Name: quantification of ethanol levels in zebrafish embryos using head space gas chromatography
Uploaded: 2020-02-11T14:00:00
Description: Watch this Scientific Journal Video about Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography at JoVE.com

The research presented in this article was supported by previous grants from National Institutes of Health/National Institute of Dental and Craniofacial Research (NIH/NIDCR) R01DE020884 to J.K.E. and National Institutes of Health/National Institute on Alcohol Abuse and Alcoholism (NIH/NIAAA) F32AA021320 to C.B.L. and by the current grant from National Institutes of Health/National Institute on Alcohol Abuse (NIH/NIAAA) R00AA023560 to C.B.L. We thank Rueben Gonzales for providing and assisting with gas chromatograph analysis. We thank Tiahna Ontiveros and Dr. Gina Nobles writing assistance.

All zebrafish embryos used in this procedure were raised and bred following established IACUC protocols27. These protocols were approved by the University of Texas at Austin and the University of Louisville.
NOTE: The zebrafish line Tg(fli1:EGFP)y1 was used in this study28. All water used in this procedure is sterile reverse osmosis water. All statistical analyses were performed using Graphpad Prism v8.2.1.
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+concentration+in+56+refillable+electronic+cigarettes+liquid+formulations+determined+by+headspace+gas+chromatography+with+flame+ionization+detector+(HS-GC-FID).">Ethanol concentration in 56 refillable electronic cigarettes liquid formulations determined by headspace gas chromatography with flame ionization detector (HS-GC-FID).</a> Drug Testing and Analysis. 9 (10), 1637-1640 (2017).</li><li>Heit, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Neural+Ethanol+and+Acetaldehyde+Using+Headspace+GC-MS.">Quantification of Neural Ethanol and Acetaldehyde Using Headspace GC-MS.</a> Alcoholism: Clinical and Experimental Research. 40 (9), 1825-1831 (2016).</li><li>Chun, H. J., Poklis, J. L., Poklis, A., Wolf, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+Validation+of+a+Method+for+Alcohol+Analysis+in+Brain+Tissue+by+Headspace+Gas+Chromatography+with+Flame+Ionization+Detector.">Development and Validation of a Method for Alcohol Analysis in Brain Tissue by Headspace Gas Chromatography with Flame Ionization Detector.</a> Journal of Analytical Toxicology. 40 (8), 653-658 (2016).</li><li>Schlatter, J., Chiadmi, F., Gandon, V., Chariot, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+determination+of+methanol,+acetaldehyde,+acetone,+and+ethanol+in+human+blood+by+gas+chromatography+with+flame+ionization+detection.">Simultaneous determination of methanol, acetaldehyde, acetone, and ethanol in human blood by gas chromatography with flame ionization detection.</a> Human and Experimental Toxicology. 33 (1), 74-80 (2013).</li><li>Schier, C. J., Mangieri, R. A., Dilly, G. A., Gonzales, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdialysis+of+ethanol+during+operant+ethanol+self-administration+and+ethanol+determination+by+gas+chromatography.">Microdialysis of ethanol during operant ethanol self-administration and ethanol determination by gas chromatography.</a> Journal of Visualized Experiments. (67), e4142 (2012).</li><li>Adalsteinsson, E., Sullivan, E. V., Mayer, D., Pfefferbaum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+quantification+of+ethanol+kinetics+in+rat+brain.">In vivo quantification of ethanol kinetics in rat brain.</a> Neuropsychopharmacology. 31 (12), 2683-2691 (2006).</li><li>Quertemont, E., Green, H. L., Grant, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+ethanol+concentrations+and+ethanol+discrimination+in+rats:+effects+of+dose+and+time.">Brain ethanol concentrations and ethanol discrimination in rats: effects of dose and time.</a> Psychopharmacology. 168 (3), 262-270 (2003).</li><li>Flentke, G. R., Klinger, R. H., Tanguay, R. L., Carvan, M. J., Smith, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evolutionarily-conserved+mechanism+of+calcium-dependent+neurotoxicity.">An evolutionarily-conserved mechanism of calcium-dependent neurotoxicity.</a> Alcoholism: Clinical and Experimental Research. 38 (5), 1255-1265 (2014).</li><li>Reimers, M. J., Flockton, A. R., Tanguay, R. 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As a developmental model system, zebrafish are ideally suited to study the impact of environmental factors on development. They produce large numbers of externally fertilized embryos, which allows for precise timing and dosage paradigms in ethanol studies. This, combined with the live imaging capabilities and the genetic and developmental conservation with humans, make zebrafish a powerful model system for teratology studies. Described is a protocol for measuring embryonic ethanol concentrations in developing zebrafish e...

Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of neurological impairments and craniofacial dysmorphologies associated with embryonic ethanol exposure1. Multiple factors, including timing and dosage of ethanol exposure and genetic background, contribute to the variation of FASD2,3. In humans, the complex relationship of these variables makes studying and understanding the etiology of FASD challenging. Animal models have proven crucial in developing our understanding of the mechanistic basis of ethanol teratogenicity. A wide variety of animal model systems has been used to study multiple aspects of FASD and results have been remarkably consistent with what is found in exposure in humans4. Rodent model systems are used to examine many aspects of FASD, with mice being the most common5,6,7. The majority of this work has focused on developmental defects to early ethanol exposure8, though later exposure to ethanol has been shown to cause developmental anomalies as well9. Moreover, the genetic capabilities of mice have greatly aided in our ability to probe the genetic underpinnings of FASD10,11. These studies in mice strongly suggest that there are gene-ethanol interactions with the sonic hedgehog pathway, retinoic acid signaling, Superoxide dismutase, Nitric oxide synthase I, Aldh2 and Fancd28,10,11,12,13,14,15,16,17,18,19,20,21. These studies show that animal models are critical to advancing our understanding of FASD and its underlying mechanisms.
The zebrafish has emerged as a powerful model system to examine many aspects of ethanol teratogenesis22,23. Due to their external fertilization, high fecundity, genetic tractability, and live imaging capabilities, zebrafish are ideally suited to study factors such as timing, dosage, and genetics of ethanol teratogenesis. Ethanol can be administered to precisely staged embryos and the embryos can then be imaged to examine the direct impact of ethanol during developmental processes. This work can be related directly to humans, because the genetic programs of development are highly conserved between zebrafish and humans and can therefore help guide FASD human studies24. While zebrafish have been used to examine ethanol teratogenesis, a lack of consensus in reporting embryonic ethanol concentrations makes comparison to humans difficult25. In mammalian systems, blood-alcohol levels correlate directly to tissue ethanol levels26. Many of the zebrafish studies treat embryos before complete formation of their circulatory system. With no maternal sample to examine, a process to assess ethanol concentrations is required to quantify ethanol levels within the embryo. Here we describe a process to quantify ethanol concentrations in a developing zebrafish embryo using head space gas chromatography.

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			<td>Air</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
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		<tr>
			<td>Analytical Balance</td>
			<td>VWR</td>
			<td>10204-962</td>
			<td></td>
		</tr>
		<tr>
			<td>AutoSampler, CP-8400</td>
			<td>Varian</td>
			<td></td>
			<td>Gas Chromatograph Autosampler</td>
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		<tr>
			<td>Calcium Chloride</td>
			<td>VWR</td>
			<td>97062-590</td>
			<td></td>
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		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatograph vial with polytetrafluoroethylene/silicone septum and plastic cap 2 mL</td>
			<td>Agilent</td>
			<td>8010-0198</td>
			<td>Can reuse the vials after cleaning, but not the caps/septa</td>
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			<td>Gas Chromatograph, CP-3800</td>
			<td>Varian</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Helium</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
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		<tr>
			<td>HP Innowax capillary column</td>
			<td>Agilent</td>
			<td>19095N-123I</td>
			<td>30 m x 0.53 mm x 1.0 &#956;m film thick</td>
		</tr>
		<tr>
			<td>Hyrdogen</td>
			<td></td>
			<td></td>
			<td>Provided by contract to the university</td>
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			<td>Magnesium Sulfate (Heptahydrate)</td>
			<td>Fisher Scientific</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube 1.5 mL</td>
			<td>Fisher Scientific</td>
			<td>2682002</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 10 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611106</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette tips 1000 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611127</td>
			<td></td>
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		<tr>
			<td>Micropipette tips 200 &#956;L</td>
			<td>Fisher Scientific</td>
			<td>13611112</td>
			<td></td>
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			<td>Petri dishes 100 mm</td>
			<td>Fisher Scientific</td>
			<td>FB012924</td>
			<td></td>
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			<td>Pipetman L p1000L Micropipette</td>
			<td>Gilson</td>
			<td>FA10006M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p200L Micropipette</td>
			<td>Gilson</td>
			<td>FA10005M</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipetman L p2L Micropipette</td>
			<td>Gilson</td>
			<td>FA10001M</td>
			<td></td>
		</tr>
		<tr>
			<td>Polytetrafluoroethylene/silicone septum and plastic cap</td>
			<td>Agilent</td>
			<td>5190-7021</td>
			<td>Replacement caps/septa for gas chromatograph vials</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Fisher Scientific</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate (Dibasic)</td>
			<td>VWR</td>
			<td>BDH9266-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>VWR</td>
			<td>97062-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Beads .5 mm</td>
			<td>Biospec Products</td>
			<td>11079105z</td>
			<td></td>
		</tr>
		<tr>
			<td>Silica Beads 1.0 mm</td>
			<td>Biospec Products</td>
			<td>11079110z</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>VWR</td>
			<td>BDH9280-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate (Dibasic)</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Solid-phase microextraction fiber assembly Carboxen/Polydimethylsiloxane</td>
			<td>Millipore Sigma</td>
			<td>57343-U</td>
			<td>Replacement fibers</td>
		</tr>
		<tr>
			<td>Star Chromatography Workstation</td>
			<td>Varian</td>
			<td></td>
			<td>Chromatography software</td>
		</tr>
		<tr>
			<td>Thermogreen Low Bleed (LB-2) Septa</td>
			<td>Millipore Sigma</td>
			<td>23154</td>
			<td>Replacement inlet septa</td>
		</tr>
	</tbody>
</table>

quantification of ethanol levels in zebrafish embryos using head space gas chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

Blood ethanol levels cannot be determined in early embryonic zebrafish, because they lack a fully formed circulatory system. To determine the level of ethanol concentration in the zebrafish embryos, the ethanol levels are measured directly from homogenized embryonic tissue. To properly measure the embryonic ethanol concentrations, the embryonic volume has to be taken into account. The embryo (yolk attached) sits inside the chorion (eggshell) surrounded by extraembryonic fluid (Figure 1). Any...

Watch this Scientific Journal Video about Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography at JoVE.com

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

This protocol is significant because it helps in understanding the pharmacokinetics of ethanol in developing zebrafish embryos and this will help standardize ethanol exposure regimes in zebrafish studies. The main advantage of our technique is that it allows researchers to rapidly and reproducibly measure ethanol levels during embryonic development, particularly in embryos where blood supply is not available. This method can be easily applied to other model systems as well as other environmental factors assuming proper technique and equipment setup.I would expect individuals performing this protocol for the first time to struggle with the speed necessary to prevent ethanol evaporation during embryo processing. Begin by transferring 10 embryos with extra embryonic fluid into 1.5 milliliter microcentrifuge tubes marked with a line at a volume of 250 microliters. Add water to the fill line.Prepare additional microcentrifuge tubes for dechorionated samples. To remove the chorion, incubate the embryos in a 100 millimeter Petri dish with two milligrams per milliliter of protease cocktail in embryo media for 10 minutes. Gently swirl the dish every few minutes to break the chorion.Once all the embryos are free of the chorion, remove them from the protease cocktail and wash them twice with fresh media. Then transfer the embryos to a fresh 100 millimeter dish. When all samples are ready for volume measurement, use a P200 micropipette with the smallest tip possible to carefully remove all liquid from around the embryos.Weigh the water with a scale with less than 0.1 milligram precision, then determine the volume of the 10 embryos by subtracting the weight of the removed water from 250 microliters. To determine the volume of a single embryo, divide the difference by 10. Gather the embryos from the mating tanks and place them in a standard 100 millimeter Petri dish then incubate the dish at 28.5 degrees Celsius.At six hours post fertilization, transfer up to 100 embryos to a new dish with embryo media and 1%ethanol or media alone. Cover but to not seal the Petri dishes and place them in a 28.5 degree Celsius incubator for 18 hours or until they reach 24 hours post fertilization. Use two P200 micropipettes set to 50 microliters and aspirate the protease cocktail solution into one and water into the second.Then quickly place 10 embryos in a 1.5 milliliter microcentrifuge tube using a glass pipette and close the cap. Repeat this process for all samples to be tested, controls and ethanol treated embryos. Working with one sample at a time, quickly open the cap and aspirate all residual embryo media with a P200 micropipette set to 200 microliters.Rapidly but gently add and remove the 50 microliters of water, then add the 50 microliters of protease cocktail and close the tube cap. Let the sample sit for 10 minutes at room temperature then quickly add 450 microliters of five molar sodium chloride and close the tube cap. Homogenize the sample by vortexing for 10 minutes and transfer two microliters of supernatant to a gas chromatography vial.Seal the vial with a polytetrafluoroethylene cap. After completing the startup method, load 436 current SPME ethanol two to five-minute RG run. meth from the methods menu in the analysis software for each sample on the sample list.Fill out the sample list starting with the air, water, and five molar sodium chloride protease cocktail blanks. Then enter in the ethanol standards in order from 0.3125 to 40 millimolar. Follow the standards with the second round of blanks.Enter all homogenized embryo supernatant samples to be tested from lowest to highest predicted ethanol concentration and finish by entering a third round of blanks. Add the gas chromatography vials to the autosampler in the order in which the samples were entered and allow them to warm for 10 to 15 minutes. Then start the sample runs using the software.After all runs are completed, activate the shutdown method by adding a final sample in the sample list and running standby.meth. Back up all data acquired during the sample run. Once the shutdown method is complete, click on open chromatogram and open the folder with the results.Samples are automatically integrated in this program. In the results, make sure the correct peaks have been integrated. Once all samples have been confirmed, print or export the results.Plot the peak height of the ethanol standards on a graph, then calculate the slope, y-intercept, and r squared values. Obtain the ethanol value for each sample by subtracting the peak height of the sample from the y-intercept of the standards and dividing this value by the slope of the standards. To calculate the ethanol concentration in the embryos, calculate the dilution factor for each sample and multiply the sample ethanol value by this dilution factor.Finally, calculate media reference samples by multiplying the media ethanol value by a dilution factor of 10. To properly calculate the embryonic ethanol concentrations, the volumes of embryos both in their chorion and removed from their chorion were measured. In this analysis, the embryo comprised 56%of the total volume inside the chorion.Based on previously published work, 73.6%was used as the embryonic water content for all the presented analyses. This resulted in water comprising 0.82 microliters of the 1.1 microliters of embryonic volume. Of the total water volume, 49%is contained in the embryo and 51%is in the extraembryonic fluid.For the gas chromatography analysis, two groups of 15 samples and the media with which they were treated measured five times per group were analyzed. Media ethanol levels were 143.6 millimolar for group one and 133.6 millimolar for group two. The ethanol concentrations of the embryos averaged 63.5 millimolar and 53.1 millimolar for groups one and two respectively.A ratio of embryonic ethanol concentration to media levels of ethanol for each sample was calculated. Group one had 44%of the media ethanol levels while group had 40%of the media ethanol levels. Untreated control embryos were measured at zero millimolar media ethanol concentration.The most important thing to remember when attempting this procedure is that processing the embryos for GC analysis properly is critical as ethanol is volatile and will evaporate quickly. The best way to perform this step is described, but it needs to be practiced to develop proper muscle memory. This procedure will help standardize ethanol treatment regimes in zebrafish and allow future work to dissect the genetic and cellular mechanisms of ethanal teratogenicity.

quantification-ethanol-levels-zebrafish-embryos-using-head-space-gas

Research

JoVE Journal

Developmental Biology

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.

This article describes a method for creating a mechanical vessel injury in zebrafish embryos. This injury model provides a platform for studying hemostasis, injury-related inflammation, and wound healing in an organism ideally suited for real-time microscopy.

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes.  External development ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

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

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

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

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry (GC-MS)-based Metabolomics

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal metabolism. By combining the vast array of Drosophila genetic tools with the ability to survey large swaths of intermediary metabolism, a metabolomics approach can reveal complex interactions between diet, genotype, life-history events, and environmental cues. In addition, metabolomics studies can discover novel enzymatic mechanisms and uncover previously unknown connections between seemingly disparate metabolic pathways. In order to facilitate more widespread use of this technology among the Drosophila community, here we provide a detailed protocol that describes how to prepare Drosophila larval samples for gas chromatography-mass spectrometry (GC-MS)-based metabolomic analysis. Our protocol includes descriptions of larval sample collection, metabolite extraction, chemical derivatization, and GC-MS analysis. Successful completion of this protocol will allow users to measure the relative abundance of small polar metabolites, including amino acids, sugars, and organic acids involved in glycolysis and the TCA cycles.

Preparation of Drosophila Larval Samples for Gas Chromatography-Mass Spectrometry GC-MS-based Metabolomics

This protocol describes how to prepare Drosophila larvae for GC-MS-based metabolomic analysis.

Recent advances in the field of metabolomics have established the fruit fly Drosophila melanogaster as a powerful genetic model for studying animal ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...