Name: isolation and characterization of single cells from zebrafish embryos
Uploaded: 2016-03-12T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

We thank Dr. C. Geoffrey Burns for fish stock. The authors are grateful to UNC Flow Cytometry Core Facility, UNC-CGIBD AAC core for resources enabling this project, and the ZAC facility for animal care. L.S. is supported by NIH T32 grant HL069768-13 (PI, Nobuyo Maeda). N.F. is supported by NSF Graduate Research Fellowship NSF-DGE-1144081. This study was supported by NIH P30DK034987 grant (to UNC Advanced Analytics Core), American Heart Association Scientist Development Grant 13SDG17060010 and Ellison Medical Foundation New Scholar Grant AG-NS-1064-13 (to Dr. Qian), and NIH R00 HL109079 grant (to Dr. Liu).

This protocol requires the use of live, adult zebrafish to produce embryos. The embryos are harvested for tissue collection. It is essential to obtain approval from appropriate ethics review boards to conduct this experiment.
1. Obtain Staged Embryos
<ol>
	<li>The day before the experiment, prepare healthy, adult zebrafish for breeding. Place one male and one female on opposite sides of a clear divider in a breeding tank.</li>
	<li>Repeat 1.1 for as many breeding tanks as necessary for suffi...

<ol>
	<li>Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+analysis:+toward+the+clinic.">Single-cell analysis: toward the clinic.</a> Genome medicine. 5, (2013).</li><li>Macaulay, I. C., Voet, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+genomics:+advances+and+future+perspectives.">Single cell genomics: advances and future perspectives.</a> PLoS genetics. 10, e1004126 (2014).</li><li>Marco, E., 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=Bifurcation+analysis+of+single-cell+gene+expression+data+reveals+epigenetic+landscape.">Bifurcation analysis of single-cell gene expression data reveals epigenetic landscape.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 5643-5650 (2014).</li><li>Yin, H., Marshall, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+single+cell+analysis.">Microfluidics for single cell analysis.</a> Current opinion in biotechnology. 23, 110-119 (2012).</li><li>Garlanda, C., Dejana, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+endothelial+cells.+Specific+markers.">Heterogeneity of endothelial cells. Specific markers.</a> Arteriosclerosis, thrombosis, and vascular biology. 17, 1193-1202 (1997).</li><li>Diaz-Cano, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+heterogeneity:+mechanisms+and+bases+for+a+reliable+application+of+molecular+marker+design.">Tumor heterogeneity: mechanisms and bases for a reliable application of molecular marker design.</a> International journal of molecular sciences. 13, 1951-2011 (2012).</li><li>Guo, G., 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=Mapping+cellular+hierarchy+by+single-cell+analysis+of+the+cell+surface+repertoire.">Mapping cellular hierarchy by single-cell analysis of the cell surface repertoire.</a> Cell stem cell. 13, 492-505 (2013).</li><li>Guo, G., 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=Resolution+of+cell+fate+decisions+revealed+by+single-cell+gene+expression+analysis+from+zygote+to+blastocyst.">Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst.</a> Developmental cell. 18, 675-685 (2010).</li><li>Treutlein, 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=Reconstructing+lineage+hierarchies+of+the+distal+lung+epithelium+using+single-cell+RNA-seq.">Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.</a> Nature. 509, 371-375 (2014).</li><li>Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+and+early+development+of+zebrafish.">Genetics and early development of zebrafish.</a> Trends in genetics : TIG. 5, 283-288 (1989).</li><li>Liu, J., 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=Zebrafish+in+the+study+of+early+cardiac+development.">Zebrafish in the study of early cardiac development.</a> Circulation research. 110, 870-874 (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> Developmental dynamics : an official publication of the American Association of Anatomists. 203, 253-310 (1995).</li><li>Zhao, S., Fung-Leung, W. P., Bittner, A., Ngo, K., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+RNA-Seq+and+microarray+in+transcriptome+profiling+of+activated+T+cells.">Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells.</a> PloS one. 9, e78644 (2014).</li><li>McCall, M. N., McMurray, H. R., Land, H., Almudevar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+non-detects+in+qPCR+data.">On non-detects in qPCR data.</a> Bioinformatics. 30, 2310-2316 (2014).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+C(T)+method.">Analyzing real-time PCR data by the comparative C(T) method.</a> Nature. 3, 1101-1108 (2008).</li><li>Lyons, I., 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=Myogenic+and+morphogenetic+defects+in+the+heart+tubes+of+murine+embryos+lacking+the+homeo+box+gene+Nkx2-5.">Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2-5.</a> Genes &amp; development. 9, 1654-1666 (1995).</li><li>Chen, J. N., 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=Zebrafish+tinman+homolog+demarcates+the+heart+field+and+initiates+myocardial+differentiation.">Zebrafish tinman homolog demarcates the heart field and initiates myocardial differentiation.</a> Development. 122, 3809-3816 (1996).</li><li>Zhou, 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=Latent+TGF-beta+binding+protein+3+identifies+a+second+heart+field+in+zebrafish.">Latent TGF-beta binding protein 3 identifies a second heart field in zebrafish.</a> Nature. 474, 645-648 (2011).</li><li>Paffett-Lugassy, N., 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=Heart+field+origin+of+great+vessel+precursors+relies+on+nkx2.5-mediated+vasculogenesis.">Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis.</a> Nature cell biology. 15, 1362-1369 (2013).</li><li>McCurley, A. T., Callard, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+housekeeping+genes+in+zebrafish:+male-female+differences+and+effects+of+tissue+type,+developmental+stage+and+chemical+treatment.">Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment.</a> BMC molecular biology. 9, 102 (2008).</li><li>Tang, R., Dodd, A., Lai, D., McNabb, W. C., Love, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+zebrafish+(Danio+rerio)+reference+genes+for+quantitative+real-time+RT-PCR+normalization.">Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization.</a> Acta biochimica et biophysica Sinica. 39, 384-390 (2007).</li><li>Serbedzija, G. N., Chen, J. N., 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=Regulation+in+the+heart+field+of+zebrafish.">Regulation in the heart field of zebrafish.</a> Development. 125, 1095-1101 (1998).</li><li>de Pater, E., 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=Distinct+phases+of+cardiomyocyte+differentiation+regulate+growth+of+the+zebrafish+heart.">Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart.</a> Development. 136, 1633-1641 (2009).</li><li>Hami, D., Grimes, A. C., Tsai, H. J., Kirby, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+cardiac+development+requires+a+conserved+secondary+heart+field.">Zebrafish cardiac development requires a conserved secondary heart field.</a> Development. 138, 2389-2398 (2011).</li><li>Yelon, D., Horne, S. A., 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=Restricted+expression+of+cardiac+myosin+genes+reveals+regulated+aspects+of+heart+tube+assembly+in+zebrafish.">Restricted expression of cardiac myosin genes reveals regulated aspects of heart tube assembly in zebrafish.</a> Developmental biology. 214, 23-37 (1999).</li><li>Molina, N., 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=Stimulus-induced+modulation+of+transcriptional+bursting+in+a+single+mammalian+gene.">Stimulus-induced modulation of transcriptional bursting in a single mammalian gene.</a> Proceedings of the National Academy of Sciences of the United States of America. 110, 20563-20568 (2013).</li></ol>

The method described herein uses expression of a fluorescent protein under control of a cell-type specific promoter to enrich a population of cardiac progenitor cells from zebrafish embryos for use in microfluidic assisted single cell capture system to assess expression of a subset of cardiac genes in single cells. Provided that FACS laser excitation and emission capabilities are compatible with the fluorophore(s) of choice, this method can be used for any fluorescent reporter line. Many zebrafish reporter lines are alre...

Most current studies of cell and molecular biology are based on population averages. However, important biological events may be masked by these traditional population-based analyses since minor populations can play major roles in biological processes and disease outcome. Understanding gene expression in heterogeneous populations at the single cell level can (and has) lead to relevant biological and clinical insights1,2. Of concern to embryonic development studies, in a larger population of cells, progenitor cells are often underrepresented, making it challenging to detect subtle changes in gene expression that ultimately initiate cell fate decisions3. Similarly, a single cell type may have different expression profiles in response to the microenvironment4. For example, resident endothelial cells in the same organ or in different organs (e.g., aorta or kidney) exhibit significant heterogeneity despite sharing common morphological and functional features5. In addition, cancer cells populating the same tumor can also have varying molecular profiles or mutations at the single cell level6.
In model systems, transcriptomics in single cells has successfully identified new cell populations, characterized intermediate states that occur during cell differentiation, and revealed differential cellular responses to stimuli7,8,9. Such insights would have been masked in conventional population-based studies. Zebrafish embryos are a tremendously under-utilized source of stem, progenitor, and differentiating cells for exploring questions of single cell heterogeneity and molecular regulation of cellular identities during development. Their highly stereotyped, ex vivo development and ease of genetic manipulation make them an excellent model system for this approach10,11. Specifically, a major limitation to interpretation of single cell gene expression data is that reliable identification of novel intermediate cell states during development requires very careful timing of tissue collection9. This is necessary to ensure that heterogeneity between captured cells represents heterogeneity within a tissue at a single time point rather than heterogeneity in gene expression presented by age-dependent cell differentiation. Compared to mice, zebrafish embryo development may be precisely synchronized across a large number of embryos12. Additionally, with large clutch sizes, zebrafish embryos can be used as an abundant source of stem and progenitor cells.
This protocol describes a method to isolate cells from zebrafish embryos and capture single cells using a commercially available integrated microfluidics circuit (IFC) chip and autoprep system for qRT-PCR gene expression analysis. This protocol can be rapidly transferrable to any high throughput multiplexing assays including whole transcriptome sequencing that allows more comprehensive analysis of cellular heterogeneity13. It also offers several advantages to traditional gene expression assays. The single cell isolation protocol yields high viability after FACS, which decreases the proportion of compromised cells that are included in downstream applications. By using an IFC, captured cells may be directly observed to evaluate capture rates and assess cell health morphologically. In addition, this protocol is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidic cell capture technologies.
As proof of principle, single cells derived from cardiac progenitors were isolated and captured on an IFC chip, and then the relative abundance of cardiac differentiation markers was measured by qRT-PCR. Gene expression analysis at the single cell level demonstrates that cardiac progenitors coexist with their differentiating progeny. The insight gained from single-cell profiling of cardiac progenitors may shed light on the heterogeneity in gene expression patterns among cardiac progenitor cells during vertebrate development, which may have been masked in traditional population-based analyses.

<table border="0" cellpadding="0" cellspacing="0" width="816">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Supplies</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont #5 forceps</td>
			<td style="width:111px;">Fine Science Tools</td>
			<td style="width:103px;">11254-20</td>
			<td>For removing the chorion from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microcentrifuge tube 2 mL</td>
			<td style="width:111px;">GeneMate</td>
			<td style="width:103px;">C-3261-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">40 um cell strainer</td>
			<td style="width:111px;">Biobasic</td>
			<td style="width:103px;">SP104151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm culture dish</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">351008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">FACS tubes topped with 35 um cell strainer</td>
			<td style="width:111px;">Falcon</td>
			<td align="right" style="width:103px;">352235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P1000 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">P20 and tips</td>
			<td style="width:111px;">Rainin</td>
			<td align="right" style="width:103px;">17005091</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">IFC chip manufacturer&#39;s protocol</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-6117</td>
			<td>Version 100-6117 E1 was used in representative experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Wide bore pastuer glass pippette</td>
			<td style="width:111px;">VWR</td>
			<td style="width:103px;">14673-010</td>
			<td>For transferring embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult wild type zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used AB line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult transgenic zebrafish</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td>We used Tg(nkx2.5:ZsYellow)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Reagents for cell dissociation</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double distilled water</td>
			<td style="width:111px;">N/A</td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Instant Ocean Sea Salt</td>
			<td style="width:111px;">Instant Ocean</td>
			<td style="width:103px;">SS15-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">S271-3</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KCl</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">P5405</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaHCO3</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">S6014</td>
			<td>make stock in water and use for de-yolking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Leibovitz&#39;s L-15</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">21083-027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:111px;">FisherScientific</td>
			<td style="width:103px;">03-600-511</td>
			<td>Heat inactivate; any brand of FBS should be fine</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 1 -TrypLE</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">12605-010</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell Dissociation Reagent 2 - FACSmax</td>
			<td style="width:111px;">Genlantis</td>
			<td style="width:103px;">T200100</td>
			<td>Store -20; thaw on ice; bring to room temp before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pronase (optional)</td>
			<td style="width:111px;">Sigma</td>
			<td style="width:103px;">P5147</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L/D Dye - Sytox Blue</td>
			<td style="width:111px;">Life Technologies</td>
			<td style="width:103px;">S34857</td>
			<td>Any live/dead stain suitable for flow cytometry will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypan Blue</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:103px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents for IFC plate use and qRT-PCR</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gene-specific probes</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>Probes will vary by experiment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe ef1a</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03432748_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe gata4</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03443262_g1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe nk2-5</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03074126_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe myl7</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03105700_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe vmhc</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03431136_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Probe isl1</td>
			<td style="width:111px;">Life Technologies</td>
			<td>Dr03425734_m1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4369016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Reagents listed in IFC manufacturer&#39;s protocol</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 Reagent Kit</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5319</td>
			<td>Reagents for loading cells onto IFC plate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ambion Single Cell-to-CTTM</td>
			<td style="width:111px;">Life Technologies</td>
			<td align="right" style="width:103px;">4458237</td>
			<td>Reagents for reverse transcription and pre-amplification steps</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Molecular Biology Quality Water</td>
			<td style="width:111px;">Corning</td>
			<td style="width:103px;">46-000CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 IFC for PreAmp (5-10 um)</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5757</td>
			<td>IFC plate for small cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name</td>
			<td style="width:111px;">Company</td>
			<td style="width:103px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C1 AutoPrep machine</td>
			<td style="width:111px;">Fluidigm</td>
			<td style="width:103px;">100-5477</td>
			<td>For IFC plate use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemocytometer&#160;</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:103px;">Z359629</td>
			<td>For counting cells and assessing cell size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For removing embryos from chorion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue culture microscope</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For assessing single cell digestion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FACS machine</td>
			<td style="width:111px;"></td>
			<td style="width:103px;"></td>
			<td>For isolating cells of interest</td>
		</tr>
	</tbody>
</table>

isolation and characterization of single cells from zebrafish embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

As proof of principle, gene expression was assessed to explore differentiation dynamics during cardiac development. In zebrafish, cardiac progenitors arise from a mesodermal population of cells that migrate to the anterior lateral plate mesoderm where they fuse to form the linear heart tube. Prior to fusion, cardiac progenitors begin to express the transcription factor nkx2.5 (NK2 homeobox 5), which is thought to be the earliest specific marker of cardiac progenitors16,17</s...

Watch this Scientific Journal Video about Isolation and Characterization of Single Cells from Zebrafish Embryos at JoVE.com

Isolation and Characterization of Single Cells from Zebrafish Embryos

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

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