Aleotti A et al. (2022), Discovery and functional characterization of ne...

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Front Neurosci 2022 Jan 01;16:1006594. doi: 10.3389/fnins.2022.1006594.

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Discovery and functional characterization of neuropeptides in crinoid echinoderms.

Aleotti A , Wilkie IC , Yañez-Guerra LA , Gattoni G , Rahman TA , Wademan RF , Ahmad Z , Ivanova DA , Semmens DC , Delroisse J , Cai W , Odekunle E , Egertová M , Ferrario C , Sugni M , Bonasoro F , Elphick MR .

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Neuropeptides are one of the largest and most diverse families of signaling molecules in animals and, accordingly, they regulate many physiological processes and behaviors. Genome and transcriptome sequencing has enabled the identification of genes encoding neuropeptide precursor proteins in species from a growing variety of taxa, including bilaterian and non-bilaterian animals. Of particular interest are deuterostome invertebrates such as the phylum Echinodermata, which occupies a phylogenetic position that has facilitated reconstruction of the evolution of neuropeptide signaling systems in Bilateria. However, our knowledge of neuropeptide signaling in echinoderms is largely based on bioinformatic and experimental analysis of eleutherozoans-Asterozoa (starfish and brittle stars) and Echinozoa (sea urchins and sea cucumbers). Little is known about neuropeptide signaling in crinoids (feather stars and sea lilies), which are a sister clade to the Eleutherozoa. Therefore, we have analyzed transcriptome/genome sequence data from three feather star species, Anneissia japonica, Antedon mediterranea, and Florometra serratissima, to produce the first comprehensive identification of neuropeptide precursors in crinoids. These include representatives of bilaterian neuropeptide precursor families and several predicted crinoid neuropeptide precursors. Using A. mediterranea as an experimental model, we have investigated the expression of selected neuropeptides in larvae (doliolaria), post-metamorphic pentacrinoids and adults, providing new insights into the cellular architecture of crinoid nervous systems. Thus, using mRNA in situ hybridization F-type SALMFamide precursor transcripts were revealed in a previously undescribed population of peptidergic cells located dorso-laterally in doliolaria. Furthermore, using immunohistochemistry a calcitonin-type neuropeptide was revealed in the aboral nerve center, circumoral nerve ring and oral tube feet in pentacrinoids and in the ectoneural and entoneural compartments of the nervous system in adults. Moreover, functional analysis of a vasopressin/oxytocin-type neuropeptide (crinotocin), which is expressed in the brachial nerve of the arms in A. mediterranea, revealed that this peptide causes a dose-dependent change in the mechanical behavior of arm preparations in vitro-the first reported biological action of a neuropeptide in a crinoid. In conclusion, our findings provide new perspectives on neuropeptide signaling in echinoderms and the foundations for further exploration of neuropeptide expression/function in crinoids as a sister clade to eleutherozoan echinoderms.

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	FIGURE 1. Anatomy of the whole-body and arms of adult Antedon mediterranea and experimental set-up used to investigate effects of neuropeptides on the mechanical behavior of arm preparations. (A) Photograph of a whole adult specimen of A. mediterranea in an 18 cm diameter petri dish. The rectangle shows the region of an arm nearest to the calyx, which corresponds to the region from which experimental preparations were dissected. (B) Photograph of lateral view of an arm, oral side up and proximal end to the right. The scale bar is 1 mm. d, diarthrosis; s, syzygy; p, pinnule. (C) A schematic drawing of a sagittal section of an arm equivalent to the portion shown in panel (B). Major anatomical structures are labeled as follows: bn, brachial nerve; c, coelom; d, diarthrosis; m, muscle; o, ossicle; s, syzygy. (D) Schematic drawing of the experimental set-up for testing the effects of neuropeptides on the mechanical properties of arm preparations. The arm preparation (1) is orientated with its oral side up, as is the natural position of the arm, and is clasped at its proximal end by a spring-loaded clamp (2) and connected at its distal end via a heart clip to a chain (3). The exact positioning of the arm preparation in relation to the apparatus can be regulated by a system of rods that are adjusted using clamp manipulators (4) and scales (5). The chain is connected to an isotonic lever transducer (6) and at the opposite end of the lever is a weight of 10 g (7), which creates a tension in the arm preparation similar to that in an arm outstretched for food collection in the animal’s natural environment (La Touche, 1978; Byrne and Fontaine, 1981). Test substances (e.g., neuropeptides) are administered to the preparation using a syringe (8). Changes in the mechanical behavior of the arm preparation are detected by the transducer and relayed via a bridge amplifier (9) to acquisition hardware (10) and then finally to a computer (11), where recordings are made and stored using LabChart software. The inset shows a close-up of an arm preparation positioned in the apparatus and a schematic representation of arm extension after application of a test substance. (E) Schematic representation of the method used to quantify effects of test substances on arm extension. The basal rate of extension is measured during the 1 min prior to application of the test substance (E1). After application of the test substance, recording is continued for a period of 5 min and the extension observed in this testing period is E2. The average extension rates (ER: mm min–1) in the 1 min before and 5 min after application are then calculated, and the effect of the test substance is quantified as ER2 – ER1.
