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

Nature Ecology & Evolution volume 5, pages 111–121 (2021)Cite this article

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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.

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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.

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Data availability

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

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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.

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Authors and Affiliations

  1. Marine Biology Department, The Leon H. Charney School of Marine Sciences, University of Haifa, Haifa, Israel

    Shani Levy, Vera Brekhman, Assaf Malik & Tamar Lotan

  2. Department of Human Biology, University of Haifa, Haifa, Israel

    Anna Bakhman & Mickey Kosloff

  3. Centre for Genomic Regulation, Barcelona Institute of Science and Technology, Universitat Pompeu Fabra, Barcelona, Spain

    Arnau Sebé-Pedrós

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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.

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Correspondence to Mickey Kosloff or Tamar Lotan.

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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.

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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

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