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/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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</ol>

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 protocol for the testing of embryos of this line in order to detect estrogen activity in vivo in pure, active ingredients.
Males and embryos of the line are also suitable for detecting estrogenic effects, but embryos have several advantages that promote their usability. In particular, the body is transparent, so the fluorescent signal in the liver can easily be observed. The zebrafish liver begins to develop 6 hours after fertilization (6 hpf) and starts working after 50 hours (50 hpf). First, the left lobe of the liver is formed, and at 96 hours (96 hpf) the right lobe of the liver also appears. The final shape of the liver is developed by around day 5 (120 hpf)28,29. The liver is able to produce endogenous vitellogenin from the age of 2&#8211;3 days of an embryo14, which coincides with the appearance of the fluorescent signal in the Tg(vtg1:mCherry) line15. Therefore, when designing experiments, it should be taken into account that a fluorescent signal can only be expected in the embryo liver of from that time. The liver of the 5 day old embryos is already well-defined in a relatively large area, where the fluorescent signal can be easily detected under a stereomicroscope. This makes the development of test protocols that are not subject to animal protection laws possible. Vitellogenin, and similarly the fluorescent protein, are produced by the left lobe of the embryos&#39; liver15. Therefore, the spatial orientation of the embryos is important for the detection of the strongest signal when examining the fluorescent signal or taking photographs. This is why embryos were laid on the left in the protocol. As can be seen from the representative results, the estrogenic effect of a test sample is clearly indicated by the fluorescent signal in the liver, so the results can be evaluated visually too. If the quantification of the results is needed, then the integrated density value defined by the ImageJ program is appropriate. However, for proper evaluation, it is indispensable that images be taken with the same settings during the experiment, and that the size of the highlighted fluorescent areas is the same in each image. Together with the precise positioning of the embryos, these are the most critical steps in the protocol. It is important to mention that in the case of embryos the expression of the transgene, similarly to the production of endogenous vitellogenin, shows a large dispersion and differences in individual sensitivity. In some cases, this can cause large variations in the results, which should be taken into account when designing the experiments.
An important aspect in determining treatment concentrations is that the cells of the embryos, and hence the liver cells, can be damaged by high concentrations of highly toxic substances, which can lead to a decline in vitellogenin induction. Therefore, tests should be performed at concentrations below LC1015.
Comparing the sensitivity of estrogen-sensitive fish lines to each other is a difficult task, because the lines described so far have been tested according to different protocols5,14,15,16. The line tested in this protocol is capable of detecting dose-effect relationships in cases of pure active ingredients, mixes, and environmental samples, and the obtained results correlated well with results with BLYES tests and HeLa cells15,20.
The utility of the embryos of the line to test agents has been proven, including zearalenone15. In this work, two metabolites of the toxin, &#945;- and &#946;-zearalenol, were tested. According to literature data, &#945;-ZOL is more toxic than &#946;-ZOL30 and its estrogenity is also higher31. These results are confirmed by our studies. Thus, studies on the embryos of the line are also suitable for comparing the estrogenic effects of other estrogenic substances.
Mycotoxin contamination in the food chain is a global problem, so several procedures have been improved to reduce mycotoxin levels in animal feed and human food25,32. One of the most promising solutions is mycotoxin biodegradation by microorganisms or by their enzymes. It may be an essential postharvest method to decrease or eliminate mycotoxin decontamination. The ZEA degrading ability of numerous bacterial strains has been tested in the literature so far, however, recent research findings that prove high degradation of the toxin rarely specify the adverse effect of metabolites33. Because the embryos of this line are theoretically suitable to test the estrogenic effects of samples with organic matter content15, a treatment protocol can be developed that can help test the biodegradation products of ZEA and the qualification of the degrading strains.
This protocol can be altered in many ways according to the planned test endpoints (e.g., exposure onset and length) and to the samples (e.g., mixtures or environmental samples) that are going to be tested and can be completed with other test methods (e.g., molecular methods). Thus, we hope that the use of the Tg(vtg1:mCherry) line will become a model of estrogenicity tests and for standard testing methods.

