Name: high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function
Uploaded: 2013-10-19T18:30:00.000+00:00
Duration: 10 min 6 s
Description: Watch this Scientific Journal Video about High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function at JoVE.com

<ol>
	<li>Lieschke, G. J., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat. Rev. Genet. 8, 353-367 (2007).</li><li>Wolman, M., Granato, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+genetics+in+larval+zebrafish:+learning+from+the+young.">Behavioral genetics in larval zebrafish: learning from the young.</a> Dev. Neurobiol. 72, 366-372 (2010).</li><li>Lawson, N. D., Wolfe, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+for+the+analysis+of+vertebrate+development+in+the+zebrafish.">Forward and reverse genetic approaches for the analysis of vertebrate development in the zebrafish.</a> Dev. Cell. 21, 48-64 (2011).</li><li>Arkhipova, V., 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=Characterization+and+regulation+of+the+hb9/mnx1+beta-cell+progenitor+specific+enhancer+in+zebrafish.">Characterization and regulation of the hb9/mnx1 beta-cell progenitor specific enhancer in zebrafish.</a> Dev. Biol. 365, 290-302 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Chitramuthu, B. 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=Molecular+cloning+and+embryonic+expression+of+zebrafish+PCSK5+co-orthologues:+functional+assessment+during+lateral+line+development.">Molecular cloning and embryonic expression of zebrafish PCSK5 co-orthologues: functional assessment during lateral line development.</a> Dev. Dyn. 239, 2933-2946 (2010).</li><li>Liao, E. 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=SCL/Tal-1+transcription+factor+acts+downstream+of+cloche+to+specify+hematopoietic+and+vascular+progenitors+in+zebrafish.">SCL/Tal-1 transcription factor acts downstream of cloche to specify hematopoietic and vascular progenitors in zebrafish.</a> Genes Dev. 12, 621-626 (1998).</li><li>Stainier, D. Y., Weinstein, B. M., Detrich, H. W., Zon, L. I., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloche,+an+early+acting+zebrafish+gene,+is+required+by+both+the+endothelial+and+hematopoietic+lineages.">Cloche, an early acting zebrafish gene, is required by both the endothelial and hematopoietic lineages.</a> Development. 121, 3141-3150 (1995).</li><li>Detrich, H. W., 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=Intraembryonic+hematopoietic+cell+migration+during+vertebrate+development.">Intraembryonic hematopoietic cell migration during vertebrate development.</a> Proc. Natl. Acad. Sci. U.S.A. 92, 10713-10717 (1995).</li><li>Liao, W., 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=The+zebrafish+gene+cloche+acts+upstream+of+a+flk-1+homologue+to+regulate+endothelial+cell+differentiation.">The zebrafish gene cloche acts upstream of a flk-1 homologue to regulate endothelial cell differentiation.</a> Development. , 124-381 (1997).</li><li>Krauss, S., Concordet, J. P., 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=A+functionally+conserved+homolog+of+the+Drosophila+segment+polarity+gene+hh+is+expressed+in+tissues+with+polarizing+activity+in+zebrafish+embryos.">A functionally conserved homolog of the Drosophila segment polarity gene hh is expressed in tissues with polarizing activity in zebrafish embryos.</a> Cell. 75, 1431-1444 (1993).</li><li>Akimenko, M. A., Ekker, M., Wegner, J., Lin, W., Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+expression+of+three+zebrafish+genes+related+to+distal-less:+part+of+a+homeobox+gene+code+for+the+head.">Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head.</a> J. Neurosci. 14, 3475-3486 (1994).</li><li>Alexander, J., Rothenberg, M., Henry, G. L., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=casanova+plays+an+early+and+essential+role+in+endoderm+formation+in+zebrafish.">casanova plays an early and essential role in endoderm formation in zebrafish.</a> Dev. Biol. 215, 343-357 (1999).</li><li>Milewski, W. M., Duguay, S. J., Chan, S. J., Steiner, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conservation+of+PDX-1+structure,+function,+and+expression+in+zebrafish.">Conservation of PDX-1 structure, function, and expression in zebrafish.</a> Endocrinology. 139, 1440-1449 (1998).</li><li>Argenton, F., Walker, M. D., Colombo, L., Bortolussi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+characterization+of+the+trout+insulin+promoter:+implications+for+fish+as+a+favorable+model+of+pancreas+development.">Functional characterization of the trout insulin promoter: implications for fish as a favorable model of pancreas development.</a> FEBS Lett. 407, 191-196 (1997).</li><li>Biemar, F., 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=Pancreas+development+in+zebrafish:+early+dispersed+appearance+of+endocrine+hormone+expressing+cells+and+their+convergence+to+form+the+definitive+islet.">Pancreas development in zebrafish: early dispersed appearance of endocrine hormone expressing cells and their convergence to form the definitive islet.</a> Dev. Biol. 230, 189-203 (2001).</li><li>Thisse, C., Thisse, B., Schilling, T. F., Postlethwait, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+zebrafish+snail1+gene+and+its+expression+in+wild-type,+spadetail+and+no+tail+mutant+embryos.">Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos.</a> Development. 119, 1203-1215 (1993).</li><li>Cadieux, B., Chitramuthu, B. P., Baranowski, D., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+progranulin+gene+family+and+antisense+transcripts.">The zebrafish progranulin gene family and antisense transcripts.</a> BMC Genomics. 6, 156 (2005).</li><li>Chitramuthu, B. P., Bennett, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+zebrafish+and+knockdown+technology+to+define+proprotein+convertase+activity.">Use of zebrafish and knockdown technology to define proprotein convertase activity.</a> Methods Mol. Biol. 768, 273-296 (2011).</li><li>Thisse, C., Thisse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+in+situ+hybridization+on+whole-mount+zebrafish+embryo.">High resolution in situ hybridization on whole-mount zebrafish embryo.</a> Nat. Protoc. 3, 59-69 (2008).</li></ol>

The authors declare that they have no competing financial interests.

We have developed improved methods to visualize RNA with high resolution. The in situ hybridization procedures are carried out using simple custom-made baskets with porous bottoms (Figure 1). Embryos are processed using RNase-free solutions in 6-well plates at room temperature under sterile conditions.
To segregate embryos baskets are made from different colored Eppendorf tubes. Baskets with or without a rim are used for easy orientation and to identify the embryos within the basket as corresponding to a particular probe.
Prehybridization at 65 &#176;C and hybridization at 70 &#176;C with 50% deionized formamide was found to be particularly important to obtain high resolution signals. In addition incubating embryos twice with alkaline Tris buffer for 15 min post PBST washes after incubation with anti-DIG antibody and prior to incubation in the presence of BCIP and NBT facilitates the appearance of high quality clear signals. In our hands we experienced that the permeabilization and fixing of embryos for the optimal time and hybridization at 70 &#176;C were very critical in obtaining clear signals. Inadequate permeabilization produced high background signals. Extended permeabilization or inadequate fixing resulted in the degradation of embryos whereas extended fixing inhibited signal detection. Hybridization between 55-60 &#176;C also produced high background signals (Figure 7).
The sense probe will detect a signal for the AS transcript if the gene of interest encodes antisense (AS) transcripts. After the staining reaction is stopped placing embryos in methanol for 30 min or longer converts the signal to a true purple color, and removes non-specific staining. Fixed embryos can be stored in the dark in stop solution at 4 &#176;C for several months.
The advantages and disadvantages of WISH
WISH is an efficient technique to characterize both the temporal and spatial expression of target genes during embryogenesis and larval stages. This protocol also permits visualization of RNA expression of two genes or co-localization of RNA and protein expression at the same time using appropriate modifications depending upon the experimental objectives. This high resolution protocol can be used to detect RNA expression in many tissues and cell types. It can be used for the detection of very low abundant RNA species with minimal background signals. However, to visualize accurate expression of genes within interior compartments of tissues requires in situ hybridization using tissue sections.

The zebrafish (Danio rerio) has emerged as a powerful animal model for the study of vertebrate development, disease, behavior, and in-drug screening1-3. Zebrafish embryos can be obtained in large numbers from a single crossing. Fertilization and development occurs ex utero and the optically clear embryos develop rapidly. Critical developmental events occur during the first 48 hr post-fertilization (hpf). This includes the appearance of organ primordia and the initiation of cytodifferentiation. Knockdown or over-expression of proteins can be achieved through the microinjection of embryos with antisense morpholino (MO) oligonucleotides or mRNA respectively at the one or two cell stage (0.75 hpf).
The WISH of zebrafish embryos facilitates the study of the spatio-temporal expression of specific genes of interest. Application of the WISH technique following microinjection of embryos with mRNA or MO to over-express or knockdown specific protein levels reveals differential regulation of other genes.
Changes in gene expression patterns can be correlated with phenotypic changes and reveal the function of target genes during early organogenesis. Since probes can be prepared and stored in advance, the ISH technique can be applied to reveal gene expression patterns for at least 20 genes at a time using six-well plates and custom made baskets.

<table><tbody><tr><td>Taq Polymerase</td><td>Invitrogen</td><td>10342053</td><td></td></tr><tr><td>TopoTA Cloning Kit- Dual Promoter pCRII-TOPO</td><td>Invitrogen</td><td>K4650-01</td><td></td></tr><tr><td>HQ Mini Plasmid Purification Kit</td><td>Invitrogen</td><td>K2100-01</td><td></td></tr><tr><td>Agarose</td><td>Roche</td><td>11 685 660 001</td><td></td></tr><tr><td>Not1 and HindIII Restriction Enzymes</td><td>Invitrogen</td><td>15441-025, 15207-012</td><td></td></tr><tr><td>Buffer Saturated Phenol, ultrapure</td><td>Invitrogen</td><td>15513039</td><td>Store at 4 &amp;deg;C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling.</td></tr><tr><td>Chloroform</td><td>Fisher</td><td>C607-1</td><td>Toxic and suspected carcinogen. Work under fume hood.</td></tr><tr><td>RNase-free DNAse I</td><td>Roche</td><td>4716728001</td><td>Prepare small aliquots and store at -20&amp;deg;C.</td></tr><tr><td>DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase</td><td>Roche</td><td>11277073910</td><td></td></tr><tr><td>RNase Inhibitor</td><td>Roche</td><td>10777-019</td><td></td></tr><tr><td>3.0 M Sodium Acetate (pH 5.5)</td><td>Fisher</td><td>50-751-7355</td><td></td></tr><tr><td>100% Ethanol</td><td>Commercial alcohols</td><td></td><td></td></tr><tr><td>Diethyl Pyrocarbonate (DEPC)</td><td>Sigma</td><td>40718-25ML</td><td>DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask.</td></tr><tr><td>NaOH</td><td>Fisher</td><td>SS255-1</td><td></td></tr><tr><td>EDTA 0.5 M (pH 8.0)</td><td>Fisher</td><td>50-751-7404</td><td></td></tr><tr><td>Instant Ocean Salt</td><td>Aquarium Systems</td><td>N/A</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher</td><td>FL-03-0900</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma</td><td>P6148-1KG</td><td>Prepare and store at -20&amp;deg;C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask.</td></tr><tr><td>Phenylthiocarbomide (PTC)</td><td>Sigma</td><td>P7629-25G</td><td>PTC is highly toxic. Use safety glasses, gloves, and dust mask.</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A947-4</td><td></td></tr><tr><td>Dumont (Watchmaker&amp;rsquo;s) Forceps pattern no. 5</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Six-well Cell Culture Cluster</td><td>Sarstedt</td><td>83.1839</td><td></td></tr><tr><td>Baskets are made of nylon mesh and Eppendorf tubes</td><td>In-house preparation</td><td></td><td>To make the baskets, cut Eppendorf tubes (1.5 ml) with or without rims to remove the conical end. Cut nylon mesh into small pieces corresponding to the size of&amp;nbsp;the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical&amp;nbsp;hot plate until both the tube and nylon mesh stick together (carry out in fume hood). Cut off the excess mesh from the baskets and store them in 100% methanol until used.</td></tr><tr><td>Polyoxyethylenesorbitan Monolaurate (Tween 20)</td><td>Sigma</td><td>P1379-500ML</td><td></td></tr><tr><td>Proteinase K</td><td>Fermentas</td><td>EO0491</td><td></td></tr><tr><td>Acetic Anhydride</td><td>Sigma</td><td>242845</td><td></td></tr><tr><td>Triethanolamine</td><td>Sigma</td><td>90279-100ML</td><td></td></tr><tr><td>Formamide, high purity grade</td><td>Sigma</td><td>F9037</td><td></td></tr><tr><td>AG501-X8 Resin</td><td>Bio Rad</td><td>142-6424</td><td></td></tr><tr><td>Citric Acid monohydrate</td><td>Sigma</td><td>C0706-500G</td><td></td></tr><tr><td>Heparin Sodium Salt</td><td>Sigma</td><td>H3393-25KU</td><td></td></tr><tr><td>Saline-sodium Citrate Buffer (SSC)</td><td>Sigma</td><td>S6639</td><td></td></tr><tr><td>tRNA from bakers yeast type X</td><td>Sigma</td><td>R5636-1ML</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>S640-500</td><td></td></tr><tr><td>Tris-HCl</td><td>Fisher</td><td>BP153-1</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher</td><td>S25403</td><td></td></tr><tr><td>Levamisole Hydrochloride</td><td>Sigma</td><td>31742</td><td></td></tr><tr><td>N,N-Dimethylformamide&lt;br/&gt; anhydrous (DMF)</td><td>Sigma</td><td>227056</td><td>Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles.</td></tr><tr><td>5-Bromo-4-chloro-3-indolyl phosphate (BCIP)</td><td>Sigma</td><td>N6639</td><td>Protect this solution from light.</td></tr><tr><td>Nitroblue tetrazolium (NBT)</td><td>Sigma</td><td>840W</td><td>Protect this solution from light.</td></tr><tr><td>Albumin from Bovine Serum, purified fraction V (BSA)</td><td>Sigma</td><td>A8806</td><td></td></tr><tr><td>Sheep Anti-digoxigenin-AP Fab Fragments</td><td>Roche</td><td>1093274910</td><td></td></tr><tr><td>Glycerol</td><td>Sigma</td><td>G5516-500ML</td><td></td></tr><tr><td>96-well cell culture plates</td><td>Sarstedt</td><td>83.1835</td><td></td></tr><tr><td>Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2</td><td>ZIRC</td><td>Tg(mnx1:GFP)ml2</td><td></td></tr><tr><td>Instant Ocean</td><td>Aquarium Systems</td><td>N/A</td><td></td></tr></tbody></table>

