We thank Stefan Materna and members of the Woo and Materna labs for helpful suggestions and comments on this protocol. We thank Anna Reade, Kevin Gardner, and Laura Motta-Mena for valuable discussion and insights while developing this protocol. This work was supported by grants from the National Institutes of Health (NIH; R03 DK106358) and the University of California Cancer Research Coordinating Committee (CRN-20-636896) to S.W.

This study was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) of the University of California Merced.
1. Zebrafish crossing and embryo collection
<ol>
	<li>Maintain separate transgenic zebrafish lines containing either the TAEL transcriptional activator or the C120-controlled reporter gene to minimize spurious activation.</li>
	<li>Cross 6-8 adult zebrafish from each line using standard methods9 to produce double transgenic embryos that contain both the TAEL and C120 components (Figure 1B). 
	NOTE: Alternatively, both components can be expressed transiently through microinjection of mRNA or plasmid DNA using standard methods10.</li>
	<li>Collect embryos in Petri dishes containing egg water (60 &#181;g/mL Instant Ocean sea salt dissolved in distilled water), with approximately 30 embryos per condition to be tested.</li>
	<li>Place the dishes in a lightproof box or cover with aluminum foil to minimize unintended activation by ambient light (see Table 1).</li>
</ol>
2. Global light induction
<ol>
	<li>Use a blue-light (465 nm) LED panel to deliver activating blue light to several embryos at once.</li>
	<li>Position the LED panel relative to the Petri dishes containing embryos so that the actual power of light received by the embryos is approximately 1.5 mW/cm2 as measured by a light power and energy meter (Figure 2A).</li>
	<li>Pulse the light at intervals of 1 h on/1 h off using a timer relay if illuminating for more than 3 h to reduce the risk of photodamage to the TAEL transcriptional activator5,8. 
	NOTE: The exact duration of illumination may need to be optimized for specific applications. In this protocol, examples of illumination duration of 30 min, 1 h, 3 h, and 6 h are provided.</li>
	<li>Remove Petri dish lids to minimize light scattering from condensation.</li>
	<li>Place Petri dishes containing control embryos in a lightproof box or cover with aluminum foil for dark controls.</li>
</ol>
3. Quantitative assessment of induction by qRT-PCR
<ol>
	<li>Remove embryos from illumination after the desired activation duration.</li>
	<li>Extract total RNA from 30-50 light-activated and 30-50 dark embryos using an RNA isolation kit following the kit&#39;s instructions.
	<ol>
		<li>Transfer embryos to a 1.5 mL microcentrifuge tube and remove excess egg water. Add lysis buffer and homogenize the embryos with a plastic pestle.</li>
		<li>Transfer the lysate to a kit-provided column and continue with the kit&#39;s instructions. Immediately proceed to step 3.3 or store purified RNA at -20 &#176;C to -80 &#176;C.</li>
	</ol>
	</li>
	<li>Use 1 &#181;g total RNA for cDNA synthesis using a cDNA synthesis kit following the kit&#39;s instructions.
	<ol>
		<li>Take a thin-walled 0.2 mL PCR tube, add 1 &#181;g of RNA to 4 &#181;L of 5x cDNA reaction master mix (containing optimized buffer, oligo-dT and random primers, and dNTPs), 1 &#181;L of 20x reverse transcriptase solution, and nuclease-free water to a total volume of 20 &#181;L.</li>
		<li>Place the tube in a thermocycler programmed as follows: 22 &#176;C for 10 min, 42 &#176;C for 30 min, 85 &#176;C for 5 min, and then hold at 4 &#176;C. Immediately proceed to step 3.4 or store cDNA at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare qPCR reactions containing SYBR green enzyme master mix, 5-fold diluted cDNA (step 3.3), and 325 nM of each primer for GFP. 
	NOTE: Primers used in this protocol as follows. GFP forward: 5&#39;-ACGACGGCAACTACAAGCACC-3&#39;; GFP reverse: 5&#39;-GTCCTCCTTGAAGTCGATGC-3&#39;; ef1a forward 5&#39;-CACGGTGACAACATGCTGGAG-3&#39;; ef1a reverse: 5&#39;-CAAGAAGAGTAGTACCGCTAGCAT-3&#39;).</li>
	<li>Carry out qPCR reactions in a real-time PCR machine. 
	NOTE: PCR program: initial activation at 95 &#176;C for 10 min, followed by 40 cycles of 30 s at 95 &#176;C, 30 s at 60 &#176;C, and 1 min at 72 &#176;C.</li>
	<li>Perform a melt curve analysis once the PCR is completed to determine the reaction specificity. Perform three technical replicates for each sample.</li>
	<li>Calculate light-activated induction as fold change relative to embryos kept in the dark using the 2-&#916;&#916;Ct method11. Statistical significance can be determined with a statistics software package.</li>
</ol>
4. Qualitative assessment of induction by fluorescence microscopy
<ol>
	<li>Remove the embryos from illumination after the desired duration of activation.</li>
	<li>Immobilize embryos for imaging in 3% methylcellulose containing 0.01% tricaine in glass depression slides.</li>
	<li>Acquire fluorescence and brightfield images on a fluorescent stereomicroscope connected to a digital camera using standard GFP filter settings. Use identical image acquisition settings for all samples.</li>
	<li>Merge brightfield and fluorescence images after the acquisition with image processing software.</li>
</ol>

<ol>
	<li>Knopf, F., 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=Dually+inducible+TetON+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetON systems for tissue-specific conditional gene expression in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (46), 19933-19938 (2010).</li><li>Halloran, M. C., 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=Laser-induced+gene+expression+in+specific+cells+of+transgenic+zebrafish.">Laser-induced gene expression in specific cells of transgenic zebrafish.</a> Development. 127 (9), 1953-1960 (2000).</li><li>Hesselson, D., Anderson, R. M., Beinat, M., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+populations+of+quiescent+and+proliferative+pancreatic+beta-cells+identified+by+HOTcre+mediated+labeling.">Distinct populations of quiescent and proliferative pancreatic beta-cells identified by HOTcre mediated labeling.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (35), 14896-14901 (2009).</li><li>Tischer, D., Weiner, O. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cell+signalling+with+optogenetic+tools.">Illuminating cell signalling with optogenetic tools.</a> Nature Reviews. Molecular Cell Biology. 15 (8), 551-558 (2014).</li><li>Reade, A., 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=TAEL:+a+zebrafish-optimized+optogenetic+gene+expression+system+with+fine+spatial+and+temporal+control.">TAEL: a zebrafish-optimized optogenetic gene expression system with fine spatial and temporal control.</a> Development. 144 (2), 345-355 (2017).</li><li>LaBelle, 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=TAEL+2.0:+An+improved+optogenetic+expression+system+for+zebrafish.">TAEL 2.0: An improved optogenetic expression system for zebrafish.</a> Zebrafish. 18 (1), 20-28 (2021).</li><li>Rivera-Cancel, G., Motta-Mena, L. B., Gardner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+natural+and+artificial+DNA+substrates+for+light-activated+LOV-HTH+transcription+factor+EL222.">Identification of natural and artificial DNA substrates for light-activated LOV-HTH transcription factor EL222.</a> Biochemistry. 51 (50), 10024-10034 (2012).</li><li>Motta-Mena, L. 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=An+optogenetic+gene+expression+system+with+rapid+activation+and+deactivation+kinetics.">An optogenetic gene expression system with rapid activation and deactivation kinetics.</a> Nature Chemical Biology. 10 (3), 196-202 (2014).</li><li>Avdesh, A., 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> Journal of visualized experiments: JoVE. (69), e4196 (2012).</li><li>Holder, N., Xu, Q. <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+DNA,+RNA,+and+Protein+into+the+Fertilized+Zebrafish+Egg+for+Analysis+of+Gene+Function.">Microinjection of DNA, RNA, and Protein into the Fertilized Zebrafish Egg for Analysis of Gene Function.</a> Molecular Embryology. , 487-490 (1999).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Wang, X., Chen, X., Yang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+control+of+gene+expression+by+a+light-switchable+transgene+system.">Spatiotemporal control of gene expression by a light-switchable transgene system.</a> Nature Methods. 9 (3), 266-269 (2012).</li><li>Mruk, K., Ciepla, P., Piza, P. A., Alnaqib, M. A., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+cell+ablation+in+zebrafish+using+optogenetic+transcriptional+control.">Targeted cell ablation in zebrafish using optogenetic transcriptional control.</a> Development. 147 (12), (2020).</li><li>Liu, H., Gomez, G., Lin, S., Lin, S., Lin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+control+of+transcription+in+zebrafish.">Optogenetic control of transcription in zebrafish.</a> PloS One. 7 (11), 50738 (2012).</li><li>Shimizu-Sato, S., Huq, E., Tepperman, J. M., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+light-switchable+gene+promoter+system.">A light-switchable gene promoter system.</a> Nature Biotechnology. 20 (10), 1041-1044 (2002).</li><li>Krueger, D., 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=Principles+and+applications+of+optogenetics+in+developmental+biology.">Principles and applications of optogenetics in developmental biology.</a> Development. 146 (20), (2019).</li></ol>

No conflicts of interest were declared.

This protocol describes the use of the optogenetic TAEL/C120 system to achieve blue light-inducible gene expression. This system consists of a transcriptional activator, TAEL, that dimerizes upon illumination with blue light and activates transcription of a gene of interest downstream of a C120 regulatory element. Induced expression of a GFP reporter can be detected after as little as 30 min of light exposure, suggesting that this approach possesses relatively fast and responsive kinetics.
Sev...

Inducible gene expression systems help control the amount, timing, and location of gene expression. However, achieving exact spatial and temporal control in multicellular organisms has been challenging. Temporal control is most commonly achieved by adding small-molecule compounds1 or activation of heat shock promoters2. Still, both approaches are vulnerable to issues of timing, induction strength, and off-target stress responses. Spatial control is mainly achieved by the use of tissue-specific promoters3, but this approach requires a suitable promoter or regulatory element, which are not always available, and it is not conducive to sub-tissue level induction.
In contrast to such conventional approaches, light-activated optogenetic transcriptional activators have the potential for finer spatial and temporal control of gene expression4. The blue light-responsive TAEL/C120 system was developed and optimized for use in zebrafish embryos5,6. This system is based on an endogenous light-activated transcription factor from the bacterium E. litoralis7,8. The TAEL/C120 system consists of a transcriptional activator called TAEL that contains a Kal-TA4 transactivation domain, a blue light-responsive LOV (light-oxygen-voltage sensing) domain, and a helix-turn-helix (HTH) DNA-binding domain5. When illuminated, the LOV domains undergo a conformational change that allows two TAEL molecules to dimerize, bind to a TAEL-responsive C120 promoter, and initiate transcription of a downstream gene of interest5,8. The TAEL/C120 system exhibits rapid and robust induction with minimal toxicity, and it can be activated by several different light delivery modalities. Recently, improvements to the TAEL/C120 system were made by adding a nuclear localization signal to TAEL (TAEL-N) and by coupling the C120 regulatory element to a cFos basal promoter (C120F) (Figure 1A). These modifications improved induction levels by more than 15-fold6.
In this protocol, a simple LED panel is used to activate the TAEL/C120 system and induce the ubiquitous expression of a reporter gene, GFP. Expression induction can be monitored qualitatively by observing fluorescence intensity or quantitatively by measuring transcript levels using quantitative real-time PCR (qRT-PCR). This protocol will demonstrate the TAEL/C120 system as a versatile, easy-to-use tool that enables robust regulation of gene expression in vivo.

<table><tbody><tr><td>BioRender web-based science illustration tool</td><td>BioRender</td><td></td><td>https://biorender.com/</td></tr><tr><td>Color CCD digital camera</td><td>Lumenara</td><td>755-107</td><td></td></tr><tr><td>Compact Power and Energy Meter Console, Digital 4&quot; LCD</td><td>Thorlabs</td><td>PM100D</td><td></td></tr><tr><td>Excitation filter, 545 nm</td><td>Olympus</td><td>ET545/25x</td><td></td></tr><tr><td>illustra RNAspin Mini kit</td><td>GE Healthcare</td><td>95017-491</td><td></td></tr><tr><td>Instant Ocean Sea Salt</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>MARS AQUA Dimmable 165 W LED Aquarium light (blue and white)</td><td>Amazon</td><td>B017GWDF7E</td><td></td></tr><tr><td>Methylcellulose</td><td>Sigma-Aldrich</td><td>M7140</td><td></td></tr><tr><td>NEARPOW Programmable digital timer switch</td><td>Amazon</td><td>B01G6O28NA</td><td></td></tr><tr><td>PerfeCTa SYBR green fast mix</td><td>Quantabio</td><td>101414-286</td><td></td></tr><tr><td>Photoshop image procesing software</td><td>Adobe</td><td></td><td></td></tr><tr><td>Prism graphing and statistics software</td><td>GraphPad</td><td></td><td></td></tr><tr><td>qScript XLT cDNA SuperMix</td><td>Quantabio</td><td>10142-786</td><td></td></tr><tr><td>QuantStudio 3 Real-Time PCR System</td><td>Applied Biosystems</td><td>A28137</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td><td>Sigma-Aldrich</td><td>E10521</td><td></td></tr><tr><td>X-Cite 120 Fluorescence LED light source</td><td>Excelitas</td><td>010-00326R</td><td>Discontinued. It has been replaced with the X-Cite mini+</td></tr></tbody></table>

light-induced gfp expression in zebrafish embryos using the optogenetic tael/c120 system

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control over gene expression timing, location, and amplitude using light as the inducing agent. In this protocol, an optogenetic expression system is used to achieve light-inducible gene expression in zebrafish embryos. This system relies on an engineered transcription factor called TAEL based on a naturally occurring light-activated transcription factor from the bacterium E. litoralis. When illuminated with blue light, TAEL dimerizes, binds to its cognate regulatory element called C120, and activates transcription. This protocol uses transgenic zebrafish embryos that express the TAEL transcription factor under the control of the ubiquitous ubb promoter. At the same time, the C120 regulatory element drives the expression of a fluorescent reporter gene (GFP). Using a simple LED panel to deliver activating blue light, induction of GFP expression can first be detected after 30 min of illumination and reaches a peak of more than 130-fold induction after 3 h of light treatment. Expression induction can be assessed by quantitative real-time PCR (qRT-PCR) and by fluorescence microscopy. This method is a versatile and easy-to-use approach for optogenetic gene expression.

For this demonstration, a C120-responsive GFP reporter line (Tg(C120F:GFP)ucm107)) was crossed with a transgenic line that expresses TAEL-N ubiquitously from the ubiquitin b (ubb) promoter (Tg(ubb:TAEL-N)ucm113)) to produce double transgenic embryos containing both elements. 24 h post-fertilization, the embryos were exposed to activating the blue light, pulsed at a frequency of 1 h on/1 h off. Induction of GFP expression was quantified by qRT-PCR at 30 min, 1 h, 3...

Watch this Scientific Journal Video about Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System at JoVE.com

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Inducible gene expression systems are an invaluable tool for studying biological processes. Optogenetic expression systems can provide precise control ...

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Research

JoVE Journal

Developmental Biology

2.9K Views.  University of California. Optogenetics is a powerful tool with wide-ranging applications. This protocol demonstrates how to achieve light-inducible gene expression in zebrafish embryos using the blue light-responsive TAEL/C120 system.

Video: Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

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.

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 ...

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific regions of developing embryos, including those of C. elegans, chick, Xenopus, zebrafish, and mouse 1. In zebrafish, in vivo electroporation has been shown to have excellent spatial and temporal resolution for the delivery of these reagents 2-7. The temporal resolution of this method is important because it allows for incorporation of these reagents at specific stages in development. Furthermore, because expression from electroporated vectors occurs within 6 hours 7, this method is more timely than transgenic approaches. While the spatial resolution can be extremely precise when targeting a single cell 2, 6, it is often preferable to incorporate reagents into a specific cell population within a tissue or structure. When targeting multiple cells, in vivo electroporation is efficient for delivery to a specific region of the embryo; however, particularly within the developing nervous system, it is difficult to target specific cell types solely through spatially discrete electroporation. Alternatively, enhancer trap transgenic lines offer excellent cell type-specific expression of transgenes 8. Here we describe an approach that combines transgenic Gal4-based enhancer trap lines 8 with spatially discrete in vivo electroporation 7, 9 to specifically target developing neurons of the zebrafish olfactory bulb. The Et(zic4:Gal4TA4,UAS:mCherry)hzm5 (formerly GA80_9) enhancer trap line previously described 8, displays targeted transgenic expression of mCherry mediated by a zebrafish optimized Gal4 (KalTA4) transcriptional activator in multiple regions of the developing brain including hindbrain, cerebellum, forebrain, and the olfactory bulb. To target GFP expression specifically to the olfactory bulb, a plasmid with the coding sequence of GFP under control of multiple Gal4 binding sites (UAS) was electroporated into the anterior end of the forebrain at 24-28 hours post-fertilization (hpf). Although this method incorporates plasmid DNA into multiple regions of the forebrain, GFP expression is only induced in cells transgenically expressing the KalTA4 transcription factor. Thus, by using the GA080_9 transgenic line, this approach led to GFP expression exclusively in the developing olfactory bulb. GFP expressing cells targeted through this approach showed typical axonal projections, as previously described for mitral cells of the olfactory bulb 10. This method could also be used for targeted delivery of other reagents including short-hairpin RNA interference expression plasmids, which would provide a method for spatially and temporally discrete loss-of-function analysis.

Targeting Olfactory Bulb Neurons Using Combined In Vivo Electroporation and Gal4-Based Enhancer Trap Zebrafish Lines

The temporal and spatial resolution of genetic manipulations determines the spectrum of biological phenomena that they can perturb. Here we use temporally and spatially discrete in vivo electroporation, combined with transgenic lines of zebrafish, to induce expression of a GFP transgene specifically in neurons of the developing olfactory bulb.

In vivo electroporation is a powerful method for delivering DNA expression plasmids, RNAi reagents, and morpholino anti-sense oligonucleotides to specific ...

Neuroscience

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 ...

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function.

Optogenetic techniques have made it possible to study the contribution of specific neurons to behavior. We describe a method in larval zebrafish for activating single somatosensory neurons expressing a channelrhodopsin variant (ChEF) with a diode-pumped solid state (DPSS) laser and recording the elicited behaviors with a high-speed video camera.

Larval  zebrafish are emerging as a model for describing the development and function  of simple neural circuits. Due to their  external fertilization, ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Biology

Investigating Development with Optogenetic Signaling Manipulation in Zebrafish

Optogenetic Signaling Activation in Zebrafish Embryos

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally manipulate signaling are required to understand how signaling is interpreted in these wide-ranging contexts. Molecular optogenetic&#160;tools can provide reversible, tunable manipulations of signaling pathway activity with a high degree of spatiotemporal control and have been applied in vitro, ex vivo, and in vivo. These tools couple light-responsive protein domains, such as the blue light homodimerizing light-oxygen-voltage sensing (LOV) domain, with signaling effectors to confer light-dependent experimental control over signaling. This protocol provides practical guidelines for using the LOV-based bone morphogenetic protein (BMP) and Nodal signaling activators bOpto-BMP and bOpto-Nodal in the optically accessible early zebrafish embryo. It describes two control experiments: A quick phenotype assay to determine appropriate experimental conditions, and an immunofluorescence assay to directly assess signaling. Together, these control experiments can help establish a pipeline for using optogenetic tools in early zebrafish embryos. These strategies provide a powerful platform to investigate the roles of signaling in development, health, and physiology.

Author Spotlight: Manipulating Signaling in Zebrafish Embryos to Decode Cell Fate Decisions 

Optogenetic manipulation of signaling pathways can be a powerful strategy to investigate how signaling is decoded in development, regeneration, homeostasis, and disease. This protocol provides practical guidelines for using light-oxygen-voltage sensing domain-based Nodal and bone morphogenic protein (BMP) signaling activators in the early zebrafish embryo.

Signaling pathways orchestrate fundamental biological processes, including development, regeneration, homeostasis, and disease. Methods to experimentally ...

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between these features may be interrogated by controlling the phase transition of a disease-associated protein in a vulnerable cell type, although this experimental approach has been limited so far. Here, we describe a protocol to induce phase transition of the RNA/DNA-binding protein TDP-43 in spinal motor neurons of zebrafish larvae for modeling cytoplasmic aggregation of TDP-43 occurring in degenerating motor neurons in amyotrophic lateral sclerosis (ALS). We describe a bacterial artificial chromosome (BAC)-based genetic method to deliver an optogenetic TDP-43 variant selectively to spinal motor neurons of zebrafish. The high translucency of zebrafish larvae allows for the phase transition of the optogenetic TDP-43 in the spinal motor neurons by a simple external illumination using a light-emitting diode (LED) against unrestrained fish. We also present a basic workflow of live imaging of the zebrafish spinal motor neurons and image analysis with freely available Fiji/ImageJ software to characterize responses of the optogenetic TDP-43 to the light illumination. This protocol enables the characterization of TDP-43 phase transition and aggregate formation in an ALS-vulnerable cellular environment, which should facilitate an investigation of its cellular and behavioral consequences.

We describe a protocol to induce phase transition of TAR DNA-binding protein 43 (TDP-43) by light in the spinal motor neurons using zebrafish as a model.

Abnormal protein aggregation and selective neuronal vulnerability are two major hallmarks of neurodegenerative diseases. Causal relationships between ...

Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Source: Asakawa, K. et al. <a href="https://www.jove.com/v/62932">Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae.</a> J. Vis. Exp. (2022)
In this video, we describe a phase separation assay, wherein intracellular proteins comprising intrinsically disordered regions fused to a photosensitive oligomerization domain are induced via blue-light exposure to self-associate into membrane-less liquid-like condensates.

Source: Asakawa, K. et al. Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (2022)
In this video, we ...

JoVE Encyclopedia of Experiments

Biological Techniques

Assay Techniques

Cell-based assays

Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses

Source: Palanca A. M. S., et al. <a href="https://www.jove.com/v/50184/" target="_blank">Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato</a>. J. Vis. Exp. (2013).
This video describes optogenetic techniques by activating genetically modified somatosensory neurons and recording elicited behavioral response with a high-speed video camera.

Source: Palanca A. M. S., et al. Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato. J. Vis. Exp. (2013).
This video describes ...

Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System

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Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae

Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses