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Purification of Fluorescent Labeled Cells from Dissociated Ciona Embryos

  • Wei Wang
  • Claudia Racioppi
  • Basile Gravez
  • Lionel Christiaen
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1029)

Abstract

Genome-wide studies in Ciona often require highly purified cell populations. In this methods chapter, we introduce multi-channel combinatorial fluorescence activated cells sorting (FACS) and magnetic-activated cell sorting (MACS) as two sensitive and efficient tools for isolating lineage-specific cell populations from dissociated Ciona embryos and larvae. We present isolation of trunk ventral cell (TVC) progeny as the test case most commonly used in our laboratory. These approaches may also be applied to purify other cell populations with the proper combination of tissue-specific reporters.

Keywords

TVC Multi-channel combinatorial FACS MACS RNA extraction Cell dissociation Cell-lineage specific population Mosaic expression 

Notes

Acknowledgements

We are grateful to Pui-Leng Ip, Ken Birnbaum, and the personnel of GenCore at NYU for their expert assistance with the FACS, ScreenTape, and high-throughput sequencing. We thank Nicole Kaplan for sharing the image of Mesp > tagRFP, MyoD905 > GFP, and Hand-r > tagBFP labelled Ciona late tailbud. Research in the laboratory of L.C. is supported by R01 awards HL108643 and GM096032 from the NIH/NHLBI and NIH/NIGMS respectively; and by grant 15CVD01 from the Leducq Foundation. C.R. has been supported by a long-term fellowship ALTF 1608-2014 from EMBO.

References

  1. Beh J, Shi W, Levine M, Davidson B, Christiaen L (2007) FoxF is essential for FGF-induced migration of heart progenitor cells in the Ascidian Ciona intestinalis. Development 134(18):3297–3305CrossRefPubMedGoogle Scholar
  2. Christiaen L, Davidson B, Kawashima T, Powell W, Nolla H, Vranizan K, Levine M (2008) The transcription/migration interface in heart precursors of Ciona intestinalis. Science 320(5881):1349–1352CrossRefPubMedGoogle Scholar
  3. Christiaen L, Wagner E, Shi W, Levine M (2009) Isolation of individual cells and tissues from electroporated sea squirt (Ciona) embryos by Fluorescence-Activated Cell Sorting (FACS). Cold Spring Harb Protoc 2009(12):db.prot5349Google Scholar
  4. Davidson B, Shi W, Levine M (2005) Uncoupling heart cell specification and migration in the simple chordate Ciona intestinalis. Development 132(21):4811–4818CrossRefPubMedGoogle Scholar
  5. Gline S, Kaplan N, Bernadskaya Y, Abdu Y, Christiaen L (2015) Surrounding tissues canalize motile Cardiopharyngeal progenitors towards collective polarity and directed migration. Development 142(3):544–554CrossRefPubMedPubMedCentralGoogle Scholar
  6. Racioppi C, Kamal AK, Razy-Krajka F, Gambardella G, Zanetti L, di Bernardo D, Sanges R, Christiaen LA, Ristoratore F (2014) Fibroblast growth factor signalling controls nervous system patterning and pigment cell formation in Ciona intestinalis. Nat Commun 5(September):4830CrossRefPubMedPubMedCentralGoogle Scholar
  7. Razy-Krajka F, Lam K, Wang W, Stolfi A, Joly M, Bonneau R, Christiaen L (2014) Collier/OLF/EBF-dependent transcriptional dynamics control pharyngeal muscle specification from primed cardiopharyngeal progenitors. Dev Cell 29(3):263–276CrossRefPubMedPubMedCentralGoogle Scholar
  8. Stolfi A, Gandhi S, Salek F, Christiaen L (2014) Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9. Development 141(21):4115–4120Google Scholar
  9. Wang W, Razy-Krajka F, Siu E, Ketcham A, Christiaen L (2013) NK4 antagonizes Tbx1/10 to promote cardiac versus pharyngeal muscle fate in the ascidian second heart field. PLoS Biol 11(12):e1001725CrossRefPubMedPubMedCentralGoogle Scholar
  10. Woznica A, Haeussler M, Starobinska E, Jemmett J, Li Y, Mount D, Davidson B (2012) Initial deployment of the cardiogenic gene regulatory network in the basal chordate, Ciona intestinalis. Dev Biol 368(1):127–139CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Wei Wang
    • 1
  • Claudia Racioppi
    • 1
  • Basile Gravez
    • 1
  • Lionel Christiaen
    • 1
  1. 1.Center for Developmental Genetics, Department of BiologyNew York UniversityNew YorkUSA

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