high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

Using the protocol with 50 embryos per basket (per gene/per experimental condition) the expression pattern of that gene can be achieved in one experiment. Almost all the embryos show similar expression patterns for a particular gene. Representative examples of the in situ hybridization staining are shown in Figures 3-6.
Both the sense and anti-sense riboprobes were synthesized from the cDNAs corresponding to PC5.1, PC5.26, SCL/tal-17, gata-18,9, flk/ kdrl10, shh11, dlx212, fkd7/foxa113, insulin14,15, and trypsin16 have been constructed by amplifying a segment of the full-length mRNA using Taq DNA polymerase, and cloned into pCRII-TOPO (Invitrogen). The authenticity of individual amplicons was confirmed by sequencing. After verification of the clone orientation, corresponding antisense riboprobes were synthesized using the protocols17 with modifications18,19 as described here using either the Sp6 or T7 RNA polymerases. Steps involved in whole-mount in situ hybridization are illustrated briefly in Figure 2. Purple staining observed after hybridization with riboprobe of interest represents both the abundance and the sites of expression of particular gene. For instance PC5.1 shows very discrete expression within the anterior and posterior part of otic vesicle and lateral line primordium at 24hpf and is strongly localized within the anterior and posterior lateral line neuromasts by 72 hpf (Figure 3). In contrast PC5.2 shows ubiquitous expression within the CNS with distinct regionalization within the somites, otic vesicle and pronephric duct and is highly expressed within the liver and intestine at 96 hpf (Figure 4). Absence of gene expression when using respective sense riboprobes serves as a negative control. (Figures 3b and 4b). Expression analyses of blood markers showed staining of SCL/tal-1 within the bilateral cranial cells and in the intermediate cell mass (ICM). Although the staining for gata-1 is also seen within the ICM the expression pattern is different. The expression of flk/ kdrl was observed within the hindbrain, main and intersegmental vessels and in the ICM (Figure 5). The staining for shh was observed in the notochord (Figure 5). The expression of dlx2 at 34 hpf is localized in the telencephalon, diencephalon, hypothalamus and cranial ectomesenchymal arches and the fkd7/foxa1 expression at 24 hpf is seen mainly in the floor plate and hypochord. Expression patterns of the pancreatic markers showed insulin staining in the endocrine pancreas and trypsin in the exocrine pancreas (Figure 6). These results showing expression pattern with high resolution represents the success of the outlined protocol for the detection of the expression of both high and low abundant genes. For further clarity images can be taken using confocal microscopy after mounting the embryos on a microscopic slide20. Some of the examples of the results obtained when the experiment was undertaken under sub-optimal conditions during optimization of the protocol are shown in Figure 7. High background signal with poor shh expression was noticed under inadequate permeabilization and hybridization between 55-60 °C.
<img alt="Cell culture plate setup; key for in vitro studies and experimental trials in biological research." fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig1large.jpg" src="/files/ftp_upload/50644/50644fig1.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Preparation and use of Eppendorf-nylon mesh baskets to hold staged embryos. One well can hold six Eppendorf-nylon mesh baskets. The image shows the 6-well sterile plates containing six Eppendorf-nylon mesh baskets used for successive dilutions of methanol in PBS.
<img alt="Embryo in situ hybridization diagram: preparation, probe synthesis, hybridization process flowchart." fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig2large.jpg" src="/files/ftp_upload/50644/50644fig2.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Diagram showing the steps involved in the whole-mount in situ hybridization technique. After fixing staged embryos can be stored in methanol at -20 °C until used. Probes can be synthesized in advance and kept at -80 °C. Storing probes in small aliquots is advisable if they are to used over long periods. In general whole-mount in situ hybridization experiments can be completed in 3 or 4 days. The staining reaction for weakly expressed genes will take up to 16 hr at room temperature or longer when the staining reaction is undertaken at 4 °C <a href="https://www.jove.com/files/ftp_upload/50644/50644fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Zebrafish lateral line development diagram; showing anterior/posterior primordium at 24hpf, 72hpf." fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig3large.jpg" src="/files/ftp_upload/50644/50644fig3.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. The expression of PC5.1 by whole-mount in situ hybridization analyses was carried out using 24 hpf and 72 hpf staged embryos. (a) Lateral view of 24 hpf embryos show very discrete expression of PC5.1 within the anterior and posterior part of the otic vesicle and lateral line primordium using PC5.1 antisense riboprobe. At 72 hpf specific staining was observed within the anterior and posterior lateral line neuromasts. (b) Absence of gene expression using PC5.1 sense riboprobe serves as a negative control. <a href="https://www.jove.com/files/ftp_upload/50644/50644fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Embryonic development stages diagram at 24-96 hpf; labels: hindbrain, otic vesicle, gut, liver." fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig4large.jpg" src="/files/ftp_upload/50644/50644fig4.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 4"> 
Figure 4. At 24 hpf and 96 hpf whole-mount in situ hybridization analyses of PC5.2 was carried out using PC5.2 anti-sense and sense riboprobes. (a) Lateral view of 24 hpf embryos show ubiquitous expression within the CNS, somites otic vesicle and pronephric duct. Specific staining in the liver and gut is seen at 96 hpf using PC5.2 antisense riboprobe. (b) Absence of gene expression using PC5.2 sense riboprobe serves as a negative control. <a href="https://www.jove.com/files/ftp_upload/50644/50644fig4large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Blood and neural markers diagram; microscope image of stem cells, cranial cells, neural features." fo:content-width="5in" fo:src="/files/ftp_upload/50644/50644fig5large.jpg" src="/files/ftp_upload/50644/50644fig5.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 5"> 
Figure 5. Whole-mount in situ hybridization analyses at 24 hpf using SCL/tal-1 riboprobe showed staining within the bilateral cranial cells and in the intermediate cell mass (ICM); gata-1 riboprobe staining is seen in the ICM; the flk/ kdrl expression within the hindbrain, main, and intersegmental vessels and in the ICM. The staining for shh was observed in the notochord.
<img alt="Zebrafish endoderm marker study; diagram with labeled embryonic development stages at 24-72 hpf." fo:content-width="5in" fo:src="/files/ftp_upload/50644/50644fig6large.jpg" src="/files/ftp_upload/50644/50644fig6.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 6"> 
Figure 6. Whole-mount in situ hybridization analyses at 34 hpf using dlx2 riboprobe revealed expression of dlx2 in the telencephalon, diencephalon, hypothalamus, and cranial ectomesenchymal arches; the fkd7/foxa1 expression at 24 hpf is seen mainly in the floor plate and hypochord; insulin staining is observed in the endocrine pancreas and trypsin expression in the exocrine pancreas.
<img alt="shh expression in zebrafish embryos; Proteinase K digestion, hybridization; microscopy result." fo:content-width="6in" fo:src="/files/ftp_upload/50644/50644fig7large.jpg" src="/files/ftp_upload/50644/50644fig7.jpg" width="500px" data-altupdatedat="1747911167000" data-alt="Figure 7"> 
Figure 7. Adequate permeabilization and appropriate hybridization temperature are critical for clear in situ hybridization signals. Whole-mount in situ hybridization analyses at 24 hpf and 48 hpf using an shh riboprobe revealed expression within the notochord and floorplate without background signal when embryos are permeabilized by proteinase K digestion for 12-15 min and hybridized at 70 °C. Incomplete expression patterns and high background signals were observed when the embryos were subjected to inadequate digestion (5 min proteinase K treatment) or hybridized at low temperature (60 °C). <a href="https://www.jove.com/files/ftp_upload/50644/50644fig7large.jpg" target="_blank"> Click here to view larger figure</a>.

Watch this Scientific Journal Video about High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function at JoVE.com

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

high-resolution-whole-mount-situ-hybridization-within-zebrafish

Nanyang Technological University

Research

JoVE Journal

Biology

22.1K Views.  Royal Victoria Hospital. The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Video: High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Chemistry

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Developmental Biology

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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 ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at 2 days post fertilization. Consisting of only two nephrons, the pronephros serves as the sole kidney during larval life until more renal function is required due to the increasing body mass. To cope with this higher demand, the mesonephros (adult kidney) begins to form during metamorphosis. The new primary nephrons fuse to the pronephros and form connected lumens. Then, secondary nephrons fuse to primary ones (and so on) to create a branching network in the mesonephros. The vast majority of research is focused on the pronephros due to the ease of using embryos. Thus, there is a need to develop techniques to study older and larger larvae and juvenile fish to better understand mesonephros development. Here, an in situ hybridization protocol for gene expression analysis is optimized for probe penetration, washing of probes and antibodies, and bleaching of pigments to better visualize the mesonephros. The Tg(lhx1a-EGFP) transgenic line is used to label progenitor cells and the distal tubules of nascent nephrons. This protocol fills a gap in mesonephros research. It is a crucial model for understanding how new kidney tissues form and integrate with existing nephrons and provide insights into regenerative therapies.

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

The zebrafish is an important model for understanding kidney development. Here, an in situ hybridization protocol is optimized to detect gene expression in zebrafish larvae and juveniles during mesonephros development.

The zebrafish forms two kidney structures in its lifetime. The pronephros (embryonic kidney) forms during embryonic development and begins to function at ...

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of specific mRNA transcripts within whole mount specimen. This technique (adapted from Albertson and Yelick, 2005) was used in an upper level undergraduate Comparative Vertebrate Biology laboratory classroom at Syracuse University. The first two thirds of the Comparative Vertebrate Biology lab course gave students the opportunity to study the embryology and gross anatomy of several organisms representing various chordate taxa primarily via traditional dissections and the use of models. The final portion of the course involved an innovative approach to teaching anatomy through observation of vertebrate development employing molecular techniques in which WISH was performed on zebrafish embryos. A heterozygous fibroblast growth factor 8 a (fgf8a) mutant line, ace, was used. Due to Mendelian inheritance, ace intercrosses produced wild type, heterozygous, and homozygous ace/fgf8a mutants in a 1:2:1 ratio. RNA probes with known expression patterns in the midline and in developing anatomical structures such as the heart, somites, tailbud, myotome, and brain were used. WISH was performed using zebrafish at the 13 somite and prim-6 stages, with students performing the staining reaction in class. The study of zebrafish embryos at different stages of development gave students the ability to observe how these anatomical structures changed over ontogeny. In addition, some ace/fgf8a mutants displayed improper heart looping, and defects in somite and brain development. The students in this lab observed the normal development of various organ systems using both external anatomy as well as gene expression patterns. They also identified and described embryos displaying improper anatomical development and gene expression (i.e., putative mutants).
For instructors at institutions that do not already own the necessary equipment or where funds for lab and curricular innovation are limited, the financial cost of the reagents and apparatus may be a factor to consider, as will the time and effort required on the part of the instructor regardless of the setting. Nevertheless, we contend that the use of WISH in this type of classroom laboratory setting can provide an important link between developmental genetics and anatomy. As technology advances and the ability to study organismal development at the molecular level becomes easier, cheaper, and increasingly popular, many evolutionary biologists, ecologists, and physiologists are turning to research strategies in the field of molecular biology. Using WISH in a Comparative Vertebrate Biology laboratory classroom is one example of how molecules and anatomy can converge within a single course. This gives upper level college students the opportunity to practice modern biological research techniques, leading to a more diversified education and the promotion of future interdisciplinary scientific research.

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Whole mount in situ hybridization (WISH) was used in an upper level undergraduate Comparative Vertebrate Biology course in addition to vertebrate dissections. This gave students the opportunity to study gene expression patterns as well as gross anatomy, linking the study of molecular and organismal biology within one course.

Whole mount in situ hybridization (WISH) is a common technique in molecular biology laboratories used to study gene expression through the localization of ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

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Chapters in this video

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

In Situ Hybridization in Zebrafish Larvae and Juveniles during Mesonephros Development

Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos