Name: high resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function
Uploaded: 2013-10-19T18:30:00
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

1. Whole-mount In Situ Hybridization of Zebrafish Embryos
1.1 Preparation of Embryos
<ol>
	<li>Collect embryos at the required developmental stages. Please see Kimmel et al.5 for embryonic stage description.</li>
	<li>Remove chorions manually using forceps (Dumont Watchmakers Forceps no. 5).</li>
	<li>Fix embryos in a 4% solution of paraformaldehyde (PFA) made up in PBS (Phosphate Buffered Saline) overnight at 4 &#176;C.</li>
	<li>Wash embryos with PBS at least 3x for 5 min ea...

<ol>
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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>

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 wi...

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 border="0" cellpadding="0" cellspacing="0" width="1117">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:440px;">Taq Polymerase</td>
			<td style="width:159px;">Invitrogen</td>
			<td align="right" style="width:137px;">10342053</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TopoTA Cloning Kit- Dual Promoter pCRII-TOPO</td>
			<td>Invitrogen</td>
			<td>K4650-01</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HQ Mini Plasmid Purification Kit</td>
			<td>Invitrogen</td>
			<td>K2100-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td>Roche</td>
			<td style="width:137px;">11 685 660 001</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Not1 and HindIII Restriction Enzymes</td>
			<td>Invitrogen</td>
			<td>15441-025, 15207-012</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="69">
			<td height="69" style="height:69px;">Buffer Saturated Phenol, ultrapure</td>
			<td>Invitrogen</td>
			<td align="right">15513039</td>
			<td>Store at 4 &#176;C. Eyes, skin, and respiratory tract irritant and suspected carcinogen. Care should be taken while handling.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Chloroform</td>
			<td>Fisher</td>
			<td>C607-1</td>
			<td>Toxic and suspected carcinogen. Work under fume hood.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase-free DNAse I</td>
			<td>Roche</td>
			<td align="right">4716728001</td>
			<td>Prepare small aliquots and store at -20&#176;C.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DIG-RNA Labeling Mix with Sp6, T7 and T3 RNA Polymerase</td>
			<td>Roche</td>
			<td align="right">11277073910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNase Inhibitor</td>
			<td>Roche</td>
			<td>10777-019</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.0 M Sodium Acetate (pH 5.5)</td>
			<td>Fisher</td>
			<td>50-751-7355</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">100% Ethanol</td>
			<td>Commercial alcohols</td>
			<td style="width:137px;"></td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Diethyl Pyrocarbonate (DEPC)</td>
			<td>Sigma</td>
			<td>40718-25ML</td>
			<td style="width:383px;">DEPC is an eye, skin, and respiratory irritant. Avoid contact with skin and eyes. Use safety glasses, gloves, and mask.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaOH</td>
			<td>Fisher</td>
			<td>SS255-1</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA 0.5 M (pH 8.0)</td>
			<td>Fisher</td>
			<td>50-751-7404</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean Salt</td>
			<td>Aquarium Systems</td>
			<td style="width:137px;">N/A</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (PBS)</td>
			<td>Fisher</td>
			<td style="width:137px;">FL-03-0900</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;">Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>P6148-1KG</td>
			<td>Prepare and store at -20&#176;C as 40 ml aliquots for later use. PFA is toxic. Use safety glasses, gloves, and dust mask.</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">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 height="21">
			<td height="21" style="height:21px;">Methanol</td>
			<td>Fisher</td>
			<td>A947-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont (Watchmaker&#8217;s) Forceps pattern no. 5</td>
			<td>Fine Science Tools</td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Six-well Cell Culture Cluster</td>
			<td>Sarstedt</td>
			<td align="right" style="width:137px;">83.1839</td>
			<td></td>
		</tr>
		<tr height="182">
			<td height="182" style="height:182px;">Baskets are made of nylon mesh and Eppendorf tubes</td>
			<td>In-house preparation</td>
			<td style="width:137px;"></td>
			<td style="width:383px;">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&#160;the cut ends of the Eppendorf tubes. Place tubes with the nylon mesh covering the cut end on an electrical&#160;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 height="23">
			<td height="23" style="height:23px;">Polyoxyethylenesorbitan Monolaurate (Tween 20)</td>
			<td>Sigma</td>
			<td>P1379-500ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteinase K</td>
			<td>Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetic Anhydride</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">242845</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triethanolamine</td>
			<td>Sigma</td>
			<td>90279-100ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formamide, high purity grade</td>
			<td>Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">AG501-X8 Resin</td>
			<td>Bio Rad</td>
			<td style="width:137px;">142-6424</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citric Acid monohydrate</td>
			<td>Sigma</td>
			<td>C0706-500G</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin Sodium Salt</td>
			<td>Sigma</td>
			<td>H3393-25KU</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saline-sodium Citrate Buffer (SSC)</td>
			<td>Sigma</td>
			<td style="width:137px;">S6639</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">tRNA from bakers yeast type X</td>
			<td>Sigma</td>
			<td>R5636-1ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:440px;">NaCl</td>
			<td>Fisher</td>
			<td>S640-500</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris-HCl</td>
			<td>Fisher</td>
			<td>BP153-1</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td>Fisher</td>
			<td>S25403</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Levamisole Hydrochloride</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">31742</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:440px;">N,N-Dimethylformamide 
			anhydrous (DMF)</td>
			<td>Sigma</td>
			<td align="right" style="width:137px;">227056</td>
			<td style="width:383px;">Irritant, toxic, combustible, and suspected teratogen. Handle with proper safety attire including gloves and goggles.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-Bromo-4-chloro-3-indolyl phosphate (BCIP)</td>
			<td>Sigma</td>
			<td style="width:137px;">N6639</td>
			<td>Protect this solution from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitroblue tetrazolium (NBT)</td>
			<td>Sigma</td>
			<td style="width:137px;">840W</td>
			<td>Protect this solution from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin from Bovine Serum, purified fraction V (BSA)</td>
			<td>Sigma</td>
			<td style="width:137px;">A8806</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sheep Anti-digoxigenin-AP Fab Fragments</td>
			<td>Roche</td>
			<td align="right">1093274910</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma</td>
			<td>G5516-500ML</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well cell culture plates</td>
			<td>Sarstedt</td>
			<td align="right" style="width:137px;">83.1835</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tg(mnx1:GFP)ml2/Tg(hb9:GFP)ml2</td>
			<td>ZIRC</td>
			<td style="width:137px;">Tg(mnx1:GFP)ml2</td>
			<td style="width:383px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Instant Ocean</td>
			<td>Aquarium Systems</td>
			<td style="width:137px;">N/A</td>
			<td style="width:383px;"></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-1...

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 ...

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JoVE Journal

Biology

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

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 ...

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 ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

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 ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

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 ...