The authors would like to thank the staff members of the Deakin University Zebrafish Facility for providing excellent ongoing husbandry care. YG is supported by an Alfred Deakin Postdoctoral Research Fellowship and a Central Research Grant from Deakin University. SLM is supported by an NHMRC Career Development Fellowship. ACW is supported by an NHMRC Enabling Grant. All authors are supported by the Molecular &amp; Medical Research Strategic Research Centre at Deakin University.

Part 1: Chemical Treatment of Zebrafish Embryos
<ol>
 <li>Collect zebrafish embryos after fertilization. Incubate at 28.5 &deg;C in E3 medium in 100&times;15 mm Petri dishes. Note: For in situ hybridization or Oil-Red-O staining, 1-phenyl-2-thiourea (PTU) at a final concentration of 0.2 mM is added to inhibit pigment formation,1 but is not necessary for Seahorse analysis.</li>
 <li>For pharmacological studies, add the required chemical at an appropriate final concentration to developing zebrafish embryos at around 26 hr post fertilization (hpf) with an appropriate vehicle-only control. Note: for light-sensitive pharmacological inhibitors, embryos can be allowed to develop in the dark prior to analysis. </li>
</ol>
Part 2: Live Metabolic Profile Using the Seahorse XF 24 Analyser
<ol>
 <li>Before each run, the Seahorse XF 24 Analyser cartridge that houses the O2 and H+ fluorophores is calibrated through an automated process performed by the analyser. </li>
 <li>Preparation of the 24-well XF 24 islet plate. The following experimental sequence is employed:
 <ol>
 <li>Since oxygen consumption is sensitive to temperature fluctuations, four wells are used to control for possible temperature fluctuations across the plate. These wells do not contain embryos, but are filled with 700 &mu;l of E3 medium (Figure 2A). </li>
 <li>The remaining 20 wells (see part 2.2) are filled with 700 &mu;l of E3 medium and one embryo each (Figure 2B).</li>
 <li>For each experiment, 10 embryos are treated with vehicle-only (control) and 10 treated with chemical inhibitor (treated). Control and treated embryos are alternated (e.g. well A2: control embryo; well A3: treated embryo: well A4: control embryo; well A5 treated embryo, etc). </li>
 <li>An islet capture screen is added on the top of each well to ensure that the specimen remains in the measurement chamber throughout the assay (Figure 2B). Note: chemically-treated embryos remain with their original chemical solution throughout the entire procedure, with no need to wash out the chemical prior running the Seahorse analyser program. </li>
 <li>Once finished, the plate is loaded into the Seahorse Analyser and the assay is started.</li>
 </ol>
 </li>
 <li>Analysis of samples. Two different assays are routinely performed. 
 <ol>
 <li>Basal respiration/Maximum respiration program (duration approx. 90 min). Alterations in basal respiration underpin metabolic dysfunction, while maximal respiration is a measure of total energy production capacity and alterations in this parameter associated with a number of both pathological and physiological states. The spare respiratory capacity, or the capacity for system to further increase ATP production, is also calculated from this test by the subtraction of basal from maximal respiration. Three repeats of each mix (2 min)/wait (1 min)/measurement (2 min) cycle is performed to first establish basal respiration. At the end of the third cycle, a final concentration of 2.5 &mu;M of FCCP (mitochondrial protonophore/uncoupler) is added to each well allowing the measurement of maximum respiration. The measurement cycle is then repeated 5 to 8 times (Figure 3). </li>
 <li>ATP turnover/Proton leak program (duration approx. 60 min). Respiration due to ATP turnover represents the major function of mitochondria in the form of ATP production, while respiration due to proton leak, or uncoupled respiration, is inextricably linked with other parameters of mitochondrial function including basal respiration and reactive oxygen species formation. Respiration due to ATP turnover is represented by the difference in respiration following the addition of 25 &mu;M oligomycin (an inhibitor of ATP synthase) compared to basal respiration. Uncoupled respiration, or respiration due to proton leak, is determined by calculating the difference between oligomycin-mediated respiration and respiration following the addition of 25 &mu;M rotenone (a complex I inhibitor). Measurement cycles are repeated 8 times after addition of each mitochondrial inhibitor. </li>
 </ol>
 </li>
</ol>
At the end of the runs, mean values of the 10 control and 10 chemically treated embryos are calculated.
Part 3: Oil-Red-O Staining
<ol>
 <li>Embryos at 50 hpf are dechorionated using fine forceps and fixed in 4% PFA overnight at 4 &deg;C. </li>
 <li>Oil-Red-O staining is performed, as previously described2 (see Figure 4). </li>
</ol>

<ol>
	<li>N&#252;sslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish. , (2002).</li><li>Schlombs, K., Wagner, T., Scheel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-1+protease+is+required+for+cartilage+development+in+zebrafish.">Site-1 protease is required for cartilage development in zebrafish.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 14024-14029 (2003).</li><li>Ingham, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+power+of+the+zebrafish+for+disease+analysis.">The power of the zebrafish for disease analysis.</a> Hum. Mol. Genet. 18, R107-R112 (2009).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Stackley, K. D., Beeson, C. C., Rahn, J. J., Chan, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioenergetic+profiling+of+zebrafish+embryonic+development.">Bioenergetic profiling of zebrafish embryonic development.</a> Plos One. 6, e25652 (2011).</li><li>McGee, S. L., Sadli, N., Morrison, S., Swinton, C., Suphioglu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DHA+protects+against+zinc+mediated+alterations+in+neuronal+cellular+bioenergetics.">DHA protects against zinc mediated alterations in neuronal cellular bioenergetics.</a> Cell Physiol. Biochem. 28, 157-162 (2011).</li><li>Sivitz, W. I., Yorek, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+diabetes:+from+molecular+mechanisms+to+functional+significance+and+therapeutic+opportunities.">Mitochondrial dysfunction in diabetes: from molecular mechanisms to functional significance and therapeutic opportunities.</a> Antioxid. Redox Signal. 12, 537-577 (2010).</li><li>Bournat, J. C., Brown, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+obesity.">Mitochondrial dysfunction in obesity.</a> Curr. Opin. Endocrinol. Diabetes Obes. 17, 446-452 (2010).</li><li>Ghafourifar, P., 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=Mitochondria+in+multiple+sclerosis.">Mitochondria in multiple sclerosis.</a> Front Biosci. 13, 3116-3126 (2008).</li><li>Winklhofer, K. F., Haass, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+Parkinson's+disease.">Mitochondrial dysfunction in Parkinson's disease.</a> Biochim. Biophys. Acta. 1802, 29-44 (2010).</li><li>Maruszak, A., Zekanowski, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+Alzheimer's+disease.">Mitochondrial dysfunction and Alzheimer's disease.</a> Prog. Neuropsychopharmacol. Biol. Psychiatry. 35, 320-330 (2011).</li><li>de Moura, M. B., dos Santos, L. S., S, L., Van Houten, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+in+neurodegenerative+diseases+and+cancer.">Mitochondrial dysfunction in neurodegenerative diseases and cancer.</a> Environ. Mol. Mutagen. 51, 391-405 (2010).</li></ol>

No conflicts of interest declared.

Zebrafish is a well-established genetic model for both forward (ENU mutagenesis) and reverse (Tilling, zinc finger nuclease targeted knock-out, morpholino knockdown) genetic approaches3,4, while gene function in zebrafish embryos can also be easily blocked or activated using selective pharmacological compounds specific for the encoded products. Due to their external development and small size, zebrafish embryos are particularly suitable for metabolic analysis. However, robust measurement of metabolic profile and mitochondrial function in vivo in zebrafish embryos has not been achieved, with only one preliminary description reported5. Seahorse analysis was originally designed for cell-based metabolic studies and has been demonstrated to give accurate and reliable results6. The application of this new methodology to zebrafish embryos is significant, and likely to augment the wider usage of this model for metabolic studies.
In this study, we demonstrate measurement of a range of respiration parameters in zebrafish embryos using the Seahorse Analyser, including basal respiration, maximal respiration, spare respiratory capacity, ATP turnover and proton leakage. We also provide an example of how such measurements can be correlated with other physiological parameters, in this case lipid accumulation, and of the use of pharmacological inhibitors in this assay system. Combined with the use of genetically-altered embryos, this provides a powerful experimental platform for understanding factors impacting on metabolism.
There are a variety of applications for this new methodology, with mitochondrial dysfunction is implicated in many human diseases, such as Diabetes mellitus7, obesity8, multiple sclerosis9, Parkinson's diseases10, Alzheimer's disease11 and some type of cancers12. Importantly, our work is performed in vivo, where all environmental influences - such as cytokines, development-related growth etc - are active, thereby providing a physiologically relevant view in vivo respiration and metabolic profile. As chemical screens are also routinely performed in wild type and mutant background zebrafish embryos (Figure 1), novel pharmaceuticals that influence respiration, mitochondria function or metabolism could be easily identified using the Seahorse analyser. The results generated using the Seahorse analyser could be used in conjunction with other assays to provide additional information. This can include physiological analysis, such as Oil-Red Staining, or molecular analysis, such as in situ hybridization for specific adipocyte markers such as cebp&alpha;, Ppar&alpha;, Ppar&gamma;, FAS etc.
There remain some limitations to this methodology. Although we and others were able to use oligomycin to measure ATP production and proton leak on young embryos5 (see Figure 3), in embryos older than 60 hpf oligomycin treatment is inefficient. We believe that in older embryos oligomycin cannot diffuse as easily than in younger embryos making the results impossible to interpret. Since the oligomycin results are mandatory for the determination of respiration due to ATP turnover and uncoupled respiration, we are currently investigating higher concentrations of oligomycin for older embryos. However, antimycin A treatments remain effective longer, with other measurements, such as basal respiration, maximum respiration and spare respiratory capacity, can be performed in older zebrafish embryos, up to 68 hpf (not shown). 
Another limitation in using the current Seahorse Analyser setup is the physical space within each islet capture plate well, such that they are only suitable for zebrafish embryos and young larvae. Therefore, performing metabolic studies on diet-induced obesity in adult fish, for example, is not yet technically feasible. However, this study may prompt the development of plates specifically designed for juvenile or adult fish, thereby extending the scope of analyses possible.

<table border="1">
<tbody>
 <tr>
 <td>Name of the 			reagent</td>
 <td>Company</td>
 <td>Catalogue 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>XF 24 			extracellular flux analyser</td>
 <td>Seahorse 			Bioscience</td>
 <td>100737-101</td>
 <td>24 well format</td>
 </tr>
 <tr>
 <td>Islet Capture 			microplate</td>
 <td>Seahorse 			Bioscience</td>
 <td>101122-100</td>
 <td>24 well format</td>
 </tr>
 <tr>
 <td>XF Calibrant 			Solution</td>
 <td>Seahorse 			Bioscience</td>
 <td>100840-000</td>
 <td></td>
 </tr>
 <tr>
 <td>XF Assay 			Medium</td>
 <td>Seahorse 			Bioscience</td>
 <td>101022-100</td>
 <td></td>
 </tr>
 <tr>
 <td>Oil-Red-O</td>
 <td>Sigma-Aldrich</td>
 <td>O0625</td>
 <td></td>
 </tr>
 <tr>
 <td>1-phenyl-2-thiourea 			(PTU)</td>
 <td>Sigma-Aldrich</td>
 <td>P7629</td>
 <td><a href="http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml51" target="_blank">http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml51</a></td>
 </tr>
 <tr>
 <td>E3 (embryonic 			medium)</td>
 <td>Self made</td>
 <td>-</td>
 <td><a href="http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml16" target="_blank">http://zfin.org/zf_info/zfbook/chapt10.html#wptohtml16</a></td>
 </tr>
 <tr>
 <td>100X15 mm 			Petri dishes</td>
 <td>Falcon</td>
 <td>35-1029</td>
 <td></td>
 </tr>
 <tr>
 <td>FCCP</td>
 <td>Sigma</td>
 <td>C2920</td>
 <td></td>
 </tr>
 <tr>
 <td>Oligomycin</td>
 <td>Sigma</td>
 <td>75351</td>
 <td></td>
 </tr>
 <tr>
 <td>Antimycin A</td>
 <td>Sigma</td>
 <td>A8674</td>
 <td></td>
 </tr>
 </tbody>
</table>

metabolic profile analysis of zebrafish embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

We are interested in understanding the role specific genes on metabolism, particularly respiration rate and lipid metabolism. Therefore, we treated wild type zebrafish embryos from 26 hpf onwards with a specific pharmacological inhibitor of enzyme encoded by one of these genes. At 50 hpf, half of the vehicle and inhibitor-treated embryos were analysed using the Seahorse for mitochondrial function, while the remaining half were fixed in 4% PFA overnight at 4 &deg;C and subsequently stained for lipid deposition with Oil-Red-O. Treatment with the specific enzyme inhibitor led a ~2 fold increase in basal respiration (Figure 4A), which was associated with an increase in lipid deposition especially in the telencephalon and underneath the otic vesicle (Figure 4B,C). 
<img src="/files/ftp_upload/4300/4300fig1.jpg" alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/4300/4300fig1highres.jpg"/> 
Figure 1. Schematic representation of chemical treatment of zebrafish embryos. Wildtype, genetically-manipulated, or pharmacologically treated zebrafish embryos are background.
<img src="/files/ftp_upload/4300/4300fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/4300/4300fig2highres.jpg"/> 
Figure 2. XF24 islet plate set up. (A) Four wells contain embryonic medium only as temperature control (marked by an X) and the others contain one embryo/well. (B) Close-up of one well (C6) showing a 50 hpf wild type embryo treated with vehicle (DMSO) covered by an islet capture screen (arrowhead). 
<img src="/files/ftp_upload/4300/4300fig3.jpg" alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/4300/4300fig3highres.jpg"/> 
Figure 3. Respiration parameters for wild type 50 hpf zebrafish embryos. (A) For each measurement, the mean of the 20 individual embryos is presented. Basal respiration (Mean: 299.7; SD: 75.7; SEM: 16.9), Maximal respiration (Mean: 387.4; SD: 93.3; SEM: 20.9), Spare Respiratory Capacity (Mean: 87.7; SD: 72.0; SEM: 16.1). (B) For each measurement, the mean of the 20 individual results is presented. Basal mitochondrial respiration; mitochondrial respiration due to ATP turnover; mitochondrial uncoupled respiration and non-mitochondrial respiration. Mt: mitochondrial; SRC: Spare Respiratory Capacity; SD: Standard Deviation, SEM: Standard Error of Mean. Results are presented in pmol of O2 consumed/min/embryo. <a href="https://www.jove.com/files/ftp_upload/4300/4300fig3large.jpg" target="_blank">Click here to view larger figure</a>.
<img src="/files/ftp_upload/4300/4300fig4.jpg" alt="Figure 4" /> 
Figure 4. Representative results of chemical exposure on basal respiration and lipid deposition. (A,B) 50 hpf embryo stained with Oil-Red O in lateral view. The embryo incubated with the selective inhibitor (B) displays more Oil-Red-O staining in the telencephalon (arrowhead) and underneath the otic vesicle (arrow) compared to a vehicle-treated control embryo. (C) Basal respiration shows an ~ 2 fold increase in chemically treated embryos (n=10 embryos per group). Results are presented in pmol of O2/min/embryo.

Watch this Scientific Journal Video about Metabolic Profile Analysis of Zebrafish Embryos at JoVE.com

Metabolic Profile Analysis of Zebrafish Embryos

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

metabolic-profile-analysis-of-zebrafish-embryos

Research

JoVE Journal

Biology

19.8K ビュー.  School of Medicine, Deakin University. ゼブラフィッシュは、代謝の研究のために十分に利用されている強力な脊椎動物のモデルを表しています。ここでは、測定するための迅速な方法を記述代謝?...

ビデオ: Metabolic Profile Analysis of Zebrafish Embryos

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Generating Chimeric Zebrafish Embryos by Transplantation

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an organism that contains cells from more than one animal; mosaics are one type of chimera in which cells from more than one genotype are mixed, usually wild-type and mutant. In the zebrafish, chimeras can be readily made by transplantation of cells from a donor embryo into a host embryo at the appropriate embryonic stage. Labeled donor cells are generated by injection of a lineage marker, such as a fluorescent dye, into the one-cell stage embryo. Labeled donor cells are removed from donor embryos and introduced into unlabeled host embryos using an oil-controlled glass pipette mounted on either a compound or dissecting microscope. Donor cells can in some cases be targeted to a specific region or tissue of the developing blastula or gastrula stage host embryo by choosing a transplantation site in the host embryo based on well-established fate maps.

A step-by-step guide to generating targeted chimeric zebrafish embryos by transplantation at the blastula or gastrula stage.

