by high-resolution microscopy from hours to days. Furthermore, its accessibility to water-soluble drugs will allow future mechanistic studies and the monitoring of cell biological events in vivo that could lead to a better understanding of the earliest stages of cancer development.
This 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 other than skin.

<table><tbody><tr><td>(Z)-4-Hydroxytamoxifen</td><td>Sigma Aldrich</td><td>H7904</td><td>dilute in ethanol and store in the dark at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>20X Objective</td><td>Zeiss</td><td></td><td>20X/0.5 Ph2 &amp;infin;/0.17</td></tr><tr><td>40X Objective</td><td>Zeiss</td><td></td><td>40X/1.2W Korr &amp;infin;/0.14-0.18</td></tr><tr><td>63X Objective</td><td>Zeiss</td><td></td><td>63X/1.4 Oil Ph3 &amp;infin;/0.17</td></tr><tr><td>BamHI</td><td>New England Biolabs</td><td>R0136S</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td></td><td>LSM 510 META</td></tr><tr><td>Cover glass</td><td>VWR</td><td>631-0171</td><td>Diameter 25 mm</td></tr><tr><td>Dimethylpolysiloxane</td><td>Sigma Aldrich</td><td>DMPSV</td><td></td></tr><tr><td>Dow Corning high-vacuum silicone grease&amp;nbsp;</td><td>Sigma Aldrich</td><td>MKBL4135V</td><td></td></tr><tr><td>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></tr><tr><td>Fluorescent stereoscope&amp;nbsp;</td><td>Leica</td><td></td><td>M205 FA</td></tr><tr><td>Kwik-Fill Brosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>Microinjection mold TU-1</td><td>Adaptive Science Tools</td><td>TU-1</td><td></td></tr><tr><td>Microloader tip</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td></td><td>Axiovert 200</td></tr><tr><td>mMESSAGE mMACHINE Kit</td><td>Ambion</td><td>AM1348</td><td></td></tr><tr><td>Nuclease-free water</td><td>Invitrogen</td><td>AM9937</td><td></td></tr><tr><td>Pneumatic pico pump</td><td>World Precision Instruments&amp;nbsp;</td><td>PV820&amp;nbsp;</td><td></td></tr><tr><td>Pneumatic pico pump</td><td>Warner Instruments</td><td>PLI-90A</td><td></td></tr><tr><td>pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP</td><td></td><td></td><td>Dr. Thomas Ramezani/Yi Feng Lab/The University of Edinburgh</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Quiagen</td><td>27106</td><td></td></tr><tr><td>Rhodamine B isothiocyanate-Dextran</td><td>Sigma Aldrich</td><td>R8881</td><td>10 mg/&amp;mu;l in water</td></tr><tr><td>Stereoscope</td><td>Leica</td><td></td><td>M80</td></tr><tr><td>&lt;em&gt;Tg(lysC:dsRed2)nz50Tg&lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;BMC Developmental Biology&lt;/em&gt; &lt;strong&gt;7&lt;/strong&gt;, 42, doi:10.1186/1471-213X-7-42 (2007)</td></tr><tr><td>&lt;em&gt;Tg(UAS:eGFP-HRASG12V)io006&lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;PloS One&lt;/em&gt; &lt;strong&gt;5&lt;/strong&gt; (12), e15170, doi:10.1371/journal.pone.0015170 (2010)</td></tr><tr><td>Tricaine/MS-222</td><td>Sigma Aldrich</td><td>A5040</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500-100</td><td></td></tr><tr><td>UltraPure Low Melting Point Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16520-050</td><td></td></tr><tr><td>UltraPure Phenol : Chloroform : Isoamyl Alcohol</td><td>Invitrogen</td><td>15593-031</td><td>25:24:1, v/v</td></tr></tbody></table>

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

Obrazowanie na żywo interakcji wrodzonej odporności i komórek przednowotworowych przy użyciu indukowalnego systemu ekspresji Gal4/UAS w skórze larw danio pręgowanego | Biologia rozwoju | Wideo

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.

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 Views.  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.

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

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Immunology and Infection

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...

Neuroscience

Elucidating Microglia Behavior in Zebrafish Optic Tectum Development

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

Author Spotlight: Using Zebrafish to Explore Microglia Migration During Brain Development

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and ...

Photoconversion-Based Leukocyte Tracking in Bacterial-Infected Transgenic Zebrafish

Source: Harvie, E. A. et al., <a href="https://app.jove.com/v/52788">Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates the tracking of leukocyte recruitment using transgenic zebrafish larvae expressing photoconvertible green fluorescent protein. Pre-injecting red fluorescent dye-labeled bacteria into the otic vesicle and inducing photoconversion enables observation of leukocytes&#39; migration, phagocytosis, and dispersal throughout the body, providing valuable insights into immune response dynamics.

Śledzenie leukocytów w oparciu o fotokonwersję u transgenicznego danio pręgowanego zakażonego bakteriami

1. Preparing Microinjection Needles

	Prepare thin wall glass capillary injection needles (1.0 OD/0.75 ID) using a micropipette puller device with the ...

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

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Śledzenie leukocytów w oparciu o fotokonwersję u transgenicznego danio pręgowanego zakażonego bakteriami