Ancient deuterostome origins of vertebrate brain signalling centres

Journal name:
Nature
Volume:
483,
Pages:
289–294
Date published:
DOI:
doi:10.1038/nature10838
Received
Accepted
Published online

Abstract

Neuroectodermal signalling centres induce and pattern many novel vertebrate brain structures but are absent, or divergent, in invertebrate chordates. This has led to the idea that signalling-centre genetic programs were first assembled in stem vertebrates and potentially drove morphological innovations of the brain. However, this scenario presumes that extant cephalochordates accurately represent ancestral chordate characters, which has not been tested using close chordate outgroups. Here we report that genetic programs homologous to three vertebrate signalling centresthe anterior neural ridge, zona limitans intrathalamica and isthmic organizerare present in the hemichordate Saccoglossus kowalevskii. Fgf8/17/18 (a single gene homologous to vertebrate Fgf8, Fgf17 and Fgf18), sfrp1/5, hh and wnt1 are expressed in vertebrate-like arrangements in hemichordate ectoderm, and homologous genetic mechanisms regulate ectodermal patterning in both animals. We propose that these genetic programs were components of an unexpectedly complex, ancient genetic regulatory scaffold for deuterostome body patterning that degenerated in amphioxus and ascidians, but was retained to pattern divergent structures in hemichordates and vertebrates.

At a glance

Figures

  1. An ANR-like signalling centre in S. kowalevskii.
    Figure 1: An ANR-like signalling centre in S. kowalevskii.

    a–h, S. kowalevskii and mouse in situ hybridizations for markers of ANR and telencephalon. S. kowalevskii embryos are at the double-groove stage (36 h), and are shown in dorsal view with the anterior (proboscis) to the top left of the image, except where noted. Embryos are optically cleared except in o, q (insets) and r–w. Mouse embryos are at approximately embryonic day 8.5. a, S. kowalevskii sfrp1/5 expression. b, Frontal view of mouse Sfrp1 expression; arrowhead denotes ANR. Inset shows lateral view. c, S. kowalevskii fgf8/17/18 expression. d, Mouse Fgf8 expression; arrowhead denotes ANR. e, S. kowalevskii hh expression. f, S. kowalevskii fgf-Sk1 expression. g, Frontal view of double FISH for hh and fgf-Sk1. h, fz5/8 expression. i, Anteroposterior expression topologies in S. kowalevskii and mouse embryos. Anterior to top. Asterisk indicates Shh is expressed in the medial ganglionic eminence, near the ANR. j–k, foxg (j) and rx (k) expression in embryos treated with SU5402. l, m, foxg (l) and rx (m) expression in embryos treated with DMSO. n, Expanded apical fgf-Sk1 expression in an embryo injected with fz5/8 siRNA. o, Retracted rx expression in an embryo injected with fz5/8 siRNA. p, fgf-Sk1 expression in a siRNA control embryo. q, rx expression in a siRNA control embryo. Insets in o, q show frontal views of uncleared embryos. r, Wild-type fgf-Sk1 expression. s, fgf-Sk1 expression in descendants of a blastomere injected with β-catenin siRNA. t, Merged darkfield and fluorescence images showing clonal descendants of the injected cell (green). u, Wild-type rx expression. v, rx is not expressed in descendants of a blastomere injected with β-catenin siRNA. w, Merged darkfield and fluorescence images showing the location of the β-catenin-deficient clone (green). x, fgf-Sk1 expression in an embryo injected with hh siRNA. y, fgf-Sk1 expression in a siRNA control embryo. Scale bars, 100 μm in S. kowalevskii, and 200 μm (b) and 500 μm (d) in mice. col, collar; di, diencephalon; hb, hindbrain; mb, midbrain; pr, proboscis; tel, telencephalon; tr, trunk.

