Synaptic and peptidergic connectome of a neurosecretory centre in the annelid brain

Neurosecretory centres in animal brains use peptidergic signalling to influence physiology and behaviour. Understanding neurosecretory centre function requires mapping cell types, synapses, and peptidergic networks. Here we use electron microscopy and gene expression mapping to analyse the synaptic and peptidergic connectome of an entire neurosecretory centre. We mapped 78 neurosecretory neurons in the brain of larval Platynereis dumerilii, a marine annelid. These neurons form an anterior neurosecretory organ expressing many neuropeptides, including hypothalamic peptide orthologues and their receptors. Analysis of peptide-receptor pairs revealed sparsely connected networks linking specific neuronal subsets. We experimentally analysed one peptide-receptor pair and found that a neuropeptide can couple neurosecretory and synaptic brain signalling. Our study uncovered extensive non-synaptic signalling within a neurosecretory centre and its connection to the synaptic brain.


Introduction
Nervous system signalling occurs either at synapses or via secreted diffusible chemicals that signal to target cells expressing specific receptors. Synapse-level connectomics using electron microscopy allows mapping synaptic networks, but fails to reveal non-synaptic signalling. In addition to acting in a neuroendocrine fashion, non-synaptic volume transmission by neuropeptides and monoamines can have neuromodulatory effects on synaptic signalling (Bargmann 2012;Marder 2012). Overlaying synaptic and peptidergic maps is challenging and requires knowledge of the expression of the modulators and their specific receptors as well as synaptic connections. Such mapping has only been achieved for relatively simple circuits, such as the stomatogastric nervous system of crustaceans where synaptic connections are known and the effect of neuropeptides and the activation of single peptidergic neurons can be analysed experimentally (Stein et al. 2007;Thirumalai and Marder 2002;Blitz et al. 1999). Likewise, connectome reconstructions combined with cellular-resolution neuropeptide and receptor mapping allow the dissection of peptidergic signalling in Drosophila (Schlegel et al. 2016). In Caenorhabditis elegans, the spatial mapping of monoamines and neuropeptides and their G protein-coupled receptors (GPCRs) revealed interconnected networks of synaptic and nonsynaptic signalling (Bentley et al. 2016). Neurosecretory centres, such as the vertebrate hypothalamus and the insect ring gland, found in the anterior brain of many animals show exceptionally high levels of neuropeptide expression (Herget and Ryu 2015;Siegmund and Korge 2001;Campbell et al. 2017) suggesting extensive non-synaptic signalling. These centres coordinate many processes in physiology, behaviour, and development, including growth, feeding, and reproduction (Sakurai et al. 1998;Bluet-Pajot et al. 2001;Sternson et al. 2013). The combined analysis of synaptic and peptidergic networks is particularly challenging using single marker approaches for neurosecretory centres that express dozens of neuropeptides. To fully understand their function, a global mapping of peptidergic networks within these brain regions and how they connect to the rest of the nervous system is required. Here, we analyse synaptic and peptidergic signalling in the anterior neurosecretory centre in larval Platynereis dumerilii, a marine annelid. Annelid and other marine larvae have an anterior sensory centre, the apical organ, involved in the detection of various environmental cues (Hadfield et al. 2000;Page 2002;Chia and Koss 1984). The apical organ is neurosecretory and expresses diverse neuropeptides that are thought to regulate various aspects of larval behaviour and physiology, including the induction of larval settlement and metamorphosis (Mayorova et al. 2016;Thorndyke et al. 1992;Tessmar-Raible et al. 2007;Conzelmann et al. 2011;Marlow et al. 2014). Apical organs have a conserved molecular fingerprint across marine larvae, suggesting that they represent a conserved sensoryneuroendocrine structure (Marlow et al. 2014). The apical organ area or apical nervous system (ANS) in Platynereis larvae shows molecular similarities to other neuroendocrine centres, including the pars intercerebralis in insects and the vertebrate hypothalamus, suggesting a common ancestry Steinmetz et al. 2010;. Molecular and developmental similarities in various protostomes and deuterostomes further suggest a more widespread conservation of neuroendocrine centres (Hartenstein 2006;Wirmer et al. 2012;Tessmar-Raible 2007). The study of marine invertebrate larval apical organs could thus inform about the evolution of neuroendocrine cell types and signalling mechanisms in metazoans. Platynereis larvae represent a powerful system to analyse gene expression and synaptic connectivity in a whole-body context, allowing linking distinct neuropeptides and other molecules to single neurons (Asadulina et al. 2012;Williams and Jékely 2016;Shahidi et al. 2015;Achim et al. 2015;Pettit et al. 2014;Vergara et al. 2017). To understand how synaptic and peptidergic signalling is integrated in the Platynereis ANS, we combine serial section electron microscopy with the cellular analysis of neuropeptide signalling. This combined analysis revealed extensive non-synaptic peptidergic signalling networks within the ANS distinguishing this area from the rest of the nervous system. Through connectomics and functional studies we also reveal how this endocrine region can interact with the synaptic nervous system by peptidergic modulation of the ciliomotor circuitry.

