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).

<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 authors have nothing to disclose.

الطريقة الموصوفة هنا يستخدم تعبير عن بروتين فلوري تحت سيطرة أحد المروجين محددة الخلية من نوع لإثراء مجموعة من الخلايا الاصلية في القلب من الأجنة الزرد لاستخدامها في نظام بمساعدة القبض على خلية واحدة ميكروفلويديك لتقييم تعبير عن مجموعة فرعية من الجينات القلب في واح?...

وتستند معظم الدراسات الحالية من الخلية والبيولوجيا الجزيئية على المعدلات السكانية. ومع ذلك، قد يكون حجب الأحداث البيولوجية الهامة من قبل هذه التحليلات التقليدية القائمة على السكان منذ السكان البسيط يمكن أن تلعب دورا رئيسيا في العمليات البيولوجية ونتيجة المرض. فهم التعبير الجيني في السكان غير متجانسة على مستوى خلية واحدة يمكن (وديه) يؤدي إلى رؤى البيولوجية والسريرية ذات الصلة 1،2. من تهم دراسات التطور الجنيني، في عدد أكبر من السكان من الخلايا، الخلايا الاولية غالبا ما تكون ممثلة تمثيلا ناقصا، مما يجعلها صعبة للكشف عن التغييرات الطفيفة في التعبير الجيني التي تبدأ في نهاية المطاف القرارات مصير الخلية 3. وبالمثل، قد يكون نوع خلية واحدة ملامح التعبير المختلفة ردا على المكروية 4. على سبيل المثال، الخلايا البطانية المقيمين في نفس الجهاز أو في أجهزة مختلفة (على سبيل المثال، الشريان الأورطي أو الكلى) يحمل تجانس كبير على الرغم من اشتراكهم morp المشتركميزات hological وظيفية 5. وبالإضافة إلى ذلك، يمكن أن الخلايا السرطانية ملء الورم نفسه أيضا متفاوتة التشكيلات الجزيئية أو الطفرات على مستوى خلية واحدة 6. في النظم نموذج، transcriptomics في الخلايا واحد وقد حددت بنجاح السكان الخلية الجديدة، تتميز الدول الوسيطة التي تحدث أثناء تمايز الخلايا، وكشفت الاستجابات الخلوية التفاضلية للمنبهات 7،8،9. قد تم ملثمين مثل هذه الأفكار في الدراسات السكانية التقليدية. الأجنة الزرد هي مصدر كبير غير المستغلة الجذعية، السلف، والتفريق خلايا لاستكشاف الأسئلة من عدم التجانس وحيد الخلية والتنظيم الجزيئي الهويات الخلوية خلال التنمية. على نمطية للغاية، خارج الحي التنمية وسهولة التلاعب الجيني جعلها نظام نموذجا ممتازا لهذا النهج 10،11. على وجه التحديد، يشكل قيدا رئيسيا على تفسير خلية واحدة جنرالبيانات البريد التعبير هو أن تحديد موثوق للدول خلية متوسطة جديدة خلال التنمية يتطلب توقيت دقيق للغاية لجمع الأنسجة 9. وهذا أمر ضروري لضمان عدم التجانس بين الخلايا والقبض يمثل عدم التجانس داخل الأنسجة عند نقطة زمنية واحدة بدلا من عدم التجانس في التعبير الجيني التي قدمها تعتمد على سن تمايز الخلايا. بالمقارنة مع الفئران، تطور الجنين الزرد قد تكون متزامنة بدقة عبر عدد كبير من الأجنة 12. بالإضافة إلى ذلك، مع أحجام مخلب كبير والأجنة الزرد يمكن استخدامها كمصدر وفرة من الخلايا الجذعية والسلف. يصف هذا البروتوكول وسيلة لعزل الخلايا من الأجنة الزرد والتقاط الخلايا واحدة باستخدام الدوائر المتكاملة على microfluidics (IFC) رقاقة وautoprep النظام المتوفرة تجاريا لQRT-PCR تحليل التعبير الجيني. هذا البروتوكول يمكن أن يكون للتحويل السريع إلى أي فحوصات عالية الإنتاجية المتنوعة بما في ذلك كلهالتسلسل Transcriptome على أن يسمح تحليل أكثر شمولا من عدم التجانس الخلوية 13. كما أنها توفر العديد من المزايا لفحوصات الجينات التقليدية. بروتوكول العزلة خلية واحدة تعطي قابلية عالية بعد FACS، مما يقلل من نسبة الخلايا الشبهة التي تم تضمينها في التطبيقات المصب. باستخدام مؤسسة التمويل الدولية، الخلايا والقبض ويمكن ملاحظة مباشرة لتقييم معدلات القبض وتقييم صحة الخلية شكليا. وبالإضافة إلى ذلك، وهذا البروتوكول هو الواجب التطبيق على نطاق واسع في الأوساط البحثية الزرد، لا تتطلب سوى خط الأسماك المعدلة وراثيا وصفت والحصول على التكنولوجيات القبض على خلية ميكروفلويديك. وكدليل على المبدأ، تم عزل الخلايا واحد مستمد من الأسلاف القلب واستولت على رقاقة مؤسسة التمويل الدولية، ومن ثم تم قياس الوفرة النسبية للعلامات التمايز في القلب من قبل QRT-PCR. تحليل التعبير الجيني على مستوى الخلية واحد يدل على أن الأسلاف القلب تتعايش مع اح بهمذرية ntiating. البصيرة المكتسبة من التنميط وحيدة الخلية من الأسلاف القلب قد تسلط الضوء على الاختلاف في أنماط التعبير الجيني بين الخلايا الاصلية القلب خلال تطوير الفقاريات، التي ربما تكون قد ملثمين في التحليلات التقليدية القائمة على السكان.

<table><tbody><tr><td>&lt;strong&gt;Supplies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11254-20</td><td>For removing the chorion from embryos</td></tr><tr><td>Microcentrifuge tube 2 ml</td><td>GeneMate</td><td>C-3261-1</td><td></td></tr><tr><td>40 um cell strainer</td><td>Biobasic</td><td>SP104151</td><td></td></tr><tr><td>35 mm culture dish</td><td>Falcon</td><td>351008</td><td></td></tr><tr><td>FACS tubes topped with 35 um cell strainer</td><td>Falcon</td><td>352235</td><td></td></tr><tr><td>P1000 and tips</td><td>Rainin</td><td>17005089</td><td></td></tr><tr><td>P20 and tips</td><td>Rainin</td><td>17005091</td><td></td></tr><tr><td>IFC chip manufacturer&#039;s protocol</td><td>Fluidigm</td><td>100-6117</td><td>Version 100-6117 E1 was used in representative experiment</td></tr><tr><td>Wide bore pastuer glass pippette</td><td>VWR</td><td>14673-010</td><td>For transferring embryos</td></tr><tr><td>Adult wild type zebrafish</td><td>N/A</td><td></td><td>We used AB line</td></tr><tr><td>Adult transgenic zebrafish</td><td>N/A</td><td></td><td>We used Tg(nkx2.5:ZsYellow)</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for cell dissociation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Double distilled water</td><td>N/A</td><td></td><td></td></tr><tr><td>Instant Ocean Sea Salt</td><td>Instant Ocean</td><td>SS15-10</td><td></td></tr><tr><td>NaCl</td><td>FisherScientific</td><td>S271-3</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>KCl</td><td>Sigma Aldrich</td><td>P5405</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>NaHCO3</td><td>Sigma Aldrich</td><td>S6014</td><td>make stock in water and use for de-yolking buffer</td></tr><tr><td>Leibovitz&#039;s L-15</td><td>Gibco</td><td>21083-027</td><td></td></tr><tr><td>FBS</td><td>FisherScientific</td><td>03-600-511</td><td>Heat inactivate; any brand of FBS should be fine</td></tr><tr><td>Cell Dissociation Reagent 1 -TrypLE</td><td>Life Technologies</td><td>12605-010</td><td>Store at room temperature.</td></tr><tr><td>Cell Dissociation Reagent 2 - FACSmax</td><td>Genlantis</td><td>T200100</td><td>Store -20; thaw on ice; bring to room temp before use</td></tr><tr><td>pronase (optional)</td><td>Sigma</td><td>P5147</td><td></td></tr><tr><td>L/D Dye - Sytox Blue</td><td>Life Technologies</td><td>S34857</td><td>Any live/dead stain suitable for flow cytometry will work</td></tr><tr><td>Trypan Blue</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for IFC plate use and qRT-PCR&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Gene-specific probes</td><td></td><td></td><td>Probes will vary by experiment</td></tr><tr><td>TaqMan Probe ef1a</td><td>Life Technologies</td><td>Dr03432748_m1</td><td></td></tr><tr><td>TaqMan Probe gata4</td><td>Life Technologies</td><td>Dr03443262_g1</td><td></td></tr><tr><td>TaqMan Probe nk2-5</td><td>Life Technologies</td><td>Dr03074126_m1</td><td></td></tr><tr><td>TaqMan Probe myl7</td><td>Life Technologies</td><td>Dr03105700_m1</td><td></td></tr><tr><td>TaqMan Probe vmhc</td><td>Life Technologies</td><td>Dr03431136_m1</td><td></td></tr><tr><td>TaqMan Probe isl1</td><td>Life Technologies</td><td>Dr03425734_m1</td><td></td></tr><tr><td>TaqMan Gene Expression Master Mix</td><td>Life Technologies</td><td>4369016</td><td></td></tr><tr><td>Reagents listed in IFC manufacturer&#039;s protocol</td><td></td><td></td><td></td></tr><tr><td>C1 Reagent Kit</td><td>Fluidigm</td><td>100-5319</td><td>Reagents for loading cells onto IFC plate</td></tr><tr><td>Ambion Single Cell-to-CT&lt;sup&gt;TM&lt;/sup&gt;</td><td>Life Technologies</td><td>4458237</td><td>Reagents for reverse transcription and pre-amplification steps</td></tr><tr><td>Molecular Biology Quality Water</td><td>Corning</td><td>46-000CM</td><td></td></tr><tr><td>C1 IFC for PreAmp (5-10 um)</td><td>Fluidigm</td><td>100-5757</td><td>IFC plate for small cells</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C1 AutoPrep machine</td><td>Fluidigm</td><td>100-5477</td><td>For IFC plate use</td></tr><tr><td>Hemocytometer&amp;nbsp;</td><td>Sigma Aldrich</td><td>Z359629</td><td>For counting cells and assessing cell size</td></tr><tr><td>Dissecting microscope</td><td></td><td></td><td>For removing embryos from chorion</td></tr><tr><td>Tissue culture microscope</td><td></td><td></td><td>For assessing single cell digestion</td></tr><tr><td>FACS machine</td><td></td><td></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.

وكدليل على المبدأ، وجرى تقييم التعبير الجيني لاستكشاف ديناميكيات التمايز خلال تطوير القلب. في الزرد، تنشأ الأسلاف القلب من السكان أرومية متوسطة من الخلايا التي تهاجر إلى لوحة جانبية الأمامي الأديم المتوسط ​​حيث تلتحم لتشكل أنبوب القلب الخطي. قبل...

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.

عزل وتوصيف الخلايا واحدة من اسماك الزرد الأجنة

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

isolation-and-characterization-of-single-cells-from-zebrafish-embryos

Research

JoVE Journal

Developmental Biology

14.5K Views.  University of North Carolina at Chapel Hill. 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. 

Video: Isolation and Characterization of Single Cells from Zebrafish Embryos

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

التوسيم وخلايا الدماغ المؤخر التصوير في اسماك الزرد

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neuroscience

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

نموذج إعداد وتحليل البيانات التعبير الجيني على أساس رناسيق من الزرد

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Genetics

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

التنميط الحمض النووي توقيت النسخ المتماثل باستخدام الزرد كنظام نموذجي في فيفو

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

يعيش تصوير الدماغ الجنينية اسماك الزرد بواسطة الميكروسكوب متحد البؤر

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biology

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

الثقافة وتعداء الخلايا الابتدائية الزرد

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Preparation of Single-Somite Explants from Zebrafish Embryos

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular prepatterns determine the initial length-scale of somites, mechanical processes have been implicated in setting their final size and shape. To better understand the intrinsic material properties of somites, a method is developed to culture single-somite explant from zebrafish embryos. Single somites are isolated by first removing the skin of embryos, followed by yolk removal and sequential excision of neighboring tissues. Using transgenic embryos, the distribution of various sub-cellular structures can be observed by fluorescent time-lapse microscopy. Dynamics of explanted somites can be followed for several hours, thus providing an experimental framework for studying tissue-scale shape changes at single-cell resolution. This approach enables direct mechanical manipulation of somites, allowing for dissection of the material properties of the tissue. Finally, the technique outlined here can be readily extended for explanting other tissues such as the notochord, neural plate, and lateral plate mesoderm.

<meta charset="utf8"></head><body>تحضير مستخلصات أحادية الجسيدات من أجنة أسماك الزرد

We present a protocol for isolating single somites from zebrafish embryos, the dynamics of which can be followed in culture for several hours by fluorescence time-lapse microscopy, thus providing a methodology to quantify tissue-scale shape changes at single-cell resolution.

تحضير مستخلصات أحادية الجسيدات من أجنة أسماك الزرد

The body axis of vertebrate embryos is periodically subdivided into 3D multicellular units called somites. While genetic oscillations and molecular ...

معالجة خلايا الأمعاء من يرقات الزرد لتسلسل الحمض النووي الريبي أحادي الخلية

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

تسليط الضوء على المؤلف: عزل وتحليل الخلايا المعوية ليرقات الزرد للتحقيق في الجوانب النسخية لتطور الجهاز الهضمي 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

عزل وتوصيف الخلايا واحدة من اسماك الزرد الأجنة

Summary

Explore More Videos

التوسيم وخلايا الدماغ المؤخر التصوير في اسماك الزرد

نموذج إعداد وتحليل البيانات التعبير الجيني على أساس رناسيق من الزرد

التنميط الحمض النووي توقيت النسخ المتماثل باستخدام الزرد كنظام نموذجي في فيفو

يعيش تصوير الدماغ الجنينية اسماك الزرد بواسطة الميكروسكوب متحد البؤر

الثقافة وتعداء الخلايا الابتدائية الزرد

تحضير مستخلصات أحادية الجسيدات من أجنة أسماك الزرد

تحضير مستخلصات أحادية الجسيدات من أجنة أسماك الزرد

عزل الأمعاء من يرقات الزرد لتسلسل الحمض النووي الريبي أحادي الخلية

عزل الأمعاء من يرقات الزرد لتسلسل الحمض النووي الريبي أحادي الخلية

عزل الأمعاء من يرقات الزرد لتسلسل الحمض النووي الريبي أحادي الخلية