  2. A ZLI-like signalling centre in S. kowalevskii.
    Figure 2: A ZLI-like signalling centre in S. kowalevskii.

    a–u, In situ hybridizations for S. kowalevskii and mouse homologues of ZLI and diencephalon markers. Arrowheads mark the proboscis–collar boundary in S. kowalevskii. Mouse images show hemisected heads at embryonic day 10.5, and dashed lines indicate the ZLI, with arrows denoting its extent. a, S. kowalevskii hh expression. b, S. kowalevskii ptch expression (see Supplementary Fig. 3). c, mouse Shh expression. d, Diagram of Shh expression showing ZLI in dark blue. e, Mouse Ptch1 expression. f, Mouse Ptch2 expression. g, S. kowalevskii fng expression. h, S. kowalevskii otx expression. i, Mouse Otx1 expression. j, Mouse Otx2 expression. k, hh (magenta) and otx (green) are co-expressed at the presumptive proboscis–collar boundary. l, S. kowalevskii wnt8 expression. m, Mouse Wnt8b expression. n, S. kowalevskii pax6 expression. o, Double in situ hybridization showing pax6 expression (fluorescence, green) anterior to hh (colorometric, black). p, Mouse Pax6 expression. q, S. kowalevskii dlx expression. r, Double FISH showing dlx expression (green) in the proboscis base anterior to hh (magenta). s, Mouse Dlx2 expression. t, S. kowalevskii foxa expression. u, Double FISH showing foxa (green) and hh (magenta) expression. v, Diagram of anteroposterior expression topologies of ZLI and forebrain marker homologues in S. kowalevskii and mice. Six3 expression based on previous data7. Anterior to top. w, x, otx siRNA downregulates hh expression at the proboscis–collar boundary (w) relative to a control siRNA (x). y, z, hh siRNA reduces dlx expression in the proboscis base (y) relative to a scrambled siRNA (z). a′, b′, cyclopamine treatment reduces dlx expression in the proboscis base (a′) relative to a control embryo treated with DMSO (b′). Insets show ventral views. Scale bars, 100 μm in S. kowalevskii embryos, 1 mm in mice. MGE, medial ganglionic eminence; pth, prethalamus; th, thalamus.

  3. An IsO-like signalling centre in S. kowalevskii.
    Figure 3: An IsO-like signalling centre in S. kowalevskii.

    a–s, In situ hybridizations for S. kowalevskii and mouse homologues of MHB markers. Arrowheads mark the S. kowalevskii collar–trunk coelom boundary and the mouse IsO. a, S. kowalevskii fgf8/17/18 expression. b, Mouse Fgf8 expression. c, S. kowalevskii wnt1 expression. d, Mouse Wnt1 expression. e, Double FISH showing S. kowalevskii wnt1 (green) expressed directly anterior to fgf8/17/18 (magenta). f, S. kowalevskii otx expression. g, Mouse Otx2 expression. h, S. kowalevskii gbx expression. Asterisks denote endodermal domains. i, Mouse Gbx2 expression. j, Double FISH for S. kowalevskii otx (green) and gbx (magenta). k, Double FISH for S. kowalevskii wnt1 (green) and en (magenta). l, Double FISH for S. kowalevskii en and otx. m, Mouse En2 expression. n, S. kowalevskii pax2/5/8 expression. o, Double FISH showing S. kowalevskii pax2/5/8 and en expression. p–r, Expression of mouse Pax2 (p), Pax5 (q) and Pax8 (r). s, Double FISH for S. kowalevskii tyrosine hydroxylase (green) and en (magenta). t, Summary of anteroposterior expression topologies in hemichordates and mice. Anterior to top. u, en expression is reduced in an embryo that has been treated with SU5402. v, en expression in a DMSO-treated control embryo. w, en expression in an embryo injected with fgf8/17/18 siRNA. x, en expression in an embryo injected with a control siRNA. y, pax2/5/8 expression in an embryo treated with SU5402. z, pax2/5/8 expression in a DMSO-treated control embryo. a′, pax2/5/8 expression in an embryo injected with fgf8/17/18 siRNA. b′, pax2/5/8 expression in an embryo injected with a control siRNA. c′, en is not expressed in descendants of a blastomere injected with β-catenin siRNA. d′, Merged darkfield and fluorescence images showing the location of the β-catenin-deficient clone (green). e′, Wild-type en expression. Scale bars, 100 μm in S. kowalevskii embryos; 1 mm in mice.