Ultrastructural reconstruction of the anterior neurosecretory centre
To comprehensively map a neurosecretory area with ultrastructural detail, we focused on the larvae of Platynereis. Due to their small size, the larvae are amenable to whole-body connectomic analysis (Randel et al. 2015;Shahidi et al. 2015). We used a full-body serial electron microscopy dataset of a 3-day-old larva (Randel et al. 2015) and reconstructed its entire apical neurosecretory nervous system ( Figure 1A-D). Platynereis and other annelids have an anterior neurosecretory plexus containing the projections of peptidergic sensory-neurosecretory neurons Aros et al. 1977). The neurosecretory plexus forms an anatomically and ultrastructurally distinct area that can be clearly distinguished from other neuropils, including the adjacent optic and nuchal organ neuropils (Randel et al. 2014;) ( Figure 1D). Neurites in this area have a high number of dense-core vesicles and very few synapses ( Figure 1E, F). Classic neurotransmitter synapses can be identified by large clusters of clear synaptic vesicles in the axons, while peptidergic synapses appear as smaller clusters of dense core vesicles (Randel et al. 2014;Shahidi et al. 2015). We reconstructed all neurons that project to this region ( Figure 1B, Supplement 1 to figure 1, Video 1) and identified 70 sensory neurons and eight projection interneurons, the latter project in and out of the neurosecretory plexus. Most of the sensory neurons are bilaterally symmetric pairs with distinct axonal projection patterns, except for a few asymmetric neurons (Supplement 1 to figure 1). The sensory neurons have diverse apical sensory specializations. Based on these morphological criteria we could distinguish at least 20 different sensory cell types with likely different sensory functions. For example, there are four ciliary photoreceptor cells (cPRC) (Arendt et al. 2004) with highly extended ciliary membranes, one asymmetric neuron with five sensory cilia (SN YFa5cil ), a pair of neurons with two long parallel cilia (SN WLD1 ), a pair with long branched sensory cilia (SN NS29 ), and three uniciliary neurons that are part of the nuchal organ (SN nuchNS ), a putatively chemosensory annelid organ (Purschke 1997). Twenty-five uniciliated neurons (23 SN DLSO cells, SN PDF-dcl2 , and SN PDF-dcr3 ) are part of a dorsolateral sensory cluster (Supplement 1 to figure 1). Most of the sensory neurons have axonal projections that are extensively branched within the neurosecretory plexus (Supplement 1 to figure 1) and that are filled with dense-core vesicles ( Figure 1F). The pairs of left-right symmetric sensory neurons project to similar areas of the neurosecretory plexus revealing a fine-scale organization within the plexus ( Figure 1G and Supplement 1 to figure 1, Video 1). We refer to all neurons that project to the neurosecretory plexus as the apical nervous system (ANS).

Comprehensive mapping of neuropeptide expression in Platynereis larvae
The Platynereis larval ANS is known to express many neuropeptides, including vasotocin, FMRFamide, and myoinhibitory peptide Conzelmann et al. 2011;. To comprehensively analyse neuropeptide expression in the whole larva, we used whole-mount in situ hybridization for 51 Platynereis proneuropeptides (of 98 total ). We used image registration to spatially map all neuropeptide expressions to a common nuclear reference template (Asadulina et al. 2012). We summed all binarized average expression domains and found that the region with the highest proneuropeptide expression corresponds to the ANS ( Figure 1H, Figure 1 -source data 1). Some voxels in the map coexpress up to 10 different neuropeptides. Neuropeptides that were expressed in the ANS include two Platynereis insulin-like peptides (IRP2, IRP3), two bursicons (bursicon-A, -B), achatin, myoinhibitory peptide (MIP), and several homologs of hypothalamic peptides (Mirabeau and Joly 2013;Jékely 2013) including NPY (three homologs, NPY1, NPY4, NKY2), orexin/allatotropin, tachykinin, galanin/allatostatin-A, and allatostatin-C/somatostatin ( Figure 1I and Supplement 2 to figure 1). The acetylated tubulin antibody we used to counterstain the in situ samples labels cilia and axonal scaffold. With this counterstaining signal, we correlated neurons expressing specific proneuropeptides and with distinct ciliation to sensory ANS neurons reconstructed from EM data ( Figure 1J and Supplement 3 to figure 1). For other ANS neurons (SN PDFdc , IN RGW ) we assigned neuropeptides based on direct immunogold labelling on the same EM series . Overall, we mapped neuropeptide expression to 25 reconstructed ANS neurons, including sensory neurons coexpressing IRP2 and FMRFamide (SN IRP2-FMRF ), IRP2 and bursicon (SN IRP2burs ), or expressing ASTC/somatostatin (SN ASTC1 ) ( Figure 1J and Supplement 3 to Figure).

Low level of synaptic connectivity within the ANS
We next analysed how the ANS neurons are synaptically connected. We found that most neurons have no or only very few synapses (Figure 2, Supplementary table 1). 33% of the neurons have 0-2 synapses despite highly branched axonal projections filled with dense core vesicles ( Figure  2). This suggests that these neurons predominantly use volume transmission. The synapses we identified in most neurons contained dense-core vesicles, indicative of their peptidergic nature. The four cPRCs, an asymmetric sensory neuron (SN 47Ach ), and the 8 projection neurons have the highest number of synapses. In addition, 15 ANS sensory neurons have 10 or more peptidergic synapses. The cPRCs express a cholinergic marker (Jékely et al. 2008) and contain large synapses with clear vesicles. The other sensory cells have ultrastructurally distinct, small peptidergic synapses characterized by dense core vesicles clustering at the membrane ( Figure 2).

