Abstract

The sudden appearance of the neural crest and neurogenic placodes in early branching vertebrates has puzzled biologists for over a century1. These embryonic tissues contribute to the development of the cranium and associated sensory organs, which were crucial for the evolution of the vertebrate “new head”2,3. A previous study suggests that rudimentary neural crest cells existed in ancestral chordates4. However, the evolutionary origins of neurogenic placodes have remained obscure owing to a paucity of embryonic data from tunicates, the closest living relatives to those early vertebrates5. Here we show that the tunicate Ciona intestinalis exhibits a proto-placodal ectoderm (PPE) that requires inhibition of bone morphogenetic protein (BMP) and expresses the key regulatory determinant Six1/2 and its co-factor Eya, a developmental process conserved across vertebrates. The Ciona PPE is shown to produce ciliated neurons that express genes for gonadotropin-releasing hormone (GnRH), a G-protein-coupled receptor for relaxin-3 (RXFP3) and a functional cyclic nucleotide-gated channel (CNGA), which suggests dual chemosensory and neurosecretory activities. These observations provide evidence that Ciona has a neurogenic proto-placode, which forms neurons that appear to be related to those derived from the olfactory placode and hypothalamic neurons of vertebrates. We discuss the possibility that the PPE-derived GnRH neurons of Ciona resemble an ancestral cell type, a progenitor to the complex neuronal circuit that integrates sensory information and neuroendocrine functions in vertebrates.

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Accessions

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

The coding sequence of SOG/Chemokine-like has been deposited in GenBank/EMBL/DDBJ under the accession KR902348.

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Acknowledgements

We thank Y. Miyamoto and M. Kotera for technical assistance and A. Stolfi for cloning Chordin>GFP. This work was supported by a grant from the National Institutes of Health (NS076542) and by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (25650118, 25290067) and from the Japan Space Forum (h160179). Portions of this study were facilitated by the National Bio-Resource Project of the Ministry of Education, Culture, Sports, Science and Technology in Japan. The work of C.H. in the laboratory of H. Yasuo was funded by the Agence Nationale de la Recherche (ANR-09-BLAN-0013-01). P.B.A. and A.N.K. were supported by predoctoral fellowships from the National Science Foundation and California Institute for Regenerative Medicine, respectively.

Author information

Author notes

    • Philip Barron Abitua
    • , T. Blair Gainous
    •  & Michael Levine

    Present addresses: Department of Molecular and Cellular Biology, Harvard University, Massachusetts 02138, USA (P.B.A.); Cardiovascular Research Institute, University of California, San Francisco, California 94158, USA (T.B.G.): Lewis-Sigler Institute of Integrative Genomics, Princeton University, New Jersey 08544, USA (M.L.).

Affiliations

  1. Center for Integrative Genomics, Division of Genetics, Genomics and Development, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA

    • Philip Barron Abitua
    • , T. Blair Gainous
    • , Angela N. Kaczmarczyk
    • , Christopher J. Winchell
    •  & Michael Levine
  2. Sorbonne Universités, Université Pierre et Marie Curie, Centre National de la Recherche Scientifique, Laboratoire de Biologie du Développement de Villefranche-sur-mer, Observatoire Océanologique, 06230 Villefranche-sur-mer, France

    • Clare Hudson
  3. Graduate School of Life Science, University of Hyogo, Kamigori, Hyogo 678-1297, Japan

    • Kaori Kamata
    • , Masashi Nakagawa
    •  & Motoyuki Tsuda
  4. Institute for Integrative Neurobiology and Department of Biology, Faculty of Science and Engineering, Konan University, Kobe 658-8501, Japan

    • Takehiro G. Kusakabe

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Contributions

P.B.A. designed and performed most experiments in consultation with M.L. T.B.G. performed the Six1/2 and Eya in situ hybridizations. A.N.K. performed the larval colorimetric in situ hybridizations. C.J.W. performed the phylogenetic analysis. C.H. performed the BMP2/4 and Chordin in situ hybridizations. T.G.K. identified and cloned Ciona CNGs and GnRHs and analysed their expression. T.G.K., M.N., K.K. and M.T. performed functional analysis of CNGs.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Michael Levine.

Extended data

Supplementary information

Videos

  1. 1.

    Six1/2 expression in a neurula stage embryo

    This video shows the neurulation of an embryo co-electroporated with Dmrt>H2B:YFP and Six1/2>mCherry from a dorsal view. The time-lapse covers a period of about 120 minutes, during which time Six1/2>mCherry becomes expressed in posterior row V cells. The video pauses to show the derivatives of rows III-IV (orange) and rows V-VI (white) at the 10th generation (i.e., a10.97, a10.98, etc.). Six1/2>mCherry is initially expressed in the derivatives of a10.77, a10.69, a10.101, and a10.103. This embryo was previously analyzed to show an extra cell division in the pigment cell lineage (Haupaix, N. et al. Ephrin-mediated restriction of ERK1/2 activity delimits the number of pigment cells in the Ciona CNS. Dev. Biol. 394, 170–180 (2014)).

  2. 2.

    Co-expression of reporter genes in PPE-derived neurons

    This video shows lateral views of the co-localization of SOG/Chemokine receptor-like>mCherry, RXFP3>mCherry, and CNGA>mCherry with GnRH>GFP in three larval stage embryos respectively. In each larva the mCherry reporter is shown first, as the plane of the Z-section pans through the left and right side of the animal. Then the GnRH>GFP signal is gradually faded in to show co-localization in the PPE-derived neurons. These neurons are located behind the oral opening (see Fig. 2c, 2d, and 3c) and are the only site of co-expression.

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DOI

https://doi.org/10.1038/nature14657

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