The authors would like to acknowledge the CIHR for fellowship support of Steve Deimling and the Department of Paediatrics, University of Western Ontario for support of Steve Deimling, Rami Halabi and Stephanie Grover. This work was supported by the NSERC grant R2654A11 and an NSERC Discovery Accelerator Supplement

1. Embryo Preparation
<ol>
	<li>If not done routinely as part of embryo culture, de-jelly the embryos using 2.5% cysteine, pH 8.0 prior to fixation 8. Although it is not absolutely necessary, it is useful to then manually remove the fertilization envelope prior to fixation using fine forceps.
	<ol>
		<li>Use glass Pasteur pipettes to transfer the embryos. The pipette is not wide enough to transfer embryos so use a diamond pen to cut the glass pipette at a point wide enough to pick up an embryo. Eliminate the...

<ol>
	<li>Ninomiya, H., 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=Cadherin-dependent+differential+cell+adhesion+in+Xenopus+causes+cell+sorting+in+vitro+but+not+in+the+embryo.">Cadherin-dependent differential cell adhesion in Xenopus causes cell sorting in vitro but not in the embryo.</a> J Cell Sci. 125, 1877-1883 (2012).</li><li>Movassagh, M., Philpott, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+differentiation+in+Xenopus+requires+the+cyclin-dependent+kinase+inhibitor,+p27Xic1.">Cardiac differentiation in Xenopus requires the cyclin-dependent kinase inhibitor, p27Xic1.</a> Cardiovasc Res. 79, 436-447 (2008).</li><li>Zhao, Y., 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+expression+of+alphaA-+and+betaB1-crystallin+during+normal+development+and+regeneration,+and+proteomic+analysis+for+the+regenerating+lens+in+Xenopus+laevis.">The expression of alphaA- and betaB1-crystallin during normal development and regeneration, and proteomic analysis for the regenerating lens in Xenopus laevis.</a> Mol Vis. 17, 768-778 (2011).</li><li>Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization:+an+improved+whole-mount+method+for+Xenopus+embryos.">In situ hybridization: an improved whole-mount method for Xenopus embryos.</a> Methods Cell Biol. 36, 685-695 (1991).</li><li>Kirilenko, P., Weierud, F. K., Zorn, A. M., Woodland, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+efficiency+of+Xenopus+primordial+germ+cell+migration+depends+on+the+germplasm+mRNA+encoding+the+PDZ+domain+protein+Grip2.">The efficiency of Xenopus primordial germ cell migration depends on the germplasm mRNA encoding the PDZ domain protein Grip2.</a> Differentiation. 76, 392-403 (2008).</li><li>Deimling, S. J., Drysdale, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+is+required+to+regulate+anterior-posterior+patterning+in+the+Xenopus+lateral+plate+mesoderm.">Fgf is required to regulate anterior-posterior patterning in the Xenopus lateral plate mesoderm.</a> Mech Dev. , (2011).</li><li>Fletcher, R. B., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+FGF+signaling+in+the+establishment+and+maintenance+of+mesodermal+gene+expression+in+Xenopus.">The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus.</a> Dev Dyn. 237, 1243-1254 (2008).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of Xenopus laevis : a laboratory manual. , (2000).</li><li>Park, E. C., Hayata, T., Cho, K. W., Han, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+cDNA+microarray+identification+of+genes+with+endodermal+organ+expression.">Xenopus cDNA microarray identification of genes with endodermal organ expression.</a> Dev Dyn. 236, 1633-1649 (2007).</li><li>Horb, M. E., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoderm+specification+and+differentiation+in+Xenopus+embryos.">Endoderm specification and differentiation in Xenopus embryos.</a> Dev Biol. 236, 330-343 (2001).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Gebhardt, D. O., Nieuwkoop, 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=The+Influence+of+Lithium+on+the+Competence+of+the+Ectoderm+in+Ambystoma+Mexicanum.">The Influence of Lithium on the Competence of the Ectoderm in Ambystoma Mexicanum.</a> J Embryol Exp Morphol. 12, 317-331 (1964).</li><li>Nieuwkoop, 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=Pattern+formation+in+artificially+activated+ectoderm+(Rana+pipiens+and+Ambystoma+punctatum).">Pattern formation in artificially activated ectoderm (Rana pipiens and Ambystoma punctatum).</a> Dev Biol. 6, 255-279 (1963).</li><li>Sato, S. M., Sargent, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+approach+to+dorsoanterior+development+in+Xenopus+laevis.">Molecular approach to dorsoanterior development in Xenopus laevis.</a> Dev Biol. 137, 135-141 (1990).</li><li>Smith, S. J., Kotecha, S., Towers, N., Latinkic, B. V., Mohun, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPOX2-peroxidase+expression+and+the+XLURP-1+promoter+reveal+the+site+of+embryonic+myeloid+cell+development+in+Xenopus.">XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus.</a> Mech Dev. 117, 173-186 (2002).</li><li>Drysdale, T. A., Tonissen, K. F., Patterson, K. D., Crawford, M. J., Krieg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+troponin+I+is+a+heart-specific+marker+in+the+Xenopus+embryo:+expression+during+abnormal+heart+morphogenesis.">Cardiac troponin I is a heart-specific marker in the Xenopus embryo: expression during abnormal heart morphogenesis.</a> Dev Biol. 165, 432-441 (1994).</li><li>Carroll, T., Wallingford, J., Seufert, D., Vize, 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=Molecular+regulation+of+pronephric+development.">Molecular regulation of pronephric development.</a> Curr Top Dev Biol. 44, 67-100 (1999).</li><li>Tonissen, K. F., Drysdale, T. A., Lints, T. J., Harvey, R. P., Krieg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XNkx-2.5,+a+Xenopus+gene+related+to+Nkx-2.5+and+tinman:+evidence+for+a+conserved+role+in+cardiac+development.">XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development.</a> Dev Biol. 162, 325-328 (1994).</li><li>Davidson, L. A., Ezin, A. M., Keller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+wound+healing+by+apical+contraction+and+ingression+in+Xenopus+laevis.">Embryonic wound healing by apical contraction and ingression in Xenopus laevis.</a> Cell Motil Cytoskeleton. 53, 163-176 (2002).</li><li>Hurtado, R., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+and+stability+in+two-color+in+situ+hybridization+by+means+of+a+novel+chromagenic+substrate+combination.">Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination.</a> Dev Dyn. 235, 2811-2816 (2006).</li></ol>

The ability to use in situ hybridization to visualize the expression pattern of specific genes remains the most commonly used method to identify specific organs or cell types in the Xenopus embryo. This is because of several advantages offered by this technique. The expression of a gene can identify specific structures well before any histological sign of differentiation such as the case for nkx2.5 expression in the heart progenitors prior to any clear demarcation of those cells 18. ...

The expression pattern of a specific gene is an important piece of information in determining the potential role for that gene in the development of a specific organ or cell type. Simply put, if it is not expressed at the right time and place it is unlikely to play a key role. In Xenopus, as in most early embryos, the most commonly used assay for detecting the expression of a gene is whole mount in situ hybridization using labeled antisense RNA probes. The use of antibody staining to assess expression of a gene in Xenopus is becoming more common as researchers discover antibodies, usually raised against mammalian proteins, that cross react to the Xenopus homologue or generate their own 1-3. However, the vast majority of studies on Xenopus organogenesis still utilize antisense RNA probes. When antibodies are used, each individual antibody often requires optimization for the primary antibody concentration or fixation protocols. In contrast, the protocol for in situ hybridizations is essentially invariant for different probes. The basic concept is relatively simple and an excellent standard protocol has been well established 4. Our protocol is a streamlined version of the original protocol 4 that still provides excellent detection of gene expression patterns in the early embryo. The embryos are fixed and then prepared for hybridization by changing solutions and temperatures such that it allows for high stringency binding of the labeled antisense RNA probe to its target mRNA. The unbound probe is washed away and the embryos are then prepared for binding of an antibody against the label on the RNA probes. Excess antibody is then washed away and an enzymatic color reaction is used to localize where the RNA probe is bound in the embryo. There are now a number of Xenopus transgenic lines that drive expression of fluorescent proteins in specific tissues and these are available at the Xenopus stock centers such as the National Xenopus Resource in Woods Hole. While very useful for many experiments that require examining organogenesis in living embryos, this option requires separate housing for the transgenic lines. 
In situ hybridization can clearly delineate where specific organs or cell types will form in the early embryo (Figure 1). The technique is remarkably sensitive given that one can detect gene expression in a small number of cells in a single embryo 5. However, in situ hybridization using the intensity of colorimetric staining is not considered quantifiable because the color reaction is not a linear one. Despite difficulty in quantifying staining intensity, changes in expression are often quite noticeable; particularly when the in situ hybridization shows quantifiable increases or decreases in the size of expression domains 6,7. 
The clear advantages of whole mount in situ hybridization make it a critical assay in the study of early development. However, it is a time consuming one that requires many steps over several days. This protocol is a simplified version of the standard protocol that eliminates several steps without reducing the quality of the in situ result. The simplification also eliminates sources of variability, making trouble shooting easier if an in situ hybridization is not optimal. Specifically, we have eliminated the use of proteinase K and RNAse treatments of the embryo, two steps that can depend on reagent quality and can also reduce signal intensity if overdone. The protocol also provides some degree of cost saving due to eliminating the use of several reagents. Finally, this protocol also provides some simple guidelines for improved capturing of images of in situ hybridization results. Although this protocol is optimized for work in Xenopus embryos, it is likely that at least some of the simplifications will be applicable to in situ hybridization work in other embryo systems.

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			<td height="41" style="height:41px;width:304px;">Name of the reagent or equipment</td>
			<td style="width:107px;">Company</td>
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			<td height="41" style="height:41px;">Labguake Tube Shakers</td>
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			<td height="36" style="height:36px;">VWR Vials</td>
			<td>VWR</td>
			<td>10-07-2012</td>
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			<td height="32" style="height:32px;">L-Cysteine</td>
			<td>BioShop</td>
			<td>CYS342.500</td>
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			<td height="31" style="height:31px;">Ribonucleoside Triphosphate Set, 100mM</td>
			<td>Roche</td>
			<td>11277057001</td>
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			<td height="35" style="height:35px;">Digoxigenin-11-UTP</td>
			<td>Roche</td>
			<td>11209256910</td>
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			<td height="36" style="height:36px;">Rnase inhibator (Rnase OUT)</td>
			<td>Invitrogen&#160;</td>
			<td>10777-019</td>
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			<td height="35" style="height:35px;">T7 RNA Polymerase</td>
			<td>Fermentas</td>
			<td>EPO111</td>
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			<td height="33" style="height:33px;">T3 RNA Polymerase</td>
			<td>Fermentas</td>
			<td>EPO101</td>
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			<td height="29" style="height:29px;">SP6 RNA Polymerase</td>
			<td>Fermentas</td>
			<td>EPO131</td>
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			<td height="32" style="height:32px;">Dnase 1</td>
			<td>Invitrogen&#160;</td>
			<td>18047-019</td>
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			<td height="33" style="height:33px;">Sheep Serum</td>
			<td>&#160;Wisent</td>
			<td>31150</td>
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			<td height="37" style="height:37px;">Blocking reagent</td>
			<td>Roche</td>
			<td>11096176001</td>
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			<td height="38" style="height:38px;">BM purple Ap Substrate</td>
			<td>Roche</td>
			<td>11442094001</td>
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			<td height="31" style="height:31px;">Anti-Digoxigenin-Ap Feb fragments</td>
			<td>Roche</td>
			<td>11093274910</td>
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			<td height="29" style="height:29px;">Methanol</td>
			<td>VWR</td>
			<td>CAMX0485-7</td>
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			<td height="29" style="height:29px;">NaCl</td>
			<td>BioShop</td>
			<td>SOD002.10</td>
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			<td>EM&#160;</td>
			<td>7910</td>
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			<td>BioShop</td>
			<td>EDT001.500</td>
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			<td height="30" style="height:30px;">Tris</td>
			<td>BioShop</td>
			<td>TRS003.5</td>
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			<td height="32" style="height:32px;">Tween-20</td>
			<td>EM&#160;</td>
			<td>9480</td>
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			<td>Sigma</td>
			<td>M-2643</td>
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			<td height="29" style="height:29px;">Mops</td>
			<td>BioShop</td>
			<td>MOP001.250</td>
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			<td height="30" style="height:30px;">EGTA</td>
			<td>Sigma</td>
			<td>E-3889-25G</td>
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			<td height="30" style="height:30px;">Paraformaldehyde</td>
			<td>BioShop</td>
			<td>PAR070.500&#160;</td>
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			<td>&#160;VWR&#160;&#160;&#160;&#160;</td>
			<td>CAFX0420-4&#160;</td>
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			<td height="31" style="height:31px;">RNA</td>
			<td>Roche</td>
			<td>10109223001</td>
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			<td>&#160;VWR&#160;&#160;&#160;&#160;</td>
			<td>CAMX0100-3</td>
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			<td>BioShop</td>
			<td>CIT001</td>
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			<td>EM&#160;</td>
			<td>HX0635-2</td>
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			<td>BioShop</td>
			<td>ALB001.100</td>
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			<td height="32" style="height:32px;">PVP-40</td>
			<td>ICN</td>
			<td>195451</td>
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			<td>GE Healthcare</td>
			<td>17-0300-10</td>
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			<td height="32" style="height:32px;">Benzyl Alcohol</td>
			<td>Sigma</td>
			<td>B-1042</td>
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			<td height="34" style="height:34px;">Benzyl Benzoate</td>
			<td>Sigma</td>
			<td>B-6630</td>
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		<tr height="32">
			<td height="32" style="height:32px;">UltraPure Agarose&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>16500-500</td>
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understanding early organogenesis using a simplified in situ hybridization protocol in xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

The use of tissue specific probes can provide outstanding information in regards to the state of development for specific organs. In the following examples, the stage of the embryo is based on the Nieuwkoop and Faber staging table 11. If one uses probes form genes expressed after differentiation, cardiac troponin I at stage 28-30, for example (Figure 1C), the presence or size of a differentiated organ can be assessed at any stage post differentiation. Years ago, embryologists were abl...

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Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

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38.9K vistas.  Hospital for Sick Children. The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.