Analysis of peptidergic signalling networks in the ANS
The high number of neuropeptides expressed in the ANS and the low degree of synaptic connectivity of the cells prompted us to further analyse peptidergic signalling networks in the Platynereis larva. We used two resources: an experimentally determined list of Platynereis neuropeptide GPCRs (Bauknecht and Jékely 2015) and a spatially-mapped single-cell transcriptome dataset (Achim et al. 2015). The GPCR list included 18 deorphanized neuropeptide receptors, to which we added a further three deorphanized neuropeptide receptors. We deorphanized a GnRH receptor activated by both Platynereis GnRH1 and GnRH2 , and second receptors for vasotocin and myomodulin (Supplement 1 to figure 3). The single-cell transcriptome data consisted of cells of the head (episphere) of 2-dayold larvae. The cells were mostly neurons (Achim et al. 2015). To comprehensively analyse potential peptide-receptor signalling networks, we first created a virtual larval brain with cells arranged in an approximate spatial map ( Figure 3A, Supplement 2 to figure 3, Supplementary Table 1, Figure 3 -source data 1, Figure 3 -source data 2). To this virtual map, we mapped the expression of all proneuropeptides and deorphanized receptors ( Figure 3C, D). The combined expression of 80 proneuropeptides showed a similar pattern to the in situ map with a highly peptidergic group of cells in the ANS region, as defined by the endocrine marker genes Phc2, dimmed, Otp and nk2.1 Tessmar-Raible et al. 2007) ( Figure 3B). Given the high level of peptide expression in the ANS, the mapping of peptidergic signalling networks will mostly reveal potential signalling partners within this region or from the ANS to the rest of the brain. Most GPCR expression was also concentrated in the ANS in peptidergic cells. Several cells expressed a unique combination of up to 9 GPCRs ( Figure 3D). We also used the spatially mapped single cell transcriptome data to map the expression of different types of sensory receptor genes in the opsin and transient receptor potential (Trp) channel families. These receptor genes are known for their function in the detection and transduction of light/pain/temperature/mechanical stimuli (Terakita 2005;Moran et al. 2004). Neurons in the ANS of 2-day-old larvae express diverse combinations of these genes (Supplement 3 to figure 3). This supports our conclusion from the morphological data that these neurons are responsible for detecting a variety of different environmental cues. Coexpression analysis with small neurotransmitter synthesis enzymes revealed 1 cell (1% of total) with only neurotransmitter markers but no neuropeptide, 76 (71% of total) purely peptidergic cells, and 21 cells that coexpress small transmitters and neuropeptides (19.6% of total)(Supplement 4 to figure 3). The remaining 9 cells expressed neither neuropeptides nor neurotransmitter markers and thus are likely non-neuronal cells (8.4% of total). Strikingly, 2 ANS cells coexpress up to 24 different proneuropeptides. Based on specific neuropeptide expression and the spatial mapping we could correlate 15 cells to ANS cells reconstructed from EM data (Supplementary Table 1). This was possible despite the two resources being derived from different larval stages, because many ANS cells are already differentiated in 2-day-old larvae and readily identifiable between stages. To establish peptidergic signalling networks, we treated peptide-expressing cells as source nodes and GPCR-expressing cells as target nodes. We define edge weights in the directed graphs as the geometric mean of normalized proneuropeptide expression in the source and normalized GPCR expression in the target. This way, we also consider the expression level of peptides and receptors. Receptor expression can correlate with neurophysiological sensitivity to a neuropeptide (Garcia et al. 2015;Root et al. 2011). For each peptide-receptor pair, we projected these networks onto the virtual map ( Figure 3E-L, Supplement 4 to figure 3, Figure 3 -source data 3). The chemical connectivity maps of individual ligand-receptor pairs show very sparse and specific chemical wiring. On average, less than 1% of all potential connections are realized (0.8% graph density averaged for all peptide-receptor pairs). Single cells expressing many different neuropeptides generally link to non-overlapping target nodes by each peptide-receptor channel ( Figure 3O and Supplement 4 to figure 3Q). Conversely, multiple signals can converge on one cell that expresses more than one GPCR ( Figure 3N). The combined multichannel peptidergic connectome of 23 receptor-ligand pairs forms a single network with an average clustering coefficient of 0.49 and an average minimum path length of 1.54, forming a small-world network (Watts and Strogatz 1998). Analysis of the combined network revealed highly connected components, including nodes that can act as both source and target as potential mediators of peptide cascades ( Figure 3N, O). The three neuron types with the highest weighted in-degree and authority value include a pair of dorsal sensory neurons, a central pair of IRP2 neurons, and a pair of RGWamide-expressing neurons ( Figure 3M and Figure 4A). The IRP2 neurons are under the influence of an NPY peptide and EFLGamide, the Platynereis homolog of thyrotropin-releasing hormone (Bauknecht and Jékely 2015) and express 20 different proneuropeptides.

Projection interneurons connect the peptidergic ANS to the synaptic nervous system
The RGWamide-expressing neurons express two NPY receptors (NPY4 and NKY) and an achatin receptor, and 17 proneuropeptides, but no markers for small neurotransmitters. By position and RGWamide-expression we identified these cells as IN RGW Table 1). The distinct anatomy and connectivity as well as the high authority values of the projection neurons in the peptidergic network indicate that these cells are important in relaying peptidergic signals from the ANS to the rest of the brain. To test how these neurons respond to neuropeptides, we used calcium-imaging experiments. The IN RGW and IN NOS neurons express 7 deorphanized GPCRs ( Figure 4F), including a receptor for achatin that is specifically activated by achatin peptide containing a D-amino acid (Bauknecht and Jékely 2015). When we treated larvae with D-achatin, four neurons in the ANS were rhythmically activated. These neurons correspond by position to the IN RGW cells ( Figure 4I). The rhythmic activation was in-phase with the activation of the Ser-h1 neurons and a ventral motorneuron (vMN async ), but out of phase with the main cholinergic motorneuron of the head ciliary band, the MC neuron (readily identifiable by calcium imaging (Verasztó et al. 2017)). We could not observe a similar effect upon L-achatin treatment (data not shown). Platynereis larvae have a cholinergic and a serotonergic circuit that oscillate out-of-phase. The cholinergic phase arrests the cilia and the serotonergic phase correlates with ciliary beating (Verasztó et al. 2017). Correlation analysis of neuronal activity patterns revealed that D-achatin coupled the activity of the IN RGW cells to the serotonergic cells, and increased the negative correlation between the activity patterns of serotonergic neurons and the MC neuron ( Figure 4I and Supplement 1 to Figure 4). This provides an example of how peptidergic signalling in the ANS could recruit neurons to a rhythmically active circuit, enhance the rhythm, and thereby potentially influence locomotor activity.

Discussion
Here we presented a comprehensive anatomical description of the ANS in the Platynereis larva. We combined this with the cellular-resolution mapping of neuropeptide signalling components to analyse potential peptidergic signalling networks. The use of scRNA-seq has a great potential to reveal such signalling networks but also has limitations. For example, we could only score proneuropeptide and receptor mRNA expression and not protein expression levels, peptide release, or the degree of neuronal activation. We also only analysed a relatively small single-cell dataset derived from the head of the larva and thus could not investigate long-range neuroendocrine signalling from the larval episphere to the rest of the body. The first analyses we present here can be extended when more data become available and can also be applied to other species (e.g., (Campbell et al. 2017)). Nevertheless, our approach can reveal all potential peptidergic connections and allows the development of hypotheses on peptidergic signalling that can be experimentally tested, as we have shown here for achatin. We found that the Platynereis larval ANS has a low degree of synaptic connectivity, a strong neurosecretory character, and shows a high diversity of neuropeptide expression. This suggests that the ANS is wired primarily chemically and not synaptically. We identified very specific peptidergic links that connect only a small subset of ANS neurons. Strikingly, it is not only the proneuropeptides that are expressed with high diversity in the ANS, but also their receptors, suggesting that most peptidergic signalling in the brain occurs within the ANS. A high diversity of neuropeptide receptor expression has also been reported for the mouse hypothalamus (Campbell et al. 2017). These observations suggest that there is more extensive peptidergic integration within these centres than was previously appreciated. Neurosecretory centres may thus also function as chemical brains wired by neuropeptide signalling where the specificity is derived not from direct synaptic connections but by peptide-receptor matching. Given a high enough number of specific peptide-receptor pairs and allowing the possibility of combinatorial signalling, it is theoretically possible to wire arbitrarily complex neural networks. This extends the concept of neurosecretory centres as organs that release peptides to influence external downstream targets. An anatomically and molecularly distinct 'chemical' and 'synaptic' brain as we found in the Platynereis larva may have originated early in the evolution of the nervous system. This idea is consistent with the chimeric brain hypothesis, which posits the fusion of a neurosecretory and a synaptic nervous system early in animal evolution (Tosches and Arendt 2013). The neurosecretory part of the brain is directly sensory in Platynereis, possibly reflecting an ancestral condition for neurosecretory centres Hartenstein 2006). The ANS also connects to the motor parts of the synaptic nervous system by specific projection neurons that are under peptidergic control. This connection may allow the translation of environmental cues that had been integrated by the chemical brain into locomotor output. Neuropeptides are ideal molecules for chemical signalling due to their small size and high diversity. Many of the neuropeptides expressed in the Platynereis ANS and the vertebrate hypothalamus belong to ancient peptide families that evolved close to the origin of the nervous system (Jékely 2013;Mirabeau and Joly 2013). Neuropeptide genes have been identified in bilaterians, cnidarians, and even the early-branching metazoan Trichoplax adhaerens, that lacks a synaptic nervous system but has neurosecretory cells (Nikitin 2015;Smith et al. 2014). Chemical signalling by neuropeptides may have been important early during nervous system evolution in small metazoans where only peptide diffusion could coordinate physiological activities and behaviour. As metazoans grew larger, the coordination of their large complex bodies required control by a synaptic nervous system (Keijzer and Arnellos 2017). The separate origins of the chemical and the synaptic nervous system may still be reflected to varying degrees in contemporary brains. The Platynereis larval nervous system, and possibly the nervous system of other marine invertebrate larvae, shows a particularly clear segregation of non-synaptic and synaptic nervous systems. The extent of non-synaptic signalling even in the relatively simple nervous system of the Platynereis larva highlights the importance of the combined study of connectomes and chemical signalling networks.

Electron microscopy reconstruction of Platynereis anterior neurosecretory plexus
We reconstructed the circuitry of cells in the anterior plexus from an existing 3-day-old Platynereis serial section transmission electron microscopy (ssTEM) dataset we generated previously (Randel et al. 2015). The dataset consists of 4845 layers of 40 nm thin sections. Preparation, imaging, montage, and alignment of the dataset is described (Randel et al. 2015). The cells were reconstructed, reviewed, 3D visualized, and the resulting synaptic network was analysed in Catmaid (Saalfeld et al. 2009;Schneider-Mizell et al. 2016).

Neuropeptide expression atlas in 2-day-old Platynereis
DIG-labelled antisense RNA probes were synthesized from clones sourced from a Platynereis directionally cloned cDNA library in pCMV-Sport6 vector . Or PCR amplified and cloned into the vectors pCR-BluntII-TOPO or pCRII-TOPO. Larvae were fixed in 4% paraformaldehyde (PFA) in 1 X PBS with 0.1% Tween-20 for 1 h at room temperature. RNA in situ hybridization using nitroblue tetrazolium (NBT)/5-bromo-4chloro-3-indolyl phosphate (BCIP) staining combined with mouse anti-acetylated-tubulin staining, followed by imaging with a Zeiss LSM 780 NLO confocal system and Zeiss ZEN2011 Grey software on an AxioObserver inverted microscope, was performed as previously described (Asadulina et al. 2012), with the following modification: fluorescence (instead of reflection) from the RNA in situ hybridization signal was detected using excitation at 633 nm in combination with a Long Pass 757 filter. Animals were imaged with a Plan-Apochromat 40x/1.3 Oil DIC objective.
We projected thresholded average gene expression patterns of >5 individuals per gene onto a common 2-day-old whole-body nuclear reference templates generated from DAPI (Asadulina et al. 2012). Thresholding was performed either manually or following (Vergara et al. 2017). Gene expression atlases were set up in the visualization software Blender (https://www.blender.org/) as described .

Deorphanization of receptor-peptide pairs
Platynereis GPCRs were identified in a reference transcriptome assembled from cDNA generated from 13 different life cycle stages . GPCRs were cloned into pcDNA3.1(+) (Thermo Fisher Scientific, Waltham, USA) and deorphanization assays were carried out as previously described (Bauknecht and Jékely 2015).

Analysis of single cell transcriptome data
Fastq files containing raw paired-end RNA-Seq data for 107 single cells from the 48 hour old Platynereis larval episphere (Achim et al. 2015) were downloaded from ArrayExpress, accession number E-MTAB-2865 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-2865/). Only those samples annotated as 'single cell' and with a corresponding spatial mapping prediction by Achim et al. 2015(Achim et al. 2015 were used in our analysis. Fastq files with raw paired end read data were loaded into CLC Genomics Workbench v6.0.4 (CLC Bio). Data was filtered to remove Illumina adapter primer sequences, low quality sequence (Quality Limit 0.05) and short fragments (less than 30 base pairs). Filtered data were mapped to the assembled Platynereis reference transcriptome (including only sequences with a BLASTX hit e-value <1e-5 to the SwissProt database, plus all previously described Platynereis genes, including 99 proneuropeptides, a total of 52,631 transcripts. Mapping was carried out in CLC Genomics Workbench v6.0.4 using the RNA-Seq Analysis function, with the following mapping parameters: paired distance 100 -800 base pairs; minimum length fraction 0.8, minimum similarity fraction 0.9, maximum number of mismatches 2. The total number of reads mapped to each gene, normalized by gene length (reads per kilobase million (RPKM)) in each sample was assembled into one spreadsheet using the 'Experiment' function, and this spreadsheet was exported as an Excel file. RPKM data for each gene and sample were converted in Excel to counts per million (cpm) by dividing RPKM by with the total number of mapped reads in each sample and multiplying by 10 6 followed by conversion to a log base 10. Single cell samples were sourced from populations consisting of dissociated cells of several individual larvae (Achim et al. 2015), therefore some of the RNA-Seq samples could represent sequencing of the same cell from different larvae. To determine a cut-off for transcriptome similarity to merge samples representing the same cell from different individuals, we calculated the all-against-all pairwise correlation coefficients. Plotting these correlation coefficients as a histogram showed a prominent peak of highly correlated samples. We interpret these as samples deriving from the re-isolation of the same cell from different larvae. We used a cut-off of 0.95 Pearson correlation as a cut-off, above which we merged samples as representing the same cell by using the mean normalized expression value for each gene. Sample names were imported as nodes into software for graph visualization and manipulation, Gephi.0.8 beta (http://gephi.org). Nodes were manually placed in position in a Gephi map in an approximate 2D representation of the 3D spatial predictions of each cell generated by Achim et al. (Achim et al. 2015) based on a whole-mount in situ hybridization gene expression atlas of 72 genes (http://www.ebi.ac.uk/~jbpettit/map_ viewer/?dataset=examples/coord_full. csv&cluster0=examples/resultsBio.csv). Samples with predicted bilateral symmetry were represented as two mirror image nodes in the map (left and right), while samples with predicted asymmetry were represented as single nodes. Node position coordinates for each sample were saved and exported as .gexf connectivity file for use in generating virtual gene expression patterns and peptide-receptor connectivity maps (Figure 3 -source data 3).
Virtual expression patterns for each gene were generated using a custom perl script that converted normalized log10 gene expression values into node colour intensity in the Gephi map. Connectivity files for each peptide-receptor pair were generated by preparation of connectivity data files as .csv files where the geometric mean of normalized log10 peptide expression from the 'sending cell' and corresponding GPCR expression from the 'receiving cell' was used as a proxy for connectivity strength. These connectivity data .csv files were imported into Gephi to generate .gexf connectivity maps, and random node positions were replaced with node position coordinates for each cell from the spatial position .gexf map described above. An 'all-by-all' connectivity map representing the potential cellular signalling generated by all known peptidereceptor pairs was generated by adding the connectivity data from each peptide-receptor pair into a single connectivity file.

Immunohistochemistry
Whole-mount triple immunostaining of 2 and 3 day old Platynereis larvae fixed with 4% paraformaldehyde were carried out using primary antibodies raised against RGWamide neuropeptide in rat (CRGWamide) and achatin neuropeptide in rabbit (CGFGD), plus a commercial antibody raised against acetylated tubulin in mouse (Sigma T7451). Double immunostaining was carried out with primary antibodies raised against MIP, RYamide or DH31 neuropeptide raised in rabbit and commercial acetylated tubulin antibody raised in mouse. The synthetic neuropeptides contained an N-terminal Cys that was used for coupling during purification. Antibodies were affinity purified from sera as previously described (Conzelmann and Jékely 2012). Immunostainings were carried out as previously described (Conzelmann and Jékely 2012).

Calcium imaging experiments
Fertilized eggs were injected as previously described ) with capped and polA-tailed GCaMP6 RNA generated from a vector (pUC57-T7-RPP2-GCaMP6s) containing the GCaMP6 ORF fused to a 169 base pair 5′ UTR from the Platynereis 60S acidic ribosomal protein P2. The injected individuals were kept at 18°C until 2-days-old in 6-well-plates (Nunc multidish no. 150239, Thermo Scientific). Calcium imaging was carried out on a Leica TCS SP8 upright confocal laser scanning microscope with a HC PL APO 40x/1.10 W Corr CS2 objective and LAS X software (Leica Microsystems) GCaMP6 signal was imaged using a 488-nm diode laser at 0.5 -4% intensity with a HyD detector in counting mode. 2-day-old Platynereis larvae were immobilized for imaging by gently holding them between a glass microscope slide and a coverslip raised with 2 layers of tape as spacer. Larvae were mounted in 10 µl sterile seawater. For peptide treatment experiments, individual larvae were imaged for 2 minutes in the plane of the RGWamide interneurons and MC cell to assess the state of the larval nervous system prior to peptide treatment. For treatment, larvae were imaged for a further 5.5 min. 10 µl 50 µM synthetic D-achatin (G{dF}GD) (final concentration 25 µM) dissolved in seawater was added to the slide at the 1 minute mark by slowly dripping it into the slide-coverslip boundary, where it was sucked under the coverslip. As a negative control, larvae were treated with synthetic Lachatin (GFGD) at the same concentration. Receptor deorphanization experiments have shown that the D-form of achatin activates the achatin receptor GPCR, whereas the L-form of achatin does not (Bauknecht and Jékely 2015). D-achatin response was recorded from 12 larvae, and Lachatin response was recorded from 6 larvae. Calcium imaging movies were analysed with a custom Fiji macro and custom Python scripts. Correlation analyses were created using Fiji and a custom Python script (Verasztó et al. 2017).
Council Grant Agreement 260821. The research was supported by a grant from the DFG -Deutsche Forschungsgemeinschaft (Reference no. JE 777/1).