Name: light-induced gfp expression in zebrafish embryos using the optogenetic tael/c120 system
Uploaded: 2021-08-19T14:30:00.000+00:00
Duration: 5 min 28 s
Description: Watch this Scientific Journal Video about Light-Induced GFP Expression in Zebrafish Embryos using the Optogenetic TAEL/C120 System at JoVE.com

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>

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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.
Several factors can affect induction levels. Most critical are the wavelength and power of activating light. In this protocol, 465 nm LED lights delivered at 1.5 W/cm2 were used. Shorter and longer wavelengths (purple and green light, respectively) and lower light power do not activate expression effectively (data not shown). On the other hand, more light power increases the risk of photodamaging the embryos. Thus, for successful use of the TAEL system, activating light must be (1) in the blue range of the visible light spectrum and (2) at sufficient power to balance effective activation of TAEL with reduced photodamage risk. Effective light power may vary depending on experimental conditions and so may need to be empirically determined. Care should also be taken to protect embryos from ambient light, containing some amount of blue light, before activation. It has been found that TAEL/C120-dependent expression can be induced by broad-spectrum ambient light, albeit at much lower levels compared to blue light only (Table 1).
While GFP expression can first be detected by qPCR after 30 min of illumination, expression levels are not steady. Still, they continue to rise until reaching a peak at 3 h of light treatment, after which these high expression levels are maintained for up to 6 h. These results suggest that, in addition to wavelength and light power, TAEL/C120-induced expression levels are also dependent on illumination duration, at least until the system reaches a maximum or saturation state. In contrast to these qPCR results, we do not qualitatively observe appreciable GFP fluorescence until after 3 h of illumination, and fluorescence intensity continues to increase for up to 6 h of illumination. The discrepancy between the qPCR and fluorescence intensity observations is likely explained by the additional time needed for GFP synthesis, folding, and maturation-factors that are likely to vary depending on the gene of interest. Therefore, some optimization of illumination duration may be needed depending on the application.
This protocol presented the most straightforward method for activating the TAEL/C120 system using a blue-light LED panel to illuminate zebrafish embryos globally. This approach has the advantages of both ease of use and cost-effectiveness. However, light activation can also be spatially controlled if needed. It was previously demonstrated that TAEL-induced expression could be spatially restricted using multiple modalities to deliver user-defined, spatially patterned blue light5. Additional spatial specificity can be achieved using tissue-specific promoters to regulate the expression of the TAEL transcriptional activator6.
Compared to drug- or heat shock-inducible expression systems, optogenetic expression systems potentially offer better spatial and temporal control overexpression by using light as the inducing agent. In addition to TAEL/C120, other light-activated transcriptional systems have been developed12,13,14,15. However, TAEL/C120 may be especially well-suited for use in zebrafish (and potentially other multicellular systems) for several reasons. First, the TAEL transcriptional activator functions as a homodimer, which simplifies the number of required components. In addition, LOV domain-containing proteins such as TAEL require a flavin chromophore for light absorption16. This cofactor is endogenously present within animal cells, removing the need to add an exogenous chromophore as with other systems. Finally, activated TAEL is predicted to have a relatively short half-life of approximately 30 s in the absence of blue light8, enabling more precise on/off control. However, this short half-life also means that long-term or chronic expression would require long-term illumination of embryos, which may or may not be desirable depending on the circumstances.
In summary, this protocol demonstrates that the TAEL/C120 system is a blue light-activated gene expression system that is easy to use, possesses fast and responsive kinetics, and is particularly well-suited for in vivo applications.

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 h, and 6 h post-activation (Figure 2B and Table 1). Compared to control&#160;sibling embryos kept in the dark, induction of GFP expression was detected as soon as 30 min after the blue light exposure. Levels of GFP expression then continued to increase up to 6 h post-activation steadily.
GFP induction was also qualitatively assessed by examining the fluorescence intensity at the same time points post-activation (Figure 2C-F). GFP fluorescence above background levels was first observed at 3 h post-activation and became noticeably brighter at 6 h post-activation. In contrast, control embryos for all time points that were kept in the dark did not exhibit any appreciable GFP fluorescence (Figure 2G-J).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62818/62818fig01.jpg" /> 
Figure 1: Schematic of TAEL/C120 function and experimental design. (A) The TAEL/C120 system consists of a transcriptional activator called TAEL fused to a nuclear localization signal (TAEL-N) and a TAEL-responsive regulatory element called C120 coupled to a cFos basal promoter (C120F) driving expression of a gene of interest. TAEL-dependent transcription is active in the presence of blue light but not in the dark. NLS, nuclear localization signal. (B) In this protocol, a transgenic line expresses TAEL-N ubiquitously (Tg(ubb:TAEL-N)) is crossed to a C120-driven GFP reporter line (Tg(C120F:GFP)) to produce double transgenic embryos. Starting at 24 hpf, the embryos are exposed to activating blue light for various durations up to 6 h-illustrations created with a web-based science illustration tool (see Table of Materials). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62818/62818fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62818/62818fig02.jpg" /> 
Figure 2: Representative results of light-activated gene expression with TAEL/C120. (A) A typical light activation setup includes a blue LED light source placed in an incubator. Petri dishes containing zebrafish embryos are positioned relative to the light source so that the received power of light is approximately 1.5 mW/cm2 (dotted line). Petri dish lids are removed during light activation to minimize light scattering. (B) Quantification of GFP mRNA levels by qRT-PCR at the indicated time points after activation with blue light. Data is presented as GFP fold induction relative to sibling control embryos kept in the dark. Dots represent biological replicates (clutches). Solid horizontal bars represent the mean. Error bars, standard deviation. *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001. One-way ANOVA determined p values. (C-J) Representative images showing GFP fluorescence intensity of embryos exposed to blue light (C-F) or kept in the dark (G-J). Fluorescent images (green) have been merged with corresponding brightfield images (grayscale): scale bars, 500 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62818/62818fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="3">GFP Fold Induction 
			Blue light (465 nm)</td>
			<td colspan="3">GFP Fold Induction 
			Ambient light</td>
			<td></td>
		</tr>
		<tr>
			<td>Time Post-illumination</td>
			<td>Mean</td>
			<td>Upper limit</td>
			<td>Lower limit</td>
			<td>Mean</td>
			<td>Upper limit</td>
			<td>Lower limit</td>
			<td>p value</td>
		</tr>
		<tr>
			<td>30 min</td>
			<td>5.363121044</td>
			<td>8.15857193</td>
			<td>3.525502696</td>
			<td>0.661534683</td>
			<td>1.097728244</td>
			<td>0.398667102</td>
			<td>0.005291</td>
		</tr>
		<tr>
			<td>1 h</td>
			<td>23.44</td>
			<td>46.35044081</td>
			<td>11.85160592</td>
			<td>2.638682529</td>
			<td>4.368971424</td>
			<td>1.593657823</td>
			<td>0.011145</td>
		</tr>
		<tr>
			<td>3 h</td>
			<td>48.09177693</td>
			<td>71.99347359</td>
			<td>32.12539822</td>
			<td>8.280376038</td>
			<td>24.86850106</td>
			<td>2.757087255</td>
			<td>0.059959</td>
		</tr>
		<tr>
			<td>6 h</td>
			<td>131.4637117</td>
			<td>163.4891638</td>
			<td>105.7116392</td>
			<td>16.66536842</td>
			<td>27.94334716</td>
			<td>9.939199585</td>
			<td>0.003102</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of TAEL/C120-induced expression by blue and ambient light. Fold induction of GFP mRNA levels after exposure to activating blue light (465 nm) or ambient light for the indicated amount of time, normalized to control sibling embryos kept in the dark. mRNA levels were quantified by qRT-PCR. Data is reported as average fold induction +/- upper and lower limits. p values were determined by multiple t-tests. n = 3 clutches for all time points.

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 ...

light-induced-gfp-expression-zebrafish-embryos-using-optogenetic

Nanyang Technological University

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

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 ...

Analysis of Zebrafish Cardiac Contraction with Virtual Reality Interaction

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

Bioengineering

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 ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

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.remotexs.ntu.edu.sg/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

Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos

Source: Silic, M. R., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57330">Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors.</a> J. Vis. Exp. (2018)
In this video, we demonstrate a procedure to generate a stable zebrafish transgenic line constitutively expressing a fluorescent voltage indicator protein following the co-injection of the Tol2 transposon system comprising the transposase mRNA and Tol2 transposon construct encoding the fluorescent voltage indicator.

Source: Silic, M. R., et al.  Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors. J. Vis. Exp. (2018)
In this video, we ...

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

Summary

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Chapters in this video

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

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

Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Optogenetic Signaling Activation in Zebrafish Embryos

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

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

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

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

Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos