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

(A) Scanning electron microscopy image of a 3-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 3-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) Volume rendering of the four INNOS neurons with incoming and outgoing synapses. (E) Expression of the NOS gene detected by in situ HCR (magenta) in a 2-day-old larva (anterior view). Antibody staining for acetylated α-tubulin (acTub: green) highlights cPRC cilia and the neuropil. (F) Expression of a membrane-targeted reporter driven by the NOS regulatory region (NOSp::palmi-3xHA-Tomato; magenta) labelled with an anti-HA antibody in a 2-day-old larva (anterior view). Antibody staining for acetylated α-tubulin (acTub; green) highlights cPRC cilia and the neuropil. (G) Immunostaining for NOS (magenta), co-stained for acetylated α-tubulin (acTub; green). (H) 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) Left: The larvae were embedded in 2% agarose seawater with the anterior side down. Right: Changes in DAF-FM fluorescence intensities were examined when the ciliary region of the cPRC was stimulated with a 405 nm laser.(B) 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. (C) DAF-FM fluorescence before and during 405 nm light stimulation. (D) 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) Schematic diagram of the set-up of the behavioural experiment. (B) Vertical displacement in 30 sec bins of wild type (10 batches) and mutant (NOSΔ11/Δ11, 5 batches and NOSΔ23/Δ23, 7 batches) 3-day-old larvae stimulated with 395 nm light from the side, 488 nm light from the top and 395 nm light from the top. One-way ANOVA with Dunnett’s multiple-comparison test are shown. *P < 0.05, **P < 0.01. (C) Swimming trajectories of wild type (WT, n=32) and NOS mutant (NOSΔ11/Δ11, n=26 and NOSΔ23/Δ23, n=47) 3-day-old larvae. All trajectories start at 0 x and y position and time 0 corresponding to 10 sec before the onset of 395 nm stimulation from the side. (D) Vertical position of batches of wild type and mutant 3-day-old larvae over time under 395 nm UV stimulation. The starting position of each larval trajectory was set to 0. (E) Comparison of vertical position of wild type and mutant 3-day-old larvae after 30 seconds of UV stimulation. One-way ANOVA with Dunnett’s multiple-comparison test are shown. The wild type (WT, n=32) and NOS mutant (NOSΔ11/Δ11, n=26 and NOSΔ23/Δ23, n=47) larvae. (F) Vertical displacement in 30 sec bins of control (15 batches) and L-NAME-treated (0.1 and 1 mM, 15 batches each) 3-day-old larvae stimulated with 395 nm light from the side, 488 nm light from the top and 395 nm light from the top. One-way ANOVA with Dunnett’s multiple-comparison test are shown. *P < 0.05. (G) Vertical position of batches of control and L-NAME-treated (0.1 and 1 mM) 3-day-old larvae over time under 395 nm UV stimulation. The starting position of each larval trajectory was set to 0. (H) Comparison of vertical position of control (n=6) and L-NAME-treated (0.1 and 1 mM, n=6 each) 3-day-old larvae after 30 seconds of UV stimulation. One-way ANOVA with Dunnett’s multiple-comparison test are shown. Figure 3 – source data 1. Vertical displacement data for wild type and NOS mutant larvae. Figure 3 – source data 2. Swimming trajectories of wild type and NOS mutant larvae. Figure 3 – source data 3. Vertical position of wild type and NOS mutant larvae. Figure 3 – source data 4. Vertical position of wild type and NOS mutant larvae after 30 sec UV exposure. Figure 3 – source data 5. Vertical displacement of control and L-NAME-treated larvae. Figure 3 – source data 6. Vertical position of control and L-NAME-treated larvae. Figure 3 – source data 7. Vertical position of control and L-NAME-treated larvae after 30 sec UV exposure.

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 3-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. GCaMP6s data for cPRCs in wild type during 405 nm stimulation. Figure 4 – source data 2. GCaMP6s data for cPRCs in NOS mutant larvae during 405 nm stimulation. Figure 4 – source data 3. Green cGull fluorescence data of cells with NIT-GC1 after SNAP treatment. Figure 4 – source data 4. Green cGull fluorescence data of cells without NIT-GC1 after SNAP treatment. Figure 4 – source data 5. Green cGull fluorescence data of cells with NIT-GC1 after DMSO treatment. Figure 4 – source data 6. Green cGull fluorescence data of cells with NIT-GC1ΔNIT after SNAP treatment. Figure 4 – source data 7. GCaMP6s data for cPRCs in NIT-GC1 morphant larvae during 405 nm stimulation. Figure 4 – source data 8. GCaMP6s data for cPRCs in NIT-GC2 morphant larvae during 405 nm stimulation.

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) GCaMP6s fluorescence in INNOS cells in wild type (WT) and NOSΔ11/Δ11 mutant larvae during 405 nm stimulation of the cPRC cilia. (D) GCaMP6s fluorescence in INNOS cells in NIT-GC2-morphant larvae during 405 nm stimulation. (E) GCaMP6s fluorescence in INRGWa cells in wild type and NOSΔ23/Δ23 mutant larvae during 405 nm stimulation. (F) GCaMP6s fluorescence in INRGWa cells in NIT-GC2-morphant larvae during 405 nm stimulation. Figure 5 – source data 1. GCaMP6s fluorescence data for INNOS in wild type and NOS mutant larvae. Figure 5 – source data 2. GCaMP6s fluorescence data for INNOS in wild type and NIT-GC2 morphant larvae. Figure 5 – source data 3. GCaMP6s fluorescence data for INRGWa in wild type and NOS mutant larvae. Figure 5 – source data 4. GCaMP6s fluorescence data for INRGWa in wild type and NIT-GC2 morphant larvae.

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) 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. (C) 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.

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

(A, B) Simulated Ca2+ traces in cPRC (A) and INNOS (B) cells in the wild-type condition. (C, D) Simulated Ca2+ traces in cPRC (C) and INNOS (D) cells in the NOS knockout condition. (E-F) Simulated Ca2+ traces in cPRC (E) and INNOS (H) cells in the NIT-GC2-morphant condition. Thin coloured curves indicate individual recordings (cPRC - purple and INNOS - blue), 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. Figure 7 – source data 1. Best fitted parameters {best/_pars.mat}. Figure 7 – source data 2. Recorded traces (as used in earlier figures) {wt/_ko/_mo/_data.mat}. Figure 7 – source data 3. script to generate the figure {fig/_all/_fits.m}./label{fig:all-fits}

Summary of the significance of global sensitivity indexes

Results of the global sensitivity analysis of the model consisting of Cp(t), B(t) and S(t) variables; fitting to NOS-knockout recordings.

Results of the global sensitivity analysis of the model consisting of S(t) and CN (t) variables; fitting to WT and NOS-knockout INNOS Ca2+ recordings.

Results of the global sensitivity analysis of the model consisting of S(t), CN (t) and N(t) variables; fitting to WT NO recordings.

Results of the global sensitivity analysis of the full WT model (remaining parameters); fitting to WT recordings.

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 3-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.(C) Expression of the NOS gene detected by in situ HCR (magenta) in a 3-day-old larva (anterior view). Antibody staining for acetylated α-tubulin (acTub: green) highlights cPRC cilia and the neuropil.

Synapse distribution in cPRCs and possynaptic interneurons (A) Axo-dendritic splitting of the INNOS neurons.

(B) Volume rendering of the four INRGWa neurons with incoming and outgoing synapses. (C) 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, C) Immunostaining for NOS antibody in NOS mutant (B, NOSΔ11/Δ11; C, NOSΔ23/Δ23) larvae. Acetylated α-tubulin staining (green) highlights the neuropil and cPRC cilia. Anterior view of a 3-day-old larva.

Generation and behavioural characterisation of NOS CRISPR knockouts.

(B) Swimming trajectories of wild type (WT, n=37) and NOS mutant (NOSΔ11/Δ11, n=18 and NOSΔ23/Δ23, n=8) 2-day-old larvae. All trajectories start at 0 x and y position and time 0 corresponding to 10 sec before the onset of 395 nm stimulation from the side. (C) Vertical displacement in 30 sec bins of wild type (WT, n=37) and mutant (NOSΔ11/Δ11, n=18 and NOSΔ23/Δ23, n=8) 2-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) Comparison of vertical position of wild type and mutant 2-day-old larvae after 30 seconds of UV stimulation. One-way ANOVA with Dunnett’s multiple-comparison test are shown. The wild type (WT, n=52) and NOS mutant (NOSΔ11/Δ11, n=30 and NOSΔ23/Δ23, n=16) larvae. (E) Vertical position of batches of wild type (12 batches) and mutant (NOSΔ11/Δ11, 8 batches and NOSΔ23/Δ23, 3 batches) 2-day-old larvae over time under 395 nm UV stimulation. The starting position of each larval trajectory was set to 0. One-way ANOVA with Dunnett’s multiple-comparison test are shown. *P < 0.05. Figure 3 – – figure supplement 2 – source data 1. Vertical displacement data for 2-day-old wild type and NOS mutant larvae. Figure 3 – – figure supplement 2 – source data 2. Swimming trajectories of 2-day-old wild type and NOS mutant larvae. Figure 3 – – figure supplement 2 – source data 3. Vertical position of 2-day-old wild wild type and NOS mutant larvae. Figure 3 – – figure supplement 2 – source data 4. Vertical position of 2-day-old wild type and NOS mutant larvae after 30 sec UV exposure.

Generation and behavioural characterisation of NOS CRISPR knockouts.

(A,B) Swimming speed of batches of wild type and NOS mutant 2-day-old (WT, n=37, NOSΔ11/ Δ11, n=18 and NOSΔ23/Δ23, n=8) (A) and 3-day-old (WT, n=32, NOSΔ11/Δ11, n=26 and NOSΔ23/Δ23, n=47) (B) larvae under 395 nm UV stimulation. (C,D) Mean swimming speed of wild type and NOS mutant 2-day-old (WT, n=37, NOSΔ11/Δ11, n=18 and NOSΔ23/Δ23, n=8) (C) and 3-day-old (WT, n=32, NOSΔ11/Δ11, n=26 and NOSΔ23/Δ23, n=47) (D) larvae during 10 seconds before (dark) and 10 seconds after the start of 395 nm UV stimulation (sideUV). Data points for the same larva are joined by lines. One-tailed paired t-test; p-values (*** <0.001) are shown. (E,F) Average ciliary beat frequency of wild type and mutant 2-day-old (E) and 3-day-old (F) single larvae during 10 seconds before (gray) and 10 seconds after the start of 395 nm UV stimulation (purple). N=6 larvae each. Data points for the same larva are joined by lines. One-tailed paired t-test; p-values (** <0.01 or * <0.05) are shown. Figure 3 – figure supplement 3 – source data 1. Swimming speed data for wild type and NOS mutant 2-day-old larvae. Figure 3 – figure supplement 3 – source data 2. Swimming speed data for wild type and NOS mutant 3-day-old larvae. Figure 3 – figure supplement 3 – source data 3. Mean swimming speed of wild type and NOS mutant 2-day-old larvae. Figure 3 – figure supplement 3 – source data 4. Mean swimming speed of wild type and NOS mutant 3-day-old larvae. Figure 3 – figure supplement 3 – source data 5. Ciliary beat frequency data for wild type and NOS mutant 2-day-old larvae. Figure 3 – figure supplement 3 – source data 6. Ciliary beat frequency data for wild type and NOS mutant 3-day-old larvae.

Lunar cycle of maturation in wild-type and NOS CRISPR knockout Platynereis worms.

Data represent the number of swimming epitoks per day over a one-year period in the Heidelberg culture. Males and females are plotted separately. Data are shown for wild type, NOSΔ11/ Δ11 and NOSΔ23/Δ23 worms as line plots and smoothed plots (with a 0.95 confidence band). Figure 3 – figure supplement 4 – source data 1. Maturation data for wild type and NOS knockout worms.

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 INRGWa 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 2-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 3-day-old larva. Nuclei were stained with DAPI (cyan). (D, E) Immunostaining for NIT-GC1 (D) and NIT-GC2 (E) antibodies in larvae injected with NIT-GC1 (D) or NIT-GC2 (E) morpholinos. Acetylated α-tubulin staining (green) highlights the neuropil and cPRC cilia. Anterior view of a 2-day-old larva. (F) The fluorescence intensities of the NIT-GC1 or NIT-GC2 in the control and morphant (MO1 and MO2) larvae (n=6 each) were normalized to the acetylated α-tubulin signals and plotted as a relative fluorescence. One-way ANOVA with Dunnett’s multiple-comparison test; p-values (‘***‘ <0.001) are shown. Figure 4 – figure supplement 3 – source data 1. Comparison NIT-GCs signals readings.

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 2-day-old larva. Nuclei were stained by DAPI (cyan).

NOS-dependent dynamics of the Ser-h1 neurons.

(A) Correlation map of neuronal activity of the cPRCs, INNOS, INRGWa, and Ser-h1 neurons. (B) GCaMP6s fluorescence in Ser-h1 cells in wild type and NOSΔ11/Δ11 mutant larvae during 405 nm stimulation. Figure 5 – figure supplement 2 – source data 1. Calcium imaging data from Ser-h1 neurons.

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

The NIT-GC2 has the same parameter values as NOS-knockout except kappaGC2 is decreased from 1 to 0.15. (A) Coloured curves indicate the mean of the recordings, thick black curves indicate simulated traces based on model parameters fitted to the mean of the recordings. (B, C) Thin grey curves (solid and dotted) represent simulated experimental conditions with different levels of efficacy of the NOS-knockout (B) and NIT-GC2 morpholino (C). To generate interpolated condition curves in (B), we multiplied kappa SGC1 in the wild-type model by values from 0.1 to 0.9 with steps of 0.1. To generate interpolated condition curves in (C), we use a set of kappaGC2 values from 0.1 to 0.9 with steps of 0.1. Figure 6 – figure supplement 1 – source data 1. Recorded traces (as used in earlier figures) wt/_ko/_mo/_data.mat. Figure 6 – figure supplement 1 – source data 2. Script to generate the figure fig/_WT/_to/_MO.

Simulated NO traces for parameter sets fitted to individual NO-recordings collected in wild type larvae.

Thin coloured curves indicate individual recordings, thin grey curves indicate simulated NO traces based on model parameters fitted independently to each recording, the thick coloured curve indicates the average of the recordings, the thick grey curve represents the average of the fits, and the thick black curve represents the fit of the model parameters to the averaged recordings. Figure 6 – figure supplement 2 – source data 1. Best fitted parameters {best/_pars.mat}. Figure 6 – figure supplement 2 – source data 2. Recorded traces (same as in Figure 2 but with removed bleaching and steady state set to 0) (NO/_data.mat). Figure 6 – figure supplement 2 – source data 3. Script to generate the figure fig/_fit/_NO.m

Distributions of the parameter values fitted to the individual recordings collected in different conditions.

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. Coloured markers represent individual parameter values, white markers indicate medians, vertical grey bars indicate interquartiles (ranges Q25 to Q75), and the dashed line indicates the value of the parameter fitted to the average of the recordings. We show histograms instead of violin plots for deltaS when fitted to NOS-knockout recordings and for deltaS and deltaC when fitted to NO recordings because these distributions are highly skewed and hence misrepresented by violin plots. Violin plots are presented in the order of our model-fitting procedure, i.e., first Ca2+ in NOS-knockout cPRC cells (yellow), then Ca2+ in wild type and NOS-knockout INNOS cells (blue), then NO in INNOS cells (green), and finally Ca2+ in wild type cPRC cells (magenta); (see Methods for details). Figure 6 – figure supplement 3 – source data 1. Best fitted parameters best/_pars.mat. Figure 6 – figure supplement 3 – source data 2. Script to generate the figure parameter/_dist.m

Distributions of the parameter values fitted to individual cPRC recordings collected in wild type larvae.

The figure is in the same format as Figure 6 – figure supplement 4. Figure 6 – figure supplement 4 – source data 1. Best fitted parameters best/_pars.mat. Figure 6 – figure supplement 4 – source data 2. Script to generate the figure parameter/_dist.m.

Pairwise correlations between parameters of the NOS-knockout model (state variables CP (t), B(t) and S(t)) fitted to NOS-knockout cPRC recordings; wild-type model (state variables S(t) and CN (t)) fitted to wild-type and NOS-knockout Ca2+ INNOS recordings.

Wild type model (state variables S(t), CN (t) and N(t)) fitted to wild type INNOS NO recordings. All visible correlation values are significant at the 5% level, p < 0.05, (trivial correlations along the diagonal are excluded). Figure 6 – figure supplement 5 – source data 1. Best fitted parameters best/_pars.mat. Figure 6 – figure supplement 5 – source data 2. Script to generate the figure correlations.m

Pairwise correlations between parameters of the wild type model fitted to wild type cPRC recordings.

All visible correlation values are significant at the 5% level, p < 0.05, (trivial correlations along the diagonal are excluded). Figure 6 – figure supplement 6 – source data 1. Best fitted parameters best/_pars.mat. Figure 6 – figure supplement 6 – source data 2. Script to generate the figure correlations.m

Validation of the wild-type model using cPRC recordings collected in wild-type larvae subject to UV stimulation with different intensity and duration.

The top row displays recorded data; the bottom row shows model simulations. Different columns show different durations of UV stimulation. Simulated Ca2+ traces use parameter sets fitted to mean of Ca2+-recordings collected in wild-type larvae subject to 20s UV stimulation at 100% power. Stimulation duration is indicated by the shaded rectangle (violet) while stimulation intensity is indicated by the shade of grey of the line plot (darker shades indicate higher power). Simulations are compared with the mean of the 50%, 75% and 100% data and their 95% confidence interval (mean pm1.96SE, here is SE = std/sqrtn and std stands for standard deviation). We use the 50%, 75% and 100% recordings to compute the average because the data indicate the existence of a non-linear relation between response and the power of the stimulation and the 25% recordings would bias the average down. Figure 7 – source data 1. Recorded traces longer_experiments_data.mat. Figure 6 – figure supplement 7 – source data 1. Script to generate the figure fig_longer_experiments.m

Simulated Ca2+ traces under stimulation with two UV pulses.

The top row shows simulations with a 10s interval between pulses; the bottom row shows simulations with a 30s interval between pulses. Different columns show different durations of UV stimulation. The duration of UV stimulation is indicated by the shaded rectangle (violet). Coloured curves indicate simulated outcome of two pulse UV stimulation; thick black curves indicate simulated reference traces with a single UV pulse. Simulations of the Ca2+ traces use parameter sets fitted to the mean of individual Ca2+-recordings collected in wild-type larvae subject to 20s UV stimulation. Figure 6 – figure supplement 8 – source data 1. Script to generate the figure fig_two_pulse.m