	FIGURE 2. Sequences of crinoid proteins that are precursors of monomeric neuropeptides. Neuropeptide precursor sequences are shown in alphabetical order and are from Antedon mediterranea (Amed), with the exception of pedal peptide/orcokinin-type precursor which was not identified in this species but which was identified in Anneissia japonica (Anjap). The predicted signal peptide is shown in blue, predicted monobasic/dibasic cleavage sites are shown in green and predicted neuropeptides are shown in red, but with C-terminal glycine residues that are potential substrates for post-translational amidation shown in orange. The underlined cysteine residues have been shown to form intramolecular disulphide bridges in homologous monomeric neuropeptides in other taxa. The neurophysin domain of the vasopressin/oxytocin-type (crinotocin) precursor is shown in purple. The DNA sequences of transcripts encoding these precursors are shown in Supplementary File 2.
	FIGURE 3. Sequences of crinoid proteins that are precursors of heterodimeric neuropeptides. Neuropeptide precursor sequences are shown in alphabetical order and are from Antedon mediterranea (Amed), with the exception of relaxin-type precursor which was not identified in this species but which was identified in Anneissia japonica (Anjap). The predicted signal peptide is shown in blue, predicted monobasic/dibasic cleavage sites are shown in green and predicted neuropeptides are shown in red. The underlined cysteine residues have been shown to form intramolecular or intermolecular disulphide bridges in homologous heterodimeric neuropeptides in other taxa. The DNA sequences of transcripts encoding these precursors are shown in Supplementary File 2.
	FIGURE 4. Summary showing in which species the monomeric and heterodimeric neuropeptide precursors have been identified in this study. Filled circles represent cases where neuropeptide precursors were identified in a given species, whereas empty circles represent cases in which the precursors were not identified. Where neuropeptide precursors were not identified, this may reflect incomplete transcriptome/genome sequence data or loss in one or more species. The DNA sequences of transcripts encoding these precursors are shown in Supplementary File 2.
	FIGURE 5. Alignments of predicted crinoid monomeric neuropeptides with homologous neuropeptides in eleutherozoan echinoderms. Neuropeptide sequences from all three crinoid species are shown, where known, and aligned with the sequences of homologous neuropeptides, where known, from two asterozoan species (starfish and brittle star) and two echinozoan species (sea urchin and sea cucumber). Species name abbreviations: Amed, Antedon mediterranea; Anjap, Anneissia japonica; Fser, Florometra serratissima; Arub, Asterias rubens (starfish); Afil, Amphiura filiformis (brittle star); Spur, Strongylocentrotus purpuratus (sea urchin) and Ajap, Apostichopus japonicus (sea cucumber). Alignments were performed using MAFFT (v7.470) with default parameters and conserved amino acids were highlighted using a threshold fraction of conserved sequences of 0.60 with Boxshade (https://manpages.ubuntu.com/manpages/bionic/man1/boxshade.1.html).
	FIGURE 6. Crinoid F-type SALMFamide precursors and comparison of gene structure with eleutherozoan F-type SALMFamide precursor genes. (A) Sequences of F-type SALMFamide precursors in three crinoid species, Antedon mediterranea (Amed), Florometra serratissima (Fser), and Anneissia japonica (Anjap). The predicted signal peptide is shown in blue, predicted monobasic/dibasic cleavage sites are shown in green and predicted neuropeptides are shown in red, but with C-terminal glycine residues that are potential substrates for post-translational amidation shown in orange. (B) Structure of the F-type SALMFamide precursor gene in the crinoid Anneissia japonica (Anjap) compared with the structures of F-type SALMFamide precursor genes in eleutherozoans, including the starfish Asterias rubens (Arub), the sea urchin Strongylocentrotus purpuratus (Spur), and the sea cucumber Apostichopus japonicus (Ajap). In all four species there are two phase 0 introns, with one preceding the start codon encoding the first methionine of the signal peptide and another located between an exon encoding the signal peptide and an exon or exons encoding the SALMFamide neuropeptides, which provides additional evidence that these genes are orthologs. The A. japonicus gene also has a second intron in phase 1 that interrupts the coding sequence for the second predicted SALMFamide neuropeptide.
	FIGURE 7. Crinoid L-type SALMFamide precursors and comparison of gene structure with eleutherozoan L-type SALMFamide precursor genes. (A) Sequences of L-type SALMFamide precursors in three crinoid species, Antedon mediterranea (Amed), Florometra serratissima (Fser), and Anneissia japonica (Anjap). The predicted signal peptide is shown in blue, predicted dibasic cleavage sites are shown in green and predicted neuropeptides are shown in red, but with C-terminal glycine residues that are potential substrates for post-translational amidation shown in orange. (B) Structure of the L-type SALMFamide precursor gene in the crinoid Anneissia japonica (Anjap) compared with the structures of L-type SALMFamide precursor genes in eleutherozoans, including the starfish Asterias rubens (Arub), the sea urchin Strongylocentrotus purpuratus (Spur), and the sea cucumber Apostichopus japonicus (Ajap). In all four species there is phase 0 intron located between an exon encoding the signal peptide and an exon or exons encoding the SALMFamide neuropeptides, which provides additional evidence that these genes are orthologs. The A. japonica gene has a second phase 0 intron preceding the start codon encoding the first methionine of the signal peptide and the A. japonicus gene also has a second phase 0 intron that interrupts the coding sequence for the second predicted SALMFamide neuropeptide.
	FIGURE 8. Other predicted crinoid neuropeptide precursors (PCNP1-10). Neuropeptide precursor sequences from Antedon mediterranea (Amed) are shown, with the exception of those precursors that were partially or not identified in this species and where sequences from Anneissia japonica (Anjap) or Florometra serratissima (Fser) are shown. The predicted signal peptide is shown in blue, predicted monobasic/dibasic cleavage sites are shown in green and predicted neuropeptides are shown in red, but with C-terminal glycine residues that are potential substrates for post-translational amidation shown in orange. The DNA sequences of transcripts encoding these precursors are shown in Supplementary File 2.
	FIGURE 9. Other predicted crinoid neuropeptide precursors (PCNP11-20). Neuropeptide precursor sequences from Antedon mediterranea (Amed) are shown, with the exception of those precursors that were partially or not identified in this species and where sequences from Anneissia japonica (Anjap) or Florometra serratissima (Fser) are shown. The predicted signal peptide is shown in blue, predicted monobasic/dibasic cleavage sites are shown in green and predicted neuropeptides are shown in red, but with C-terminal glycine residues that are potential substrates for post-translational amidation shown in orange. The DNA sequences of transcripts encoding these precursors are shown in Supplementary File 2.
	FIGURE 10. Summary showing the species in which the sequences of other predicted crinoid neuropeptide precursors (PCNP) have been identified in this study. Filled circles represent cases where neuropeptide precursors were identified in a given species, whereas empty circles represent cases in which the precursors were not identified. The DNA sequences of transcripts encoding these precursors are shown in Supplementary File 2. Multiple sequence alignments of PCNP sequences from the three crinoid species are shown in Supplementary File 3.
	FIGURE 11. F-type SALMFamide precursor expression across different life stages of Antedon mediterranea revealed using mRNA in situ hybridization. (A–C) Doliolaria larva from dorsal (A), ventral (B), and lateral (C) views, showing strong staining in two symmetric dorso-lateral clusters of cells located between the second and third ciliary bands (asterisks) and weak staining in sparsely distributed cells located in an anterior-ventral position (arrowheads in panel B). (D–H) A pentacrinoid juvenile (D), showing stained cells in both the calyx and stalk. High magnification images of the boxed regions in (D) show stained cells in mouth (E), stalk nerve (F) and aboral nerve center (G,H). (I–K) Longitudinal (I,J) and transverse (K) sections of arms from adult animals, revealing stained cells in the brachial nerve (I), branches of the brachial nerve that project into the pinnules (J) and flexor muscles (K). Scale bars: 150 μm for (A–D,I); 75 μm for (F,G,J,K); 25 μm for (E,H). A, anterior side; Ab, aboral side; ad, adhesive pit; bn, brachial nerve; c, calyx; D, dorsal side; m, muscle; in, intestine; Or, oral side; P, posterior side; p, pinnule; s, saccules, sn, stalk nerve; st, stalk; tf, tube feet; V, ventral side; v, vestibulum.
	FIGURE 12. Localization of calcitonin-type neuropeptide expression in post-metamorphic stages of Antedon mediterranea. (A–Di) Immunofluorescent labeling of calcitonin-type neuropeptide expression in the pentacrinoid stage using an antiserum to the A. rubens calcitonin-type neuropeptide ArCT. Immunolabeling is observed in the calyx, stalk, and tube feet (A). High magnification images show five tracts containing strongly labeled fibers in the stalk nerve (B,Bi), and fibers and cell bodies (arrowheads) in the aboral nerve center (B,Bi), from which five immunolabeled brachial nerve primordia project orally (A,B) to the circumoral nerve ring [(A), resliced for oral view in panel (C,Ci)]. Labeled fibers in the tube feet (D,Di) appear to originate in the circumoral nerve ring [white arrow in panel (D)]. (E,F) Localization of calcitonin-type neuropeptide expression in sections of the adult calyx (E,F) and arm (G,H) as revealed by immunohistochemistry using an antiserum to the A. rubens neuropeptide ArCT (E–H) and by mRNA in situ hybridization using probes for A. mediterranea calcitonin-type precursor (AmCT) transcripts (I,J). In the calyx, immunostaining is located at the base of the mucosa of the esophagus (E), where high magnification images show staining located in cell bodies and fibers (F). In a sagittal section of an arm (oral side uppermost), immunostained fibers can be seen running longitudinally in the brachial nerve (G). In a transverse section of an arm (oral side uppermost), immunostained cells/fibers can be seen in the ectoneural region of the nervous system at the base of the ambulacral groove (H) and in fibers located aboro-laterally to the ambulacral groove (black arrow in panel H), which may be derived from hyponeural neurons. Consistent with immunohistochemical analysis of calcitonin-type neuropeptide expression, mRNA in situ hybridization reveals AmCT precursor transcripts in cells located in the brachial nerve (I) (oral side uppermost) and at the base of the ectoneural region of the ambulacral groove (J) (oral side uppermost). Scale bar is 100 μm for (A,E,G,I); 75 μm for (B–Di,H,J); 25 μm for (F). Ab, aboral side; ag, ambulacral groove; anc, aboral nerve center; bn, brachial nerve; conr, circumoral nerve ring; in, intestine; bnp, brachial nerve primordia; Or, oral side; rp, radial plates; s, saccules; sn, stalk nerve; tf, tube foot.
	FIGURE 13. Crinotocin precursor expression in Antedon mediterranea and in vitro effects of crinotocin on arm preparations. (A–C) Arm sections of A. mediterranea labeled with antisense probes for crinotocin precursor transcripts using mRNA in situ hybridization. bn, brachial nerve; l, ligament; m, muscle; p, pinnule; o, ossicle. s, syzygy; Scale bars: 120 μm for (A,B); 210 μm for (C). (A) Transverse section of the arm, with the square showing the region containing the brachial nerve, which is shown at higher magnification in panel (B). (B) High magnification image of the brachial nerve, showing the presence of crinotocin precursor-expressing cells in the core and margins of the nerve. (C) Sagittal section of an arm, showing labeled cells in the brachial nerve along its length and in branches of the brachial nerve that innervate ligaments and muscles (arrow). (D) Effects of crinotocin (10–5 M) on arm preparations, with five tests on different preparations from two representative animals shown. After addition of crinotocin (arrow), recording was continued for several minutes. Variability in responses both within and between animals is observed. For example, with animal 11 crinotocin caused a gradual and small flexion in some arm preparations, whereas in most animals (e.g., animal 15) crinotocin caused an abrupt and large flexion in most arm preparations. (E) Concentration-dependent effect of crinotocin on mean arm flexion rate. Five different concentrations were tested (10–4 M, 10–5 M, 10–6 M, 10–7 M, 10–8 M), and an example test per concentration for a representative animal is shown. Responses are greatest at the highest concentrations tested (10–4 M and 10–5 M), and visibly decrease at intermediate concentrations until little or no visible effect is observed at the lowest concentrations tested (10–7 M and 10–8 M). (F) Concentration-response curve for the effect of crinotocin on arm preparations. Data from 143 arm preparations from four animals were used to generate this concentration-response curve, but one data point for the 10–4 M concentration was excluded as an outlier. See Supplementary File 5. Error bars represent standard error of the mean (SEM). The graph was generated using GraphPad Prism. (G) Anesthetization of arm preparations blocks the response of arms to crinotocin. ASW: pretreated in ASW alone then treated with ASW alone; CRIN: pretreated in ASW alone then treated with 10–5 M crinotocin; CRIN+PRO: anesthetized in 10–3 M procaine hydrochloride then treated with 10–5 M crinotocin in anesthetic solution. Numbers of preparations are shown in parentheses. Error bars represent SEM. The graph was generated using GraphPad Prism.
	FIGURE 14. Summary of neuropeptide expression in Antedon mediterranea. The distribution of cells expressing F-type SALMFamide (purple, mRNA in situ); calcitonin-type neuropeptide (green, immunohistochemistry and mRNA in situ); and crinotocin (cyan, mRNA in situ) is summarized here for doliolaria larva, pentacrinoid and adult stages and related to the major compartments of the nervous system (blue). ag, ambulacral groove; anc, aboral nerve center; ao, apical organ; bn, brachial nerve; bnp, brachial nerve primordia; cc, coelomic canal; conr, circumoral nerve ring; hns, hyponeural system i, intestine; m, mouth; mu, muscles; sn, stalk nerve; tf, tube feet.

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