Advertisement

Practical Guide for Ascidian Microinjection: Phallusia mammillata

  • Hitoyoshi Yasuo
  • Alex McDougall
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1029)

Abstract

Phallusia mammillata has recently emerged as a new ascidian model. Its unique characteristics, including the optical transparency of eggs and embryos and efficient translation of exogenously introduced mRNA in eggs, make the Phallusia system suitable for fluorescent protein (FP)-based imaging approaches. In addition, genomic and transcriptomic resources are readily available for this ascidian species, facilitating functional gene studies. Microinjection is probably the most versatile technique for introducing exogenous molecules such as plasmids, mRNAs, and proteins into ascidian eggs/embryos. However, it is not practiced widely within the community; presumably, because the system is rather laborious to set up and it requires practice. Here, we describe in as much detail as possible two microinjection methods that we use daily in the laboratory: one based on an inverted microscope and the other on a stereomicroscope. Along the stepwise description of system setup and injection procedure, we provide practical tips in the hope that this chapter might be a useful guide for introducing or improving a microinjection setup.

Keywords

Microinjection Ascidian Phallusia mammillata Live imaging Fluorescent protein 

Supplementary material

418806_1_En_3_MOESM1_ESM.avi (48.3 mb)
Movie 3.1 Unfertilized Phallusia egg during microinjection with mRNA. Images created using an inverted microscope (Olympus IX70) and a ×10 objective lens. To show the difference between a dead Phallusia egg and a live Phallusia egg, a live egg is moved next to a dead egg (which appears dark in the movie) using the injection needle. The needle is inserted into the live egg a little more than half way; then, using the high-pressure injection system, a 100-ms air pulse is activated, which forces some mRNA into the egg, causing a small displacement of the cytoplasm at the tip of the needle (a transient clear zone appears, arrow at 6 s). Using the stage control, the needle is then removed rapidly from the egg at the end of the movie. The dark zone to the right is the VALAB of the wedge. Scale bar = 50 μm (AVI 49468 kb)

References

  1. Brozovic M, Martin C, Dantec C, Dauga D, Mendez M, Simion P, Percher M, Laporte B, Scornavacca C, Di Gregorio A, Fujiwara S, Gineste M, Lowe EK, Piette J, Racioppi C, Ristoratore F, Sasakura Y, Takatori N, Brown TC, Delsuc F, Douzery E, Gissi C, McDougall A, Nishida H, Sawada H, Swalla BJ, Yasuo H, Lemaire P (2016) ANISEED 2015: a digital framework for the comparative developmental biology of ascidians. Nucleic Acids Res 44:D808–D818CrossRefPubMedGoogle Scholar
  2. Christiaen L, Wagner E, Shi W, Levine M (2009) Electroporation of transgenic DNAs in the sea squirt Ciona. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.prot5345
  3. Corbo JC, Levine M, Zeller RW (1997) Characterization of a notochord-specific enhancer from the Brachyury promoter region of the ascidian, Ciona intestinalis. Development 124:589–602PubMedGoogle Scholar
  4. Gregory C, Veeman M (2013) 3D-printed microwell arrays for Ciona microinjection and timelapse imaging. PLoS One 8:e82307CrossRefPubMedPubMedCentralGoogle Scholar
  5. Haupaix N, Stolfi A, Sirour C, Picco V, Levine M, Christiaen L, Yasuo H (2013) p120RasGAP mediates ephrin/Eph-dependent attenuation of FGF/ERK signals during cell fate specification in ascidian embryos. Development 140:4347–4352CrossRefPubMedPubMedCentralGoogle Scholar
  6. Levasseur M, McDougall A (2000) Sperm-induced calcium oscillations at fertilisation in ascidians are controlled by cyclin B1-dependent kinase activity. Development 127:631–641PubMedGoogle Scholar
  7. Matsuoka T, Awazu S, Shoguchi E, Satoh N, Sasakura Y (2005) Germline transgenesis of the ascidian Ciona intestinalis by electroporation. Genes 41:67–72CrossRefGoogle Scholar
  8. McDougall A, Chenevert J, Pruliere G, Costache V, Hebras C, Salez G, Dumollard R (2015) Centrosomes and spindles in ascidian embryos and eggs. Methods Cell Biol 129:317–339CrossRefPubMedGoogle Scholar
  9. Negishi T, Yasuo H (2015) Distinct modes of mitotic spindle orientation align cells in the dorsal midline of ascidian embryos. Dev Biol 408:66–78CrossRefPubMedGoogle Scholar
  10. Prodon F, Chenevert J, Hébras C, Dumollard R, Faure E, Gonzalez-Garcia J, Nishida H, Sardet C, McDougall A (2010) Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors. Development 137:2011–2021CrossRefPubMedGoogle Scholar
  11. Roure A, Rothbächer U, Robin F, Kalmar E, Ferone G, Lamy C, Missero C, Mueller F, Lemaire P (2007) A multicassette Gateway vector set for high throughput and comparative analyses in ciona and vertebrate embryos. PLoS One 2:e916CrossRefPubMedPubMedCentralGoogle Scholar
  12. Sardet C, McDougall A, Yasuo H, Chenevert J, Pruliere G, Dumollard R, Hudson C, Hebras C, Le Nguyen N, Paix A (2011) Embryological methods in ascidians: the Villefranche-sur-Mer protocols. Methods Mol Biol 770:365–400CrossRefPubMedGoogle Scholar
  13. Zeller RW, Virata MJ, Cone AC (2006) Predictable mosaic transgene expression in ascidian embryos produced with a simple electroporation device. Dev Dyn 235:1921–1932CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Sorbonne Universités, UPMC Univ Paris 06CNRS, Laboratoire de Biologie du Développement de Villefranche-sur-mer, Observatoire OcéanologiqueVillefranche-sur-merFrance

Personalised recommendations