Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis

Abstract

The metabotropic gamma-aminobutyric acid B receptor (GABABR) is a G protein-coupled receptor that mediates neuronal inhibition by the neurotransmitter GABA. While GABABR-mediated signalling has been suggested to play central roles in neuronal differentiation and proliferation across evolution, it has mostly been studied in the mammalian brain. Here, we demonstrate that ectopic activation of GABABR signalling affects neurogenic functions in the sea anemone Nematostella vectensis. We identified four putative Nematostella GABABR homologues presenting conserved three-dimensional extracellular domains and residues needed for binding GABA and the GABABR agonist baclofen. Moreover, sustained activation of GABABR signalling reversibly arrests the critical metamorphosis transition from planktonic larva to sessile polyp life stage. To understand the processes that underlie the developmental arrest, we combined transcriptomic and spatial analyses of control and baclofen-treated larvae. Our findings reveal that the cnidarian neurogenic programme is arrested following the addition of baclofen to developing larvae. Specifically, neuron development and neurite extension were inhibited, resulting in an underdeveloped and less organized nervous system and downregulation of proneural factors including NvSoxB(2), NvNeuroD1 and NvElav1. Our results thus point to an evolutionarily conserved function of GABABR in neurogenesis regulation and shed light on early cnidarian development.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: GABA and a specific GABABR agonist and modulator cause reversible inhibition of Nematostella metamorphosis.
Fig. 2: Sequence comparison of the extracellular regions in bilaterian and putative Nematostella GABAB1R homologues.
Fig. 3: Three-dimensional visualization of the human and Nematostella GABABR extracellular region bound to baclofen.
Fig. 4: Transcriptomic analysis of control and baclofen-treated planulae.
Fig. 5: Autoregulation of GABA synthesis following baclofen treatment.
Fig. 6: Baclofen inhibits Nematostella neurogenesis.

Data availability

Transcriptome datasets used in this study are available via the SRA database with accession no. SRP140400.

References

  1. Ulrich, D. & Bettler, B. GABAB receptors: synaptic functions and mechanisms of diversity. Curr. Opin. Neurobiol. 17, 298–303 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Kaupmann, K. et al. GABA B-receptor subtypes assemble into functional heteromeric complexes. Nature 396, 683–687 (1998).

    Article  Google Scholar 

  3. Jones, K. A. et al. GABAB receptors function as a heteromeric assembly of the subunits GABABR1 and GABABR2. Nature 396, 674–679 (1998).

    Article  CAS  PubMed  Google Scholar 

  4. White, J. H. et al. Heterodimerization is required for the formation of a functional GABA B receptor. Nature 396, 679–682 (1998).

    Article  CAS  Google Scholar 

  5. Geng, Y., Bush, M., Mosyak, L., Wang, F. & Fan, Q. R. Structural mechanism of ligand activation in human GABAB receptor. Nature 504, 254–259 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Galvez, T. et al. Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding. J. Biol. Chem. 274, 13362–13369 (1999).

  7. Margeta-Mitrovic, M., Jan, Y. N. & Jan, L. Y. A trafficking checkpoint controls GABAB receptor heterodimerization. Neuron 27, 97–106 (2000).

    Article  CAS  PubMed  Google Scholar 

  8. Robbins, M. J. et al. GABAB2 is essential for G-protein coupling of the GABAB receptor heterodimer. J. Neurosci. 21, 8043–8052 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Kniazeff, J., Galvez, T., Labesse, G. & Pin, J. P. No ligand binding in the GB2 subunit of the GABA(B) receptor is required for activation and allosteric interaction between the subunits. J. Neurosci. 22, 7352–7361 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Liu, J. et al. Molecular determinants involved in the allosteric control of agonist affinity in the GABAB receptor by the GABAB2 subunit. J. Biol. Chem. 279, 15824–15830 (2004).

    Article  CAS  PubMed  Google Scholar 

  11. Felder, C. B., Graul, R. C., Lee, A. Y., Merkle, H. P. & Sadee, W. The Venus flytrap of periplasmic binding proteins: an ancient protein module present in multiple drug receptors. AAPS PharmSciTech. 1, 7–26 (1999).

    Article  Google Scholar 

  12. Kilb, W., Kirischuk, S. & Luhmann, H. Role of tonic GABAergic currents during pre- and early postnatal rodent development. Front. Neural Circuits 7, 139 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Fukui, M. et al. Modulation of cellular proliferation and differentiation through GABAB receptors expressed by undifferentiated neural progenitor cells isolated from fetal mouse brain. J. Cell. Physiol. 216, 507–519 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Gaiarsa, J.-L., Kuczewski, N. & Porcher, C. Contribution of metabotropic GABAB receptors to neuronal network construction. Pharmacol. Ther. 132, 170–179 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Bony, G. et al. Non-hyperpolarizing GABAB receptor activation regulates neuronal migration and neurite growth and specification by cAMP/LKB1. Nat. Commun. 4, 1800 (2013).

    Article  PubMed  CAS  Google Scholar 

  16. Giachino, C. et al. GABA suppresses neurogenesis in the adult hippocampus through GABAB receptors. Development 141, 83–90 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Sibbe, M. & Kulik, A. GABAergic regulation of adult hippocampal neurogenesis. Mol. Neurobiol. 54, 5497–5510 (2017).

    Article  CAS  PubMed  Google Scholar 

  18. Dittman, J. S. & Kaplan, J. M. Behavioral impact of neurotransmitter-activated G-protein-coupled receptors: muscarinic and GABAB receptors regulate Caenorhabditis elegans locomotion. J. Neurosci. 28, 7104–7112 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mezler, M., Müller, T. & Raming, K. Cloning and functional expression of GABAB receptors from Drosophila. Eur. J. Neurosci. 13, 477–486 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Colombo, G. (ed.) GABAB Receptor Vol. 29 (Springer, 2016).

  21. Blankenburg, S. et al. Cockroach GABAB receptor subtypes: molecular characterization, pharmacological properties and tissue distribution. Neuropharmacology 88, 134–144 (2015).

    Article  CAS  PubMed  Google Scholar 

  22. Rentzsch, F., Layden, M. & Manuel, M. The cellular and molecular basis of cnidarian neurogenesis. Wiley Interdiscip. Rev. Dev. Biol. 6, e257 (2016).

    Article  PubMed Central  Google Scholar 

  23. Ryan, J. F. & Chiodin, M. Where is my mind? How sponges and placozoans may have lost neural cell types. Phil. Trans. R. Soc. B 370, 20150059 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Nickel, M. Evolutionary emergence of synaptic nervous systems: what can we learn from the non-synaptic, nerveless Porifera? Invertebr. Biol. 129, 1–16 (2010).

    Article  Google Scholar 

  25. Watanabe, H. in Brain Evolution by Design: From Neural Origin to Cognitive Architecture (eds Shigeno, S. et al.) 45–75 (Springer Japan, 2017).

  26. Moroz, L. L. & Kohn, A. B. Independent origins of neurons and synapses: insights from ctenophores. Phil. Trans. R. Soc. Lond. B. 371, 20150041 (2016).

    Article  CAS  Google Scholar 

  27. Kelava, I., Rentzsch, F. & Technau, U. Evolution of eumetazoan nervous systems: insights from cnidarians. Phil. Trans. R. Soc. Lond. B. 370, 20150065 (2015).

    Article  CAS  Google Scholar 

  28. Galliot, B. & Quiquand, M. A two-step process in the emergence of neurogenesis. Eur. J. Neurosci. 34, 847–862 (2011).

    Article  PubMed  Google Scholar 

  29. Layden, M. J., Rentzsch, F. & Röttinger, E. The rise of the starlet sea anemone Nematostella vectensis as a model system to investigate development and regeneration. Wiley Interdiscip. Rev. Dev. Biol. 5, 408–428 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Rentzsch, F., Juliano, C. & Galliot, B. Modern genomic tools reveal the structural and cellular diversity of cnidarian nervous systems. Curr. Opin. Neurobiol. 56, 87–96 (2019).

    Article  CAS  PubMed  Google Scholar 

  31. Rentzsch, F. & Technau, U. Genomics and development of Nematostella vectensis and other anthozoans. Curr. Opin. Genet. Dev. 39, 63–70 (2016).

    Article  CAS  PubMed  Google Scholar 

  32. Putnam, N. et al. Sea anemone genome reveals ancestral eumetazoan gene repertoire and genomic organization. Science 317, 86–94 (2007).

    Article  CAS  PubMed  Google Scholar 

  33. Darling, J. A. et al. Rising starlet: the starlet sea anemone, Nematostella vectensis. Bioessays 27, 211–221 (2005).

    Article  CAS  PubMed  Google Scholar 

  34. Anctil, M. Chemical transmission in the sea anemone Nematostella vectensis: a genomic perspective. Comp. Biochem. Physiol. D 4, 268–289 (2009).

    Google Scholar 

  35. Bosch, T. C. G. et al. Back to the basics: cnidarians start to fire. Trends Neurosci. 40, 92–105 (2017).

    Article  CAS  PubMed  Google Scholar 

  36. Marlow, H. Q., Srivastava, M., Matus, D. Q., Rokhsar, D. & Martindale, M. Q. Anatomy and development of the nervous system of Nematostella vectensis, an anthozoan cnidarian. Dev. Neurobiol. 69, 235–254 (2009).

    Article  CAS  PubMed  Google Scholar 

  37. Faltine-Gonzalez, D. Z. & Layden, M. J. Characterization of nAChRs in Nematostella vectensis supports neuronal and non-neuronal roles in the cnidarian–bilaterian common ancestor. EvoDevo 10, 27 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Adams, C. & Lawrence, A. CGP7930: a positive allosteric modulator of the GABAB receptor. CNS Drug Rev. 13, 308–316 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Lee, P. N., Pang, K., Matus, D. Q. & Martindale, M. Q. A. WNT of things to come: evolution of Wnt signaling and polarity in cnidarians. Semin. Cell Dev. Biol. 17, 157–167 (2006).

    Article  PubMed  CAS  Google Scholar 

  40. Trevino, M., Stefanik, D. J., Rodriguez, R., Harmon, S. & Burton, P. M. Induction of canonical Wnt signaling by alsterpaullone is sufficient for oral tissue fate during regeneration and embryogenesis in Nematostella vectensis. Dev. Dyn. 240, 2673–2679 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kusserow, A. et al. Unexpected complexity of the Wnt gene family in a sea anemone. Nature 433, 156–160 (2005).

    Article  CAS  PubMed  Google Scholar 

  42. Gurevich, V. V. & Gurevich, E. V. How and why do GPCRs dimerize? Trends Pharmacol. Sci. 29, 234–240 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Calver, A. R. et al. The C-terminal domains of the GABAB receptor subunits mediate intracellular trafficking but are not required for receptor signaling. J. Neurosci. 21, 1203–1210 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Grünewald, S. et al. Importance of the γ-aminobutyric acidB receptor C-termini for G-protein coupling. Mol. Pharmacol. 61, 1070–1080 (2002).

    Article  PubMed  Google Scholar 

  45. Pagano, A. et al. C-terminal interaction is essential for surface trafficking but not for heteromeric assembly of GABAB receptors. J. Neurosci. 21, 1189–1202 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Sebé-Pedrós, A. et al. Cnidarian cell type diversity revealed by whole-organism single-cell RNA-seq analysis. Cell 173, 1520–1534 (2018).

    Article  PubMed  CAS  Google Scholar 

  47. Karabulut, A., He, S., Chen, C.-Y., McKinney, S. A. & Gibson, M. C. Electroporation of short hairpin RNAs for rapid and efficient gene knockdown in the starlet sea anemone, Nematostella vectensis. Dev. Biol. 448, 7–15 (2019).

    Article  CAS  PubMed  Google Scholar 

  48. He, S. et al. An axial Hox code controls tissue segmentation and body patterning in Nematostella vectensis. Science 361, 1377–1380 (2018).

    Article  CAS  PubMed  Google Scholar 

  49. Benke, D. in Advances in Pharmacology (ed. Blackburn, T. P.) 93–111 (Elsevier, 2010).

  50. Kantamneni, S. et al. GISP binding to TSG101 increases GABAB receptor stability by down‐regulating ESCRT‐mediated lysosomal degradation. J. Neurochem. 107, 86–95 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kantamneni, S., Holman, D., Wilkinson, K. A., Nishimune, A. & Henley, J. M. GISP increases neurotransmitter receptor stability by down-regulating ESCRT-mediated lysosomal degradation. Neurosci. Lett. 452, 106–110 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Magie, C., Pang, K. & Martindale, M. Genomic inventory and expression of Sox and Fox genes in the cnidarian Nematostella vectensis. Dev. Genes Evol. 215, 618–630 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Richards, G. S. & Rentzsch, F. Transgenic analysis of a SoxB gene reveals neural progenitor cells in the cnidarian Nematostella vectensis. Development 141, 4681–4689 (2014).

    Article  CAS  PubMed  Google Scholar 

  54. Layden, M. J., Boekhout, M. & Martindale, M. Q. Nematostella vectensis achaete-scute homolog NvashA regulates embryonic ectodermal neurogenesis and represents an ancient component of the metazoan neural specification pathway. Development 139, 1013–1022 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Richards, G. S. & Rentzsch, F. Regulation of Nematostella neural progenitors by SoxB, Notch and bHLH genes. Development 142, 3332–3342 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Layden, M. J. et al. MAPK signaling is necessary for neurogenesis in Nematostella vectensis. BMC Biol. 14, 61 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Watanabe, H. et al. Sequential actions of β-catenin and Bmp pattern the oral nerve net in Nematostella vectensis. Nat. Commun. 5, 5536 (2014).

  58. Mazza, M. E., Pang, K., Martindale, M. Q. & Finnerty, J. R. Genomic organization, gene structure, and developmental expression of three clustered otx genes in the sea anemone Nematostella vectensis. J. Exp. Zool. B 308, 494–506 (2007).

    Article  CAS  Google Scholar 

  59. Nakanishi, N., Renfer, E., Technau, U. & Rentzsch, F. Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms. Development 139, 347–357 (2012).

    Article  CAS  PubMed  Google Scholar 

  60. Marlow, H., Roettinger, E., Boekhout, M. & Martindale, M. Q. Functional roles of Notch signaling in the cnidarian Nematostella vectensis. Dev. Biol. 362, 295–308 (2012).

    Article  CAS  PubMed  Google Scholar 

  61. Babonis, L. S. & Martindale, M. Q. PaxA, but not PaxC, is required for cnidocyte development in the sea anemone Nematostella vectensis. EvoDevo 8, 14 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Zenkert, C., Takahashi, T., Diesner, M.-O. & Özbek, S. Morphological and molecular analysis of the Nematostella vectensis cnidom. PLoS ONE 6, e22725 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Sunagar, K. et al. Cell type-specific expression profiling unravels the development and evolution of stinging cells in sea anemone. BMC Biol. 16, 108 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  64. Matus, D. Q., Pang, K., Daly, M. & Martindale, M. Q. Expression of Pax gene family members in the anthozoan cnidarian, Nematostella vectensis. Evol. Dev. 9, 25–38 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Zatylny-Gaudin, C. & Favrel, P. Diversity of the RFamide peptide family in mollusks. Front. Endocrinol. (Lausanne) 5, 178 (2014).

    Article  Google Scholar 

  66. Bause, M., van der Horst, R. & Rentzsch, F. Glypican1/2/4/6 and sulfated glycosaminoglycans regulate the patterning of the primary body axis in the cnidarian Nematostella vectensis. Dev. Biol. 414, 108–120 (2016).

    Article  CAS  PubMed  Google Scholar 

  67. Rentzsch, F., Fritzenwanker, J. H., Scholz, C. B. & Technau, U. FGF signalling controls formation of the apical sensory organ in the cnidarian Nematostella vectensis. Development 135, 1761–1769 (2008).

    Article  CAS  PubMed  Google Scholar 

  68. Hozumi, A. et al. GABA-Induced GnRH release triggers chordate metamorphosis. Curr. Biol. 30, 1555–1561 (2020).

    Article  CAS  PubMed  Google Scholar 

  69. Biscocho, D., Cook, J. G., Long, J., Shah, N. & Leise, E. M. GABA is an inhibitory neurotransmitter in the neural circuit regulating metamorphosis in a marine snail. Dev. Neurobiol. 78, 736–753 (2018).

    Article  CAS  PubMed  Google Scholar 

  70. Joyce, A. & Vogeler, S. Molluscan bivalve settlement and metamorphosis: neuroendocrine inducers and morphogenetic responses. Aquaculture 487, 64–82 (2018).

  71. Rahmani, M. & Uehara, T. Induction of metamorphosis and substratum preference in four sympatric and closely related species of sea urchins (Genus Echinometra) in Okinawa. Zool. Stud. 40, 29–43 (2001).

    Google Scholar 

  72. Scappaticci, A. A. & Kass-Simon, G. NMDA and GABAB receptors are involved in controlling nematocyst discharge in hydra. Comp. Biochem. Physiol. A 150, 415–422 (2008).

    Article  CAS  Google Scholar 

  73. Lauro, B. M. & Kass-Simon, G. Hydra’s feeding response: effect of GABAB ligands on GSH-induced electrical activity in the hypostome of H. vulgaris. Comp. Biochem. Physiol. A 225, 83–93 (2018).

    Article  CAS  Google Scholar 

  74. Schaeffer, J. M. & Hsueh, A. J. Identification of gamma-aminobutyric acid and its binding sites in the rat ovary. Life Sci. 30, 1599–1604 (1982).

    Article  CAS  PubMed  Google Scholar 

  75. Shen, W., Nan, C., Nelson, P. T., Ripps, H. & Slaughter, M. M. GABAB receptor attenuation of GABAA currents in neurons of the mammalian central nervous system. Physiol. Rep. 5, e13129 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Shim, J. et al. Olfactory control of blood progenitor maintenance. Cell 155, 1141–1153 (2013).

    Article  CAS  PubMed  Google Scholar 

  77. Sarkar, A. & Hochedlinger, K. The Sox family of transcription factors: versatile regulators of stem and progenitor cell fate. Cell Stem Cell 12, 15–30 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Bylund, M., Andersson, E., Novitch, B. G. & Muhr, J. Vertebrate neurogenesis is counteracted by Sox1–3 activity. Nat. Neurosci. 6, 1162–1168 (2003).

    Article  CAS  PubMed  Google Scholar 

  79. Pevny, L. & Placzek, M. SOX genes and neural progenitor identity. Curr. Opin. Neurobiol. 15, 7–13 (2005).

    Article  CAS  PubMed  Google Scholar 

  80. Wegner, M. SOX after SOX: SOXession regulates neurogenesis. Genes Dev. 25, 2423–2428 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Whittington, N., Cunningham, D., Le, T.-K., De Maria, D. & Silva, E. M. Sox21 regulates the progression of neuronal differentiation in a dose-dependent manner. Dev. Biol. 397, 237–247 (2015).

    Article  CAS  PubMed  Google Scholar 

  82. Royo, J. L. et al. Transphyletic conservation of developmental regulatory state in animal evolution. Proc. Natl Acad. Sci. USA 108, 14186–14191 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Jager, M., Quéinnec, E., Le Guyader, H. & Manuel, M. Multiple Sox genes are expressed in stem cells or in differentiating neuro-sensory cells in the hydrozoan Clytia hemisphaerica. EvoDevo 2, 12 (2011).

  84. Schnitzler, C. E., Simmons, D. K., Pang, K., Martindale, M. Q. & Baxevanis, A. D. Expression of multiple Sox genes through embryonic development in the ctenophore Mnemiopsis leidyi is spatially restricted to zones of cell proliferation. EvoDevo 5, 15 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  85. Steinmetz, P. R. H., Aman, A., Kraus, J. E. M. & Technau, U. Gut-like ectodermal tissue in a sea anemone challenges germ layer homology. Nat. Ecol. Evol. 1, 1535–1542 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  86. Busengdal, H. & Rentzsch, F. Unipotent progenitors contribute to the generation of sensory cell types in the nervous system of the cnidarian Nematostella vectensis. Dev. Biol. 431, 59–68 (2017).

    Article  CAS  PubMed  Google Scholar 

  87. Elran, R. et al. Early and late response of Nematostella vectensis transcriptome to heavy metals. Mol. Ecol. 23, 4722–4736 (2014).

    Article  CAS  PubMed  Google Scholar 

  88. Fritzenwanker, J. & Technau, U. Induction of gametogenesis in the basal cnidarian Nematostella vectensis (Anthozoa). Dev. Genes Evol. 212, 99–103 (2002).

    Article  PubMed  Google Scholar 

  89. Bordoli, L. et al. Protein structure homology modeling using SWISS-MODEL workspace. Nat. Protoc. 4, 1–13 (2008).

    Google Scholar 

  90. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  91. Love, M.I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15, 550 (2014).

  92. Khomtchouk, B. B., Hennessy, J. R. & Wahlestedt, C. shinyheatmap: ultra fast low memory heatmap web interface for big data genomics. PLoS ONE 12, e0176334 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  93. Young, M. D., Wakefield, M. J., Smyth, G. K. & Oshlack, A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 11, R14 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  94. Wolenski, F. S., Layden, M. J., Martindale, M. Q., Gilmore, T. D. & Finnerty, J. R. Characterizing the spatiotemporal expression of RNAs and proteins in the starlet sea anemone, Nematostella vectensis. Nat. Protoc. 8, 900–915 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  95. Genikhovich, G. & Technau, U. Anti-acetylated tubulin antibody staining and phalloidin staining in the starlet sea anemone Nematostella vectensis. Cold Spring Harb. Protoc. 2009, pdb.prot5283 (2009).

  96. Gassmann, M. & Bettler, B. Regulation of neuronal GABAB receptor functions by subunit composition. Nat. Rev. Neurosci. 13, 380–394 (2012).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank F. Rentzsch for providing the NvElav1 reporter line. We thank the Bioinformatics Service Unit at the University of Haifa and, specifically, N. Sher and M. Lalzar for their assistance. We thank S. Ben-Tabou de-Leon for helpful comments. This work was supported by the Israel Ministry of Science and Technology (grant no. 3-8774), the Israel Science Foundation (grant nos. 1454/13, 2155/15 and 3512/19) and the DS Research Centre at the University of Haifa.

Author information

Authors and Affiliations

Authors

Contributions

S.L. designed and performed experiments. V.B. performed gene cloning, shRNA knockdown and assisted in experiments. S.L., A.B. and M.K. performed sequence and structure analysis. A.M. performed bioinformatics analyses. A.S.-P. analysed GABABR homologue and TF expression in the single-cell dataset. S.L., V.B., M.K. and T.L. analysed the data. M.K. supervised sequence and structure analysis. T.L. conceived and supervised the project and wrote the manuscript with M.K. and input from all authors. All authors discussed the results and commented on the manuscript. All authors read and approved the final version of the manuscript.

Corresponding authors

Correspondence to Mickey Kosloff or Tamar Lotan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 GABABR modulators inhibit planulae-to-polyp transformation.

Confocal sections at 5 dpf (a-d) and 8 dpf (i-l) labeled with antibodies against phalloidin (green) and DAPI (blue). DIC images of the aboral/apical tuft at 5 dpf (e–h) and 8 dpf (m-p). Control planulae (a,i) and primary polyps (i, m) are shown, as are planulae treated with GABA (b, f, j, n), baclofen (c, g, k, o) or CGP-7930 (d, h, I, p). While at 5 dpf all planulae possessed an apical tuft (numbers are not shown), in 8 dpf control primary polyps after metamorphosis (m), the apical tuft was lost, whereas treated 8 dpf planulae (n–p) still maintained it. The fraction of similar phenotypes from the total number of analyzed samples is given in the lower right-hand corner. Scale bars, 50 µm.

Extended Data Fig. 2 Schematic representation of predicted domains in putative Nematostella GABABR homologs in comparison to human GABAB1R.

The eight Nematostella proteins contain a conserved signal peptide, an extracellular ‘Periplasmic Binding Protein type1 (PBP1) GABAB ligand-binding domain’ (the structural VFT module that in mammals binds GABA), predicted helical transmembrane domains, and coiled-coil domains. One Nematostella homolog (v1g206093) contained two extracellular domains, each corresponding to a separate predicted VFT module. These two domains are 26% identical in sequence, suggesting that they serve dissimilar functions (Supplementary Fig. 2). v1g243252 contains eight predicted TM helices and a ~300 residue domain of unknown function (DUF4475) located after these TM domains. However, the intracellular C-terminal domains of the Nematostella homologs present low similarity to the corresponding regions of human sequences. Coiled-coil motifs found in the C-terminus of human GABABR were predicted in three Nematostella homologs. The mammalian GABABR C-terminal domain mediate processes such as trafficking out of the ER or modulation of receptor activity44, but it has also been suggested as non-essential for functional GABABR heterodimers42,43,45. GABABR C-termini therefore differ dramatically between mammals and cnidarians, suggesting they do not affect essential functions, and were excluded from the full comparison. Protein domains were predicted as detailed in Methods.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5, Methods and Table 3.

Reporting Summary

Supplementary Table 1

Transcriptome analysis of differentially expressed transcripts of control and baclofen-treated planulae.

Supplementary Table 2

GOSeq analysis of differentially expressed transcripts of control and baclofen-treated planulae.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Levy, S., Brekhman, V., Bakhman, A. et al. Ectopic activation of GABAB receptors inhibits neurogenesis and metamorphosis in the cnidarian Nematostella vectensis. Nat Ecol Evol 5, 111–121 (2021). https://doi.org/10.1038/s41559-020-01338-3

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41559-020-01338-3

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing