Name: technique to target microinjection to the developing xenopus kidney
Uploaded: 2016-05-03T11:00:00
Description: Watch this Scientific Journal Video about Technique to Target Microinjection to the Developing Xenopus Kidney at JoVE.com

This work was supported by a National Institutes of Health NIDDK grant (K01DK092320) and startup funding from the Department of Pediatrics at the University of Texas McGovern Medical School.

The following protocol has been approved by the University of Texas Health Science Center at Houston&#39;s Center for Laboratory Animal Medicine Animal Welfare Committee, which serves as the Institutional Care and Use Committee (protocol #: HSC-AWC-13-135).
1. Identification and Selection of Blastomeres for Kidney-targeted Injections&#160;
<ol>
	<li>Prior to generating embryos, use the Normal Table of Xenopus Development21 to understand the orientation of the early cell divisions in the embry...

<ol>
	<li>Vize, P. D., Seufert, D. W., Carroll, T. J., Wallingford, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Model+systems+for+the+study+of+kidney+development:+Use+of+the+pronephros+in+the+analysis+of+organ+induction+and+patterning.">Model systems for the study of kidney development: Use of the pronephros in the analysis of organ induction and patterning.</a> Dev. Biol. 188 (2), 189-204 (1997).</li><li>Brandli, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+a+molecular+anatomy+of+the+Xenopus+pronephric+kidney.">Towards a molecular anatomy of the Xenopus pronephric kidney.</a> Int. J. Dev. Biol. 43 (5), 381-395 (1999).</li><li>Hensey, C., Dolan, V., Brady, 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+Xenopus+pronephros+as+a+model+system+for+the+study+of+kidney+development+and+pathophysiology.">The Xenopus pronephros as a model system for the study of kidney development and pathophysiology.</a> Nephrol. Dial. Transplant. 17, 73-74 (2002).</li><li>Carroll, T., 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=Synergism+between+Pax-8+and+lim-1+in+embryonic+kidney+development.">Synergism between Pax-8 and lim-1 in embryonic kidney development.</a> Dev. Biol. 214 (1), 46-59 (1999).</li><li>Zhou, X., 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=Proximo-distal+specialization+of+epithelial+transport+processes+within+the+Xenopus+pronephric+tubules.">Proximo-distal specialization of epithelial transport processes within the Xenopus pronephric tubules.</a> Dev. Biol. 271 (2), 322-338 (2004).</li><li>Raciti, D., 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=Organization+of+the+pronephric+kidney+revealed+by+large-scale+gene+expression+mapping.">Organization of the pronephric kidney revealed by large-scale gene expression mapping.</a> Genome Biol. 9 (5), 84 (2008).</li><li>Moody, S. A., Kline, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+fate+during+cleavage+of+frog+(Xenopus+laevis)+blastomeres.">Segregation of fate during cleavage of frog (Xenopus laevis) blastomeres.</a> Anat. Embryol. Berl. 182 (4), 347-362 (1990).</li><li>Moody, 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=Fates+of+the+blastomeres+of+the+16-cell+stage+of+Xenopus+embryo.">Fates of the blastomeres of the 16-cell stage of Xenopus embryo.</a> Dev. Biol. 119 (2), 560-578 (1987).</li><li>Moody, 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=Fates+of+the+blastomeres+of+the+32-cell+stage+of+Xenopus+embryo.">Fates of the blastomeres of the 32-cell stage of Xenopus embryo.</a> Dev. Biol. 122 (2), 300-319 (1987).</li><li>Bauer, D. V., Huang, S., Moody, 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=The+cleavage+stage+origin+of+Spemann's+Organizer:+Analysis+of+the+movements+of+blastomere+clones+before+and+during+gastrulation+in+Xenopus.">The cleavage stage origin of Spemann's Organizer: Analysis of the movements of blastomere clones before and during gastrulation in Xenopus.</a> Dev. 120 (5), 1179-1189 (1994).</li><li>Karpinka, J. B., 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=Xenbase,+the+Xenopus+model+organism+database;+new+virtualized+system,+data+types+and+genomes.">Xenbase, the Xenopus model organism database; new virtualized system, data types and genomes.</a> Nucleic Acids Res. 43, 756-763 (2015).</li><li>Davidson, L. A., Marsden, M., Keller, R., Desimone, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+alpha5beta1+and+fibronectin+regulate+polarized+cell+protrusions+required+for+Xenopus+convergence+and+extension.">Integrin alpha5beta1 and fibronectin regulate polarized cell protrusions required for Xenopus convergence and extension.</a> Curr. Biol. 16 (9), 833-844 (2006).</li><li>Wallingford, J. B., Carroll, T. J., 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=Precocious+expression+of+the+Wilms'+tumor+gene+xWT1+inhibits+embryonic+kidney+development+in+Xenopus+laevis.">Precocious expression of the Wilms' tumor gene xWT1 inhibits embryonic kidney development in Xenopus laevis.</a> Dev. Biol. 202, 103-112 (1998).</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>Miller, R. K., Lee, M., McCrea, P. D., Kloc, M., Kubiak, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Xenopus+pronephros:+A+kidney+model+making+leaps+toward+understanding+tubule+development.">The Xenopus pronephros: A kidney model making leaps toward understanding tubule development.</a> Xenopus Development. , 215-238 (2014).</li><li>Lienkamp, S. S., 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=Vertebrate+kidney+tubules+elongate+using+a+planar+cell+polarity-dependent+rosette-based+mechanism+of+convergent+extension.">Vertebrate kidney tubules elongate using a planar cell polarity-dependent rosette-based mechanism of convergent extension.</a> Nat. Genet. 44 (12), 1382-1387 (2012).</li><li>Lyons, J. 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=Requirement+of+Wnt/&#946;-catenin+signaling+in+pronephric+kidney+development.">Requirement of Wnt/&#946;-catenin signaling in pronephric kidney development.</a> Mech. Dev. 126 (3-4), 142-159 (2009).</li><li>Miller, R. K., 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=Pronephric+tubulogenesis+requires+Daam1-mediated+planar+cell+polarity+signaling.">Pronephric tubulogenesis requires Daam1-mediated planar cell polarity signaling.</a> J. Am. Soc. Nephrol. 22, 1654-1667 (2011).</li><li>Vize, P. D., Jones, E. A., Pfister, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Xenopus+pronephric+system.">Development of the Xenopus pronephric system.</a> Dev. Biol. 171 (2), 531-540 (1995).</li><li>Miller, R. K., Lee, M., McCrea, P. D., Kloc, M., Kubiak, J. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+12:+The+Xenopus+Pronephros:+A+Kidney+Model+Making+Leaps+toward+Understanding+Tubule+Development.">Chapter 12: The Xenopus Pronephros: A Kidney Model Making Leaps toward Understanding Tubule Development.</a> Xenopus Development. , (2014).</li><li>Taira, M., Otani, H., Jamrich, M., Dawid, I. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+LIM+class+homeobox+gene+Xlim-1+in+pronephros+and+CNS+cell+lineages+of+Xenopus+embryos+is+affected+by+retinoic+acid+and+exogastrulation.">Expression of the LIM class homeobox gene Xlim-1 in pronephros and CNS cell lineages of Xenopus embryos is affected by retinoic acid and exogastrulation.</a> Development. 120, 1525-1536 (1994).</li><li>Weber, 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=Regulation+and+function+of+the+tissue-specific+transcription+factor+HNF1+alpha+(LFB1)+during+Xenopus+development.">Regulation and function of the tissue-specific transcription factor HNF1 alpha (LFB1) during Xenopus development.</a> Int. Dev. Biol. 40 (1), 297-304 (1996).</li><li>Carroll, T. J., 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=Synergism+between+Pax-8+and+lim-1+in+embryonic+kidney+development.">Synergism between Pax-8 and lim-1 in embryonic kidney development.</a> Dev. Biol. 214 (1), 46-59 (1999).</li><li>Etkin, L. D., Pearman, B., Ansah-Yiadom, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+injected+DNA+templates+in+Xenopus+embryos.">Replication of injected DNA templates in Xenopus embryos.</a> Exp. Cell Res. 169 (2), 468-477 (1987).</li><li>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=The+chloride+conductance+channel+ClC-K+is+a+specific+marker+for+the+Xenopus+pronephric+distal+tubule+and+duct.">The chloride conductance channel ClC-K is a specific marker for the Xenopus pronephric distal tubule and duct.</a> Gene Expr. Patterns. 3 (3), 347-350 (2003).</li><li>Uochi, T. S., Takahashi, S., Ninomiya, H., Fukui, A., Asashima, 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+Na+,+K+-ATPase+alpha+subunit+requires+gastrulation+in+the+Xenopus+embryo.">The Na+, K+-ATPase alpha subunit requires gastrulation in the Xenopus embryo.</a> Dev. Growth Differ. 39 (5), 571-580 (1997).</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 &amp; chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Ubbels, G. A., Hara, K., Koster, C. H., Kirschner, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+a+functional+role+of+the+cytoskeleton+in+determination+of+the+dorsoventral+axis+in+Xenopus+laevis+eggs.">Evidence for a functional role of the cytoskeleton in determination of the dorsoventral axis in Xenopus laevis eggs.</a> J. Embryol. Exp. Morphol. 77, 15-37 (1983).</li><li>Huang, S., Johnson, K. E., Wang, H. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blastomeres+show+differential+fate+changes+in+8-cell+Xenopus+laevis+embryos+that+are+rotated+90+degrees+before+the+first+cleavage.">Blastomeres show differential fate changes in 8-cell Xenopus laevis embryos that are rotated 90 degrees before the first cleavage.</a> Dev. Growth Differ. 40 (2), 189-198 (1998).</li><li>Colman, A., Drummond, 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+stability+and+movement+of+mRNA+in+Xenopus+oocytes+and+embryos.">The stability and movement of mRNA in Xenopus oocytes and embryos.</a> J. Embryol. Exp. Morph. 97, 197-209 (1986).</li></ol>

Targeting the pronephros of developing Xenopus embryos relies on identifying and injecting the correct blastomere. Injection of the V2 blastomere of 8-cell embryos targets the left pronephros18. This leaves the contralateral right pronephros as an internal control. If morpholino knockdown or RNA overexpression is used to alter kidney development, the contralateral right pronephros can be used to compare the effects of gene knockdown or overexpression on the left pronephros. In this case, proper contro...

The Xenopus embryonic kidney, the pronephros, is a good model for studying kidney development and disease. The embryos develop externally, are large in size, can be produced in large numbers, and are easily manipulated through microinjection or surgical procedures. In addition, the genes governing kidney development in mammals and amphibians are conserved. Mammalian kidneys progress through three stages: the pronephros, mesonephros, and metanephros1, while embryonic amphibians have a pronephros and adult amphibians have a metanephros. The basic filtering unit of these kidney forms is the nephron, and both mammals and amphibians require the same signaling cascades and inductive events to undergo nephrogenesis2, 3. The Xenopus pronephros contains a single nephron composed of proximal, intermediate, distal and connecting tubules, and&#160;a glomus (analogous to the mammalian glomerulus)1, 4-6 (Figure 1). The single, large nephron present in the Xenopus pronephros makes it suitable as a simple model for the study of genes involved in kidney development and disease processes.
Cell fate maps have been established for early Xenopus embryos, and are freely available online at Xenbase7-11. Here, we describe a technique for microinjection of lineage tracers to target the developing Xenopus pronephros, although the same technique can be adapted to target other tissues such as the heart or eyes. Lineage tracers are labels (including vital dyes, fluorescently labeled dextrans, histochemically detectable enzymes, and mRNA encoding fluorescent proteins) that can be injected into an early blastomere, allowing the visualization of the progeny of that cell during development. This protocol utilizes MEM-RFP mRNA, encoding membrane targeted red fluorescent protein12, as a lineage tracer. The targeted microinjection techniques for individual blastomeres in 4- and 8-cell embryos described here can be utilized for injection with morpholinos to knock down gene expression, or with exogenous RNA to overexpress a gene of interest. By injecting into the ventral, vegetal blastomere, primarily the pronephros of the embryo will be targeted, leaving the contralateral pronephros as a developmental control. Co-injection of a tracer verifies that the correct blastomere was injected, and shows which tissues in the embryo arose from the injected blastomere, verifying targeting of the pronephros. Immunostaining of the pronephros allows visualization of how well the pronephric tubules have been targeted. Overexpression and knockdown effects can then be scored against the contralateral side of the embryo, which serves as a developmental control, and can be used to calculate the pronephric index13. The availability of cell fate maps allows this targeted microinjection technique to be used to target tissues other than the pronephros, and co-injection of a fluorescent tracer allows the targeted microinjection to each tissue to be verified prior to analysis.
During embryo microinjection, developmental temperature should be regulated tightly, given that the rate of Xenopus development is highly dependent upon it14. Embryos should be incubated at cooler temperatures (14 - 16 &#176;C) for 4- and 8-cell injections because the development time is slowed down. At 22 &#176;C, development time from stage 1 (1 cell) to stage 3 (4 cells) is approximately 2 hours, while at 16 &#176;C development time to stage 3 is approximately 4 hours. It takes approximately 15 minutes to go from a 4-cell embryo to an 8-cell (stage 4) embryo at 22 &#176;C, but takes approximately 30 minutes at 16 &#176;C. Similarly, at 22 &#176;C, it only takes 30 minutes for an 8-cell embryo to progress to a 16-cell embryo (stage 5). This time is increased to 45 minutes at 16 &#176;C. Therefore, it is useful to slow the development rate of the embryos to enable enough time for injections at the 8-cell stage before the embryos progress to the 16-cell stage. Additionally, growth temperatures can be modulated to speed or slow embryonic development until the kidney has fully developed.
The epidermis of tadpole-stage Xenopus embryos is relatively transparent, allowing for easy imaging of the developing pronephros without dissection or clearing of the tissue15. Due to the relative transparency of Xenopus embryos, live cell imaging is also feasible16,17. Whole-mount immunostaining to visualize the pronephros is possible with established antibodies that label the proximal, intermediate, distal and connecting tubules of stage 38 - 40 embryos that allow for assessment of pronephric development after targeted manipulation of gene expression in Xenopus embryos18-20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate&#160;</td>
			<td>Fisher</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher</td>
			<td>S311-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin solution, 50 mg/mL</td>
			<td>Amresco</td>
			<td>E737-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine, 99%+</td>
			<td>Acros Organics</td>
			<td>52-90-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>Fisher</td>
			<td>BP525-100</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm x 15 mm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Fridge II</td>
			<td>Boekel</td>
			<td>260009</td>
			<td>Benchtop incubator for embryos.</td>
		</tr>
		<tr>
			<td>Gap43-RFP plasmid</td>
			<td></td>
			<td></td>
			<td>For making tracer RNA. Ref: Davidson et al., 2006.</td>
		</tr>
		<tr>
			<td>Rhodamine dextran, 10,000 M.W.</td>
			<td>Invitrogen</td>
			<td>D1817</td>
			<td>Tracer.</td>
		</tr>
		<tr>
			<td>Molecular biology grade, USP sterile purified water</td>
			<td>Corning</td>
			<td>46-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>7&#34; Drummond replacement tubes</td>
			<td>Drummond</td>
			<td>3-000-203-G/XL</td>
			<td>Microinjection needles.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Sutter Instruments</td>
			<td>P-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>11252-30</td>
			<td>For breaking microinjection needle tip.</td>
		</tr>
		<tr>
			<td>Nanoject II</td>
			<td>Drummond</td>
			<td>3-000-204</td>
			<td>Microinjector.</td>
		</tr>
		<tr>
			<td>Mineral oil, heavy</td>
			<td>Fisher</td>
			<td>CAS 8042-47-5</td>
			<td>Oil for needles.</td>
		</tr>
		<tr>
			<td>27G monoject hypodermic needle</td>
			<td>Covidien</td>
			<td>8881200508</td>
			<td>For loading mineral oil into microinjection needle.</td>
		</tr>
		<tr>
			<td>5 mL Luer-Lok syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td>For loading mineral oil into microinjection needle.</td>
		</tr>
		<tr>
			<td>60 mm x 15 mm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>800 micron polyester mesh</td>
			<td>Small Parts</td>
			<td>CMY-0800-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td>For transferring embryos. Cut off the tip so that the embryos are easily taken up by the pipette.</td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>Nest Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Fisher</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros Organics</td>
			<td>67-42-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher</td>
			<td>BP531-500</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm x 45 mm screw thread vial</td>
			<td>Fisher</td>
			<td>03-339-25B</td>
			<td>For fixing, staining, and storing embryos.</td>
		</tr>
		<tr>
			<td>24-well cell culture plate</td>
			<td>Nest Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Spectrum</td>
			<td>BE130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1X powder</td>
			<td>Fisher</td>
			<td>BP661-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumen</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>3D mini rocker, model 135</td>
			<td>Denville Scientific</td>
			<td>57281</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum, New Zealand origin</td>
			<td>Invitrogen</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher</td>
			<td>S227I-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 3G8 antibody</td>
			<td>European Xenopus Resource Centre</td>
			<td></td>
			<td>Primary antibody to label proximal tubules.</td>
		</tr>
		<tr>
			<td>Monoclonal 4A6 antibody</td>
			<td>European Xenopus Resource Centre</td>
			<td></td>
			<td>Primary antibody to label distal tubule and duct.</td>
		</tr>
		<tr>
			<td>anti-RFP pAb, purified IgG/rabbit</td>
			<td>MBL</td>
			<td>PM005</td>
			<td>Primary antibody to label Gap43-RFP tracer.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A11001</td>
			<td>Secondary antibody to label distal tubule and duct.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A21428</td>
			<td>Secondary antibody to label Gap43-RFP tracer.</td>
		</tr>
		<tr>
			<td>9 cavity spot plate</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl benzoate</td>
			<td>Fisher</td>
			<td>105862500</td>
			<td>Optional - for clearing embryos</td>
		</tr>
		<tr>
			<td>Benzyl alcohol</td>
			<td>Fisher</td>
			<td>A396-500</td>
			<td>Optional - for clearing embryos</td>
		</tr>
	</tbody>
</table>

technique to target microinjection to the developing xenopus kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Microinjections of 4- and 8-cell Xenopus embryos with MEM-RFP mRNA show different levels of targeting to the pronephros. Figure 4 shows stage 40 embryos with correct MEM-RFP mRNA expression patterns. Embryos were injected in the left ventral blastomere (Figure 4A), and sorted for the proper expression pattern of MEM-RFP mRNA. In addition to expressing MEM-RFP in the proximal, intermediate, distal and connecting tubules of the kidney, properly inj...

Watch this Scientific Journal Video about Technique to Target Microinjection to the Developing Xenopus Kidney at JoVE.com

Technique to Target Microinjection to the Developing Xenopus Kidney (Video) | JoVE

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

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