One of the most powerful tools used to gain insight into complex developmental processes is the analysis of chimeric embryos. A chimera is defined as an ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

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

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

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

Eukaryotic Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase or the elongation phase by a variety of cellular stresses such as amino acid starvation or growth factor withdrawal. Alternatively, translation of individual mRNAs can be regulated by mRNA localization or the presence of cognate microRNAs. Studies of protein synthesis frequently utilize polyribosome analysis to shed light on the mechanisms of translation regulation or defects in protein synthesis. In this assay, mRNA/ribosome complexes are isolated from eukaryotic cells.  A sucrose density gradient separates mRNAs bound to multiple ribosomes known as polyribosomes from mRNAs bound to a single ribosome or monosome. Fractionation of the gradients allows isolation and quantification of the different ribosomal populations and their associated mRNAs or proteins. Differences in the ratio of polyribosomes to monosomes under defined conditions can be indicative of defects in either translation initiation or elongation/termination. Examination of the mRNAs present in the polyribosome fractions can reveal whether the cohort of individual mRNAs being translated changes with experimental conditions. In addition, ribosome assembly can be monitored by analysis of the small and large ribosomal subunit peaks which are also separated by the gradient. In this video, we present a method for the preparation of crude ribosomal extracts from yeast cells, separation of the extract by sucrose gradient and interpretation of the results.  This procedure is readily adaptable to mammalian cells.

This article describes a protocol for the extraction of translating ribosomes from eukaryotic cells. Once extracted, ribosomes are separated into monosomes and polyribosomes by sucrose gradient fractionation to allow different ribosomal populations to be analyzed. As such, this method is the gold standard for examining the regulation of translation.

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Imaging Glycans in Zebrafish Embryos by Metabolic Labeling and Bioorthogonal Click Chemistry

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or alkyne-tagged monosaccharides1, 2. The modified monosaccharides, processed by the glycan biosynthetic machinery, are incorporated into cell surface glycoconjugates. The bioorthogonal azide or alkyne tags then allow covalent conjugation with fluorescent probes for visualization, or with affinity probes for enrichment and glycoproteomic analysis. This protocol describes the procedures typically used for noninvasive imaging of fucosylated glycans in zebrafish embryos, including: 1) microinjection of one-cell stage embryos with GDP-5-alkynylfucose (GDP-FucAl), 2) labeling fucosylated glycans in the enveloping layer of zebrafish embryos with azide-conjugated fluorophores via biocompatible Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), and 3) imaging by confocal microscopy3. The method described here can be readily extended to visualize other classes of glycans, e.g. glycans containing sialic acid4 and N-acetylgalactosamine5, 6, in developing zebrafish and in other living organisms.

A click-chemistry based method that allows for the rapid, noninvasive, and robust labeling of alkyne-tagged glycans in zebrafish embryos is described. Fucosylated glycans in the enveloping layer of zebrafish embryos in the late gastrulation stage were imaged in this study.

Imaging glycans in vivo has recently been enabled using a bioorthogonal chemical reporter strategy by treating cells or organisms with azide- or ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

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 ...

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for forward genetic analysis of developmental processes due to their external fertilization, embryonic size, rapid ontogeny, and optical clarity &#8211; a constellation of traits that enable the direct observation of events ranging from gastrulation to organogenesis with a basic stereomicroscope. Further, zebrafish embryos can survive for several days in the haploid state. The production of haploid embryos in vitro is a powerful tool for mutational analysis, as it enables the identification of recessive mutant alleles present in first generation (F1) female carriers following mutagenesis in the parental (P) generation. This approach eliminates the necessity to raise multiple generations (F2, F3, etc.) which involves breeding of mutant families, thus saving the researcher time along with reducing the needs for zebrafish colony space, labor, and the husbandry costs. Although zebrafish have been used to conduct forward screens for the past several decades, there has been a steady expansion of transgenic and genome editing tools. These tools now offer a plethora of ways to create nuanced assays for next generation screens that can be used to further dissect the gene regulatory networks that drive vertebrate ontogeny. Here, we describe how to prepare haploid zebrafish embryos. This protocol can be implemented for novel future haploid screens, such as in enhancer and suppressor screens, to address the mechanisms of development for a broad number of processes and tissues that form during early embryonic stages.

Production of Haploid Zebrafish Embryos by In Vitro Fertilization

The zebrafish is a powerful model system for developmental biology and human disease research due to their genetic similarity with higher vertebrates. This protocol describes a methodology to create haploid zebrafish embryos that can be utilized for forward screen strategies to identify recessive mutations in genes essential for early embryogenesis.

The zebrafish has become a mainstream vertebrate model that is relevant for many disciplines of scientific study. Zebrafish are especially well suited for ...