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A Simple Method to Identify Ascidian Brain Lineage Cells at Neural Plate Stages Following In Situ Hybridization

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Brain Development

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2047))

Abstract

The technique of in situ hybridization can be used to visualize the spatial and temporal pattern of gene expression during development. Ascidians are invertebrate chordates that develop with a fixed cell cleavage pattern into a tadpole larvae. The knowledge of the cell lineage allows the earliest steps of cell fate specification to be followed at a single cell resolution. This protocol describes preparation of Ciona intestinalis embryos, classical in situ hybridization protocol coupled with nuclear staining, and a guide to identify gene expression in specific precursors of the developing brain at neural plate stages of development.

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References

  1. Satoh N, Rokhsar D, Nishikawa T (2014) Chordate evolution and the three-phylum system. Proc Biol Sci 281:20141729

    Article  Google Scholar 

  2. Hashimoto H, Robin FB, Sherrard KM et al (2015) Sequential contraction and exchange of apical junctions drives zippering and neural tube closure in a simple chordate. Dev Cell 32:241–255

    Article  CAS  Google Scholar 

  3. Cole AG, Meinertzhagen IA (2004) The central nervous system of the ascidian larva: mitotic history of cells forming the neural tube in late embryonic Ciona intestinalis. Dev Biol 271:239–262

    Article  CAS  Google Scholar 

  4. Taniguchi K, Nishida H (2004) Tracing cell fate in brain formation during embryogenesis of the ascidian Halocynthia roretzi. Develop Growth Differ 46:163–180

    Article  Google Scholar 

  5. Brozovic M, Martin C, Dantec C et al (2016) ANISEED 2015: a digital framework for the comparative developmental biology of ascidians. Nucleic Acids Res 44:D808–D818

    Article  CAS  Google Scholar 

  6. Brozovic M, Dantec C, Dardaillon J et al (2018) ANISEED 2017: extending the integrated ascidian database to the exploration and evolutionary comparison of genome-scale datasets. Nucleic Acids Res 46:D718–D725

    Article  CAS  Google Scholar 

  7. Gilchrist MJ, Sobral D, Khoueiry P et al (2015) A pipeline for the systematic identification of non-redundant full-ORF cDNAs for polymorphic and evolutionary divergent genomes: application to the ascidian Ciona intestinalis. Dev Biol 404:149–163

    Article  CAS  Google Scholar 

  8. Lemaire P (2011) Evolutionary crossroads in developmental biology: the tunicates. Development 138:2143–2152

    Article  CAS  Google Scholar 

  9. Satou Y, Kawashima T, Shoguchi E et al (2005) An integrated database of the ascidian, Ciona intestinalis: towards functional genomics. Zool Sci 22:837–843

    Article  CAS  Google Scholar 

  10. Hudson C (2016) The central nervous system of ascidian larvae. Wiley Interdiscip Rev Dev Biol. https://doi.org/10.1002/wdev.239

    Article  Google Scholar 

  11. Satoh N (2014) Developmental genomics of Ascidians. Wiley-Blackwell, New York

    Google Scholar 

  12. Satou Y, Imai KS (2015) Gene regulatory systems that control gene expression in the Ciona embryo. Proc Jpn Acad Ser B Phys Biol Sci 91:33–51

    Article  CAS  Google Scholar 

  13. Delsuc F, Philippe H, Tsagkogeorga G et al (2018) A phylogenomic framework and timescale for comparative studies of tunicates. BMC Biol 16:39

    Article  Google Scholar 

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

    Article  Google Scholar 

  15. Stolfi A, Lowe EK, Racioppi C et al (2014) Divergent mechanisms regulate conserved cardiopharyngeal development and gene expression in distantly related ascidians. elife 3:e03728

    Article  Google Scholar 

  16. Ryan K, Lu Z, Meinertzhagen IA (2016) The CNS connectome of a tadpole larva of Ciona intestinalis (L.) highlights sidedness in the brain of a chordate sibling. Elife 5

    Google Scholar 

  17. Nicol D, Meinertzhagen IA (1991) Cell counts and maps in the larval central nervous system of the ascidian Ciona intestinalis (L.). J Comp Neurol 309:415–429

    Article  CAS  Google Scholar 

  18. Tsuda M, Sakurai D, Goda M (2003) Direct evidence for the role of pigment cells in the brain of ascidian larvae by laser ablation. J Exp Biol 206:1409–1417

    Article  Google Scholar 

  19. Eakin RM, Kuda A (1971) Ultrastructure of sensory receptors in Ascidian tadpoles. Z Zellforsch 112:287–312

    Article  CAS  Google Scholar 

  20. Oonuma K, Tanaka M, Nishitsuji K et al (2016) Revised lineage of larval photoreceptor cells in Ciona reveals archetypal collaboration between neural tube and neural crest in sensory organ formation. Dev Biol 420:178–185

    Article  CAS  Google Scholar 

  21. Yoshida K, Saiga H (2011) Repression of Rx gene on the left side of the sensory vesicle by Nodal signaling is crucial for right-sided formation of the ocellus photoreceptor in the development of Ciona intestinalis. Dev Biol 354:144–150

    Article  CAS  Google Scholar 

  22. Veeman MT, Newman-Smith E, El-Nachef D et al (2010) The ascidian mouth opening is derived from the anterior neuropore: reassessing the mouth/neural tube relationship in chordate evolution. Dev Biol 344:138–149

    Article  CAS  Google Scholar 

  23. Wada S, Katsuyama Y, Yasugi S et al (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  CAS  Google Scholar 

  24. Nicol D, Meinertzhagen IA (1988) Development of the central nervous system of the larva of the ascidian, Ciona intestinalis L. I. The early lineages of the neural plate. Dev Biol 130:721–736

    Article  CAS  Google Scholar 

  25. Nicol D, Meinertzhagen IA (1988) Development of the central nervous system of the larva of the ascidian, Ciona intestinalis L. II. Neural plate morphogenesis and cell lineages during neurulation. Dev Biol 130:737–766

    Article  CAS  Google Scholar 

  26. Haupaix N, Abitua PB, Sirour C et al (2014) Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS. Dev Biol 394:170–180

    Article  CAS  Google Scholar 

  27. Navarrete IA, Levine M (2016) Nodal and FGF coordinate ascidian neural tube morphogenesis. Development 143:4665–4675

    Article  CAS  Google Scholar 

  28. Ikuta T, 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  CAS  Google Scholar 

  29. Imai KS, Stolfi A, Levine M et al (2009) Gene regulatory networks underlying the compartmentalization of the Ciona central nervous system. Development 136:285–293

    Article  CAS  Google Scholar 

  30. Abitua PB, Wagner E, Navarrete IA et al (2012) Identification of a rudimentary neural crest in a non-vertebrate chordate. Nature 492:104–107

    Article  CAS  Google Scholar 

  31. Stolfi A, Levine M (2011) Neuronal subtype specification in the spinal cord of a protovertebrate. Development 138:995–1004

    Article  CAS  Google Scholar 

  32. Hotta K, Mitsuhara K, Takahashi H et al (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  Google Scholar 

  33. Jing L (2012) Preparation of Torula yeast RNA for Hybe solutions. BIO-Protoc 2

    Google Scholar 

  34. Lauter G, Söll I, Hauptmann G (2011) Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems. BMC Dev Biol 11:43

    Article  CAS  Google Scholar 

  35. Thisse B, Thisse C (2014) In situ hybridization on whole-mount zebrafish embryos and young larvae. In: Nielsen BS (ed) In situ hybridization protocols. Springer New York, New York, NY, pp 53–67

    Chapter  Google Scholar 

  36. Shimeld SM, Levin M (2006) Evidence for the regulation of left-right asymmetry in Ciona intestinalis by ion flux. Dev Dyn 235:1543–1553

    Article  CAS  Google Scholar 

  37. Conklin EG (1905) The organisation and cell lineage of the ascidian egg. Science 23:340–344

    Google Scholar 

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Acknowledgments

The author is a CNRS researcher in the laboratory of Hitoyoshi Yasuo. I would like to thank H. Yasuo and Cathy Sirour for their help and for critical reading of the manuscript. Many thanks to Hiroki Nishida and Shih Yu (Osaka University) for kindly sharing their DAPI staining and mounting protocol and agreeing to publish it in this chapter. This work was supported by Central National de la Recherche Scientifique (CNRS), Sorbonne Université, Sorbonne Université Emegence project (2016), and the Agence Nationale de la Recherche (ANR-09-BLAN-0013-01, ANR-17-CE13-0003).

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Authors and Affiliations

  1. Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Sorbonne Université, CNRS, Villefranche-sur-mer, France

    Clare Hudson

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  1. Clare Hudson
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Correspondence to Clare Hudson .

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Editors and Affiliations

  1. Department of Biology, University of Fribourg, Fribourg, Switzerland

    Simon G. Sprecher

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Hudson, C. (2020). A Simple Method to Identify Ascidian Brain Lineage Cells at Neural Plate Stages Following In Situ Hybridization. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_18

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  • DOI: https://doi.org/10.1007/978-1-4939-9732-9_18

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9731-2

  • Online ISBN: 978-1-4939-9732-9

  • eBook Packages: Springer Protocols

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