ECB-ART-51049
Cell Tissue Res
2023 Mar 01;3913:441-456. doi: 10.1007/s00441-023-03738-w.
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Immunohistochemical localisation of vasopressin/oxytocin-type, corazonin-type and luqin-type neuropeptide expression in the starfish Asterias rubens using antibodies to the C-terminal region of precursor proteins.
Tinoco AB
,
Egertová M
,
Elphick MR
.
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Neuropeptides derived from larger precursor proteins are secreted as signalling molecules by neurons and regulate diverse physiological and behavioural processes in animals. Recently, we reported the discovery of ArCRZ (HNTFTMGGQNRWKAG-NH2) and ArLQ (EEKTRFPKFMRW-NH2)-novel neuropeptides in the starfish Asterias rubens that are orthologs of arthropod corazonins and molluscan luqins, respectively. However, our efforts to generate antibodies to ArCRZ and ArLQ have been unsuccessful, precluding immunohistochemical analysis of their expression. Here, we investigated an alternative experimental approach for neuropeptide immunohistochemistry by generating antibodies to peptides corresponding to the C-terminal region of the precursor proteins. As proof of principle, we generated antibodies to the C-terminal region of the precursor of the vasopressin/oxytocin-type neuropeptide asterotocin and show that these reveal immunostaining in A. rubens that is very similar to that observed with asterotocin antibodies. Furthermore, antibodies to the C-terminal region of the ArCRZ precursor (ArCRZP) and the ArLQ precursor (ArLQP) produced patterns of immunostaining consistent, respectively, with the distribution of ArCRZP and ArLQP transcripts revealed by mRNA in situ hybridisation. Detailed immunohistochemical analysis revealed widespread expression of ArCRZP and ArLQP in A. rubens, including the central nervous system, digestive system and the body wall and its associated appendages (e.g. tube feet), providing a neuroanatomical framework for investigation and interpretation of the pharmacological actions of ArCRZ and ArLQ in A. rubens. Furthermore, our findings provide a basis for use of antibodies to the C-terminal region of neuropeptide precursor proteins in other species where the production of antibodies to the bioactive neuropeptides is unsuccessful.
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Fig. 1. Amino acid sequences of a vasopressin/oxytocin-type (asterotocin) precursor (ArASTP), b corazonin-type precursor (ArCRZP) and c luqin-type precursor (ArLQP) in Asterias rubens. Predicted signal peptides are shown in blue, neuropeptides are shown in red but with C-terminal glycine residues that are substrates for amidation shown in orange, dibasic cleavage sites are shown in green and the neurophysin domain of ArASTP is shown in pink. The cysteine residues in ArASTP that form a disulphide bridge in the mature neuropeptide are underlined. The sequences of the C-terminal peptides that were used as antigens for antibody production are shown in bold yellow. Note, however, that the underlined cysteine residue in ArLQP was replaced with a lysine residue at the N-terminus of the antigen peptide to provide reactive sites for glutaraldehyde-mediated coupling to a carrier protein (thyroglobulin). GenBank Accession numbers: a ALJ99953.1 (Semmens et al. 2016; Odekunle et al. 2019); b ALJ99955.1 (Semmens et al. 2016; Tian et al. 2017, 2016); c ALJ99961.1 (Semmens et al. 2016; Yañez-Guerra et al. 2018) |
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Fig. 2. Graphical representation of starfish anatomy showing a vertical section of the central disk and the proximal region of an adjoining arm. Colour key: body wall skeleton, green; digestive system, orange; hemal system, brown; muscles, purple; nervous system, pink; perihemal system, yellow; reproductive system, grey; water vascular system, blue. Abbreviations: a, anus; amp, ampulla; am, apical muscle; cs, cardiac stomach; conr, circumoral nerve ring; gcc, general coelomic cavity; gon, gonad; m, mouth; ma, madreporite; oa, organ axial; os, ossicle; pa, papullae; pm, peristomial membrane; pc, pyloric caecum; pd, pyloric duct; ps, pyloric stomach; rc, rectal caecum; rnc, radial nerve cord; rw, radial water vascular canal; sa, sinus of axial organ; sc, stone canal; tm, tourniquet muscle; tb, Tiedemann’s body; tf, tube foot. Diagram was modified from Yañez-Guerra et al. (2018) |
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Fig. 3. Characterisation of rabbit antibodies to ArASTP, ArCRZP and ArLQP using an enzyme-linked immunosorbent assay (ELISA). a 0.1 nmol of the ArASTP antigen peptide is detected by the ArASTP antiserum (black line) at dilutions between 1:500 and 1:16,000, with the presence and titre of antibodies to the ArASTP antigen peptide being inferred by comparison with pre-immune serum (grey line). b 0.1 nmol of the ArCRZP antigen peptide is detected by the ArCRZP antiserum (black line) at dilutions between 1:500 and 1:16,000, with the presence and titre of antibodies to the ArCRZP antigen peptide being inferred by comparison with pre-immune serum (grey line). c 0.1 nmol of the ArLQP antigen peptide is detected by the ArLQP antiserum (black line) at dilutions between 1:500 and 1:16,000, with the presence and titre of antibodies to the ArLQP antigen peptide being inferred by comparison with pre-immune serum (grey line). All data points are mean values from three replicates |
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Fig. 4. Immunostaining in the radial nerve cord of A. rubens observed with neuropeptide precursor antisera is abolished by pre-absorption of antisera with antigen peptides. a ArASTP antiserum, b ArASTP antiserum pre-absorbed with antigen peptide, c ArACRZP antiserum, d ArCRZP antiserum pre-absorbed with antigen peptide, e ArLQP antiserum, f ArLQP antiserum pre-absorbed with antigen peptide. Immunostaining can be seen in transverse sections of the V-shaped radial nerve cords, with regions of the ectoneural neuropile containing a high density of immunostained fibres (white asterisks in a, c, e), while these (grey asterisks) and other regions of the neuropile are void of staining in adjacent sections in b, d, f. Selected immunostained cell bodies are labelled with black arrowheads in a, c and e, whilst in adjacent sections, there is an absence of immunostained cell bodies in these (grey arrowheads) and other regions of the ectoneural epithelium in b, d, f. Abbreviations: rhs, radial hemal strand; rnc, radial nerve cord; tf, tube foot. Scale bars: a—f = 25 μm |
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Fig. 5. Comparative immunohistochemical analysis of vasopressin/oxytocin-type neuropeptide expression in the starfish Asterias rubens using antibodies to asterotocin (ArAST) and antibodies to the C-terminal region of the asterotocin precursor (ArASTP). In different regions of the starfish body shown here, similar patterns of immunostaining are observed in adjacent sections labelled with ArAST antibodies or ArASTP antibodies. a, b Radial nerve cord (V-shaped in transverse section); the patterns of immunostaining are similar, with the exception of the apical region of the nerve cord, which exhibits ArASTP-immunoreactivity (ArASTP-ir; *) but is largely void of ArAST-immunoreactivity (ArAST-ir). c, d Circumoral nerve ring; immunostained cells can be seen in the epithelium of the ectoneural region (arrowheads) and a region of the ectoneural neuropile containing a higher density of immunostained fibres is highlighted with a white asterisk. e, f Marginal nerve and adjacent tube foot; ArASTP-ir, but not ArAST-ir, is revealed in the subepithelial nerve plexus of the tube foot (arrowhead). g, h Disk region of a tube foot, showing ArAST-ir and ArASTP-ir in the basal nerve ring (white arrowheads), whereas in the subepithelial nerve plexus only ArASTP-ir is detected (grey arrowhead). i, j Lateral pouches of the cardiac stomach, with insets showing boxed regions at higher magnification. Immunostaining is observed in cells located in the mucosal epithelium (black arrowheads) and in the basiepithelial nerve plexus (white arrowheads). k, l Intestine. Immunostaining is present in the basiepithelial nerve plexus of the intestine (white arrowheads), but the rectal caecum is void of immunostaining. m, n Apical muscle and pyloric stomach. Immunostaining is present in the basiepithelial nerve plexus located beneath the coelomic epithelium of the apical muscle (white arrowheads) and in the basiepithelial nerve plexus of the pyloric stomach (grey arrowheads). o, p Body wall. Immunostaining is present in cells located in the external epithelium (black arrowheads) and in the subepithelial nerve plexus (white arrowhead). Abbreviations: am, apical muscle; conr, circumoral nerve ring; Ect, ectoneural; Hyp, hyponeural; int, intestine; mn, marginal nerve; pm, peristomial membrane; ps, pyloric stomach; rc, rectal caecum; rhs, radial hemal strand; rnc, radial nerve cord; tf, tube foot. Scale bars: i, j = 60 μm; a, b, c, d, g, h, k, l, m, n, o, p = 32 μm; e, f, i-inset, j-inset = 16 μm |
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Fig. 6. Imunohistochemical localisation of ArCRZP in Asterias rubens. a ArCRZP-immunoreactivity (ArCRZP-ir) in a transverse section of the V-shaped radial nerve cord, with bilaterally symmetrical regional variation in the density of immunostaining in the ectoneural neuropile. The boxed regions are at higher magnification in the insets, with regions of the ectoneural neuropile containing a high density of immunostained fibres labelled with asterisks. Immunostained cell bodies can be seen in the ectoneural epithelium (black arrowheads) and punctate immunostained fibres can be seen projecting across the ectoneural neuropile in different orientations (grey arrowheads). Immunostaining can also be seen in a lateral nerve emanating from the radial nerve cord and in the subepithelial nerve plexus of an adjacent tube foot. b ArCRZP-ir in the circumoral nerve ring, showing regional variation in the density of immunostaining in the ectoneural neuropile; the boxed regions are at higher magnification in the insets, showing immunostained cells in the ectoneural epithelium (black arrowheads) and in the external epithelial layer of the peristomial membrane (white arrowhead) near to the junction with the circumoral nerve ring. c High magnification image of the junction between the radial nerve cord and an adjacent tube foot, showing ArCRZP-ir in the ectoneural neuropile of the radial nerve cord and in the subepithelial nerve plexus of the adjacent tube foot. d ArCRZP-ir in the marginal nerve. e ArCRZP-ir in the basal nerve ring (black arrowheads) of a tube foot. f Horizontal section of an arm tip showing ArCRZP-ir in the terminal tentacle (black arrowhead), lateral lappet (white arrowhead) and subepithelial nerve plexus of the body wall surrounding the terminal tentacle (grey arrowhead). g Horizontal section of an arm tip showing ArCRZP-ir in bipolar-shaped cells (black arrowheads) located in the photosensory epithelium and in the underlying neuropile (*) of the optic cushion. h High magnification transverse section of the oesophagus showing an immunostained cell (black arrowhead) in the mucosal layer and immunostained processes (grey arrowhead) in the basiepithelial nerve plexus. i ArCRZP-ir in the lateral pouches of the cardiac stomach; note that the density of immunostained fibres is highest (grey arrowheads) in regions of the basiepithelial nerve plexus adjacent to the retractor strand and nodule, which are unstained. j, k High magnification images of cardiac stomach tissue showing ArCRZP-ir in cell bodies (black arrowheads) and in the basiepithelial nerve plexus (grey arrowheads). l ArCRZP-ir in the pyloric stomach showing immunostaining in cells (black arrowheads) of the mucosa layer and in the basiepithelial nerve plexus (grey arrowhead). m Longitudinal section of an arm showing ArCRZP-ir in the basiepithelial nerve plexus of a pyloric duct (grey arrowheads). n ArCRZP-ir in a cell body (black arrowhead) and in the basiepithelial nerve plexus (grey arrowheads) of a pyloric caecum. o ArCRZP-ir in the intestine, with immunostaining located in cells of the mucosal layer (black arrowhead) and in the basiepithelial nerve plexus (grey arrowheads). p Immunostained processes (grey arrowheads) in a Tiedemann’s body, which is a specialised organ involved in the regulation of the composition of the coelomic fluid. q ArCRZP-ir located beneath the coelomic epithelium of an apical muscle (white arrowheads) and in the circular muscle layer of the coelomic lining of the body wall (grey arrowheads). r, s ArCRZP-ir in the subepithelial nerve plexus of the external epithelium of the body wall (grey arrowheads) on both aboral (r) and oral (s) sides of the body. On the oral side, the stained cells (black arrowheads) can be seen to be located between the spines of the body wall. On the aboral side of the body wall, the external epithelium is naturally pigmented, and this is labelled with an asterisk to highlight that this is not ArCRZP-ir. Abbreviations: am, apical muscle; bnp, basiepithelial nerve plexus; bw, body wall; conr, circumoral nerve ring; cs, cardiac stomach; Ect, ectoneural; Hyp, hyponeural; int, intestine; ln, lateral nerve; ll, lateral lappet; lu, lumen; mn, marginal nerve; mu, mucosa; no, nodule; oc, optic cushion; ohr, oral hemal ring; pd, pyloric duct; pm, peristomial membrane; ps, pyloric stomach; rhs, radial hemal strand; rnc, radial nerve cord; tb, Tiedeman’s body; tf, tube foot; tt, terminal tentacle. Scale bars: a = 120 μm; b = 60 μm; d, e, f, g, i, l, m, n, o, q, r, s = 32 μm; a-insets, c, h, j, k, p = 16 μm; b-insets = 6 μm |
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Fig. 7. Immunohistochemical localisation of ArLQP in Asterias rubens. a ArLQP-immunoreactivity (ArLQP-ir) in a transverse section of the V-shaped radial nerve cord, with bilaterally symmetrical regional variation in the density of immunostaining in the ectoneural neuropile. The boxed region is shown at higher magnification in the inset, showing stained cell bodies in the hyponeural (grey arrowhead) and ectoneural (black arrowheads) regions of the radial nerve cord. b High magnification image of a longitudinal section of radial nerve cord showing an immunostained cell with a stained process in the hyponeural region (grey arrowhead) and an immunostained cell with a stained process in the ectoneural region (black arrowhead). Immunostained fibres can also be seen throughout the ectoneural neuropile. c ArLQP-ir in the circumoral nerve ring, showing regional variation in the intensity of immunostaining in the ectoneural neuropile and with a strongly stained region highlighted with an asterisk; the boxed regions are shown at higher magnification in the insets, where immunostained cells can be seen in the hyponeural region (grey arrowhead) and the ectoneural region (black arrowhead). d High magnification image of the junction between the radial nerve cord and an adjacent tube foot, showing ArLQP-ir in the ectoneural neuropile of the radial nerve cord and in the subepithelial nerve plexus of the adjacent tube foot. e ArLQP-ir in the marginal nerve and the subepithelial nerve plexus of an adjacent tube foot. f ArLQP-ir in the subepithelial nerve plexus (grey arrowheads) and basal nerve ring (black arrowheads) of a tube foot. g Horizontal section of an arm tip showing ArLQP-ir in cells/fibres located in the terminal tentacle (black arrowheads), a lateral lappet (white arrowheads) and the body wall surrounding the terminal tentacle (grey arrowheads). h Horizontal section of an arm tip showing ArLQP-ir in bipolar-shaped cells (black arrowheads) located in the photosensory epithelium and in the underlying neuropile (*) of the optic cushion. Immunostaining in adjacent tube feet can also be seen here. i Horizontal section of the central disc showing ArLQP-ir in the peristomial membrane and oesophagus. Immunostaining can be seen in the subepithelial nerve plexus of the peristomial membrane (grey arrowhead) and the oesophagus (white arrowhead). Immunostained cells can also be seen in the oesophagus (black arrowhead) and the peristomial membrane (boxed region). The inset shows the boxed region at higher magnification, with the cell body and process of an immunostained cell labelled with black and grey arrowheads, respectively. j ArLQP-ir in the lateral pouches of the cardiac stomach; note that the density of immunostained fibres is highest (grey arrowheads) in regions of the basiepithelial nerve plexus adjacent to the retractor strand and nodule, which are unstained. Immunostained bipolar-shaped cells (black arrowheads) can be seen in the mucosal layer of the cardiac stomach. k, l High magnification images of cardiac stomach tissue showing ArLQP-ir in cell bodies (black arrowheads) and processes in the basiepithelial nerve plexus (grey arrowheads); note that in l a process emanating from an immunostained cell body can be seen projecting into the plexus (white arrowhead). m ArLQP-ir in the pyloric stomach showing immunostaining in cells (black arrowheads) of the mucosa layer and processes in the basiepithelial nerve plexus (grey arrowhead). n ArLQP-ir in bipolar-shaped cells (black arrowheads) and in the basiepithelial nerve plexus (grey arrowheads) of the pyloric duct; intense immunostaining of the basiepithelial nerve plexus of the pyloric stomach can also be seen here (white arrowhead). o Horizontal section of an arm showing ArLQP-ir in the pyloric duct (white arrowheads) and in cells (black arrowheads) and the basiepithelial nerve plexus (grey arrowheads) of the pyloric caeca. p ArLQP-ir in the intestine, with immunostaining located in the basiepithelial nerve plexus (grey arrowheads). q ArLQP-ir in a rectal caecum, with an immunostained cell (black arrowhead) and a process emanating from it (grey arrowhead) highlighted. r ArLQP-ir in the basiepithelial nerve plexus (grey arrowhead) underlying the external epithelium of the body wall. A region of the external epithelium which is naturally pigmented and this is labelled with an asterisk to highlight that this is not ArLQP-ir. Abbreviations: bnp, basiepithelial nerve plexus; conr, circumoral nerve ring; cs, cardiac stomach; Ect, ectoneural; Hyp, hyponeural; ll, lateral lappet; mn, marginal nerve; mu, mucosa; nod, nodule; oc, optic cushion; oes, oesophagus; ohr, oral hemal ring; pm, peristomial membrane; pc, pyloric caecum; pd, pyloric duct; ps, pyloric stomach; rhs, radial hemal strand; rnc, radial nerve cord; tf, tube foot; tt, terminal tentacle. Scale bars: a, c, j, o, p = 60 μm; e, f, g, h, i, m, n, r = 32 μm; a-inset, b, c-right inset, d, k, l, q = 16 μm; c-left inset, i-insect = 6 μm |
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