Fig. 5.

Localization of ArSS1 in A. rubens using immunohistochemistry. (A) Transverse section of a radial nerve cord showing ArSS1 immunoreactivity (immunostaining) in the hyponeural (arrow) and ectoneural (arrowhead) regions. The Inset shows that no staining is observed in sections of radial nerve cord incubated with affinity-purified ArSS1 antibodies preabsorbed with ArSS1 peptide, demonstrating the specificity of immunostaining observed in sections incubated with affinity-purified ArSS1 antibodies. (B) Transverse section of the circumoral nerve ring showing immunostained cells in the hyponeural region (arrow) and in the subcuticular epithelial layer of the ectoneural region (arrowheads); note the regional variation in the density of immunostained fibers in the ectoneural neuropile (asterisks). Immunostaining can also be seen here in the basiepithelial nerve plexus of the adjacent peristomial membrane. (C) Higher-magnification image of the boxed region in B, showing round-shaped monopolar immunostained cells (arrows) in the hyponeural region and immunostained fibers (white arrows) parallel to the collagenous tissue layer (white arrowhead), which is located between the hyponeural and ectoneural regions. (D) Immunostaining can be seen here in a marginal nerve; note the strongly immunostained fibers (arrowhead) emanating from the marginal nerve. (E) Parasagittal longitudinal section of a radial nerve cord showing immunostained cells in the hyponeural region (arrow) and in the subcuticular epithelial layer of the ectoneural region (arrowheads); note also the regional variation in the density of immunostained fibers in the ectoneural neuropile (asterisks). (F) Transverse section through the central disk region showing ArSS1 immunoreactivity (immunostaining) in the peristomial membrane and the adjacent circumoral nerve ring. (G) Higher-magnification image of the boxed region in F, showing immunostained cells in the coelomic epithelium (white arrowhead) and immunostained fibers in the underlying nerve plexus (black arrowhead). Immunostained fibers can also be seen here in the basiepithelial nerve plexus (arrow) underlying the external epithelial layer of the peristomial membrane. The collagenous tissue layer that separates the two nerve plexi is unstained. (H) Transverse section through the central disk region showing immunostaining in a longitudinal section of the esophagus and in the peristomial membrane and circumoral nerve ring. More specifically, immunostaining can be seen in the coelomic epithelium (arrowhead) and the basiepithelial nerve plexus (arrow). (I) Horizontal section of the cardiac stomach showing immunostained fibers in the basiepithelial nerve plexus (arrow) with regional variation in staining intensity. The Inset shows a higher-magnification image of the boxed region, showing immunostained bipolar shaped cells present in the mucosal layer (arrowhead) and immunostained fibers in the basiepithelial nerve plexus (arrow). (J) Horizontal section of the pyloric stomach showing immunostained fibers in the basiepithelial nerve plexus (arrow) with regional variation in staining intensity. The Inset shows a higher-magnification image of the boxed region, showing immunostained bipolar shaped cells in the mucosal layer (arrowhead) and immunostained fibers in the basiepithelial nerve plexus (arrow). (K) Transverse section through the central disk region showing intense immunostaining in the region of the digestive system linking the cardiac stomach with the pyloric stomach. (L) Higher-magnification image of the boxed region in K, showing immunostained bipolar-shaped cells present in the mucosal layer (arrowhead) and intense immunostaining in the basiepithelial nerve plexus (arrow). (M) Transverse section of a pyloric duct showing immunostaining in the basiepithelial nerve plexus on both the aboral and oral sides but with denser staining on the oral side (arrow). (N) Higher-magnification image of the boxed region in M, showing immunostained bipolar-shaped cells (arrowhead) present in the mucosal layer and immunostained fibers in the basiepithelial nerve plexus (arrow). (O) Transverse section of a starfish arm showing regional variation in the intensity of immunostaining in a pyloric cecum, with immunostaining most intense in the oral region that is continuous with the pyloric ducts. (P) Higher-magnification image of the boxed region in O, showing immunostained bipolar-shaped cells in the mucosal layer (arrowhead) and immunostaining in the basiepithelial nerve plexus (arrow). (Q) Longitudinal section of a tube foot showing ArSS1 immunoreactivity in the subepithelial nerve plexus along the length of the stem and extending into the basal nerve ring of the disk region. The Inset shows a higher-magnification image of the boxed region, showing immunostaining in the basal nerve ring of the disk region. (R) Parasagittal longitudinal section of an ampulla of a tube foot showing immunostaining in the coelomic epithelial lining of the ampulla. The Inset shows a higher-magnification image of the boxed region, showing immunostaining in the coelomic epithelial layer (arrowhead). (S) Transverse section of the aboral body wall showing immunostaining in the subepithelial nerve plexus beneath the external epithelial layer of aboral body wall. (T) Transverse section of the aboral body wall showing immunostaining (arrow) in a pedicellaria. (U) Transverse section of an arm tip, showing immunostaining in the terminal tentacle, lateral lappet, optic cushion, and the surrounding body wall epithelium. Abbreviations: Amp, ampulla; BNP, basi-epithelial nerve plexus; BW, body wall; CE, coelomic epithelium; CONR, circumoral nerve ring; CS, cardiac stomach; CT, collagenous tissue; Di, disk; Ec, ectoneural region; Ep, epithelium; Es, esophagus; Hy, hyponeural region; LL, lateral lappet; Lu, lumen; MN, marginal nerve; Mu, mucosa; OC, optic cushion; OS, ossicle; Pe, pedicellaria; PM, peristomial membrane; PS, pyloric stomach; RHS, radial hemal strand; RNC: radial nerve cord; SNP, subepithelial nerve plexus; St, stem; TF, tube foot. THS, transverse hemal strand; TT, terminal tentacle. (Scale bars, 12 μm in Q, Inset; 20 μm in C, G, I, Inset, and R, Inset; 32 μm in D, J, Inset, N, P, S, and T; 60 μm in A, A, Inset, B, E, F, L, M, O, Q, R, and U; 120 μm in H, I, and K; and 160 μm in J.)