Abstract
The evolutionary transition from diffusion-mediated cell-cell communication to faster, targeted synaptic signaling in animal nervous systems is still unclear. Genome sequencing analyses have revealed a widespread distribution of synapse-related genes among early-diverging metazoans, but how synaptic machinery evolved remains largely unknown. Here, we examine the function of neurexins (Nrxns), a family of presynaptic cell adhesion molecules with critical roles in bilaterian chemical synapses, using the cnidarian model, Nematostella vectensis. Delta-Nrxns are expressed mainly in neuronal cell clusters that exhibit both peptidergic and classical neurotransmitter signaling. Knockdown of δ-Nrxn reduces spontaneous peristalsis of N. vectensis polyps. Interestingly, gene knockdown and pharmacological studies suggest that δ-Nrxn is involved in glutamate- and glycine-mediated signaling rather than peptidergic signaling. Knockdown of the epithelial α-Nrxn reveals a major role in cell adhesion between ectodermal and endodermal epithelia. Overall, this study provides molecular, functional, and cellular insights into the pre-neural function of Nrxns, as well as key information for understanding how and why they were recruited to the synaptic machinery.
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Introduction
The nervous system takes center stage among key innovations in animal evolution. Its appearance drastically shaped how animals perceive signals from the environment, integrate and store information, and mount appropriate responses. Chemical synapses are one of the major modes of network-dependent signaling in the nervous system. In recent years, comparative genomic analyses across various metazoan species have revealed the conservation and deep evolutionary origins of synapse constituent genes. Many proteins that have synaptic functions in Bilateria are found in non-bilaterian genomes1,2,3, including neuron-less animals4,5,6, and in unicellular close relatives of metazoans7,8,9. It remains a mystery, however, how synapse-dependent neural signaling emerged early during animal evolution and how synaptic protein machinery was later refined.
During the early evolution of neural systems, diffusion-based volume transmission by peptides may have been the primary mode of cell–cell communication10,11,12,13,14. Later, faster and more targeted synaptic signaling was needed, as animal bodies and behaviors increased in complexity15. One key factor that sets chemical synaptic signaling apart from diffusible signaling is the establishment of precise cell–cell contacts between the presynaptic neurons and the postsynaptic cells. These contacts are facilitated and stabilized by Synaptic Adhesion Molecules (SAMs). Several SAMs are also involved in assembly of intracellular protein complexes, effectively contributing to maintenance, specificity, and plasticity of synapses16,17,18. Disruption of SAMs cause synaptic dysfunction, leading to various neurodegenerative and neurodevelopmental diseases19,20.
Among all SAMs, neurexins (Nrxns) are major presynaptic hub molecules at chemical synapses in Bilateria21,22,23. Depending on which postsynaptic ligand is bound, Nrxns may promote excitatory or inhibitory synapse development24. Furthermore, Nrxns are involved in Ca2+-triggered neurotransmitter release25 and proper organization of synaptic molecules26,27,28,29. Alterations of Nrxn gene sequence or expression have been linked to neuropsychiatric and neurodegenerative disorders22,30. Previous studies reported that Nrxns are expressed mainly in neurons and astrocytes31,32. However, several phylum-wide gene surveys demonstrated that Nrxns predated neuron and synapse appearance and that they are conserved in all metazoan lineages33,34, suggesting that Nrxns served functions unrelated to synapses in early metazoan ancestors. Still, neither pre-neural functions of Nrxns nor why they became major components of neural synapses during metazoan evolution are well known.
In this paper, we first compared structures of Nrxn proteins and their features required for synaptic function, mainly targeting early divergent lineages of metazoans. Interestingly, bilaterians and cnidarians are the only lineages in which Nrxns are abundantly expressed in the nervous system. In ctenophores, the earliest branching lineage with a nervous system, Nrxns are expressed in a wide range of cell types, including epithelial cells, as they are in neuron-less poriferans and placozoans. Then, we examined functions of the two types of Nrxns—neuronal and non-neuronal Nrxns—in the sea anemone, Nematostella vectensis, a cnidarian that is the closest outgroup to the Bilateria. We show that neuronal Nrxns are involved in motor control of N. vectensis polyps and that this function is independent of neuropeptide-mediated neurotransmission. Pharmacological testing indicated instead the potential involvement of small-molecule neurotransmitters, glutamate and glycine. Furthermore, we observed that the widely expressed and more ancestral non-neuronal Nrxn is involved in proper heterophilic cell-cell adhesion. Altogether, these results enhance our understanding of putative synaptically mediated neurotransmission in non-bilaterians and their non-neuronal ancestral functions.
Results
Identification and characterization of non-bilaterian Nrxns
At synapses in bilaterians, Nrxn exerts its functions by binding to various proteins through its intracellular and extracellular domains (Fig. 1a). To explore possible roles of Nrxns during early nervous system evolution, we first surveyed the presence of Nrxn homologs and their domain organization in metazoans and their closest unicellular relatives. In contrast to a previous phylogenetic study of Nrxn superfamily members33, we performed a detailed comparison focused on the organization of Laminin/Neurexin/Sex hormone-binding globulin (LNS) repeated domains. Despite low sequence identity (20%), LNS domains show high structural similarity as revealed by the crystal structure of Nrxns35. Therefore, their structural information is useful for obtaining ideal alignments between phylogenetically distant bilaterian and prebilaterian Nrxns sequences. We first performed a structural prediction based on Bos taurus alpha-Nrxn1 structures (PDB IDs: 3QCW and 3POY) and found a conserved LNS domain organization in prebilaterian and bilaterian Nrxns (Fig. 1b). Our molecular phylogenetic tree using alignments based on structural information confirmed the presence of Nrxn genes in all metazoan species surveyed, with the exception of some poriferan species (Fig. 1c, Supplementary Figs. 1, 2). Bilaterian Nrxns grouped in a single clade with bilaterian Casprs (contactin-associated proteins), also members of the Nrxn superfamily. However, no non-bilaterian Nrxn-like homologs contained the N-terminal discoidin domain typical of Caspr proteins (Supplementary Fig. 2). Hence, it seems that Caspr genes first appeared in the bilaterian lineage.
a Overview of synaptic protein complexes mediated by Nrxns in Bilateria. b 3D structure models of bilaterian and non-bilaterian Nrxns. Domain organization of bovine α-Neurexin 1 (PDB ID:3POY) used as a template for homology modeling (upper panel). The crystal structure of bovine NRXN1A includes seven contiguous domains of the extracellular region (LNS2 to LNS6 domains and intervening EGF2 and EGF3 domains) (lower panels, top left). Predicted 3D structures of Nrxns of Mus musculus (Bilateria), Nematostella vectensis (Cnidaria), Bolinopsis mikado (Ctenophora), Trichoplax adhaerens H2 (Placozoa), and Sycon ciliatum (Porifera) are shown. The root-mean-square deviation (RMSD) values in angstroms between the template and modeled structures were also calculated. c Distribution of Neurexin (Nrxn), its binding partners, and other associated synaptic proteins in metazoan lineages and their closest unicellular relatives. d Domain organizations of the Nrxn proteins. Bilaterian α-Nrxn (full-length) consists of an N-terminal signal peptide, followed by three repeats of LNS(A)-EGF-LNS(B), a single transmembrane domain, and a short cytoplasmic tail. Shorter splice variants (β-Nrxn (vertebrates only) and γ-Nrxn) of bilaterian Nrxns are also shown. Non-bilaterian Nrxns are classified into three types: α-, δ-, and ε-Nrxns. Source data are provided as a Source Data file.
A survey of known binding partners of Nrxns demonstrated that many postsynaptic interacting factors, e.g., Dystroglycan, appeared after the last common ancestor of Placozoa/Cnidaria/Bilateria (Fig. 1c). Meanwhile, among the major Nrxn partner candidates, only Latrophilin and Neuroligin have homologs in the Ctenophora and Porifera. The paucity of Nrxn apparatus components in early-branching metazoans contrasts with the full deployment of other membrane signaling proteins, such as Semaphorin/Plexin and Ephrin/EphrinR (Supplementary Fig. 3).
There are three Nrxn protein products in vertebrates, named α, β, and γ, but only α and β types contain LNS domains36,37. Therefore, γ-Nrxn was excluded from our analysis. All investigated members of Porifera (other than demosponges Amphimedon queenslandica and Haliclona amboinensis) and Ctenophora have a single α-Nrxn homolog. Poriferan and ctenophoran α-Nrxns possess six LNS domains and three epidermal growth factor (EGF) domains, similar to the full-length α-Nrxn of bilaterians. Cnidarians and placozoans also harbor an α-like Nrxn which contains only four or five LNS domains (Fig. 1d, Supplementary Fig. 1). In addition to the α-Nrxns, cnidarians also possess other Nrxn genes. Firstly, a new type, named here as “δ-Nrxn”, is a short Nrxn sequence that is observed only in cnidarians. This type is a reminiscent of the short β and γ isoforms found in bilaterians. Secondly, another cnidarian-specific type named “ε-Nrxn” is supposedly sister to cnidarian α-Nrxns (Supplementary Fig. 2). However, some regions, in particular at the C-terminus, are rather divergent and cannot be annotated with any protein domains. Unlike the β- and γ-Nrxns in bilaterians, which are produced by alternative splicing from the same gene as α-Nrxn, the cnidarian α-, ε-, and δ-Nrxns are encoded by separate genes. It seems that the α-Nrxns are ancestral, δ- and ε-Nrxns were acquired and expanded independently in cnidarians, and β- and γ-splice variants of α-Nrxns emerged only in bilaterians (Supplementary Fig. 2).
Our detailed comparative analysis showed further potential conservation of non-bilaterian Nrxns. The evolution of Nrxn involved the assembly of a single LNS-EGF-LNS (LEL) cassette and became triplicated to create the ancestral α-Nrxn with three copies of LEL36,38. This hypothesis was supported by our analysis of α-Nrxn sequences showing that LNS1/3/5 and LNS2/4/6 form distinct clades, respectively (Supplementary Fig. 4). Among the multiple LNS domains, LNS2 and LNS6 stand out due to the large number of known binding partners. In vertebrates, Neuroligin, Dystroglycan, GABA(A)R are known to bind to LNS6, while Latrophilin and Dystroglycan bind to LNS2. It should be noted that these Nrxn binding partner gene homologs exist in cnidarians (Fig. 1). Crystal structures of Nrxns demonstrated a calcium ion coordinated by four amino acid residues and two water molecules39. Our modeling demonstrated that majority of cnidarian δ-Nrxns have conserved geometry for Ca2+ coordination. Even when some residues are not conserved, the amino acid substitute might still be able to chelate Ca2+ (Supplementary Fig. 5), suggesting the ability of cnidarian δ-Nrxns to bind Neurologin and Dystroglycan in a calcium-dependent manner, as it is for bilaterian Nrxns. On the other hand, non-bilaterian α-Nrxns may not require Ca2+. These data, taken together with the specific function of δ-Nrxns in cnidarian neurons (see below), suggest that Ca-dependent Nrxn function may have arisen in conjunction with the evolution of the nervous system. Notably, the PDZ-binding motif at the C-terminus of bilaterian Nrxns crucial for intracellular signaling26,40 is absent in α-Nrxns of Porifera and Ctenophora (Supplementary Fig. 1). The preservation of the core domain, in contrast to the increased Nrxn gene repertoire in prebilaterians, suggests evolutionary conservation in the mode of action of Nrxns, including partner binding.
To gain insights into the function of Nrxn proteins in early metazoans, we next examined the expression profile of Nrxns in known cell types41,42. In N. vectensis, δ-Nrxns (NvNrxnδ1,−2,−3, and –4) and ε-Nrxns (NvNrxnε1, −2, and −3) are highly enriched in neurons, while the two homologs of α-Nrxns (NvNrxnα1 and −2) are broadly expressed in different cell types including epithelial cells (Fig. 2a, Supplementary Fig. 6). Likewise, the α-Nrxns of Trichoplax adhaerens (Placozoa) and of Mnemiopsis leidyi (Ctenophora) also show broad expression patterns (Supplementary Fig. 7).
a Abundance of Nrxn homologs across cell type categories of adult Nematostella vectensis. b Abundance of δ-Nrxns across neuronal cell clusters within neuron categories (peptidergic and uncharacterized). Cell clusters 62 and 63 (green box) express both δ-Nrxns and neuropeptide NvPRGamide. c Representative images of hybridization chain reaction staining of NvNrxnδ2 and NvPRGamide mRNAs in primary polyps (7 dpf). Arrowheads denote the neurons expressing both NvNrxnδ2 and NvPRGamide genes. d qPCR analysis of δ-Nrxns (n = 4) and NvPRGamide (n = 3) expression at 7 dpf after triple knockdown of δ-Nrxn genes (NvNrxnδ1, −2, −4). Shown is the mean ± SD; ** and *** denote the statistical significance p < 0.01 and p < 0.001 in two-sided unpaired t test of at least three independent experiments, respectively. e Representative images of NvPRGamide-positive neurons visualized by immunostaining of 7 dpf polyps transfected with control siRNA (left) or siRNAs for NvNrxnδ1 (#2 + #3), NvNrxnδ2 (#3), and NvNrxnδ4 (#1 + #2) (right). In (a, b), dots correspond to normalized UMI count abundance scaled per gene. Dots scale from smallest to largest, corresponding to lowest and highest expression, respectively. Scale bars in (c) top, (c) middle and bottom and (e), 50, 20 and 100 µm, respectively. Statistics of (d) Control vs TKD p = 0.002238 (NvNrxnδ1), Control vs TKD p = 0.000520 (NvNrxnδ2), Control vs TKD p = 0.001978 (NvNrxnδ4), Control vs TKD p = 0.379941 (NvPRGa). Source data are provided as a Source Data file.
A population of PRGamide-expressing neurons is δ-Nrxn-positive
We then analyzed whether non-bilaterian Nrxns and their putative membrane-bound partners are expressed in neurons using single-cell expression data. As with Nrxns, many putative partners are expressed in neurons of adult N. vectensis (Fig. 2a, Supplementary Fig. 6). In T. adhaerens, expression is observed in epithelial and lipophil cells, as well as in some peptide-expressing cells (Supplementary Fig. 7). In M. leidyi, Latrophilin-like and Neuroligin-like genes, the only Nrxn partner candidates, are mainly localized in non-neuronal cells, consistently with the broad expression profile of the ctenophore α-Nrxn. In N. vectensis, δ-Nrxns (NvNrxnδ1,−2,−3, and −4) are enriched in neuronal clusters C62 and C63, suggesting a specific requirement for this type of Nrxns in functions of this subpopulation of neurons (Fig. 2b). Remarkably, the C62/C63 neuronal cluster also exclusively expresses the PRGamide neuropeptide. We then focused on NvNrxnδ1,−2, and −4, since NvNrxnδ3 has an incomplete C-terminus. Expressions of NvNrxnδ1,−2, and −4 increased during the period when neural networks develop after the planula stage (Supplementary Fig. 8) and are expressed in neurons in polyp stage (Fig. 2a, b, Supplementary Fig. 6). Since we were not successful in producing specific antibodies to detect NvNrxnδ1,−2, and −4 proteins, we then performed mRNA detection by in situ hybridization chain reaction (HCR). Although expression of δ-Nrxn mRNAs was weak, clear expression of NvNrxnδ2 could be detected in at least some PRGamide-expressing neurons (Fig. 2c).
Neuron development is not affected by δ-Nrxn knockdown
In bilaterians, Nrxns are expressed early in development long before synapse formation begins and they are required for neurite development in Drosophila melanogaster 43. To verify Nrxn function in cnidarians, we first tested the effects of Nrxn knockdown on the developmental process of δ-Nrxn-positive neurons. We performed small interfering RNA (siRNA)-mediated gene knockdown by delivering siRNAs targeting each δ-Nrxn mRNA into N. vectensis embryos44. Loss of any of the three δ-Nrxns (NvNrxnδ1,−2, and −4) was non-lethal, and caused no visible defects in polyp development (Supplementary Fig. 9). The effect of knockdown of δ-Nrxns on development of the neural network was further evaluated by immunostaining for PRGamide in the primary polyps. Single-gene knockdown showed no obvious changes in development, projection patterning, and distribution of the PRGamide+ neurons (Supplementary Fig. 10). The expression level of the PRGamide gene was also unaffected. To test the possibility that loss of a single δ-Nrxn could be compensated by functional redundancy by other δ-Nrxn proteins, we performed triple gene knockdown targeting all three δ-Nrxns. However, even in embryos in which all three δ-Nrxns were knocked down, no significant defects in neuron development and assembly were observed, nor was deregulation of PRGamide gene expression (Fig. 2d, e).
δ-Nrxn knockdown impairs locomotor activity
In bilateral animals, such as mice and Drosophila, where Nrxn functions as a synaptic member, loss of Nrxns negatively influences locomotor activity27,45. Similarly, we carried out behavioral assays to assess whether the reduction of δ-Nrxns has the same effect in N. vectensis. Among the several behaviors of N. vectensis polyps, we observed and quantified peristalsis, a rhythmic radial contraction of the body column of the polyp that starts from the region just below the pharynx and propagates to the foot. This peristaltic movement, which is actively observed even in petri dishes, is a major spontaneous behavior of burrowing sea anemone polyps. Knockdown of a single δ-Nrxn significantly decreased the number of peristaltic waves (Fig. 3a). Similarly, triple-knockdown polyps displayed fewer waves compared to control animals, but unexpectedly, it did not elicit further wave reduction compared to single-knockdown animals (Fig. 3a–c). A previous study showed that reduced peristaltic waves in juvenile N. vectensis is correlated to a reduced number of neurons46, indicating that peristalsis is under the control of neural activities. Our results suggest that the function of δ-Nrxns expressed in specific PRGamide+ neuronal subpopulation play an essential role in the neuronal activities responsible for controlling peristalsis.
a Number of peristaltic waves of polyps transfected with siRNA #2 + #3 for NvNrxnδ1, #3 for NvNrxnδ2, or #1 + #2 for NvNrxnδ4, respectively. b Number of peristaltic waves of polyps after triple knockdown of NvNrxnδ1/2/4 genes by co-transfection of siRNAs (δ1 #3, δ2 #3, and δ4 #2). c Representative image time series of peristalsis of control (upper panels) or NvNrxnδ1/2/4 siRNAs-transfected (lower panels) polyps. Green, black, and red arrowheads indicate the 1st, 2nd and 3rd propagated wave, respectively. d qPCR analysis of NvPRGamide mRNA expression of NvPRGamide siRNA-transfected polyps (n = 3). e Representative immunostaining images of NvPRGamide peptide in NvPRGamide siRNA-transfected polyps. Scale bar, 100 µm. f Peristalsis rate of PRGamide-depleted polyps. g Expression of NvNrxnδ genes in PRGamide-depleted polyps (n = 3 per gene). Number of peristaltic waves of polyps upon an increasing concentration (1, 10, and 100 µM) of dizocilpine, an ionotropic glutamate receptor NMDA inhibitor (h, i) (n = 10 for 1 and 100 µM, n = 11 for 10 µM), or glycine, an inhibitory neurotransmitter (j, k) (n = 11 for 1, n = 12 for 10 µM, n = 13 for 100 µM). Two image time series are shown as examples (h); 10 µM dizocilpine, (j); 10 µM glycine. Below the images, wave initiation events are marked as a wave icon on the timeline. mRNA/protein expression and peristalsis were analyzed on 7 dpf and 8 dpf, respectively. a, b, d, f, g, i, k Statistical data are presented as means ± SDs for barplots and as interquartiles with minimum and maximum data for boxplots; ** and ***, p < 0.01 and p < 0.001, respectively. P values were obtained using two-sided unpaired t tests of at least three independent experiments in (a, b, d, f, g). Statistics of (a) Control vs NvNrxnδ1 KD p = 2.10735E–20, Control vs NvNrxnδ2 KD p = 2.44847E–21, Control vs NvNrxnδ3 KD p = 2.36523E–22; (b) Control vs TKD p = 5.90799E–09; (d) control vs NvPRGamide KD p = 0.003816; (f) Control vs NvPRGamide KD p = 0.537192; (g) Control vs NvPRGamide KD p = 0.579649 (NvNrxnδ1), Control vs NvPRGamide KD p = 0.665634 (NvNrxnδ2), Control vs NvPRGamide KD p = 0.151328 (NvNrxnδ4). For (i, k), two independent experiments we performed. Source data are provided as a Source Data file.
So how do δ-Nrxns control polyp peristalsis? They do not appear to be expressed at high levels in all PRGamide+ neurons (Fig. 2c), an indication that they might not participate in PRGamide-mediated peptide neurotransmission. To examine whether peristalsis is modulated by PRGamide neuropeptide-mediated signaling, we decreased PRGamide precursor mRNAs by gene knockdown. Although a significant reduction of PRGamide mRNA levels and immunofluorescent signaling of mature PRGamide peptides was observed upon knockdown (Fig. 3d, e), we did not see a significant effect on the peristaltic wave rate (Fig. 3f). Moreover, knockdown of PRGamide did not affect expression of δ-Nrxns (Fig. 3g). These data indicate that PRGamide peptidergic signaling is not involved in the control of δ-Nrxn-dependent spontaneous peristalsis and support the idea that neuropeptide signals act through synapse-independent volume transmission.
Since δ-Nrxns may not be required in peptidergic signaling, these synaptic cell adhesion molecules may be crucial for signaling mediated by other neurotransmitters. In cnidarians, evidence is accumulating to suggest that glutamate and glycine are responsible for signaling through ionotropic receptors47,48,49. δ-Nrxn+ neurons are not only equipped with peptidergic signaling-related genes, but also with molecules necessary for chemical transmission by glutamate and glycine14 (Supplementary Fig. 11). We therefore carried out a series of pharmacological assays to determine whether peristalsis is regulated by glutamate and glycine signaling. Interestingly, systemic treatment with L-glutamate or NMDA at concentrations previously used in other cnidarians47,50 had no clear effects (Supplementary Fig. 12), but the NMDA antagonist, Dizocilpine, resulted in a significant decrease in N. vectensis peristalsis (Fig. 3h, i). The inhibitory effect on ionotropic GluRs was specific to Dizocilpine, and no significant effect was observed with the Kainate/AMPA receptor antagonist, NBQX (Supplementary Fig. 12). Also, treatment with glycine, a major inhibitory neurotransmitter in bilaterians, resulted in a reduction in wave number, similar to inhibition of NMDA-mediated glutamate signaling (Fig. 3j, k) and is consistent with the glycine’s inhibitory function observed in other cnidarians49,51. The above data indicate that spontaneous polyp peristalsis observed in our experimental conditions may be caused by high glutamate signaling activity. Our detection by HCR of genes involved in glutamate and glycine signaling (vesicular transporters: SLC17, SLC32, and SLC6; ionotropic receptors: iGluRs and GlyR alphas) was not successful, probably due to their low expression levels. Therefore, physiological details of the glutamatergic and glycinergic neuronal types in N. vectensis remain unclear. Furthermore, it should be noted that the effects of Nrxn KD and glutamate signal inhibition may result in similar phenotypes through different mechanisms. Nevertheless, our results from gene expression patterns, knockdown experiments, and pharmacology suggest that δ-Nrxns regulate peristalsis in N. vectensis through classical neurotransmitters such as glutamate, rather than PRGamide peptide signals.
α-Nrxn knockdown disrupts proper epithelial organization
After we demonstrated involvement of Nrxn in prebilaterian neuronal function, we decided to examine the ancestral function of Nrxns, which was acquired before the emergence of the nervous system. In cnidarians, α-Nrxns are a group of broadly-expressed Nrxns that offer insight into preneural functions of Nrxns. In this analysis, we focused on NvNrxnα1, which is mainly expressed in epithelial cells of adult N. vectensis (Fig. 2a, Supplementary Fig. 6). Visualization of mRNA confirmed the epithelial expression of NvNrxnα1 during planula and polyp stages (Fig. 4a, b). Knockdown of NvNrxnα1 caused a significant decrease in mRNA and protein levels without affecting polyp viability (Fig. 4c, Supplementary Fig. 13). However, knockdown animals displayed decreased epithelial integrity and abnormal morphogenesis (Fig. 4d–h). Cortical F-actin staining showed that by the planula stage, ectoderm and endoderm layers were not properly attached to each other (Fig. 4d, e, Supplementary Fig. 14). On the other hand, staining of Cadherin 1 (NvCdh1), located in the apical and basal junctions of both ectoderm and endoderm layers of N. vectensis planula larva52, suggested that homophilic cell-cell adhesion in the epithelial layers remains intact (Supplementary Fig. 15). The detachment between ectoderm and endoderm caused by NvNrxnα1 knockdown became more pronounced at the polyp stage, and an abnormal “blister” appearance of the tentacles was observed (Fig. 4f–h). Additionally, some knockdown individuals were unable to achieve normal body size. These findings indicate that NvNrxnα1 is involved in proper organization of epithelial tissue.
Representative images of hybridization chain reaction staining of NvNrxnα1 mRNA at planula (a) and primary polyp (b) stages. Lower panels, higher magnification. Dotted line in (a) denotes the ecto/endo boundary. Filled and open arrowheads indicate ectoderm and endoderm, respectively. c qPCR and western blotting analysis of 7 dpf polyps transfected with control siRNA or NvNrxnα1 siRNAs (#1 + #2). α-tubulin as loading control (right) (n = 8 for qpcr, n = 3 for western blotting). d Representative images of phalloidin staining (black) of control (left) or NvNrxnα1-depleted (right) planulae (4 dpf). e Quantification of the epithelial detachment length measured by confocal longitudinal sections of phalloidin-stained planulae (4 dpf) (n = 23 for siControl, n = 31 for siNvNrxnα1). f Representative images of typical phenotypes in NvNrxnα1-depleted polyps: normal, abnormal tentacles, and abnormal body. Polyps with abnormal tentacles have crooked-, or rigid-looking tentacles (middle). Polyps with abnormal bodies have short and underdeveloped body column (right). g Representative images of phalloidin staining (black) of control (left) or NvNrxnα1-depleted (right) polyps. h Quantification of the percentages of NvNrxnα1 knockdown polyps showing the three phenotypes described in (f). In (c) statistical data are presented as mean ± SD, and in (e) as median with interquartile range; ** and ***, p < 0.01 and p < 0.001, respectively. P values were obtained using two-sided unpaired t test of at least three independent experiments in (c). Scale bars in (a, b) top, (a, b) bottom, (d, f, g), 50, 20, 20, 100, 25 µm, respectively. Statistics of (c) left Control vs NvNrxnα2 KD p = 1.71908E–10; (c) right Control vs NvNrxnα2 KD p = 0.003351. For (e), two independent experiments were performed. Source data are provided as a Source Data file.
Discussion
Previous studies of non-bilaterian nervous systems began with electron microscopic observations of neurons and synapse-like structures. In recent years, the repertoire of neural-related gene homologs in early diverging metazoan genomes and molecular mechanisms of neural development have been analyzed1,53,54,55. Additionally, our understanding of the function of the cnidarian nervous system is rapidly advancing, primarily due to application of molecular biological tools44,56,57,58,59,60. However, despite the high evolutionary conservation of synapse-related molecules in their genomes, there is little direct experimental data on their function in cnidarians; thus, little is clear about pre-bilaterian synaptic functions. Nrxn is a major cell adhesion molecule at presynapses of neurons in bilateral animals and participates in synapse assembly through adhesion with various postsynaptic membrane proteins. Our present study of Nrxns in N. vectensis characterizes the potential function of the synaptic molecule in the cnidarian nervous system. We showed that knockdown of neuronally expressed δ-Nrxns resulted in abnormal polyp behavior and demonstrated that Nrxns are involved in regulating motor activity in animals with relatively simple nervous systems.
A genomic survey identified full-length and several lineage-specific short Nrxn genes in early-branching metazoans, further confirming conservation of the presence of putative Nrxn partners (Fig. 1c, Supplementary Figs. 3, 6, 7). In cnidarians, Heliopora coerulea (Octocorallia) and Hydra vulgaris (Hydrozoa), as well as in early-branching lineages other than cnidarians, sequence conservation of putative Nrxn partners is low, so Nrxn-binding membrane protein candidates could not be predicted. However, the richness of candidate protein repertoires in a wide range of metazoan lineages suggests that their interactions were established before the emergence of neural synapses. Further insights were also obtained regarding the ability of Nrxn to form presynaptic complexes via its C-terminus. In N. vectensis (Anthozoa), short Nrxns (δ- and ε-Nrxns) are the major group of Nrxns bearing a clear C-terminal PDZ motif (Supplementary Fig. 1), which is thought to be required for formation of presynaptic complexes in bilaterian neuronal synapses. Our analyses of δ-Nrxn expression and function demonstrated their neural roles in N. vectensis, suggesting conservation of presynaptic function mediated by the C-terminus. This neurospecific function may also be served by another short Nrxn, the ε type. In all cnidarian species examined, including N. vectensis, the ε type also has a PDZ motif (Supplementary Fig. 1). Interestingly, ε-Nrxn, the only short Nrxn with a PDZ motif in Hydra, is expressed in their neurons (Supplementary Fig. 16). These findings may suggest that acquisition of a PDZ binding motif coincided with the neural functions of Nrxns. In bilaterians, the PDZ binding motif is responsible for forming a complex with the scaffolding proteins like CASK and Mint26,40,61. However, CASK and Mint are not exclusively expressed in δ-Nrxn+ neurons of N. vectensis. This may indicate that the complex was not fully deployed yet in the common ancestor of Cnidaria/Bilateria. Indeed, PDZ motifs are present in α-Nrxns even in neuron-free placozoa (Supplementary Fig. 1). Additionally, in Hydra, the PDZ motif is conserved in Nrxnα1 and −2, which are mainly expressed in ectoderm cells (Supplementary Fig. 16). Therefore, the presence of the PDZ motif alone is not sufficient to infer the ancestral function of Nrxn at synapses. Attempts to identify the repertoire of Nrxn protein complexes formed in early-branching metazoans, including N. vectensis, and the cell types in which they are located, will be important research directions to understand evolutionary processes of synaptic organization and function.
From our results, we could deduce that like the canonical roles of Nrxns in bilaterian neuronal synapses, cnidarian δ-Nrxns are involved in neurotransmission of fast chemical transmitters, e.g., glutamate and glycine, rather than in release of other transmitters, e.g., neuropeptides, which is consistent with previous reports on regulation of peristalsis by acetylcholine signaling62. However, much remains to be discovered about mechanisms of action of neurotransmitter signals in cnidarians. For example, the neurophysiological details of glutamate and glycine signaling and their Nrxn dependence require further analysis. Despite the high conservation of nAChR genes, cnidarians appear unable to synthesize acetylcholine54,63,64,65. Although the rich repertoire of GABA(A) receptor genes that are candidate binding partners for Nrxn in N. vectensis and their neuronal localization are interesting (Fig. 2a), in cnidarians GABA appears to be synthesized primarily in non-neuronal cells14; therefore, its function as a neurotransmitter is still uncertain. Molecular phylogenetic analyzes have also shown that the ability to synthesize many monoamine molecules may have been acquired after bilaterian cladogenesis66. Interestingly, we observed a significant decrease in the mRNA expression of postsynaptic protein PSD95 upon knockdown of δ-Nrxns (NvNrxnδ1 and −2) (Supplementary Fig. 17), suggesting a possible interaction between pre- and post-synaptic protein complexes. A clear understanding of the chemical neurotransmission in cnidarians may reveal the hidden complexity of their nervous systems, which have been considered simple based on their morphology. Actually, our data provided further insight into the specificity and complexity of cell types or synaptic connections in controlling cnidarian behavior. As seen in the knockdown phenotype, the absence of one or more δ-Nrxns results in the same level of dysregulation. This suggests that the three δ-Nrxn homologs either act cooperatively or have different binding partners and specific pathways to which they belong. It is reasonable to imagine that the diversification of Nrxn has also led to the diversification of its binding proteins. For example, bilaterian γ-Nrxns has been reported to interact with Frizzled67.
Single-cell transcriptome (SCT) and in situ hybridization chain reaction (HCR) also provided insights into early-branching metazoan cell types that express the most evolutionarily conserved α-Nrxns. In N. vectensis polyps, NvNrxnα1, are expressed in various cell types, but are most abundant in epithelial cells. Multiple SCT data42,68 have shown neuron clusters that weakly express N. vectensis α-Nrxns. However, in our HCR analysis, we observed undetectable level of α-Nrxn expression in neurons. This is consistent with experimental data showing that α-Nrxn protein is abundantly expressed in the epithelia of other sea anemones33. The fact that α-Nrxn is expressed in epithelial cells in early-branching metazoans (Supplementary Figs. 6, 7) suggests that the ancestral function of this cell adhesion apparatus is related to organization of epithelial cell layers. As shown in Fig. 4, functional loss of NvNrxnα1 in N. vectensis caused a wider gap between the ectoderm and endoderm layers, whereas cell adhesion between homogeneous cells in each epithelial cell layer appeared intact. This data seems to suggest that NvNrxnα1 is involved mainly in regulation of adhesion between heterogeneous cell types, rather than between homologous cell types within each epithelial cell layer. However, we cannot exclude the possibility that α-Nrxns also contributes to homologous cell-cell adhesion, but that its role is complemented by other mechanisms. It has been postulated that α-Nrxns function as a key component of septate junctions (SJs) formed between homogeneous cells33. Since the distribution of SJ structures before cnidarians is not clear yet, the actual nature of the organization of SJs and Nrxn involvement in early-branching metazoans requires further analysis. The formation of inter-epithelial gap caused by α-Nrxns deletion was observed not only in developing planula larvae, but also at the polyp stage, where mesoglea filled with extracellular matrix (ECM) develops between both epithelia. This may indicate that cnidarian α-Nrxns may interact with ECM proteins in addition to cell adhesion molecules. Indeed, in bilaterians, ECM components such as thrombospondin, Hevin, and Sparc are known to interact with Nrxns69,70.
Molecular mechanisms of functions of Nrxns in N. vectensis requires further analysis in the future. Figure 5a shows one possible evolutionary scenario for emergence and placement of the Nrxn complex in neurons. If α-Nrxns serve an important function in cell adhesion between heterologous cells, our data that demonstrate the ancestry and subsequent recruitment of non-neuronal Nrxns into the neural synaptic machinery may offer a hypothesis about the evolution of Nrxn-mediated intercellular contact (Fig. 5b). Nrxns were acquired in multicellular ancestral animals and organize adhesion between different cell types, such as ectoderm and endoderm. This new mechanism for regulating cell adhesion between different cell types has diversified in function as the number of cell types increases due to “division of labor” and as multicellular systems become more complex. For example, in the nervous system, division of labor such as sensory and effector cells created a need for specific cell-to-cell communication. Further sophistication of heterogeneous cell adhesion mechanisms in the nervous system through the co-option of Nrxn genes may have been a necessary adaptation toward the evolution from peptide-mediated volume transmission to synaptic transmission mediated by chemical neurotransmitters such as glutamate.
a Nrxns appeared in the last common ancestor of Metazoa. Early Nrxns, which lack the PDZ motif and calcium-binding ability, functioned in a variety of cell types including epithelial cells. Nrxns acquired the PDZ motif in the last common Placozoa/Cnidaria/Bilateria ancestor. Specific neuronal expression and synaptic functions of Nrxn subtypes occurred both in Cnidaria and Bilateria. b Proposed hypothesis of the repurposing of Nrxns from heterologous cell-cell adhesion molecules to the chemical synapse organizer. As inferred from the current findings in choanoflagellates, multicellularity in ancestral animals is thought to have begun with colonization of homologous cells. At this evolutionary stage, cells began to be assembled by homophilic binding molecules such as cadherins. The development of the ECM and the increase in the repertoire of cell types facilitated the diversification of adhesion abilities between heterologous cells and with the ECM through the employment of heterophiloc adhesion molecules such as Nrxn. In the nervous system, deployment of Nrxns transformed peptide-mediated volume transmission into synaptic transmission, which enables more specific communication with various cell types.
Methods
Protein search
Protein sequences of representative non-bilaterian species from genome or transcriptome studies were obtained (Supplementary Table 1). Synaptic protein sequences were retrieved from UniProtKB/Swiss-Prot database and used as query sequences for Blastp searches. Blastp was run locally using BLAST+ (v2.7.1) or on the NCBI BLAST webpage with an e-value cutoff of 1 × 10−5. Redundant protein sequences that originated from the same gene model were identified and only the longest sequence was included for downstream analysis. Conserved protein domains were identified using consensus predictions of four databases: Conserved Domain Database (CD-Search), ScanProsite (ExPASy), Simple Modular Architecture Research Tool (SMART), and Comprehensive Domain Visualization Tool (CDvist)71. Conserved motifs and functional sites within a particular domain were checked using ScanProsite. For specific domains, such as PDZ domain, MoDPepInt Server72 was used to predict PDZ-binding motifs at the C-terminal region of each protein. Transmembrane domains and signal peptides were confirmed with SignalP 4.073 and Phobius74 algorithms. All protein accession IDs retrieved are listed in Supplementary Data 1.
Protein three-dimensional (3D) modeling
Homology modeling was used to computationally predict the three-dimensional (3D) structures of non-bilaterian Nrxn proteins. Each protein sequence was used as input to the HHPred server (https://toolkit.tuebingen.mpg.de/hhpred)75. Searches were done on the PDB_mmCIF70 database using default settings. The top query-template alignment was converted to PIR file format and forwarded to MODELLER76 for model building. 3D structures were visualized using the UCSF Chimera package (v1.15)77. Superimposition was done with the Chimera’s matchmaker command. Amino acid residues for Ca2+-binding were identified after superimposition. In addition to MODELLER, the recently available AlphaFold78 algorithm was also used to further confirm the models.
Sequence alignments and phylogenetic analyses
Sequence-based analysis of proteins containing repeated domains is particularly difficult. Because of the repeated, shuffled, missing, and highly degenerate domains found in repeat domain proteins, conventional multiple alignment algorithms often fail to yield accurate homology between related proteins. Thus, to avoid possible erroneous output of aligning full-length amino acid sequences containing multiple domain repeats, individual LNS domain sequences were retrieved from each Nrxn homolog. A phylogenetic analysis of LNS domains from full-length α-Nrxn homologs were first performed to inspect the relationship among them. Meanwhile, for the phylogenetic analysis of Nrxn proteins, only the two functional domains, LNS2 and LNS6 domains, were used for building the multiple sequence alignments. To do this, the LNS domains of each non-bilaterian α-Nrxn that aligned against the 3D structure of bilaterian LNS#2 or LNS#6 domain were retrieved after the superimposition in Chimera. The LNS domains of shorter type δ- and ε-Nrxns were structurally aligned to the cnidarian α-Nrxns. The LNS#2 and LNS#6 domain sequences from all species were aligned separately using Clustal Omega or the online version of MAFFT-DASH (https://mafft.cbrc.jp/alignment/server/) with default parameters and refinement algorithm L-INS-i79. A python script was written to merge the two alignments. The final alignment containing the LNS#2 and LNS#6 domains (Supplementary Data 2, 3) was used to build the phylogenetic tree. Maximum-likelihood analyses were implemented on IQ-TREE (v2.0.6)80 with standard model selection and 5,000 bootstraps or on RAxML (v8.2.11) with automated prediction of best protein substitution model and 1,000 bootstraps. Bayesian inference analysis was executed on the MPI version of MrBayes (v3.2.3)81 using two independent Markov chain Monte Carlo (MCMC) runs with four chains per run for 20,000 generations and sample every 100 generations. Phylogenetic trees were visualized in FigTree (v1.4.3) (http://tree.bio.ed.ac.uk/software/figtree/).
Single-cell expression analysis
Single-cell expression matrices for N. vectensis and M. leidyi were obtained previously14. The same method was performed to obtain the expression matrices for T. adhaerens. Briefly, protein models were annotated by Blastp alignment to the the C. elegans and human UniProt/NCBInr databases with an e-value cutoff of 1 × 10−5. Raw UMI counts, metacell assignments, and metacell annotations were downloaded from http://compgenomics.weizmann.ac.il/tanay/?page_id=724. A global-scaling normalization method was employed to normalize gene expression measurements for each cell by the total expression, multiplied with a scale factor (1,000 for N. vectensis; 10,000 for M. leidyi and T. adhaerens). A single-cell expression plot for H. vulgaris was obtained from https://research.nhgri.nih.gov/HydraAEP/ Single Cell Browser.
Nematostella culture
Animal care, spawning induction, and de-jellying were carried out as previously described82. Briefly, animals were maintained in culture boxes containing 1/3-strength artificial seawater (SEALIFE), hereafter called Nematostella medium, and kept in the dark at 18 °C. Animals were fed twice per week with freshly hatched Artemia (Ocean Star International, Inc.). Spawning was induced by incubating the animals at 26 °C for 12-14 hr. Gelatinous mass surrounding the eggs was removed using 4% (wt/vol) L-cysteine (Nacalai Tesque, Inc., cat. 10309-12) in Nematostella medium, pH ~7.5.
siRNA-mediated knockdown experiments
Custom siRNA design service was provided by Sigma-Aldrich. For each gene, two or three siRNA duplexes were custom synthesized as 21-mers with 3’ dTdT overhangs. To determine the non-specific effects related to siRNA delivery, a non-targeting negative control siRNA with the same modification was also synthesized. All siRNAs used in this study are listed in Supplementary Table 2. siRNA-mediated knockdown was performed as previously described60 with modifications. Briefly, concentrated fertilized eggs were washed three times with fresh Nematostella medium. Eggs were transferred to a 2-mL microcentrifuge tube. Then, medium was removed and replaced with 6% (wt/vol) Ficoll PM400 (GE Healthcare, cat. GE17-0300-10) in Nematostella medium. Each electroporation mixture, containing 200 μL of eggs and siRNAs suspended in 6% Ficoll/Nematostella medium, was transferred to a 4-mm-gap sterile cuvette (Bio-Rad, cat. 1652088). Cuvettes were gently tapped for 10 sec on each side to ensure complete mixing of eggs and siRNAs. Eggs were subjected to experimental electroporation parameters (voltage = 50 V; pulse = 25 ms; number of pulses = 1) using the Gene Pulser Xcell Eukaryotic System (Bio-Rad, cat. 1652661). Immediately, cuvettes were filled with Nematostella medium and then gently poured into a 150 mm × 15 mm petri dish containing Nematostella sperm water. Two control electroporation conditions were included per experiment: (1) with nuclease-free water instead of siRNAs and (2) a non-electroporated condition. Plates were incubated at room temperature (RT).
RNA extraction, cDNA synthesis, and RT-qPCR
Total RNA was extracted from 5- or 7-day post-fertilization (dpf) stage animals using RNeasy Mini Kit (Qiagen, cat. 74104) according to the manufacturer’s instructions. Genomic DNA residues were removed using an RNase-Free DNase Set (Qiagen, cat. 79254). RNA concentrations were determined using a NanoDrop 2000c spectrophotometer (Thermo Scientific). First strand cDNA synthesis was carried out using the SuperScript IV First-Strand Synthesis System (Thermo Scientific, cat. 18091050). To check siRNA knockdown efficiency, quantitative real-time PCR (qPCR) was conducted on a StepOnePlus Real-Time PCR System (Applied Biosystems) using the PowerUP SYBR Green Master Mix (Applied Biosystems, cat. A25741). All qPCR experiments were performed with a minimum of three technical replicates. Quantification was performed using the –ΔΔCT method with N. vectensis Gapdh (XM_001632855.1) as normalization control. Expression levels obtained using Gapdh were comparable with the results using Elongation factor 1-alpha (EF1α) and 18 S as control genes. All qPCR primer pairs used in this study are listed in Supplementary Table 3.
Production of antibodies
For antibodies against NvNrxnα1 protein and PRGamide peptides, custom-made polyclonal antibodies were produced in rabbit. The following NvNrxnα1 peptides were used for immunization: SFRTYKSSGTVL, LVSQRQMFQYA, MRPPSADLPQN. Each peptide was conjugated with KHL carrier protein. Pooled peptides for NvNrxnα1 and the C-terminal structure of amidated peptide (Cys-PRGamide) were used to immunize rabbits with FCA (Freund’s Complete Adjuvant) for the first injection and FIA (Freund’s Incomplete Adjuvant) for boost (Scrum Inc.). After checking immunoreactivity by ELISA assay, anti-sera were affinity-purified using peptide conjugated beads.
Western blot analysis
Protein extracts were prepared from primary polyps (5 or 7 dpf). Tissues were solubilized in lysis buffer (125 mM Tris-HCl pH 6.8, 2% SDS, 0.01% β-mercaptoethanol, cOmplete Mini EDTA-free (Roche, cat. 04 693 159 001)). After manual homogenization using a plastic pestle, samples were boiled at 95 °C for 5 min and sonicated for 15 min. Undissolved samples were separated from the supernatant by centrifugation at 8,000 x g for 1 min. Protein lysates were stored at −20 °C until western blotting. Prior to SDS-PAGE, protein lysates were prepared by heating at 95 °C for 5 min in sample buffer (Nacalai Tesque, Inc, cat. 09499-14). Samples were separated by SDS-PAGE using 7.5% gels or Any kD Mini-PROTEAN TGX Precast Protein Gels (Biorad, cat. 4569033) and transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Biorad, cat. 1704150). Membranes were incubated with blocking solution (50 mM Tris-HCl pH 6.8, 2 mM CaCl2, 80 mM NaCl, 5% skim milk powder, 0.2% NP40) for 1 h at RT. Incubation with primary antibodies (anti-NvNrxnα1—1:1000; anti-alpha-tubulin—1:1000 (Sigma Aldrich, cat. T6074)) was performed overnight at 4 °C. Membranes were washed with wash buffer (PBSTw 0.1%) (3 × 10 min each) before incubation with secondary antibody (anti-rabbit—1:10,000 (Jackson’s ImmunoResearch, cat. 111-035-003)) for 1 h at RT. After washing (5 × 10 min each), detection was performed using the ImmunoStar Zeta (FUJIFILM Wako Pure Chemical Corporation, cat. 291-72401). Band signal quantification was performed using Image Lab software (Biorad, v6.1).
Neuropeptide and phalloidin staining
For detection of PRGamide peptides, primary polyps were relaxed using 7% MgCl2 in Nematostella medium for 10 min and fixed in Zamboni’s fixative (2% paraformaldehyde (PFA), 0.2% picric acid, 0.1 M Phosphate buffer, pH 7.2) overnight at 4 °C. After fixation, animals were washed in PBS with 0.1% Tween (PBSTw 0.1%) at RT (3 × 20 min each), followed by inactivation of residual formaldehyde by a single wash with 0.1 M Glycine–HCl pH 7.0 for 15 min at RT. Samples were permeabilized with PBS containing 0.2% Triton X-100 (PBSTr 0.2%) at RT (3 × 20 min each). Samples were blocked in blocking buffer (1% BSA, 5% Normal goat serum, 0.1% sodium azide in PBSTw 0.2%) for 1 h at RT. Anti-PRGamide primary antibody was diluted in blocking solution (1:200) and incubation was performed overnight at 4 °C, followed by washing in PBSTr 0.2% at RT (3 × 10 min each). Animals were then placed in a secondary antibody solution of Alexa Flour 488 AffiniPure Goat Anti-Rabbit IgG (1:500, Jackson ImmunoResearch, cat. 111-545-144) for 1 h at RT. After washing in PBSTr 0.2% at RT (6 × 10 min each), drops of SlowFade Gold Antifade Mountant (Invitrogen, cat. S36937) were applied. For F-actin staining with phalloidin, animals were fixed with 4% PFA in PBS for 2 h at 4 °C followed by washing with PBSTr 0.2% (4 x 20 min each) at RT. Phalloidin (Cayman Chemical iFluor 488 Conjugate, cat. 20549) and DAPI (Sigma-Aldrich, cat. MBD0015-1ML) were diluted in PBS with 1% BSA and incubated with the samples overnight at 4 °C. After washing in PBSTr 0.2% at RT (5 × 15 min each), drops of SlowFade Gold Antifade Mountant were applied. Antibodies against N. vectensis Cadherin1 (Cdh1) (v1g244010) were graciously shared by Prof. Ulrich Technau (University of Vienna). Planula larvae (3 dpf) were fixed and stained with Cdh1 antibody following52 with few modifications. Samples were fixed for 1 h at 4 °C with Lavdovsky’s fixative (3.7% formaldehyde (FA), 50% ethanol, 4% acetic acid). After fixation, samples were incubated in ice-cold acetone for 7 min on ice followed by washing with PBSTr 0.2% (5 × 10 min each). Samples were then incubated in blocking solution (1% BSA, 5% normal goat serum, 0.1% Sodium azide in PBSTw 0.2%) for 2 h at RT. Anti-Cdh1 antibodies were diluted in blocking solution (1:300) and incubation was performed overnight at 4 °C, followed by washing in PBSTr 0.2% at RT (8 ×15 min each). After incubation in blocking solution for 2 h at RT, samples were placed in a secondary antibody solution of Alexa Flour 488 AffiniPure Goat Anti-Rat IgG (1:300, Jackson ImmunoResearch, cat. 112-035-003) and DAPI overnight at 4 °C. Samples were washed in PBSTr 0.2% at RT (8 × 15 min each) and infiltrated with VECTASHIELD PLUS Antifade Mounting Medium (Vector Laboratories, cat. H-1900-2).
HCR
HCR was performed according to the manufacturer’s instructions (Nepagene, HCR kit, ISHpalette Short hairpin amplifier), with slight modifications. Ten split probes, which were concatenated with initiator sequences for chain reaction, were designed against coding sequences of NvNrxnδ1,−2, and −4, NvNrxnα1 and NvPRGamide (Supplementary Data 4). The 4 dpf planulae or 7 dpf polyps of N. vectensis were fixed with 4% PFA 4 °C overnight and washed with PBSTw 0.1% (3 × 10 min each). They were kept in 100% methanol and stored at −20 °C until use. These samples were rehydrated with a series of graded MeOH/PBSTw 0.1% washes for 15 min each: 75% MeOH:25% PBSTw 0.1%, 50% MeOH:50% PBSTw 0.1%, 25% MeOH:75% PBSTw 0.1%, and two washes with PBSTw 0.1%. After 30-min treatment with the hybridization solution (Nepagene) for prehybridization treatment, samples were incubated in hybridization buffer containing 20 nM of probes overnight at 37 °C. Samples were washed with 0.5x SSC 0.1% Tween20 at 37 °C (3 × 15 min each), incubated with signal amplification buffer (Nepagene) for 30 min, and with amplification buffer containing hairpin amplifiers (ISH short hairpin amplifier SaraFluor488-S45 and/ or ISH short hairpin amplifier ATTO647N-A161: Nepagene) and DAPI for 2 h. Samples were washed with PBSTw 0.1% (3 × 15 min each) and mounted on glass slide with SlowFade Gold Antifade Mountant.
Observation of morphology and behavior
Animal conditions were examined at various developmental stages. For behavior, occurrences of peristaltic waves were counted during the primary polyp stage. A peristaltic wave was defined as a radial contraction of the body column from oral to aboral direction. All waves were counted, including those in progress at the start of the recording and those that did not finish by the end of data collection. Prior to recording, polyps were left to relax their bodies and tentacles. After this, animals were recorded for 5 min and the number of propagated radial contractions during this time was counted. Animals that failed to maintain their orientation, to adhere to the plate, or underwent sudden full-body or tentacle retraction during the 5-min video recording were excluded from these measurements.
Pharmacology
All experiments were carried out on young polyps (7 to 8 dpf). All reagents were purchased either from Sigma-Aldrich or from Tocris Bioscience. All stock concentrations were prepared by dissolving reagents in Milli-Q water and diluted to their final concentration with Nematostella medium. To perform pharmacological assays, polyps were placed in a cell culture plate well (1.9 cm2) and left to relax their tentacles. Recordings began 5 min prior to addition of the reagent and continued for an additional 10 min after addition. The number of waves performed by each animal before and after treatment was counted. Only complete, uninterrupted waves were counted, except for those waves at the start and end of recordings. A complete wave is considered when the propagation starts from a position level with pharynx and ends at the foot/physa of the polyp. Control experiments were performed by adding a volume of Nematostella medium instead of reagent.
Imaging
Live images and video recordings were acquired using a stereo microscope (Olympus SZX16). Stained samples were imaged with a fluorescent microscope ECLIPSE Ni (Nikon) equipped with Ds-Ri2 (Nikon) or with SD-OSR microscope system (Olympus). Image and video files were processed with ImageJ (v1.53) or ImageJ/Fiji software (v2.14.0).
F-actin quantification
Calculation of the gap between the ectodermal and endodermal layers was processed using ImageJ/Fiji software as follows. Using phalloidin-stained confocal images of longitudinal sections around the midline of each sample, the length of the basal surface of the ectodermal layer sealed and unsealed from the endodermal epithelium was measured, and the percentage of unsealed length was calculated for each larval sample.
Statistics and reproducibility
In all experiments, neither randomization, blinding, nor pre-specified sample size determination were performed. Statistical comparisons were performed using two-tailed unpaired Student’s t tests. Data are presented as means ± SDs, scatterplots showing medians with interquartile ranges, or as box plots showing the median (center line), upper and lower quartiles (box limits), and minimum and maximum values (whiskers). P values were calculated in GraphPad Prism 9 and Microsoft Excel, and are designated as *P < 0.05, **P < 0.01, and ***P < 0.001. Immunostaining and western blot experiments were repeated at least twice, and representative images and blots are shown. All behavior and pharmacological experiments were conducted between 13:00 and 17:00 hr.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
Published single cell data used in this study can be accessed using BioProject accessions: PRJNA378226 [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE95723] (N. vectensis)42 and PRJNA435744 (M. leidyi and T. adhaerens)41. Source data are provided with this paper.
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Acknowledgements
We are grateful to Akiko Tanimoto and Junko Higuchi for maintaining the Nematostella culture. We thank the OIST Imaging Section for providing access to their microscopes. We also thank Prof. Ulrich Technau (University of Vienna) for the generous gift of anti-NvCdh1 antibodies. This work was supported by JSPS KAKENHI Grant Numbers JP20K06662 for H.W. and JP23K14244 for R.N., and by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED Grant Number JP21am0101110 (support number 0883) for K.T. C.G. and M.M. were supported by a fellowship from JSPS Research Fellowship for Young Scientists—DC1 JP19J20655 and JP23KJ2140, respectively.
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C.G. and H.W. conceived and designed the study. C.G. performed bioinformatic analyses, knockdown experiments, western blotting, immunostaining, qPCR, and pharmacological assays. K.M. performed HCR experiments. C.G., K.M., R.N. and M.M. performed imaging. Y.T. and K.T. helped with 3D modeling and structure-based sequence alignments. C.G., K.M. and H.W. interpreted the results and wrote the manuscript.
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Guzman, C., Mohri, K., Nakamura, R. et al. Neuronal and non-neuronal functions of the synaptic cell adhesion molecule neurexin in Nematostella vectensis. Nat Commun 15, 6495 (2024). https://doi.org/10.1038/s41467-024-50818-8
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DOI: https://doi.org/10.1038/s41467-024-50818-8
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