  4. Evolutionary gain and loss of ANR, ZLI and IsO-like genetic programs.
    Figure 4: Evolutionary gain and loss of ANR, ZLI and IsO-like genetic programs.

    Schematic diagrams depicting the expression of Fgf8, Sfrp1, Shh and Wnt1 homologues in the mouse brain and ectoderm of C. intestinalis, amphioxus and S. kowalevskii. Embryos are oriented with their anterior side to the left of the image and their dorsal side to top. Bar diagrams are oriented with the anterior side to the left of the image. Diagrams depict only expression domains that are related to signalling components of vertebrate CNS signalling centres. cv, cerebral vesicle; n, neck; nc, nerve cord; sv, sensory vesicle; vg, visceral ganglion. Diagrams are not to scale. Single asterisk indicates that Shh is expressed in the medial ganglionic eminence, near the ANR. Double asterisk indicates that sfrp1/5 is expressed in the C. intestinalis anterior ectoderm from the 64-cell stage up to neurulation but is then downregulated in the anterior ectoderm and CNS (shown as yellow stripes).

References

  1. Echevarria, D., Vieira, C., Gimeno, L. & Martinez, S. Neuroepithelial secondary organizers and cell fate specification in the developing brain. Brain Res. Brain Res. Rev. 43, 179191 (2003)
  2. Wilson, S. W. & Houart, C. Early steps in the development of the forebrain. Dev. Cell 6, 167181 (2004)
  3. Wurst, W. & Bally-Cuif, L. Neural plate patterning: upstream and downstream of the isthmic organizer. Nature Rev. Neurosci. 2, 99108 (2001)
  4. Wicht, H. & Lacalli, T. C. The nervous system of amphioxus: structure, development, and evolutionary significance. Can. J. Zool. 150, 122150 (2005)
  5. Lacalli, T. C. Prospective protochordate homologs of vertebrate midbrain and MHB, with some thoughts on MHB origins. Int. J. Biol. Sci. 2, 104109 (2006)
  6. Meinertzhagen, I. A., Lemaire, P. & Okamura, Y. The neurobiology of the ascidian tadpole larva: recent developments in an ancient chordate. Annu. Rev. Neurosci. 27, 453485 (2004)
  7. Lowe, C. J. et al. Anteroposterior patterning in hemichordates and the origins of the chordate nervous system. Cell 113, 853865 (2003)
  8. Holland, L. Z. & Short, S. Gene duplication, co-option and recruitment during the origin of the vertebrate brain from the invertebrate chordate brain. Brain Behav. Evol. 72, (2008)
  9. Holland, L. Z. Chordate roots of the vertebrate nervous system: expanding the molecular toolkit. Nature Rev. Neurosci. 10, 736746 (2009)
  10. Irimia, M. et al. Conserved developmental expression of Fezf in chordates and Drosophila and the origin of the Zona Limitans Intrathalamica (ZLI) brain organizer. Evodevo. 1, 7 (2010)
  11. Tomer, R., Denes, A. S., Tessmar-Raible, K. & Arendt, D. Profiling by image registration reveals common origin of annelid mushroom bodies and vertebrate pallium. Cell 142, 800809 (2010)
  12. Urbach, R. A procephalic territory in Drosophila exhibiting similarities and dissimilarities compared to the vertebrate midbrain/hindbrain boundary region. Neural Dev. 2, 23 (2007)
  13. Crossley, P. H., Martinez, S. & Martin, G. R. Midbrain development induced by FGF8 in the chick embryo. Nature 380, 6668 (1996)
  14. Reifers, F. et al. Fgf8 is mutated in zebrafish acerebellar (ace) mutants and is required for maintenance of midbrain-hindbrain boundary development and somitogenesis. Development 125, 23812395 (1998)
  15. Houart, C. et al. Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling. Neuron 35, 255265 (2002)
  16. Paek, H., Gutin, G. & Hebert, J. M. FGF signaling is strictly required to maintain early telencephalic precursor cell survival. Development 136, 24572465 (2009)
  17. Kiecker, C. & Lumsden, A. Hedgehog signaling from the ZLI regulates diencephalic regional identity. Nature Neurosci. 7, 12421249 (2004)
  18. Scholpp, S., Wolf, O., Brand, M. & Lumsden, A. Hedgehog signalling from the zona limitans intrathalamica orchestrates patterning of the zebrafish diencephalon. Development 133, 855864 (2006)
  19. Meulemans, D. & Bronner-Fraser, M. Insights from amphioxus into the evolution of vertebrate cartilage. PLoS ONE 2, e787 (2007)
  20. Imai, K. S., Stolfi, A., Levine, M. & Satou, Y. Gene regulatory networks underlying the compartmentalization of the Ciona central nervous system. Development 136, 285293 (2009)
  21. Shimeld, S. M. The evolution of the hedgehog gene family in chordates: insights from amphioxus hedgehog. Dev. Genes Evol. 209, 4047 (1999)
  22. Takatori, N., Satou, Y. & Satoh, N. Expression of hedgehog genes in Ciona intestinalis embryos. Mech. Dev. 116, 235238 (2002)
  23. Scholpp, S. & Lumsden, A. Building a bridal chamber: development of the thalamus. Trends Neurosci. 33, 373380 (2010)
  24. Bertrand, S. et al. Amphioxus FGF signaling predicts the acquisition of vertebrate morphological traits. Proc. Natl Acad. Sci. USA 108, 91609165 (2011)
  25. Holland, L. Z., Holland, N. N. & Schubert, M. Developmental expression of AmphiWnt1, an amphioxus gene in the Wnt1/wingless subfamily. Dev. Genes Evol. 210, 522524 (2000)
  26. Bourlat, S. J. et al. Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida. Nature 444, 8588 (2006)
  27. Darras, S., Gerhart, J., Terasaki, M., Kirschner, M. & Lowe, C. J. β-catenin specifies the endomesoderm and defines the posterior organizer of the hemichordate Saccoglossus kowalevskii. Development 138, 959970 (2011)
  28. Gillis, J. A., Fritzenwanker, J. H. & Lowe, C. J. A stem-deuterostome origin of the vertebrate pharyngeal transcriptional network. Proc. R. Soc. B 279, 237246 (2012)
  29. Lowe, C. J. et al. Dorsoventral patterning in hemichordates: insights into early chordate evolution. PLoS Biol. 4, e291 (2006)
  30. Shimamura, K. & Rubenstein, J. L. Inductive interactions direct early regionalization of the mouse forebrain. Development 124, 27092718 (1997)
  31. Fukuchi-Shimogori, T. & Grove, E. A. Neocortex patterning by the secreted signaling molecule FGF8. Science 294, 10711074 (2001)
  32. Walshe, J. & Mason, I. Unique and combinatorial functions of Fgf3 and Fgf8 during zebrafish forebrain development. Development 130, 43374349 (2003)
  33. Garel, S., Huffman, K. J. & Rubenstein, J. L. Molecular regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants. Development 130, 19031914 (2003)
  34. Mohammadi, M. et al. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 276, 955960 (1997)
  35. Lagutin, O. V. et al. Six3 repression of Wnt signaling in the anterior neuroectoderm is essential for vertebrate forebrain development. Genes Dev. 17, 368379 (2003)
  36. Crossley, P. H., Martinez, S., Ohkubo, Y. & Rubenstein, J. L. Coordinate expression of Fgf8, Otx2, Bmp4, and Shh in the rostral prosencephalon during development of the telencephalic and optic vesicles. Neuroscience 108, 183206 (2001)
  37. Hébert, J. M. & Fishell, G. The genetics of early telencephalon patterning: some assembly required. Nature Rev. Neurosci. 9, 678685 (2008)
  38. Zeltser, L. M., Larsen, C. W. & Lumsden, A. A new developmental compartment in the forebrain regulated by Lunatic fringe. Nature Neurosci. 4, 683684 (2001)
  39. Scholpp, S. et al. Otx1l, Otx2 and Irx1b establish and position the ZLI in the diencephalon. Development 134, 31673176 (2007)
  40. Chen, J. K., Taipale, J., Cooper, M. K. & Beachy, P. A. Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. Genes Dev. 16, 27432748 (2002)
  41. Simon, H. H., Thuret, S. & &Alberi, L. Midbrain dopaminergic neurons: control of their cell fate by the engrailed transcription factors. Cell Tissue Res. 318, 5361 (2004)
  42. McMahon, A. P., Joyner, A. L., Bradley, A. & McMahon, J. A. The midbrain-hindbrain phenotype of Wnt-1Wnt-1 mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum. Cell 69, 581595 (1992)
  43. Nomaksteinsky, M. et al. Centralization of the deuterostome nervous system predates chordates. Curr. Biol. 19, 12641269 (2009)
  44. Gavino, M. A., Reddien, P. W. & A Bmp/Admp regulatory circuit controls maintenance and regeneration of dorsal-ventral polarity in planarians. Curr. biol. 21, 294299 (2011)
  45. Grande, C. & Patel, N. H. Nodal signalling is involved in left–right asymmetry in snails. Nature 457, 10071011 (2009)
  46. Campo-Paysaa, F., Marletaz, F., Laudet, V. & Schubert, M. Retinoic acid signaling in development: tissue-specific functions and evolutionary origins. Genesis 46, 640656 (2008)
  47. Freeman, R. M., Jr et al. cDNA sequences for transcription factors and signaling proteins of the hemichordate Saccoglossus kowalevskii: efficacy of the expressed sequence tag (EST) approach for evolutionary and developmental studies of a new organism. Biol. Bull. 214, 284302 (2008)
  48. Larkin, M. A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 29472948 (2007)
  49. Ronquist, F. & Huelsenbeck, J. P. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 15721574 (2003)
  50. Huelsenbeck, J. P. & Ronquist, F. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754755 (2001)

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Author information

  1. These authors contributed equally to this work.

    • Erin E. Mullarkey &
    • Jochanan Aronowicz

Affiliations

  1. Committee on Evolutionary Biology, The University of Chicago, 1025 East 57th Street, Chicago, Illinois 60637, USA

    • Ariel M. Pani &
    • Christopher J. Lowe
  2. Hopkins Marine Station, Department of Biology, Stanford University, 120 Oceanview Boulevard, Pacific Grove, California 93950, USA

    • Ariel M. Pani &
    • Christopher J. Lowe
  3. Committee on Neurobiology, The University of Chicago, 947 East 58th Street, Chicago, Illinois 60637, USA

    • Erin E. Mullarkey &
    • Elizabeth A. Grove
  4. Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, Illinois 60637, USA

    • Jochanan Aronowicz,
    • Elizabeth A. Grove &
    • Christopher J. Lowe
  5. Department of Neurobiology, The University of Chicago, 947 East 58th Street, Chicago, Illinois 60637, USA

    • Stavroula Assimacopoulos &
    • Elizabeth A. Grove

Contributions

A.M.P., C.J.L. and J.A. conceived the project. A.M.P. and C.J.L. performed the hemichordate experiments and wrote the paper. E.E.M. and S.A. performed mouse experiments, and E.A.G. edited the paper. All authors discussed and commented on the data.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

S. kowalevskii gene sequences have been deposited in GenBank, and accession numbers are provided in Supplementary Table 2.

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Supplementary information

PDF files

  1. Supplementary Information (2.3M)

    This file contains Supplementary Figures 1-4 and Supplementary Tables 1-2. Please note that Supplementary Figure 1 shows that fz5/8 siRNA affects proboscis patterning specifically and that Supplementary Figure 2 shows Ptch expression in wild-type S. kowalevskii embryos and spectrum of phenotypes after hh siRNA injection. Ptch expression indicates that hh can signal to numerous body regions. Hh siRNA injection causes pleiotropic effects on AP and DV patterning.

Additional data