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/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/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, both substances induced expression of the transgene by the end of the exposure time at those test concentrations at which individuals survived. In the cases of untreated control fish, no fluorescent signal was visible.
In the case of &#945;-ZOL, at the highest test concentration (8 &#181;M) all individuals died, so in this case the fluorescent signal could not be examined. At lower concentrations (0.5 &#181;M&#8211;4 &#181;M), a strong fluorescent signal was observed in the liver of the embryos (Figure 4A). No significant difference was observed in the fluorescence intensity and the size of the fluorescent areas (p &#60; 0.05). The &#945;-ZOL integrated density values (Figure 4C), show that the substance induced the appearance of a fluorescent signal. No significant difference was found between the integrated density values and between treatments (p &#60; 0.05). The average integrated density varied between 31.26 &#177; 13.95 (0.5 &#181;M) and 34.25 &#177; 15.36 (4 &#181;M).
No mortality was documented during the treatment with &#946;-ZOL, and the substance induced transgene activity at all treatment concentrations. The fluorescence intensity and the size of the fluorescent area increased as the concentration increased, as seen in the fluorescent images (Figure 4B). Comparing the fluorescent images of &#945;- and &#946;-ZOL visually, both the signal strength and the size of the fluorescent area were visibly weaker for &#946;-ZOL at the same treatment concentrations of the two substances. Studying the integrated values of &#946;-ZOL (Figure 4D), the average integrated density value almost doubled between the lowest and the highest treatment concentrations. However, in the case of &#946;-ZOL there was no significant difference between the integrated density values of the individual concentrations (p &#60; 0.05). The average integrated density varied between 15.86 &#177; 4.08 (0.5 &#181;M) and 21.73 &#177; 5.94 (8 &#181;M).
<img alt="Figure 4" class="xfigimg bigfig" src="/files/ftp_upload/60462/60462fig04.jpg" /> 
Figure 4: Presentation of integrated density values derived from the intensity of fluorescent signals in the liver and from the size of the affected area caused by &#945;- and &#946;-zearalenol treatment on 5 day old Tg(vtg1:mCherry) transgenic zebrafish embryos.&#160;In the experiment, estrogen-sensitive embryos of the biomarker zebrafish line (20 larvae per groups in three replicates in every treatment concentration) were treated with 0.5 &#181;M&#8211;8 &#181;M concentrations of &#945;- and &#946;-ZOL from fertilization onwards for 5 days. Images of the fish livers for &#945;-ZOL (A), and &#946;-ZOL (B) clearly show that the substances induced the appearance of the fluorescent signal. Integrated density data are presented as mean &#177; standard deviation (SD = error bar). Data were analyzed with the iterative Grubbs&#8217; for identify outliers, which were excluded. Data were checked for normality with the Shapiro-Wilk normality test and compliance with the requirements of parametric methods was established. Statistical analyses were performed using a one-way ANOVA followed a Dunnett&#8217;s test. Studying the integrated density values, no significant difference was found between the treatments in the cases of &#945;-ZOL (C) and &#946;-ZOL (D) (p &#60; 0.05). Scale bar = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60462/60462fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
By examining the integrated density values obtained from the same treatment concentrations of the two substances (Figure 5), &#945;-ZOL presented higher integrated density averages in each case relative to &#946;-ZOL, which is consistent with the differences between signal strengths observed in the fluorescent images. In the cases of all treatment concentrations, significant differences (0.5 &#956;M, p = 0.0011; 1 &#956;M, p = 0.0003; 2 &#956;M, p = 0.0329; and 4 &#956;M, p = 0.0325) were found.
<img alt="Figure 5" class="xfigimg bigfig" src="/files/ftp_upload/60462/60462fig05.jpg" /> 
Figure 5: Comparison of &#945;- and &#946;-zearalenol integrated density values.&#160;Integrated density data are presented as mean &#177; standard deviation SD = error bar. Data were analyzed with iterative Grubbs&#8217; to identify outliers, which were excluded. Data were checked for normality with the Shapiro-Wilk normality test and compliance with the requirements of parametric methods was established. Significant differences were verified with unpaired t-test between &#945;-ZOL and &#946;-ZOL in the case of each concentration (0.5 &#956;M, p = 0.0011; 1 &#956;M, p = 0.0003; 2 &#956;M, p = 0.0329; and 4 &#956;M, p = 0.0325). <a href="https://www.jove.com/files/ftp_upload/60462/60462fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 Científica del Sur

Research

JoVE Journal

Environment

6.3K 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

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

Developmental Biology

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Cancer Research

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

The interrenal gland in zebrafish is the teleostean counterpart of the mammalian adrenal gland. This protocol introduces how to perform the 3-&#946;-hydroxysteroid dehydrogenase (&#916;5-4 isomerase; 3&#946;-Hsd) enzymatic activity assay, which detects differentiated steroidogenic cells in the developing zebrafish.

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Biology

Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non coding sequences. One prominent function carried out by the non coding fraction of the genome is to regulate gene transcription; however, there are no effective methods to broadly predict cis-regulatory elements from primary DNA sequence. We have developed an efficient protocol to functionally evaluate potential cis-regulatory elements through zebrafish transgenesis. Our approach offers significant advantages over cell-culture based techniques for developmentally important genes, since it provides information on spatial and temporal gene regulation. Conversely, it is faster and less expensive than similar experiments in transgenic mice, and we routinely apply it to sequences isolated from the human genome. Here we demonstrate our approach to selecting elements for testing based on sequence conservation and our protocol for cloning sequences and microinjecting them into zebrafish embryos.

We demonstrate our approach to finding potential enhancer elements from developmentally regulated genes and evaluating their function through mosaic zebrafish transgenesis.

The completion of the human genome sequence, along with that of many other species, has highlighted the challenge of ascribing specific function to non ...

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 ...

Embryo-Based Chemical Toxicity Screen: Assessing Effects on Developing Zebrafish Embryos

Source: Hao J. et al., <a href="https://www.jove.com/v/2243/" target="_blank">Large Scale Zebrafish-Based In vivo Small Molecule Screen</a>. J. Vis. Exp. (2010)
In this article, zebrafish embryos are used for chemical toxicity screening. Chemical toxicity is correlated with phenotype readouts. The sample protocol shows a methodological approach of performing the chemical toxicity screening.

Source: Hao J. et al., Large Scale Zebrafish-Based In vivo Small Molecule Screen. J. Vis. Exp. (2010)
In this article, zebrafish embryos are used for ...

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

Summary

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Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences

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

Embryo-Based Chemical Toxicity Screen: Assessing Effects on Developing Zebrafish Embryos