Skip to main content
SpringerLink
Log in

Microinjection of Exogenous DNA into Eggs of Halocynthia roretzi

  • Chapter
  • First Online:
Transgenic Ascidians

Part of the book series: Advances in Experimental Medicine and Biology ((AEMB,volume 1029))

Abstract

Exogenous gene expression assays during development, including reporters under the control of 5′ upstream enhancer regions of genes, constitute a powerful technique for understanding the mechanisms of tissue-specific gene expression regulation and determining the characteristics, behaviors, and functions of cells that express these genes. The simple marine chordate Halocynthia roretzi has been used for these transgenic analyses for a long time and is an excellent model system for such studies, especially in comparative analyses with other ascidians. In this study, I describe simple methods for microinjecting H. roretzi eggs with exogenous DNA, such as a promoter construct consisting of a 5′ upstream region and a reporter gene, which are prerequisites for transgenic analyses. I also describe basic knowledge regarding this ascidian species, providing reasons why it is an ideal subject for developmental biology studies.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.00
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • Araki I, Satoh N (1996) Cis-regulatory elements conserved in the proximal promoter region of an ascidian embryonic muscle myosin heavy-chain gene. Dev Genes Evol 206:54–63

    Article  CAS  PubMed  Google Scholar 

  • Berna L, Alvarez-Valin F (2014) Evolutionary genomics of fast evolving tunicates. Genome Biol Evol 6:1724–1738. https://doi.org/10.1093/gbe/evu122

    Article  PubMed  PubMed Central  Google Scholar 

  • Berrill NJ (1947) The development and growth of Ciona intestinalis. J Mar Biol Assoc 26:616–625

    Article  CAS  Google Scholar 

  • Castle WE (1896) The early embryology of Ciona intestinalis Flemming (L.) Bull Mus Comp Zool, Harvard 27:201–280

    Google Scholar 

  • Cloney RA (1961) Observations on the mechanism of tail resorption in ascidians. Am Zool 1:67–87

    Article  Google Scholar 

  • Cloney RA (1987) Ascidian metamorphosis: review and analysis. In: Chia FS, Rice ME (eds) Settlement and metamorphosis of marine invertebrate larvae. Elsevier, Amsterdam, pp 255–282

    Google Scholar 

  • Conklin EG (1905) The organization and cell lineage of the ascidian egg. J Acad Nat Sci Philadelphia 13:1–119

    Google Scholar 

  • Costello DP et al. (1957) Methods for obtaining and handling marine eggs and embryos. Marine Biological Laboratory, Woods Hole

    Google Scholar 

  • Delsuc F, Brinkmann H, Chourrout D, Philippe H (2006) Tunicates and not cephalochordates are the closest living relatives of vertebrates. Nature 439:965–968

    Article  CAS  PubMed  Google Scholar 

  • Fuke MT (1983) Self and non-self recognition between gametes of the ascidian, Halocynthia roretzi. Rouxs Arch Dev Biol 192:347–352

    Article  Google Scholar 

  • Herzog RO, Gonell HW (1924) Ueber den feinbau der kunstseide. Kolloid-Zeitschrift 35:201–202

    Article  CAS  Google Scholar 

  • Hirai E (1941) The early development of Cynthia roretzi. Sci Rep Tohoku Imp Univ Biol 16:217–232

    Google Scholar 

  • Hirai E, Tsubata B (1956) On the spawning of an ascidian, Halocynthia roretzi. Bull Mar Biol Stat Asamushi, Tohoku Univ 8:1–4

    Google Scholar 

  • Hudson C, Yasuo H (2008) Similarity and diversity in mechanisms of muscle fate induction between ascidian species. Biol Cell 100:265–277. https://doi.org/10.1042/BC20070144

    Article  PubMed  Google Scholar 

  • Hunt S (1970) Polysaccharide-protein complexes in invertebrates. Academic Press, London, p 149

    Google Scholar 

  • Hŭus J (1939) The effect of light on the spawning in ascidians. Avhandlinger utgitt av Det Norske Videnskaps-Akademi l Oslo I. Mat-Naturv Klasse 4:5–49

    Google Scholar 

  • Ishida K, Satoh N (1999) Genomic organization and the 5′ upstream sequences associated with the specific spatio-temporal expression of HrEpiC, an epidermis-specific gene of the ascidian Halocynthia roretzi. Cell Mol Biol 45:523–536

    CAS  PubMed  Google Scholar 

  • Jeffery WR, Meier S (1983) A yellow crescent cytoskeletal domain in ascidian eggs and its role in early development. Dev Biol 96:125–143

    Article  CAS  PubMed  Google Scholar 

  • Karaiskou A, Swalla BJ, Sasakura Y, Chambon JP (2015) Metamorphosis in solitary ascidians. Genesis 53:34–47

    Article  PubMed  Google Scholar 

  • Katsuyama Y, Matsumoto J, Okada T, Ohtsuka Y, Chen L, Okado H, Okamura Y (2002) Regulation of synaptotagmin gene expression during ascidian embryogenesis. Dev Biol 244:293–304

    Article  CAS  PubMed  Google Scholar 

  • Kim GJ, Yamada A, Nishida H (2000) An FGF signal from endoderm and localized factors in the posterior-vegetal egg cytoplasm pattern the mesodermal tissues in the ascidian embryo. Development 127:2853–2862

    CAS  PubMed  Google Scholar 

  • Kobayashi K, Sawada K, Yamamoto H, Wada S, Saiga H, Nishida H (2003) Maternal macho-1 is an intrinsic factor that makes cell response to the same FGF signal differ between mesenchyme and notochord induction in ascidian embryos. Development 130:5179–5190

    Article  CAS  PubMed  Google Scholar 

  • Kowalevsky A (1861) Entwicklungsgeschichte der einfachen Ascidien. Mem l’Acad St Petersbourg Ser 7(10):1–19

    Google Scholar 

  • Kowalevsky A (1871) Weitere studjen uber dje Entwicklung der einfachen Ascidien. Arch Mikr Anat 7:101–130

    Article  Google Scholar 

  • Kumano G, Nishida H (2007) Ascidian embryonic development: an emerging model system for the study of cell fate specification in chordates. Dev Dyn 236:1732–1747

    Article  CAS  PubMed  Google Scholar 

  • Kumano G, Nishida H (2009) Patterning of an ascidian embryo along the anterior-posterior axis through spatial regulation of competence and induction ability by maternally localized PEM. Dev Biol 331:78–88. https://doi.org/10.1016/j.ydbio.2009.04.024

    Article  CAS  PubMed  Google Scholar 

  • Kumano G, Negoro N, Nishida H (2014) Transcription factor Tbx6 plays a central role in fate determination between mesenchyme and muscle in embryos of the ascidian, Halocynthia roretzi. Develop Growth Differ 56:310–322. https://doi.org/10.1111/dgd.12133

    Article  CAS  Google Scholar 

  • Kusakabe T, Hikosaka A, Satoh N (1995) Coexpression and promoter function in two muscle actin gene complexes of different structural organization in the ascidian Halocynthia roretzi. Dev Biol 169:461–472

    Article  CAS  PubMed  Google Scholar 

  • Lemaire P (2009) Unfolding a chordate developmental program, one cell at a time: invariant cell lineages, short-range inductions and evolutionary plasticity in ascidians. Dev Biol 332:48–60

    Article  CAS  PubMed  Google Scholar 

  • Lemaire P, Piette J (2015) Tunicates: exploring the sea shores and roaming the open ocean. A tribute to Thomas Huxley. Open Biol 5:150053. https://doi.org/10.1098/rsob.150053

    Article  PubMed  PubMed Central  Google Scholar 

  • Lemaire P, Smith WC, Nishida H (2008) Ascidians and the plasticity of the chordate developmental program. Curr Biol 18:R620–R631. https://doi.org/10.1016/j.cub.2008.05.039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Makabe KW, Nishida H (2012) Cytoplasmic localization and reorganization in ascidian eggs: role of postplasmic/PEM RNAs in axis formation and fate determination. Wiley Interdiscip Rev Dev Biol 1:501–518

    Article  CAS  PubMed  Google Scholar 

  • Matsumoto J, Kumano G, Nishida H (2007) Direct activation by Ets and Zic is required for initial expression of the Brachyury gene in the ascidian notochord. Dev Biol 306:870–882

    Article  CAS  PubMed  Google Scholar 

  • Matsumoto J, Katsuyama Y, Ohtsuka Y, Lemaire P, Okamura Y (2008) Functional analysis of synaptotagmin gene regulatory regions in two distantly related ascidian species. Develop Growth Differ 50:543–552. https://doi.org/10.1111/j.1440-169X.2008.01049.x.

    CAS  Google Scholar 

  • Mitani Y, Takahashi H, Satoh N (2001) Regulation of the muscle-specific expression and function of an ascidian T-box gene, as-T2. Development 128:3717–3728

    CAS  PubMed  Google Scholar 

  • Monroy A (1979) Introductory remarks on the segregation of cell lines in the embryo. In: Le Douarin N (ed) In cell lineage, stem cells and cell determination. North-Holland Biomedical Press, Amsterdam, pp 3–13

    Google Scholar 

  • Morgan TH (1923) Removal of the block to self-fertilization in the ascidian Ciona. Proc Nat Acad Sci USA 9:170–171

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Morgan TH (1938) The genetic and the physiological problems of self-sterility in Ciona. I. Data on self- and cross-fertilization. J Exp Zool 78:271–318

    Article  Google Scholar 

  • Negishi T, Takada T, Kawai N, Nishida H (2007) Localized PEM mRNA and protein are involved in cleavage-plane orientation and unequal cell divisions in ascidians. Curr Biol 17:1014–1025

    Article  CAS  PubMed  Google Scholar 

  • Nishida H (1987) Cell lineage analysis in ascidian embryos by intracellular injection of a tracer enzyme. III. Up to the tissue restricted stage. Dev Biol 121:526–541

    Article  CAS  PubMed  Google Scholar 

  • Nishida H (1994) Localization of determinants for formation of the anterior-posterior axis in eggs of the ascidian Halocynthia roretzi. Development 120:3093–3104

    CAS  Google Scholar 

  • Nishida H (1997) Cell lineage and timing of fate restriction, determination and gene expression in ascidian embryos. Semin Cell Dev Biol 8:359–365

    Article  CAS  PubMed  Google Scholar 

  • Nishida H (2005) Specification of embryonic axis and mosaic development in ascidians. Dev Dyn 233:1177–1193

    Article  CAS  PubMed  Google Scholar 

  • Nishida H, Sawada K (2001) Macho-1 encodes a localized mRNA in ascidian eggs that specifies muscle fate during embryogenesis. Nature 409:724–729

    Article  CAS  PubMed  Google Scholar 

  • Numakunai T, Hoshino Z (1973) Biology of the ascidian, Halocynthia roretzi (Drasche), in Mutsu Bay. I. Differences of spawning time and external features. Bull Mar Biol Stat Asamushi, Tohoku Univ 14:191–196

    Google Scholar 

  • Numakunai T, Hoshino Z (1974) Biology of the ascidian, Halocynthia roretzi (Drasche), in Mutsu Bay II. One of the three types which has the spawning season and the different time from two others. Bull Mar Biol Stat Asamushi, Tohoku Univ, 15:23–27

    Google Scholar 

  • Numakunai T, Ishikawa M, Hirai E (1964) Changes of structure stainable with modified Gomori’s aldehyde-fuchsin method in the tadpole larvae of the ascidian, Halocynthia roretzi (V. Drasche), relating to tail resorption. Bull Mar Biol Stat Asamushi Tohoku Univ 12:161–172

    Google Scholar 

  • Oda-Ishii I, Saiga H (2003) Genomic organization and promoter and transcription regulatory regions for the expression in the anterior brain (sensory vesicle) of Hroth, the otx homologue of the ascidian, Halocynthia roretzi. Dev Dyn 227:104–113

    Article  CAS  PubMed  Google Scholar 

  • Oda-Ishii I, Bertrand V, Matsuo I, Lemaire P, Saiga H (2005) Making very similar embryos with divergent genomes: conservation of regulatory mechanisms of Otx between the ascidians Halocynthia roretzi and Ciona intestinalis. Development 132:1663–1674

    Article  CAS  PubMed  Google Scholar 

  • Okada T, Katsuyama Y, Ono F, Okamura Y (2002) The development of three identified motor neurons in the larva of an ascidian, Halocynthia roretzi. Dev Biol 244:278–292

    Article  CAS  PubMed  Google Scholar 

  • Prodon F, Yamada L, Shirae-Kurabayashi M, Nakamura Y, Sasakura Y (2007) Postplasmic/PEM RNAs: a class of localized maternal mRNAs with multiple roles in cell polarity and development in ascidian embryos. Dev Dyn 236:1698–1715

    Article  CAS  PubMed  Google Scholar 

  • Rosati F, De Santis R (1978) Studies on fertilization in ascidians. I. Self-sterility and specific recognition between gametes of Ciona intestinalis. Exp Cell Res 121:111–119

    Article  Google Scholar 

  • Rose MS (1939) Embryonic induction in ascidia. Biol Bull 77:216–232

    Article  Google Scholar 

  • Sardet C, Speksnidjer J, Inoue S, Jaffe L (1989) Fertilization and ooplasmic movements in the ascidian eggs. Development 105:237–249

    CAS  PubMed  Google Scholar 

  • Sardet C, Paix A, Prodon F, Dru P, Chenevert J (2007) From oocyte to 16-cell stage: cytoplasmic and cortical reorganizations that pattern the ascidian embryo. Dev Dyn 236:1716–1731

    Article  CAS  PubMed  Google Scholar 

  • Satoh N (1994) Developmental biology of ascidians. Cambridge University Press, New York

    Google Scholar 

  • Satoh N (2003) The ascidian tadpole larva: comparative molecular development and genomics. Nat Rev Genet 4:285–295

    Article  CAS  PubMed  Google Scholar 

  • Sawada T, Osanai K (1981) The cortical contraction related to the ooplasmic segregation in Ciona intestinalis eggs. Wilhelm Roux’s Arch Dev Biol 190:208–214

    Article  Google Scholar 

  • Stolfi A, Lowe E, Racioppi C, Ristoratore F (2014) Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians. elife 3:e03728. https://doi.org/10.7554/eLife.03728

    Article  PubMed  PubMed Central  Google Scholar 

  • Takahashi H, Mitani Y, Satoh G, Satoh N (1999) Evolutionary alterations of the minimal promoter for notochord-specific Brachyury expression in ascidian embryos. Development 126:3725–3734

    CAS  PubMed  Google Scholar 

  • Takatori N, Kumano G, Saiga H, Nishida H (2010) Segregation of germ layer fates by nuclear migration-dependent localization of Not mRNA. Dev Cell 19:589–598. https://doi.org/10.1016/j.devcel.2010.09.003

    Article  CAS  PubMed  Google Scholar 

  • Toyoda R, Kasai A, Sato S, Wada S, Saiga H, Ikeo K, Gojobori T, Numakunai T, Yamamoto H (2004) Pigment cell lineage-specific expression activity of the ascidian tyrosinase-related gene. Gene 332:61–69

    Article  CAS  PubMed  Google Scholar 

  • Tsagkogeorga G, Turon X, Hopcroft RR, Tilak M-K, Feldstein T, Shenkar N, Loya Y, Huchon D, Douzery EJ, Delsuc F (2009) An updated 18S rRNA phylogeny of tunicates based on mixture and secondary structure models. BMC Evol Biol 9:187. https://doi.org/10.1186/1471-2148-9-187

    Article  PubMed  PubMed Central  Google Scholar 

  • Vienne A, Pontarotti P (2006) Metaphylogeny of 82 gene families sheds a new light on chordate evolution. Int J Biol Sci 2:32–37

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wada S, Toyoda R, Yamamoto H, Saiga H (2002) Ascidian otx gene Hroth activates transcription of the brain-specific gene HrTRP. Dev Dyn 225:46–53

    Article  CAS  PubMed  Google Scholar 

  • Willey A (1893a) Studies on the Protochordata. I. On the origin of the branchial stigmata, praeoral lobe, endostyle, arterial cavities etc. in Ciona intestinalis Linn, with remarks on Clavelina lepadiformis. Q J Microbiol Sci 34:317–360

    Google Scholar 

  • Willey A (1893b) Studies on the Protochordata. II. The development of the neuro-hypophyseal system in Ciona intestinalis and Clavelina lepadiformis, with an account of the origin of the sense organ in Ascidia mentula. Q J Microbiol Sci 35:295–334

    Google Scholar 

  • Yamada A, Nishida H (1999) Distinct parameters are involved in controlling the number of rounds of cell division in each tissue during ascidian embryogenesis. J Exp Zool 284:379–391

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

I would like to thank Dr. Takaharu Numakunai for providing the image in Fig. 4.1b, Dr. Hiroki Nishida for providing the data on the developmental timetable in Fig. 4.2, and Ms. Kaori Miyaoku and Mr. Tao Zheng for their help in preparing the images in Fig. 4.4a–f.

Author information

Authors and Affiliations

  1. Asamushi Research Center for Marine Biology, Graduate School of Life Sciences, Tohoku University, Asamushi, Aomori, Japan

    Gaku Kumano

Authors
  1. Gaku Kumano
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gaku Kumano .

Editor information

Editors and Affiliations

  1. Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, Japan

    Yasunori Sasakura

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Nature Singapore Pte Ltd.

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Kumano, G. (2018). Microinjection of Exogenous DNA into Eggs of Halocynthia roretzi . In: Sasakura, Y. (eds) Transgenic Ascidians . Advances in Experimental Medicine and Biology, vol 1029. Springer, Singapore. https://doi.org/10.1007/978-981-10-7545-2_4

Download citation

Publish with us

Policies and ethics

Search

Navigation