Identification of NOS-expressing interneurons (INNOS) within the cPRC circuit.

(A) Scanning electron microscopy image of a three-day-old Platynereis larva. (B, C) Volume rendering of the neuron types (cPRC, INNOS, INRGWa, Ser-h1 and MC) in the cPRC circuit reconstructed from a whole-body transmission electron microscopy volume of a three-day-old larva. Neurite skeletons are shown with cell-body positions represented by spheres. Projections of all neurons in the body are shown in grey to highlight the neuropils. The outline of the yolk is also indicated in grey. In B, nuclei positions of the prototroch head ciliary band are shown as grey spheres. (D) Expression of the NOS gene detected by in situ HCR (magenta) in a two-day-old larva (anterior view). Antibody staining for acetylated α-tubulin (acTub: green) highlights cPRC cilia and the neuropil. (E) Expression of a NOS reporter (NOSp::palmi-3xHA; magenta) labelled with an anti-HA antibody in a two-day-old larva (anterior view). Antibody staining for acetylated α-tubulin (acTub; green) highlights cPRC cilia and the neuropil. (F) Synaptic wiring diagram of the cPRC circuit. Hexagons represent cell groups, with the number of cells per group shown in square brackets. Arrows represent the summed number of synaptic contacts between cell groups. Arrow thickness is proportional to the number of synapses.

NO produced by UV/violet stimulation to cPRCs.

(A) DAF-FM fluorescence in the region of the neurosecretory neuropil. The white line indicates the outline of the larva. The dashed line corresponds to the area where fluorescence was quantified. The circles indicate the location of cPRC and control stimulation. The cPRCs are marked by thin lines. (B) DAF-FM fluorescence before and during 405 nm light stimulation. (C) Changes in DAF-FM fluorescence over time during 405 nm stimulation of the cPRCs or a control area (ctr stim.). The purple box indicates the duration of 405 nm stimulation. Individual normalized traces (ΔF/F0) are shown as thin lines. Thick lines show the mean value with 0.95 confidence intervals. N = 9 larvae for control and 11 for cPRC stimulation.

Figure 2 – source data 1. DAF-FM fluorescence reads.

NOS is required for UV avoidance in Platynereis larvae.

(A) Swimming trajectories of wild type (WT, n=32) and NOS mutant (NOSΔ11/Δ11, n=26 and NOSΔ23/Δ23, n=47) three-day-old larvae. All trajectories start at 0 x and y position and time 0 corresponding to 10 sec after the onset of 395 nm stimulation from the side. (B) Vertical position of batches of wild type and mutant three-day-old larvae over time under 395 nm UV stimulation. The starting position of each larval trajectory was set to 0. (C) Vertical position of batches of control and L-NAME-treated (0.1 and 1 mM) three-day-old larvae over time under 395 nm UV stimulation. The starting position of each larval trajectory was set to 0. (D) Vertical displacement in 30 sec bins of wild type and mutant (NOSΔ11/Δ11 and NOSΔ23/Δ23) three-day-old larvae stimulated with 395 nm light from the side, 488 nm light from the top and 395 nm light from the top. (E) Vertical displacement in 30 sec bins of control and L-NAME-treated (0.1 and 1 mM) three-day-old larvae stimulated with 395 nm light from the side, 488 nm light from the top and 395 nm light from the top.

Figure 3 – source data 1-5. Source data for panels A-E.

NOS and two NIT-GCs shape Ca2+ signals during cPRC UV/violet response.

(A, B) GCaMP6s signals in cPRCs in wild type and NOS mutant (A, NOSΔ11/Δ11, B, NOSΔ23/Δ23) larvae during 405 nm light stimulation. (C, D) In situ HCR for (C) NIT-GC1 and (D) NIT-GC2 (magenta) in three-day-old Platynereis larvae. Larvae were co-stained with an antibody against acetylated α-tubulin to label cPRC cilia and the neuropil (green). (E, F) Immunostaining for (E) NIT-GC1 and (F) NIT-GC2 (magenta), co-stained for acetylated α-tubulin (green). (G) The domain structure of Platynereis NIT-GC1 and the truncated NIT-GC1ΔNIT protein lacking the NIT domain. A predicted transmembrane region (TM) is shown in grey. (H) Schematic of the cell-based assay to detect cGMP production following the addition of an NO donor SNAP or DMSO as control. (I-L) Green cGull fluorescence over time for the four conditions tested. Individual responses and their mean with 0.95 confidence interval are shown (n > 6 cells). Intensities are normalized (ΔF/F0). The indicated chemicals were added at 2 min after the start of imaging (grey bars). (M, N) GCaMP6s signals in cPRCs in (M) NIT-GC1 and (N) NIT-GC2 morphant larvae during 405 nm light stimulation. Individual responses and their mean with 0.95 confidence interval are shown.

Figure 4 – source data 1-8. Source data for panels A, B and I-N.

NOS- and NIT-GC2-dependent dynamics of the cPRC circuit.

(A, B) GCaMP6s imaging from cPRCs and INNOS cells (left panels) followed by on-slide immunostaining for (A) RYamide to label INNOS and (B) RGWamide+serotonin to label INRGWa and Ser-h1 (red). Nuclei are stained with DAPI (cyan). Asterisks indicate cPRC nuclei. Numbers mark the same cells in the GCaMP and immunostaining images matched by position. (C) Correlation map of neuronal activity of the cPRCs, INNOS, INRGWa, Ser-h1 and MC neurons. (D) GCaMP6s fluorescence in INNOS cells in wild type (WT) and NOSΔ11/Δ11 mutant larvae during 405 nm stimulation of the cPRC cilia. (E) GCaMP6s fluorescence in INNOS cells in NIT-GC2 morphant larvae during 405 nm stimulation. (F) GCaMP6s fluorescence in INRGWa cells in wild type and NOSΔ23/Δ23 mutant larvae during 405 nm stimulation. (G) GCaMP6s fluorescence in INRGWa cells in NIT-GC2 morphant larvae during 405 nm stimulation. (H, I) GCaMP6s fluorescence in (H) Ser-h1 cells and (I) the MC cell in wild type and NOSΔ11/Δ11 mutant larvae during 405 nm stimulation.

Figure 5 – source data 1-6. Source data for panels D-I.

Mathematical modelling and signalling mechanisms of the cPRC circuit.

(A) Diagram of the mathematical model with the componenets, interactions, parameters and equations used to model Ca2+ dynamics. (B) Average Ca2+ traces (upper) and traces from model simulations (lower) fitted to the data. (C) Dot plot of genes (columns) expressed in three types of cells (rows) in the cPRC circuit using single cell RNA-Seq. The size of the dots is expressed in proportion to the percentage of cells expressing that gene relative to all cells. The colours represent the normal logarithm of the number of transcripts in the cells expressing the gene. (D) Schematic diagram of the signalling pathway of the cPRC circuit, focusing on the NO feedback.

Figure 6 – source data 1. TPM values for each gene and the percentage of expressed genes.

Magnitude of cPRC activation as a function of stimulus duration and intensity.

Median values of the AUP are presented as a heatmap with dark blue indicating flat CP (t) traces with median AUP close to 0 and red indicating that the CP (t) traces have a clearly defined delayed activation peak.

Maximum-likelihood phylogenetic tree of NOS protein sequences.

Individual branches are coloured by taxonomy. Branch support values indicate UFBoot and aLRT-SH-like values.

Figure 1 – figure supplement 1 – source data 1. NOS sequences used for the phylogenetic reconstruction.

Figure 1 – figure supplement 1 – source data 2. Aligned and trimmed NOS sequences used for the phylogenetic reconstruction.

Figure 1 – figure supplement 1 – source data 3. Tree file of the reconstructed NOS phylogeny.

Expression of NOS in three-day-old Platynereis larva.

(A) HCR in situ for NOS (magenta), dorsal view. (B) HCR in situ for NOS (magenta), ventral view. Inset in (B) shows close-up of two NOS-positive cells (INNOS_dl and INNOS_vl) on the left side. Samples were also stained for DAPI (cyan) to label nuclei.

Synapse distribution in cPRCs and possynaptic interneurons

(A) Volume rendering of the four INNOS neurons with incoming and outgoing synapses. (B) Axo-dendritic splitting of the INNOS neurons. (C) Volume rendering of the four INRGW neurons with incoming and outgoing synapses. (D) Volume rendering of the four cPRC neurons with incoming and outgoing synapses. All images are anterior views. Asterisk marks the same position across images for reference. Grey speheres indicate the position of cell nuclei. NS plexus; neurosecretory plexus. The radius of INNOS nuclei is set to 2 μm for scale.

Generation and behavioural characterisation of NOS CRISPR knockouts.

(A) Top: The domain organisation of Platynereis NOS protein and exon/intron structure of the NOS gene. Bottom: The genomic locus of NOS with the CRISPR target site, wild-type and knockout (NOSΔ11, NOSΔ23) sequences and predicted protein sequences. The PAM sequence is shown in grey, stop codons in red. (B) Swimming trajectories of wild type (WT, n=37) and NOS mutant (NOSΔ11/Δ11, n=18 and NOSΔ23/Δ23, n=8) two-day-old larvae. All trajectories start at 0 x and y position and time 0 corresponding to 10 sec after the onset of 395 nm stimulation from the side. (C) Vertical displacement in 30 sec bins of wild type and mutant (NOSΔ11 and NOSΔ23) two-day-old larvae stimulated with 395 nm light from the side, 488 nm light from the top and 395 nm light from the top. (D) Vertical position of batches of wild type and mutant two-day-old larvae over time under 395 nm UV stimulation. The starting position of each larval trajectory was set to 0. (E-G) Swimming speed of batches of wild type and mutant two-day old (E) and three-day-old (F) larvae and L-NAME-treated (NOS inhibitor) three-day-old larvae under 395 nm UV stimulation.

Figure 3 – figure suppmenet 1 – source data 1-6. Source data for panels B-G.

Cluster analysis of guanylate and adenylate cyclase sequences.

Each node represents one sequence, colour-coded by taxonomy. Connections represent BLAST P-values of <1e-16. NIT-GCs, NIT domain containing guanylate cyclases; membrane-bound GCs, membrane-bound guanylate cyclases; sGCs, soluble guanylate cyclases; ACs, adenylate cyclases.

Maximum-likelihood phylogenetic tree of NIT-domain-containing guanylate cyclases

Membrane-bound and soluble guanylate cyclases (sGC) were included as outgroups. Guanylate cyclases with NIT domains are found in most animal phyla except Porifera, Ctenophora, Urochordata and Chordata. Branch support values indicate UFBoot and aLRT-SH-like values. The expression of Platynereis NIT-GC genes in cPRC, INNOS and INRGW cells is indicated on the right side of the tree. The values represent expression based on single-cell sequencing data. Dot size indicates specificity (percent of transcripts expressed in the indicated cell across all cells). Dot colour represents the logarithm of the number of transcripts in the expressing cells.

Figure 4 – figure supplement 2 – source data 1. GC sequences used for the phylogenetic reconstruction.

Figure 4 – figure supplement 2 – source data 2. Aligned and trimmed GC sequences used for the phylogenetic reconstruction.

Figure 4 – figure supplement 3 – source data 3. Tree file of the reconstructed GC phylogeny.

Expression of NIT-GC1 and NIT-GC2 in the cPRCs

(A) HCR co-expression analysis of NIT-GC1 (magenta) and MLD/pedal-peptide-2 proneuropeptide (green). Anterior view of a two-day-old larva. Nuclei were stained with DAPI (cyan). (B, C) Expression of NIT-GC2 (magenta) detected by in situ HCR. Anterior (B) and ventral (C) views of a three-day-old larva. Nuclei were stained with DAPI (cyan). (D) Immunostaining for NIT-GC1 (magenta) and NOS (green). Anterior view of a two-day-old larva. Acetylated α-tubulin staining (blue) highlights the neuropil and cPRC cilia. (E, F) Immunostaining for NIT-GC1 (E) and NIT-GC2 (F) antibodies in larvae injected with NIT-GC1 (E) or NIT-GC2 (F) morpholinos. Acetylated α-tubulin staining (green) highlights the neuropil and cPRC cilia. Anterior view of a two-day-old larva.

On-slide immunostaining and coexpression analysis of NOS and RYamide proneuropeptide

(A) Schematic diagram of the on-slide immunostaining procedure after Ca2+ imaging.(B-D) Co-expression analysis by HCR in situ of NOS (magenta) and RYamide proneuropeptide (green). Anterior view of a two-day-old larva. Nuceli were stained by DAPI (cyan).

Simulated Ca2+ traces for parameter sets fitted to individual Ca2+-recordings collected in wild type, NOS knockout, and NIT-GC2 morphant larvae.

(A-C) Simulated Ca2+ traces in cPRC (A), INNOS (B) and INRGW (C) cells in the wild-type condition. (D-F) Simulated Ca2+ traces in cPRC (D), INNOS (E) and INRGW (F) cells in the NOS-knockout condition. (G-I) Simulated Ca2+ traces in cPRC (G), INNOS (H) and INRGW (I) cells in the NIT-GC2-morphant condition. Thin coloured curves indicate individual recordings (cPRC - purple, INNOS - blue and INRGW - green), thin grey curves indicate simulated Ca2+ traces based on model parameters fitted to the individual recording, thick coloured curves indicate averages of the recordings and thick black curves represent the average of the fits.

Comparison of fits with different fixed parameter sets when fitting the WT model to individual recordings from the WT INRGW cells.

Panels A-B, D-E, G-H (column fixed parameters) show simulated traces of the Ca2+ levels in cPRC and INNOS cells produced by different fixed parameter sets. Panels C, F and I show individual recordings fitted to the data from INRGW cells.

Comparison of fits to the cPRC recordings from NIT-GC2 morphant larvae using the NIT-GC2 morpholino model or the NOS-knockout model.

Thin coloured curves indicate individual recordings, thin grey curves are simulated Ca2+ traces, thick coloured curves indicate averages of the recordings and thick black curves represent the mean of the fits.

Distributions of the parameter values fitted to the cPRC recordings.

Violin plots (grey lines) are kernel density estimates of the underlying distributions; computed using the Matlab function ksdensity with default settings, i.e., using normal kernel function, plots are trimmed to the observed range of data. Markers represent individual parameter values, white markers indicate medians, vertical grey bars indicate interquartile range (Q25 to Q75), and the dashed line indicates the value 1. Where violin plots are not shown, these parameters are associated with terms/equations that are excluded from the fitting procedure (see Methods for details).

Pairwise correlations between parameters of the WT model fitted to WT cPRC recordings

Parameters associated with the CR equation are not included (see Methods for details). Panels on the diagonal show kernel density estimates of the distributions of the individual parameters; computed using Matlab function ksdensity with default settings, i.e., using normal kernel function, plots are trimmed to the observed range of data. The other panels show kernel density estimates of bivariate distributions of pairs of parameters (heatmaps; darker colours indicate higher density). Also shown using markers are the values of the individual parameter combinations.