Odekunle EA , Semmens DC , Martynyuk N , Tinoco AB , Garewal AK , Patel RR , Blowes LM , Zandawala M , Delroisse J , Slade SE , Scrivens JH , Egertová M , Elphick MR .

BB/M001644/1 Biotechnology and Biological Sciences Research Council , BB/M001032/1 Biotechnology and Biological Sciences Research Council

	Fig. 1. Asterotocin: a vasopressin/oxytocin (VP/OT)-type neuropeptide in the starfish Asterias rubens. a Amino acid sequence of the A. rubens asterotocin precursor with the predicted signal peptide shown in blue, a predicted cleavage site shown in green, the asterotocin peptide shown in red with a C-terminal glycine that is a putative substrate for amidation shown in orange and the neurophysin domain shown in purple. b Structure of the mature asterotocin peptide as determined by mass spectrometry (Additional file 1), with a disulphide bridge between the two cysteine residues and with C-terminal amidation. c Clustal-X alignment of asterotocin (A_rub) with VP/OT-type peptides from other animals reveals that the pair of cysteine residues (asterisks) is conserved in all of the peptides, whereas other residues are conserved in a subset of the peptides. Full species names within each taxonomic group (Ambulacraria, Chordata and Protostomia) and corresponding accession numbers for the peptide sequences are listed in Table S1 of Additional file 3
	Fig. 2. Phylogenetic identification and deorphanisation of an A. rubens VP/OT-type receptor. a Maximum likelihood tree showing that a VP/OT-type receptor identified by BLAST analysis of A. rubens transcriptome sequence data (boxed) is positioned within a clade comprising VP/OT-type receptors from other taxa. NPS/NG peptide/CCAP-type receptors are paralogs of VP/OT-type receptors [25]. Phylogenetic analyses of bilaterian neuropeptide receptors have shown that GnRH/AKH/ACP/CRZ-type receptors are closely related to VP/OT-type receptors and NPS/NG peptide/CCAP-type receptors [6]. Therefore, GnRH/AKH/ACP/CRZ-type receptors were included as an outgroup in the phylogenetic tree. The scale bar indicates amino acid substitutions per site, and bootstrap values are shown at nodes. Species where activation of the VP/OT-type receptor by a cognate VP/OT-type neuropeptide has been demonstrated experimentally (including A. rubens, see b) are labelled with an asterisk. Full species names and accession numbers for the receptor sequences are listed in Table S2 of Additional file 3. b Asterotocin (black circles) causes concentration-dependent activation of the A. rubens VP/OT-type receptor, demonstrated by measuring Ca2+-induced luminescence in CHO-K1 cells expressing aequorin, and transfected with the promiscuous G-protein Gα16 and the A. rubens VP/OT-type receptor (CHO-K1/G5A-ArVPOTR cells); EC50 = 5.7 × 10−8 M. CHO-K1 cells transfected with empty pcDNA vector (black square) were used as a negative control and do not exhibit luminescence when exposed to asterotocin. c Comparison of the relative luminescence responses of CHO-K1/G5A-ArVPOTR cells when exposed to asterotocin or the related peptides NGFFYamide (a paralog of asterotocin in A. rubens), human vasopressin or human oxytocin, all at a concentration of 10−4 M. Luminescence responses in the presence of vasopressin, oxytocin and NGFFYamide were not significantly higher than the basal media control (P = 0.5, P = 0.25, P = 0.25, respectively; Wilcoxon signed-rank test; n = 9)
	Fig. 3. Starfish anatomy. a Schematic vertical section of the central disc and the proximal region of an arm. b Schematic transverse section of an arm. c Schematic transverse section of a radial nerve cord. Abbreviations: a, anus; am, apical muscle; amp, ampulla; conr, circumoral nerve ring; cs, cardiac stomach; cut, cuticle; ec, ectoneural region; g, gonad; gcc, general coelomic cavity; hy, hyponeural region; m, mouth; md, madreporite; mn, marginal nerve; o, ossicle; p, papula; pc, pyloric caecum; pd., pyloric duct; ped, pedicellaria; pm, peristomial membrane; ps, pyloric stomach; rc, rectal caecum; rca, ring canal; rn, radial nerve; rw, radial water vascular canal; sp., spine; sc, stone canal; tb, Tiedemann’s body; tf, tube foot
	Fig. 4. Localisation of asterotocin in A. rubens using in situ hybridisation (ISH) and immunohistochemistry (IHC). a Asterotocin precursor transcript-expressing cells (AstPtc) in a radial nerve cord (black arrowheads) and tube feet (white arrowheads). Inset shows the absence of staining with sense probes. b Asterotocin-immunoreactive (Ast-ir) cells (arrowheads in inset) and fibres (white asterisks) in a radial nerve cord. c AstPtc in the circumoral nerve ring (arrowheads and inset). d Ast-ir cells (arrowheads) and fibres (white asterisks) in the circumoral nerve ring. e AstPtc in marginal nerve. f Ast-ir fibres in marginal nerve. g AstPtc in tube foot disc. h Ast-ir in the basal nerve ring (black arrow) and longitudinal nerve tract (grey arrow) of a tube foot. i AstPtc in the peristomial membrane. j AstPtc in the oesophagus. k Ast-ir in the peristomial membrane (square bracket) and oesophagus. l AstPtc in the cardiac stomach. m Ast-ir in the cardiac stomach. n AstPtc in the pyloric stomach. o AstPtc in a pyloric duct. p Ast-ir in the pyloric stomach. q AstPtc in the coelomic epithelium of the body wall. r Ast-ir in the coelomic basiepithelial nerve plexus of body wall. s Ast-ir in the basiepithelial nerve plexus of the apical muscle. t AstPtc in a papula. u Ast-ir in a papula. v AstPtc in the body wall. w Ast-ir in the body wall. x AstPtc in a pedicellaria. y Ast-ir in a pedicellaria. z AstPtc in a spine. z’ Ast-ir in an ambulacral spine. Abbreviations: AM, apical muscle; BNR, basal nerve ring; BNP, basiepithelial nerve plexus; CMLNP, circular muscle layer nerve plexus; CBNP, coelomic basiepithelial nerve plexus; CE, coelomic epithelium; CT, collagenous tissue; Di, disc; Ec, ectoneural region; EE, external epithelium; Hy, hyponeural region; LNT, longitudinal nerve tract; Lu, lumen; MN, marginal nerve; ML, mucosal layer; MuL, muscle layer; Oes, oesophagus; Pa, papula; RHS, radial hemal sinus; TF, tube foot; VML, visceral muscle layer. Scale bars: a, a inset, h, l, m, n, o, p, u = 60 μm; b, d, g, z’ = 40 μm; i, j, l inset, p inset, t, w, x = 30 μm; B inset, c, d inset, e, k, n inset, o inset, s, v, y, z = 20 μm; c inset, f, m inset, q, r = 10 μm
	Fig. 5. Localisation of asterotocin receptor in A. rubens using in situ hybridisation (ISH) and immunohistochemistry (IHC). a Asterotocin receptor transcript-expressing cells (AstRtc) in a radial nerve cord (black arrowheads) and tube feet (white arrowheads). Inset shows the absence of staining with sense probes. b AstR-immunoreactive (AstR-ir) cells (black arrowhead) and processes (arrows and in inset) in a radial nerve cord. Stained fibres in the adjacent tube foot (white arrow). c AstRtc in the circumoral nerve ring. d AstR-ir cells (arrowheads) and fibres (arrow) in the circumoral nerve ring. e AstRtc in a marginal nerve. f AstR-ir cell (arrowhead) and stained process (black arrow) in a marginal nerve. Stained fibres in the adjacent tube foot (white arrow). g AstRtc in a tube foot (black arrowhead). h Ast-ir in a tube foot. i AstRtc in the cardiac stomach. j AstR-ir in the mucosal layer (white arrowhead), basiepithelial nerve plexus (black arrowhead) and visceral muscle layer (black arrow; inset) of the cardiac stomach. k AstRtc in the coelomic epithelium of the body wall. l AstR-ir cell in the coelomic epithelium of the body wall. m AstRtc in the external epithelium of the body wall. n AstR-ir cell in the external epithelium of the body wall. o AstRtc in a papula. p AstR-ir cells in an ambulacral spine. q AstR-ir cells in a pedicellaria. Abbreviations: BNR, basal nerve ring; BNP, basiepithelial nerve plexus; CMLNP, circular muscle layer nerve plexus; Ce, coelom; CBNP, coelomic basiepithelial nerve plexus; CT, collagenous tissue; Di, disc; Ec, ectoneural region; EE, external epithelium; Hy, hyponeural region; Lu, lumen; MN, marginal nerve; ML, mucosal layer; Pa, papula; RHS, radial hemal sinus; TF, tube foot; VML, visceral muscle layer. Scale bars: a, a inset, b, c, g, h, o, p, q = 60 μm; d, e, j = 30 μm; c inset, f, g inset, n = 20 μm; b inset, e inset, f inset, i, i inset, j inset, k, l, m, o = 10 μm
	Fig. 6. Comparison of asterotocin and asterotocin receptor expression in A. rubens using double-labelling fluorescence immunohistochemistry. Comparison of the distribution of asterotocin immunoreactivity (Ast-ir; green) and asterotocin receptor immunoreactivity (AstR-ir; red) in A. rubens reveals ‘salt and pepper’ patterns of labelling consistent with expression largely in different, but often adjacent, populations of cells/processes. In the few instances where labelling appears yellow/orange, this could be due to the co-localisation of asterotocin and the asterotocin receptor in processes or alternatively it may simply reflect where asterotocin-containing fibres happen to be positioned directly above asterotocin receptor containing fibres. a Radial nerve cord showing Ast-ir cells (yellow arrowheads) and AstR-ir cells (white arrowheads) in the ectoneural epithelium and Ast-ir processes (yellow arrow) and AstR processes (white arrow) in the ectoneural neuropile. b Marginal nerve; AstR-ir cells (white arrowhead) in the epithelial layer and both Ast-ir processes (yellow arrow) and AstR-ir processes (white arrow) in the underlying neuropile. c Disc region of a tube foot; Ast-ir processes (yellow arrow) and AstR-ir processes (white arrow) in the basal nerve ring. d Cardiac stomach; Ast-ir processes (white arrowhead) and AstR-ir processes (blue arrow) in the basiepithelial nerve plexus and mucosa, while in the visceral muscle layer, only AstR-ir is present (white arrow). Abbreviations: BNR, basal nerve ring; BNP, basiepithelial nerve plexus; CS, cardiac stomach; CT, collagenous tissue; Ec, ectoneural; Lu, lumen; ML, mucosal layer; TF, tube foot; VML, visceral muscle layer. Scale bars: d = 30 μm; c = 20 μm; a, b = 10 μm
	Fig. 7. Asterotocin causes relaxation of in vitro cardiac stomach and apical muscle preparations from A. rubens. a Representative recording showing that asterotocin causes relaxation of a cardiac stomach preparation. Seawater supplemented with KCl (3 × 10−2 M) was used to induce contraction of the cardiac stomach prior to the application of asterotocin. The relaxing effect of asterotocin is reversed when the preparation is washed with KCl-supplemented seawater. b Graph showing the concentration-dependent relaxing effect of asterotocin on cardiac stomach preparations at concentrations ranging from 3 × 10−11 M to 10−6 M. The responses are expressed as the mean relative percentage (± SEM; n = 16) of the maximal relaxing effect of asterotocin in each preparation. c Representative recording from a cardiac stomach preparation that compares the relaxing effects of asterotocin and the SALMFamide-type neuropeptide S2, both at 10−7 M. As in a, the cardiac stomach preparation was pre-contracted with KCl-supplemented seawater prior to the application of the neuropeptides. Inset compares the effects of asterotocin and S2 on cardiac stomach preparations at a concentration of 10−7 M, expressed as mean percentages (± SEM; n = 5) with the relaxing effect of S2 defined as 100%. The relaxing effect of asterotocin is significantly larger than the effect S2 (Mann-Whitney U test; P = 0.0079; n = 5). d Representative recording showing that asterotocin (10−6 M) causes relaxation of an apical muscle preparation. 10−6 M acetylcholine (ACh) was used to induce contraction of the apical muscle preparation prior to the application of asterotocin. Following washing of the preparation with artificial seawater, it returns to its basal relaxed state. Inset shows the mean percentage (± SEM; n = 4) reversal of 10−6 M ACh-induced contraction of apical muscle preparations caused by 10−6 M asterotocin over a 50-s period after peptide administration
	Fig. 8. In vivo injection of asterotocin triggers cardiac stomach eversion in A. rubens. a Dose-dependent effect of asterotocin in inducing cardiac stomach eversion. The graph shows the percentage of animals (n = 5 per group) that exhibit cardiac stomach eversion when injected with 10 μl asterotocin at concentrations ranging from 10−6 M to 10−3 M, by comparison with 10 μl of the starfish neuropeptide S2 (10−3 M) or 10 μl of water. Stomach eversion occurred in 100%, 100% and 80% of the animals injected with 10−5 M, 10−4 M and 10−3 M asterotocin, respectively, but stomach eversion was not observed in any of the animals injected with 10−6 M asterotocin, 10−3 M S2 or water. b Temporal dynamics of asterotocin-induced (10 μl of 10−3 M) cardiac stomach eversion. The graph shows mean area (± SEM; n = 13) of the cardiac stomach everted expressed as the percentage of the area of the central disc region at 30-s intervals over a 10-min period following injection of asterotocin. c Images from video recordings of the experiment in b showing a representative water-injected (control) starfish (i–iii) and a representative asterotocin-injected starfish (iv–vi) at 0 min (immediately after injection), after 5 min and after 10 min. The area of the cardiac stomach everted is shown with a dashed line in v and vi. The representative video recordings used to generate the images in c are in Additional file 7

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