This work was supported by the National Research, Development and Innovation Office (NKFIH) from the National Research, Development and Innovation Fund (NKFIA); Grant Agreement: NVKP_16-1-2016-0003, EFOP-3.6.3-VEKOP-16-2017-00008 project co-financed by the European Union, and the Thematic Excellence Program NKFIH-831-10/2019 of Szent Istv&#225;n University, awarded by Ministry for Innovation and Technology.

The Animal Protocol was approved under the Hungarian Animal Welfare Law and all studies were completed before the treated individuals would have reached the free feeding stage.
1. Embryo harvest and treatment
<ol>
	<li>Maintain Tg(vtg1:mCherry) zebrafish at 25.5 &#177; 0.5 &#176;C, pH = 7 &#177; 0.2, conductivity between 525 &#177; 50 &#956;S/m, oxygen level &#8805;80% of saturation, and 14 h light and 10 h dark cycle.</li>
	<li>Fill the mating tanks with system water and set up the fish for mating the afternoon before harvesting eggs.</li>
	<li>Place the male and female fish into the tank and separate them with the help of a divider.</li>
	<li>Remove the divider from the tanks as the light switches on next morning. Check the mating tanks for eggs every 15&#8211;20 min.</li>
	<li>Harvest all embryos using a tea strainer or densely woven fine mesh and combine them into one large Petri dish with E3 buffer (5 mM sodium chloride, 0.17 mM potassium chloride, 0.33 mM calcium chloride, 0.33 mM magnesium sulfate) or clear system water.</li>
	<li>Place the embryos in the incubator set to 25.5 &#177; 0.5 &#176;C.</li>
	<li>After 1&#8211;1.5 h, remove and discard unfertilized or inadequately dividing eggs with a plastic transfer pipet under a dissecting microscope. 
	NOTE: Unfertilized eggs are opaque; fertilized eggs are transparent. To start the treatment in a later stage of development, pay attention to the normal development of the individuals being treated.</li>
	<li>Place the selected embryos in the treatment vessels (e.g., in Petri dishes or tissue culture plates) that have already been labeled and filled with different concentrations of the test substance.</li>
	<li>Incubate the embryos at 25.5 &#177; 0.5 &#176;C until the end of the experiment. 
	NOTE: Embryos can react with significant individual sensitivity to various estrogenic compounds, so it is recommended to use at least 15 embryos in at least three repeat treatments in order to evaluate the experiment properly. When selecting the container, keep in mind that the development of an embryo requires at least 200 &#181;L of water26. In this experiment the embryos were incubated until 120 hpf at 25.5 &#177; 0.5 &#176;C.</li>
	<li>Refresh the test solution if it is neccesary to maintain the treatment concentration. Be careful when changing the test solution in order to avoid damaging the embryos. 
	NOTE: It is also possible to investigate mortality or sublethal symptoms during the experiment. The concentrations used should remain as stable as possible to obtain reliable results. This can be easily achieved by refreshing the test solutions. The frequency of refreshing may vary depending on the test substance. Therefore, it is advisable to check the concentration of the test substance by analytical measurements to determine the frequency of the solution updates.</li>
</ol>
2. Larvae preparation for photography
<ol>
	<li>Prepare 4% methylcellulose with MS-222 (tricaine-methane-sulfonate) beforehand.
	<ol>
		<li>To do so, add 4 mg/mL MS-222 to 100 mL double distilled water to a final concentration of 0.168 mg/mL, and bring the solution to 4 &#730;C. Then add 4 g of methylcellulose. Stir it with a magnetic stirrer, then leave it in 4 &#730;C overnight.</li>
		<li>Next day, stir it again and the solution should be ready to use. If the methylcellulose is not completely dissolved put it back at 4 &#730;C and wait a few more hours.</li>
	</ol>
	</li>
	<li>Place 5 day old larvae into a 5 cm Petri dish per treatment group with a Pasteur pipette.</li>
	<li>Remove the treatment solution from the larvae with a plastic pipette, then fill the Petri dish with 2 mL of 0.02% MS-222 anesthetic solution. 
	NOTE: Anesthesia is usually effective in less than a minute. The larvae are anesthetized if they do not swim away in response to touch. An overdose of MS-222 can kill the larvae.</li>
	<li>Fill each square of a specially designed 10 cm Petri dish with 4% methylcellulose with MS-222 (Figure 3). 
	NOTE: Glue two 1.5 x 1.5 cm square areas at the bottom of the Petri dish using plastic sheets (1 mm height, 5 mm wide). Twenty larvae can be placed next to each other in a square. Instead of a specially designed Petri dish, another low edge container can be used to fix the position of the embryos.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60462/60462fig03.jpg" /> 
Figure 3: A 10 cm Petri dish with glued 1.5 x 1.5 cm wide, 1 mm thick plastic sheet squares for larvae preparation for photography. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60462/60462fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Transfer the anesthetized larvae to methylcellulose with a little water in one of the two squares. From the first square transfer the larvae into the second square. 
	NOTE: With the help of this transfer, the 4% methylcellulose used for photography will not be diluted and the larvae will not rotate during imaging.</li>
	<li>In the second square, rotate and orient larvae to their left side and gently press them down to the bottom of the cellulose with a microloader pipette tip cut up to 2 cm. 
	NOTE: Do not use other pipette tips because they cause injury to the larvae.</li>
</ol>
3. Microscopy
NOTE: Photography does not kill the animals. Animals can be awakened by removing them from the methylcellulose and placing them in fresh system water or treatment solution, so the same individual can be examined several times during the treatment.
<ol>
	<li>In order to evaluate the signal of the expressed reporter, image the embryos with the same view and settings. 
	NOTE: See step 2.6 for optimal embryo positioning. The photos in the manuscript were taken under the conditions described. Bright field: 60x magnification, 6 ms exposition, Iris 100%, Gain:1.1, fluorescence photos: 60x magnification, 300 ms exposition, Iris: 100%, Gain:1, mCherry filter, fluorescent light source: mercury metal halide bulb. For photo taking a fluorescent stereomicroscope, dedicated camera, and software for microscope were used.</li>
	<li>Place the Petri dish on the stage of the microscope.</li>
	<li>Focus on the liver of the embryo and capture a bright field image using the associated software.</li>
	<li>Switch the microscope to the mCherry filter and take a fluorescent image of the liver under fluorescent light using the associated software. 
	NOTE: Perform all the fluorescence steps in the dark. Do not change the magnification, the focus, or the position of the embryo between capturing the bright field and the fluorescent images, as this will assist in the analysis of the images.</li>
	<li>Repeat steps 3.3 and 3.4 until all the embryos in the experiment have been imaged.</li>
</ol>
4. Determining integrated density
NOTE: One of the best indicators for comparing fluorescent signal strength is the integrated density value (i.e., the product of the area and mean gray value). One of the easiest ways to determine integrated density is to use the ImageJ program27. The program is available on the internet and can be installed on the computer.
<ol>
	<li>Open ImageJ then upload the fluorescent image to be analyzed by either dragging and droping the image or clicking on File|Open.</li>
	<li>Click on Image|Color|Split Channels to split the image made by the fluorescent filter according to the RGB color chart.</li>
	<li>Work with the red channel image spectrum, close the other channels.</li>
	<li>Designate a similarly sized elliptical area in the image so false signals do not interfere with the evaluation. Using the Oval tools, draw an ellipse over the highlighted liver area as accurately as possible.</li>
	<li>If the signal is weak use light microscopy images (i.e., the bright field pair of the fluorescent image) to determine the location of the liver.</li>
	<li>Click on Analyze|Measure to determine the signal strength and the size of the affected area. The integrated density value is automatically calculated by the software (IntDen column in the chart).</li>
	<li>Continue the analysis by repeating steps 4.1&#8211;4.6 until all the fluorescent images of all embryos in the treatment group are analyzed.</li>
	<li>Save the data and then analyze the integrated density values. 
	NOTE: Always select the same area size in the images during analysis. The size of the area to be analyzed depends on the magnification, image resolution, other settings for shooting, etc. The analysis can be accelerated or made more accurate by using personal macros for analysis. For example, macros can be used to ensure that the selected area is always the same in the examined images. Detailed descriptions for creating macros that allow the best image analysis according to particular photo settings are found on the ImageJ website.</li>
</ol>

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The authors have nothing to disclose.

The use of biomonitors/bioindicators for estrogenic effects has been spreading in toxicological studies. In vivo models play an outstanding role, because unlike in vitro tests, they not only provide information about the response of a cell or a receptor, but also allow the investigation of complex processes in the organism. Several transgenic lines for studying estrogenic effects have been produced from zebrafish, one of which Tg(vtg1:mCherry) was used for these studies. The method described here illustrates a p...

There is a significant number of endocrine disrupting compounds (EDC) that are among the most hazardous substances in our environment. These are mainly estrogenic compounds that contaminate water from natural resources. The chemical diversity of the substances belonging to the group makes testing for their presence difficult, as different analytical methods are required for their detection. Based on their chemical structure it is very difficult to determine whether a substance is actually able to act as an estrogen. In addition, these substances are never present in a pure form in the environment, so their effects may be affected by other compounds, too1. This problem can be solved by effect-detecting methods, such as the use of biomonitor/bioindicator organisms that show estrogenic effects2,3,4,5.
Recently, a variety of cell line6 and yeast-based test systems2,3 have been developed to detect estrogenic effects. However, these are generally only able to detect the binding of the substance to the estrogen receptor2,3. In addition, they are unable to model complex physiological processes in the organism, or to detect hormone-sensitive phases of life stages; thus, they often lead to false results.
It is known that certain genes react sensitively to estrogen in living organisms7. The detection of gene products by molecular biology methods is also possible at the protein or mRNA level8,9, but usually involves animal sacrifice. Animal protection laws have become stricter, and there is a growing demand for alternative test systems that minimize the number and suffering of animals used in experiments or the replacement of the animal model with another model system10. With the discovery of fluorescent proteins and the creation of biomarker lines, transgenic technologies provide a good alternative11. With these lines, the activation of an estrogen-sensitive gene can be tested in vivo.
Among vertebrates, the potential of fish in environmental risk assessment is outstanding. They offer many advantages over mammalian models: being aquatic organisms, they are able to absorb pollutants through their entire body, produce a large number of offspring, and some of their species are characterized by short generation time. Their endocrine system and physiological processes show great similarities with other vertebrates and even with mammals, including humans12.
Several genes for the detection of estrogenic effects in fish are also known. The most important are the estrogen receptors aromatase-b, choriogenin-H, and vitellogenin (vtg)7,13. Recently, several estrogen-producing biosensor lines have also been created from fish models used in the laboratory, such as from zebrafish (Danio rerio)4,5,14,15,16,17. The main advantage of zebrafish in creating biosensor lines is the transparent body of the embryos and larvae, because the fluorescent reporter signal can then be easily studied in vivo without sacrificing the animal10. In addition to animal protection, it is also a valuable feature as it allows for studying the reaction of the same individual at different times of the treatment18.
These experiments use a vitellogenin reporter transgenic zebrafish line15. The transgene construct used for the development of Tg(vtg1:mCherry) has a long (3.4 kbp) natural vitellogenin-1 promoter. The estrogen receptor (ER) is an enhancer protein activated by ligands that is a representative of the steroid/nuclear receptor superfamily. ER binds to specific DNA sequences called estrogen response elements (EREs) with high affinity and transactivates gene expression in response to estradiol and other estrogenic substances, so the more ERE in the promoter causes a stronger response19. There are 17 ERE sites in the promoter region of the Tg(vtg1:mCherry) transgene construct and they are expected to mimic the expression of the native vtg gene15. There is a continuous expression of the fluorescent signal in sexually matured females. However, in males and embryo the expression in the liver is only visible upon treatment with estrogenic substances (Figure 1).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60462/60462fig01.jpg" /> 
Figure 1: Red fluorescent signal in the liver of vtg1:mCherry transgenic adult zebrafish and 5 dpf embryos, following 17-&#223;-estradiol (E2) induction.&#160;In female and in male treated with E2 (25 &#181;g/L exposure time:48hrs) strong fluorescence of the liver is visible even through the pigmented skin. No fluorescent signal is visible in untreated male (A). Following E2 induction (50 &#181;g/L exposure time: 0-120 hpf), a red fluorescent signal in the liver of 5 dpf embryos can also be observed, which is not visible in control embryos (B). While the fluorescent signal is continuously present in adult females, primarily males and embryos of the line are suitable for detecting estrogenic effects. (BF: bright field, mCherry: red fluorescent filter view, single plain images, Scale bar A: 5mm, scale bar B: 250 &#181;m) <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60462/60462fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Similar to the endogenous vitellogenin, the mCherry reporter is only expressed in the liver. Because vitellogenin is only produced in the presence of estrogen, there is no fluorescent signal in the controls. Because the expression is only in the liver, the evaluation of the results is much easier15.
The sensitivity and usability of this line&#39;s embryos have been investigated on various estrogenic compound mixtures and also on environmental samples15,20, and in most cases dose-response relationships were documented (Figure 2). However, in the case of highly toxic, mainly hepatotoxic, substances (e.g., zearalenone), only a very weak fluorescent signal may be visible in the liver of treated embryos and the maximum intensity fluorescent signal caused can be reached within a very small concentration range, which makes it difficult to establish dose-effect relationships20.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60462/60462fig02.jpg" /> 
Figure 2: Dose-response diagram (A) and fluorescent images (mCherry) of the liver (B) exposed to 17-&#945;-ethynilestradiol (EE2), in 5 dpf vtg1:mCherry larvae.&#160;Results are expressed as integrated density generated from the signal strength and the size of the affected area (&#177;SEM, n = 60). 100% refers to the observed maximum. Fluorescent signal intensity increased gradually with concentration. Scale bar = 250 &#181;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60462/60462fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
There are several estrogenic substances present in the environment, such as 17-&#946;-estradiol (environmental concentration: 0.1&#8211;5.1 ng/L)21, 17-&#945;-ethynylestradiol (environmental concentration: 0.16&#8211;0.2 &#181;g/L)22, zearalenone (environmental concentration: 0.095&#8211;0.22 &#181;g/L)23, bisphenol-A (environmental concentration: 0.45&#8211;17.2 mg/L)24. When testing these substances in a pure active form with the help of mCherry transgenic embryos, the lowest observed effect concentrations (LOEC) for fluorescent sign detection were 100 ng/L for 17-&#223;-estradiol, 1 ng/L for 17-&#945;-ethynilestradiol, 100 ng/L for zearalenone, and 1 mg/L for bisphenol-A (96&#8211;120 hpf treatment), which is very close to or within the range of environmental concentrations of the substances15. The Tg(vtg1:mCherry) transgenic line can help detect estrogenicity in wastewater samples after direct exposure. The line is as sensitive as the commonly used yeast estrogen test, the bioluminiscent yeast estrogen (BLYES) assay15. With the help of this line, the protective effects of beta-cyclodextrins against zearalenone-induced toxicity has been confirmed using chemical mixtures20.
In a recent report, the in vivo use of the transgenic line was demonstrated with the help of two estrogenic zearalenone (ZEA) metabolites, &#945;- and &#946;-zearalenol (&#945;-ZOL and &#946;-ZOL)25. The protocol baseline is appropriate to study the estrogenic effects of several compounds or environmental samples on Tg(vtg1:mCherry) embryos.

<table><tbody><tr><td>24 well tissue culture plate</td><td>Jet Biofil</td><td>TCP011024</td><td></td></tr><tr><td>Calcium-chloride (CaCl2)</td><td>Reanal Laborvegyszer Ltd.</td><td>16383-0-27-39</td><td></td></tr><tr><td>GraphPad Prism 6.01 software</td><td>GraphPad Software Inc.</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>National Institutes of Health, USA</td><td></td><td>Public access software, downloadable from: http://imagej.nih.gov/</td></tr><tr><td>Leica Application Suite X calibrated software</td><td>Leica Microsystems GmbH.</td><td></td><td>We used the softver described in the experiments, but any photographic software complies with the tests</td></tr><tr><td>Leica M205 FA stereomicroscope, Leica DFC 7000T camera</td><td>Leica Microsystems GmbH.</td><td></td><td>We used the equipments described in the experiments, but any fluorescent stereomicroscope is suitable for the tests</td></tr><tr><td>Magnesium-sulphate (MgSO4)</td><td>Reanal Laborvegyszer Ltd.</td><td>20342-0-27-38</td><td></td></tr><tr><td>mCherry filter</td><td>Leica Microsystems GmbH.</td><td></td><td></td></tr><tr><td>Mehyl-cellulose</td><td>Sigma Aldrich Ltd.</td><td>274429</td><td></td></tr><tr><td>Microloader pipette tip</td><td>Eppendorf GmbH.</td><td>5242956003</td><td></td></tr><tr><td>Pasteur pipette</td><td>VWR International LLC.</td><td>612-1684</td><td></td></tr><tr><td>Petri-dish</td><td>Jet Biofil</td><td>TCD000060</td><td></td></tr><tr><td>Potassium-chloride (KCl)</td><td>Reanal Laborvegyszer Ltd.</td><td>18050-0-01-33</td><td></td></tr><tr><td>Sodium-chloride (NaCl)</td><td>Reanal Laborvegyszer Ltd.</td><td>24640-0-01-38</td><td></td></tr><tr><td>Tricane-methanesulfonate (MS-222)</td><td>Sigma Aldrich Ltd.</td><td>E10521</td><td></td></tr></tbody></table>

using tg(vtg1:mcherry) zebrafish embryos to test the estrogenic effects of endocrine disrupting compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult due to their chemical diversity; therefore, increasingly more effect-detecting methods are used, such as estrogenic effect-sensitive biomonitor/bioindicator organisms. These biomonitoring organisms include several fish models. This protocol covers the use of zebrafish Tg(vtg1: mCherry) transgenic line as a biomonitoring organism, including the propagation of fish and the treatment of embryos, with an emphasis on the detection, documentation, and evaluation of fluorescent signals induced by EDC. The goal of the work is the demonstration of the use of the Tg(vtg1: mCherry) transgenic line embryos to detect estrogenic effects. This work documents the use of transgenic zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects by testing two estrogenic substances, &#945;- and &#946;-zearalenol. The described protocol is only a basis for designing assays; the test method can be varied according to the test endpoints and the samples. Moreover, it can be combined with other assay methods, thereby facilitating the future use of the transgenic line.

In the experiment presented in this manuscript, the effects of two estrogenic substances were tested at five concentrations starting at fertilization for 5 days on Tg(vtg1:mCherry) zebrafish embryos. We investigated whether fluorescent signals appeared in the liver of fish by the end of the exposure time because of the substances and whether there were differences in the estrogenicity of the two substances. Results were evaluated on the basis of the fluorescent images and integrated density values. In general, b...

Watch this Scientific Journal Video about Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds at JoVE.com

Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

There are many endocrine disrupting compounds (EDC) in the environment, especially estrogenic substances. The detection of these substances is difficult ...

using-tg-vtg1mcherry-zebrafish-embryos-to-test-estrogenic-effects

Universidad San Sebastian

Research

JoVE Journal

Environment

6.2K Views.  Szent István University. Present here is a detailed protocol for the use of zebrafish embryos Tg(vtg1: mCherry) for the detection of estrogenic effects. The protocol covers the propagation of the fish and treatment of embryos, and emphasizes the detection, documentation, and the evaluation of fluorescent signals induced by endocrine disrupting compounds (EDC).

Video: Using TgVtg1:mcherry Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Developmental Biology

Development of automated imaging and analysis for zebrafish chemical screens. 

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK pathway in zebrafish embryos. The zebrafish embryo is an ideal system for in vivo high-content chemical screens. The 1-day old embryo is approximately 1mm in diameter and can be easily arrayed into 96-well plates, a standard format for high throughput screening. During the first day of development, embryos are transparent with most of the major organs present, thus enabling visualization of tissue formation during embryogenesis. The complete automation of zebrafish chemical screens is still a challenge, however, particularly in the development of automated image acquisition and analysis. We previously generated a transgenic reporter line that expresses green fluorescent protein (GFP) under the control of FGF activity and demonstrated their utility in chemical screens 1. To establish methodology for high throughput whole organism screens, we developed a system for automated imaging and analysis of zebrafish embryos at 24-48 hours post fertilization (hpf) in 96-well plates 2. In this video we highlight the procedures for arraying transgenic embryos into multiwell plates at 24hpf and the addition of a small molecule (BCI) that hyperactivates FGF signaling 3. The plates are incubated for 6 hours followed by the addition of tricaine to anesthetize larvae prior to automated imaging on a Molecular Devices ImageXpress Ultra laser scanning confocal HCS reader. Images are processed by Definiens Developer software using a Cognition Network Technology algorithm that we developed to detect and quantify expression of GFP in the heads of transgenic embryos. In this example we highlight the ability of the algorithm to measure dose-dependent effects of BCI on GFP reporter gene expression in treated embryos.

Development of automated imaging and analysis for zebrafish chemical screens.

We report the development of a system for automated imaging and analysis of zebrafish transgenic embryos in multiwell plates. This demonstrates the ability to measure dose dependent effects of a small molecule, BCI, on Fibroblast Growth Factor reporter gene expression and provide technology for establishing high-throughput zebrafish chemical screens.

We demonstrate the application of image-based high-content screening (HCS) methodology to identify small molecules that can modulate the FGF/RAS/MAPK ...

Biology

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

Immunology and Infection

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. Despite a broad genetic heterogeneity, germline gain-of-function mutations in key regulators of the RAS-MAPK pathway underlie the majority of the cases, and, thanks to advanced sequencing techniques, potentially pathogenic variants affecting the RAS-MAPK pathway continue to be identified. Functional validation of the pathogenicity of these variants, essential for accurate diagnosis, requires fast and reliable protocols, preferably in vivo. Given the scarcity of effective treatments in early childhood, such protocols, especially if scalable in cost-effective animal models, can be instrumental in offering a preclinical ground for drug repositioning/repurposing. 
Here we describe step-by-step the protocol for rapid generation of transient RASopathy models in zebrafish embryos and direct inspection of live disease-associated ERK activity changes occurring already during gastrulation through real-time multispectral F&#246;rster resonance energy transfer (FRET) imaging. The protocol uses a transgenic ERK reporter recently established and integrated with the hardware of commercial microscopes. We provide an example application for Noonan syndrome (NS) zebrafish models obtained by expression of the Shp2D61G. We describe a straightforward method that enables registration of ERK signal change in the NS fish model before and after pharmacological signal modulation by available low-dose MEK inhibitors. We detail how to generate, retrieve, and assess ratiometric FRET signals from multispectral acquisitions before and after treatment and how to cross-validate the results via classical immunofluorescence on whole embryos at early stages. We then describe how, via examining standard morphometric parameters, to query late changes in embryo shape, indicative of a resulting impairment of gastrulation, in the same embryos whose ERK activity is assessed by live FRET at 6 h post fertilization.

RASopathies are multisystem genetic syndromes caused by RAS-MAPK pathway hyperactivation. Potentially pathogenic variants awaiting validation emerge continuously while poor preclinical evidence limits therapy. Here, we describe our in vivo protocol to test and cross-validate RASopathy-associated ERK activation levels and its pharmacological modulation during embryogenesis by live FRET imaging in Teen-reporter zebrafish.

RASopathies are genetic syndromes caused by ERK hyperactivation and resulting in multisystemic diseases that can also lead to cancer predisposition. ...

Biochemistry

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

Medicine

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 ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Startle Assay for Toxicity Evaluation in Zebrafish Embryo

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of electrodynamic transducers above which tissue culture dishes with embryos can be placed. Multiple such loudspeaker-tissue culture dish pairs can be combined. Vibrational stimuli generated by the electrodynamic transducers induce a characteristic startle and escape response in the embryos. A belt-driven linear drive sequentially positions a camera above each loudspeaker to record the movement of the embryos. In this way, alterations to the startle response due to lethality or neuromuscular toxicity of chemical compounds can be visualized and quantified. We present an example of the workflow for chemical compound screening using this system, including the preparation of embryos and treatment solutions, operation of the recording system, and data analysis to calculate benchmark concentration values of compounds active in the assay. The modular assembly based on commercially available simple components makes this system both economical and flexibly adaptable to the needs of particular laboratory setups and screening purposes.

Author Spotlight: High-Throughput Toxicity Screening Using Zebrafish Embryo Startle Response Assay

We describe a screening system's workflow and data analysis for evaluating chemical compound toxicity based on the zebrafish embryo vibration startle response. The system records the movements of zebrafish embryos upon exposure to a vibration stimulus and allows for an integrated evaluation of general toxicity/lethality and neuromuscular toxicity.

We developed a simple screening system for the evaluation of neuromuscular and general toxicity in zebrafish embryos. The modular system consists of ...

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, therapeutic drug discovery, and other biomedical research applications. The presented protocol aims to provide a clear guide for drug screening and toxicity testing in early-stage zebrafish larvae (aged 3-7 days post fertilization, dpf). Several methodologies describing toxicity testing in zebrafish larvae have been published, albeit lacking a standardized protocol for exposure durations, rate of media changes, and morphology scoring, amongst others. We experimentally determined the ideal larvae ages for conducting the experiments, the plate sizes that work best for those ages, the rate of media changes that provide reliable results while limiting wastage&#160;of test compound, and a morphology scoring system that is easily quantifiable in a statistical manner. The manuscript describes in detail a protocol for toxicity testing in zebrafish larvae aged between&#160;3 to 7 dpf, aiming to target the gap in drug toxicity testing and taking the first step towards a standardized method similar to the Fish Embryo Acute Toxicity test.

This protocol describes a drug screening procedure in zebrafish larvae 3-7 days post fertilization followed by morphological assessment.

Zebrafish have become a prominent model organism for human translational research, toxicity testing of small molecules and environmental pollutants, ...

Using Tg(Vtg1:mcherry) Zebrafish Embryos to Test the Estrogenic Effects of Endocrine Disrupting Compounds

Summary

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Chapters in this video

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Development of automated imaging and analysis for zebrafish chemical screens.

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

An Efficient Protocol to Assess ERK Activity Modulation in Early Zebrafish Noonan Syndrome Models via Live FRET Microscopy and Immunofluorescence

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

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

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Evaluating Toxicity of Chemicals using a Zebrafish Vibration Startle Response Screening System

Small Molecule Screening and Toxicity Testing in Early-stage Zebrafish Larvae