Skip to main content
SpringerLink
Log in

Embryological Methods in Ascidians: The Villefranche-sur-Mer Protocols

  • Protocol
  • First Online:
Vertebrate Embryogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 770))

Abstract

Ascidians (marine invertebrates: urochordates) are thought to be the closest sister groups of vertebrates. They are particularly attractive models because of their non-duplicated genome and the fast and synchronous development of large populations of eggs into simple tadpoles made of about 3,000 cells. As a result of stereotyped asymmetric cleavage patterns all blastomeres become fate restricted between the 16- and 110 cell stage through inheritance of maternal determinants and/or cellular interactions. These advantageous features have allowed advances in our understanding of the nature and role of maternal determinants, inductive interactions, and gene networks that are involved in cell lineage specification and differentiation of embryonic tissues. Ascidians have also contributed to our understanding of fertilization, cell cycle control, self-recognition, metamorphosis, and regeneration. In this chapter we provide basic protocols routinely used at the marine station in Villefranche-sur-Mer using the cosmopolitan species of reference Ciona intestinalis and the European species Phallusia mammillata. These two models present complementary advantages with regard to molecular, functional, and imaging approaches. We describe techniques for basic culture of embryos, micro-injection, in vivo labelling, micro-manipulations, fixation, and immuno-labelling. These methods allow analysis of calcium signals, reorganizations of cytoplasmic and cortical domains, meiotic and mitotic cell cycle and cleavages as well as the roles of specific genes and cellular interactions. Ascidians eggs and embryos are also an ideal material to isolate cortical fragments and to isolate and re-associate individual blastomeres. We detail the experimental manipulations which we have used to understand the structure and role of the egg cortex and of specific blastomeres during development.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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

  1. Raff, R. A. and Love, A. C. (2004) Kowalevsky, comparative evolutionary embryology, and the intellectual lineage of evo-devo. J. Exp. Zool. B Mol. Dev. Evol. 302, 19–34.

    PubMed  Google Scholar 

  2. Fol, H. (1879) Recherches sur la fécondation et le commencement de l‘hénogénie chez divers animaux. Mém. Soc. de Phys. et d’Hist. Nat. Genève 26, 92–397.

    Google Scholar 

  3. Chabry, L. (1887) Contribution à l‘embryologie normale et tératologique des Ascidies simples. Paris: Felix Alcan.

    Google Scholar 

  4. Conklin, E. G. (1905) The organization and cell lineage of the ascidian egg. J. Acad. Sci. Philadelphia 13, 1–119.

    Google Scholar 

  5. Reverberi, G. (1956) The mitochondrial pattern in the development of the ascidian egg. Experientia (Basel) 12, 55–56.

    Article  Google Scholar 

  6. Nishida, H. (1997) Cell fate specification by localized cytoplasmic determinants and cell interactions in ascidian embryos. Int. Rev. Cytol. 176, 245–306.

    Article  PubMed  CAS  Google Scholar 

  7. Satoh, N. (1994) Developmental Biology of Ascidians. Cambridge University Press, New York.

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  10. Negishi, T., Takada, T., Kawai, N., and 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  PubMed  CAS  Google Scholar 

  11. Momose, T. and Houliston, E. (2007) Two Oppositely Localised Frizzled RNAs as Axis Determinants in a Cnidarian Embryo. PLoS Biol. 5, e70.

    Article  PubMed  CAS  Google Scholar 

  12. Kloc, M. and Etkin, L. D. (2005) RNA localization mechanisms in oocytes. J. Cell. Sci. 118, 269–282.

    Article  PubMed  CAS  Google Scholar 

  13. St Johnston, D. (2005) Moving messages: the intracellular localization of mRNAs. Nat. Rev. Mol. Cell. Biol. 6, 363–375.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  15. Lemaire, P., Smith, W. C., and Nishida, H. (2008) Ascidians and the plasticity of the chordate developmental program. Curr. Biol. 18, R620–31.

    Article  PubMed  CAS  Google Scholar 

  16. Satou, Y., Satoh, N., and Imai, K. S. (2009) Gene regulatory networks in the early ascidian embryo. Biochim. Biophys. Acta 1789, 268–273.

    Article  PubMed  CAS  Google Scholar 

  17. Satoh, N. (2007) Toward a new paradigm for studying ascidian developmental dynamics. Dev. Dyn. 236, 1695–1697.

    Article  CAS  Google Scholar 

  18. Imai, K. S., Levine, M., Satoh, N., and Satou, Y. (2006) Regulatory blueprint for a chordate embryo. Science 312, 1183–1187.

    Article  PubMed  CAS  Google Scholar 

  19. Hudson, C. and Yasuo, H. (2008) Similarity and diversity in mechanisms of muscle fate induction between ascidian species. Biol. Cell 100, 265–277.

    Article  PubMed  Google Scholar 

  20. Picco, V., Hudson, C., and Yasuo, H. (2007) Ephrin-Eph signalling drives the asymmetric division of notochord/neural precursors in Ciona embryos. Development 134, 1491–1497.

    Article  PubMed  CAS  Google Scholar 

  21. Sardet, C., Swalla, B. J., Satoh, N., Sasakura, Y., Branno, M., Thompson, E. M., Levine, M., and Nishida, H. (2008) Euro chordates: Ascidian community swims ahead. The 4th International Tunicate meeting in Villefranche sur Mer. Dev. Dyn. 237, 1207–1213.

    Article  PubMed  Google Scholar 

  22. Fujii, S., Nishio, T., and Nishida, H. (2008) Cleavage pattern, gastrulation, and neurulation in the appendicularian, Oikopleura dioica. Dev. Genes Evol. 218, 69–79.

    Article  PubMed  Google Scholar 

  23. Dehal, P., Satou, Y., Campbell, R. K., Chapman, J., Degnan, B., De Tomaso, A., Davidson, B., Di Gregorio, A., Gelpke, M., Goodstein, D. M., Harafuji, N., Hastings, K. E., Ho, I., Hotta, K., Huang, W., Kawashima, T., Lemaire, P., Martinez, D., Meinertzhagen, I. A., Necula, S., Nonaka, M., Putnam, N., Rash, S., Saiga, H., Satake, M., Terry, A., Yamada, L., Wang, H. G., Awazu, S., Azumi, K., Boore, J., Branno, M., Chin-Bow, S., DeSantis, R., Doyle, S., Francino, P., Keys, D. N., Haga, S., Hayashi, H., Hino, K., Imai, K. S., Inaba, K., Kano, S., Kobayashi, K., Kobayashi, M., Lee, B. I., Makabe, K. W., Manohar, C., Matassi, G., Medina, M., Mochizuki, Y., Mount, S., Morishita, T., Miura, S., Nakayama, A., Nishizaka, S., Nomoto, H., Ohta, F., Oishi, K., Rigoutsos, I., Sano, M., Sasaki, A., Sasakura, Y., Shoguchi, E., Shin-i, T., Spagnuolo, A., Stainier, D., Suzuki, M. M., Tassy, O., Takatori, N., Tokuoka, M., Yagi, K., Yoshizaki, F., Wada, S., Zhang, C., Hyatt, P. D., Larimer, F., Detter, C., Doggett, N., Glavina, T., Hawkins, T., Richardson, P., Lucas, S., Kohara, Y., Levine, M., Satoh, N., and Rokhsar, D. S. (2002) The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science 298, 2157–2167.

    Article  PubMed  CAS  Google Scholar 

  24. Nishida, H. (1996) Vegetal egg cytoplasm promotes gastrulation and is responsible for specification of vegetal blastomeres in embryos of the ascidian Halocynthia roretzi. Development 122, 1271–1279.

    PubMed  CAS  Google Scholar 

  25. Erives, A. and Levine, M. (2000) Characterization of a maternal T-Box gene in Ciona intestinalis. Dev. Biol. 225, 169–178.

    Article  PubMed  CAS  Google Scholar 

  26. Hudson, C., Lotito, S., and Yasuo, H. (2007) Sequential and combinatorial inputs from Nodal, Delta2/Notch and FGF/MEK/ERK signalling pathways establish a grid-like organisation of distinct cell identities in the ascidian neural plate. Development 134, 3527–3537.

    Article  PubMed  CAS  Google Scholar 

  27. Tiozzo, S., Voskoboynik, A., Brown, F. D., and De Tomaso, A. W. (2008) A conserved role of the VEGF pathway in angiogenesis of an ectodermally-derived vasculature. Dev. Biol. 315, 243–255.

    Article  PubMed  CAS  Google Scholar 

  28. Nishiyama, A. and Fujiwara, S. (2008) RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryo. Dev. Growth Differ. 50, 521–529.

    Article  PubMed  CAS  Google Scholar 

  29. Sasakura, Y., Konno, A., Mizuno, K., Satoh, N., and Inaba, K. (2008) Enhancer detection in the ascidian Ciona intestinalis with transposase-expressing lines of Minos. Dev. Dyn. 237, 39–50.

    Article  PubMed  CAS  Google Scholar 

  30. Dahlberg, C., Auger, H., Dupont, S., Sasakura, Y., Thorndyke, M., and Joly, J. S. (2009) Refining the Ciona intestinalis model of central nervous system regeneration. PLoS ONE 4, e4458.

    Article  PubMed  CAS  Google Scholar 

  31. Joly, J. S., Kano, S., Matsuoka, T., Auger, H., Hirayama, K., Satoh, N., Awazu, S., Legendre, L., and Sasakura, Y. (2007) Culture of Ciona intestinalis in closed systems. Dev. Dyn. 236, 1832–1840.

    Article  PubMed  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  33. Prodon, F., Dru, P., Roegiers, F., and Sardet, C. (2005) Polarity of the ascidian egg cortex and relocalization of cER and mRNAs in the early embryo. J. Cell. Sci. 118, 2393–2404.

    Article  PubMed  CAS  Google Scholar 

  34. Dumollard, R. and Sardet, C. (2001) Three different calcium wave pacemakers in ascidian eggs. J. Cell. Sci. 114, 2471–2481.

    PubMed  CAS  Google Scholar 

  35. Prodon, F., Chenevert, J., Hebras, C., Dumollard, R., Faure, E., Gonzalez-Garcia, J., Nishida, H., Sardet, C., and McDougall, A. (2010) Dual mechanism controls asymmetric spindle position in ascidian germ cell precursors. Development 137, 2011–2021.

    Google Scholar 

  36. Nishikata, T., Hibino, T., and Nishida, H. (1999) The centrosome-attracting body, microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo. Dev. Biol. 209, 72–85.

    Article  PubMed  CAS  Google Scholar 

  37. Johnson, D. S., Davidson, B., Brown, C. D., Smith, W. C., and Sidow, A. (2004) Noncoding regulatory sequences of Ciona exhibit strong correspondence between evolutionary constraint and functional importance. Genome Res. 14, 2448–2456.

    Article  PubMed  CAS  Google Scholar 

  38. Hill, M. M., Broman, K. W., Stupka, E., Smith, W. C., Jiang, D., and Sidow, A. (2008) The C. savignyi genetic map and its integration with the reference sequence facilitates insights into chordate genome evolution. Genome Res. 18, 1369–1379.

    Article  PubMed  CAS  Google Scholar 

  39. Sobral, D., Tassy, O., and Lemaire, P. (2009) Highly Divergent Gene Expression Programs Can Lead to Similar Chordate Larval Body Plans. Curr. Biol. 19, 2014–2019.

    Article  PubMed  CAS  Google Scholar 

  40. Corbo, J. C., Di Gregorio, A., and Levine, M. (2001) The ascidian as a model organism in developmental and evolutionary biology. Cell 106, 535–538.

    Article  PubMed  CAS  Google Scholar 

  41. Manni, L., Zaniolo, G., Cima, F., Burighel, P., and Ballarin, L. (2007) Botryllus schlosseri: a model ascidian for the study of asexual reproduction. Dev. Dyn. 236, 335–352.

    Article  PubMed  CAS  Google Scholar 

  42. Lamy, C. and Lemaire, P. (2008) Ascidian embryos: from the birth of experimental embryology to the analysis of gene regulatory networks. Med. Sci. (Paris) 24, 263–269.

    Article  Google Scholar 

  43. Swalla, B. J. (2004) Procurement and culture of ascidian embryos. Methods Cell Biol. 74, 115–141.

    Article  PubMed  Google Scholar 

  44. Paix, A., Chenevert, J., and Sardet, C. (2011) Localization and anchorage of maternal mRNAs to cortical structures of ascidian eggs and embryos using high resolution in situ hybridization. Messages on the move: Techniques in RNA visualization. Methods Mol Biol. 714, 49–70.

    Google Scholar 

  45. Hendrickson, C., Christiaen, L., Deschet, K., Jiang, D., Joly, J. S., Legendre, L., Nakatani, Y., Tresser, J., and Smith, W. C. (2004) Culture of adult ascidians and ascidian genetics. Methods Cell Biol. 74, 143–170.

    Article  PubMed  CAS  Google Scholar 

  46. Jaffe, L. A. and Terasaki, M. (2004) Quantitative microinjection of oocytes, eggs, and embryos. Methods Cell Biol. 74, 219–242.

    Article  PubMed  Google Scholar 

  47. Lemaire, P., Garrett, N., and Gurdon, J. B. (1995) Expression cloning of Siamois, a Xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis. Cell 81, 85–94.

    Article  PubMed  CAS  Google Scholar 

  48. Roure, A., Rothbacher, U., Robin, F., Kalmar, E., Ferone, G., Lamy, C., Missero, C., Mueller, F., and Lemaire, P. (2007) A multicassette Gateway vector set for high throughput and comparative analyses in ciona and vertebrate embryos. PLoS ONE 2, e916.

    Article  PubMed  CAS  Google Scholar 

  49. Turner, D. L. and Weintraub, H. (1994) Expression of achaete-scute homolog 3 in Xenopus embryos converts ectodermal cells to a neural fate. Genes Dev. 8, 1434–1447.

    Article  PubMed  CAS  Google Scholar 

  50. Prodon, F., Chenevert, J., and Sardet, C. (2006) Establishment of animal-vegetal polarity during maturation in ascidian oocytes. Dev. Biol. 290, 297–311.

    Article  PubMed  CAS  Google Scholar 

  51. Zalokar, M. and Sardet, C. (1984) Tracing of cell lineage in embryonic development of Phallusia mammillata (Ascidia) by vital staining of mitochondria. Dev. Biol. 102, 195–205.

    Article  PubMed  CAS  Google Scholar 

  52. Speksnijder, J. E., Jaffe, L. F., and Sardet, C. (1989) Polarity of sperm entry in the ascidian egg. Dev. Biol. 133, 180–184.

    Article  PubMed  CAS  Google Scholar 

  53. Dumollard, R., Hammar, K., Porterfield, M., Smith, P. J., Cibert, C., Rouviere, C., and Sardet, C. (2003) Mitochondrial respiration and Ca2+ waves are linked during fertilization and meiosis completion. Development 130, 683–692.

    Article  PubMed  CAS  Google Scholar 

  54. Roegiers, F., McDougall, A., and Sardet, C. (1995) The sperm entry point defines the orientation of the calcium-induced contraction wave that directs the first phase of cytoplasmic reorganization in the ascidian egg. Development 121, 3457–3466.

    PubMed  CAS  Google Scholar 

  55. Roegiers, F., Djediat, C., Dumollard, R., Rouviere, C., and Sardet, C. (1999) Phases of cytoplasmic and cortical reorganizations of the ascidian zygote between fertilization and first division. Development 126, 3101–3117.

    PubMed  CAS  Google Scholar 

  56. Speksnijder, J. E., Terasaki, M., Hage, W. J., Jaffe, L. F., and Sardet, C. (1993) Polarity and reorganization of the endoplasmic reticulum during fertilization and ooplasmic segregation in the ascidian egg. J. Cell Biol. 120, 1337–1346.

    Article  PubMed  CAS  Google Scholar 

  57. McDougall, A. and Sardet, C. (1995) Function and characteristics of repetitive calcium waves associated with meiosis. Curr. Biol. 5, 318–328.

    Article  PubMed  CAS  Google Scholar 

  58. Paix, A., Yamada, L., Dru, P., Lecordier, H., Pruliere, G., Chenevert, J., Satoh, N., and Sardet, C. (2009) Cortical anchorages and cell type segregations of maternal postplasmic/PEM RNAs in ascidians. Dev. Biol. 336, 96–111.

    Article  PubMed  CAS  Google Scholar 

  59. Patalano, S., Pruliere, G., Prodon, F., Paix, A., Dru, P., Sardet, C., and Chenevert, J. (2006) The aPKC-PAR-6-PAR-3 cell polarity complex localizes to the centrosome attracting body, a macroscopic cortical structure responsible for asymmetric divisions in the early ascidian embryo. J. Cell. Sci. 119, 1592–1603.

    Article  PubMed  CAS  Google Scholar 

  60. Sardet, C., Prodon, F., Dumollard, R., Chang, P., and Chenevert, J. (2002) Structure and function of the egg cortex from oogenesis through fertilization. Dev. Biol. 241, 1–23.

    Article  PubMed  CAS  Google Scholar 

  61. Sardet, C., Speksnijder, J., Terasaki, M., and Chang, P. (1992) Polarity of the ascidian egg cortex before fertilization. Development 115, 221–237.

    PubMed  CAS  Google Scholar 

  62. Tassy, O., Daian, F., Hudson, C., Bertrand, V., and Lemaire, P. (2006) A quantitative approach to the study of cell shapes and interactions during early chordate embryogenesis. Curr. Biol. 16, 345–358.

    Article  PubMed  CAS  Google Scholar 

  63. Satou, Y., Kawashima, T., Shoguchi, E., Nakayama, A., and Satoh, N. (2005) An integrated database of the ascidian, Ciona intestinalis: towards functional genomics. Zoolog. Sci. 22, 837–843.

    Article  PubMed  CAS  Google Scholar 

  64. Sierro, N., Kusakabe, T., Park, K. J., Yamashita, R., Kinoshita, K., and Nakai, K. (2006) DBTGR: a database of tunicate promoters and their regulatory elements. Nucleic Acids Res. 34, D552–5.

    Article  PubMed  CAS  Google Scholar 

  65. Hotta, K., Mitsuhara, K., Takahashi, H., Inaba, K., Oka, K., Gojobori, T., and Ikeo, K. (2007) A web-based interactive developmental table for the ascidian Ciona intestinalis, including 3D real-image embryo reconstructions: I. From fertilized egg to hatching larva. Dev. Dyn. 236, 1790–1805.

    Article  PubMed  Google Scholar 

  66. Ikuta, T. and Saiga, H. (2007) Dynamic change in the expression of developmental genes in the ascidian central nervous system: revisit to the tripartite model and the origin of the midbrain-hindbrain boundary region. Dev. Biol. 312, 631–643.

    Article  PubMed  CAS  Google Scholar 

  67. Sardet, C., Nishida, H., Prodon, F., and Sawada, K. (2003) Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex. Development 130, 5839–5849.

    Article  PubMed  CAS  Google Scholar 

  68. Wada, S., Katsuyama, Y., Yasugi, S., and Saiga, H. (1995) Spatially and temporally regulated expression of the LIM class homeobox gene Hrlim suggests multiple distinct functions in development of the ascidian, Halocynthia roretzi. Mech. Dev. 51, 115–126.

    Article  PubMed  CAS  Google Scholar 

  69. Levasseur, M. and McDougall, A. (2000) Sperm-induced calcium oscillations at fertilisation in ascidians are controlled by cyclin B1-dependent kinase activity. Development 127, 631–641.

    PubMed  CAS  Google Scholar 

  70. Speksnijder, J. E., Sardet, C., and Jaffe, L. F. (1990b) The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation. J. Cell Biol. 110, 1589–1598.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

We thank personnel of the Developmental Biology unit (BioDev, Villefranche-sur-Mer Marine Station, CNRS/UPMC UMR7009, France) for helpful discussions and previous members of the Villefranche lab, Fabrice Roegiers and François Prodon. We thank M. Khamla for help with graphics. The laboratories of C. Sardet, A. McDougall and H. Yasuo are supported by ANR (Agence Nationale de la Recherche), CNRS-ACI (Centre National de la Recherche Scientifique—Action Concertée Incitative), UPMC (Université Pierre et Marie Curie, Paris 06), ARC (Association pour la Recherche sur le Cancer) and AFM (Association Française contre les Myopathies).

Author information

Authors and Affiliations

  1. Biologie du Développement, UMR 7009 CNRS/UPMC, Observatoire Océanologique, Villefranche sur Mer, 06230, France

    Christian Sardet, Alex McDougall, Hitoyoshi Yasuo, Janet Chenevert, Gérard Pruliere, Rémi Dumollard, Clare Hudson, Celine Hebras, Ngan Le Nguyen & Alexandre Paix

  2. Univ Nice-Sophia Antipolis, Nice, 0600, France

    Alexandre Paix

Authors
  1. Christian Sardet
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alex McDougall
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hitoyoshi Yasuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Janet Chenevert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gérard Pruliere
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rémi Dumollard
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Clare Hudson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Celine Hebras
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Ngan Le Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Alexandre Paix
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Christian Sardet .

Editor information

Editors and Affiliations

  1. Dept. Genetics, University of Wisconsin, Madison, Henry Mall 425-G, Madison, 53706-1574, Wisconsin, USA

    Francisco J. Pelegri

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Sardet, C. et al. (2011). Embryological Methods in Ascidians: The Villefranche-sur-Mer Protocols. In: Pelegri, F. (eds) Vertebrate Embryogenesis. Methods in Molecular Biology, vol 770. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-210-6_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-210-6_14

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-209-0

  • Online ISBN: 978-1-61779-210-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation