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Fig 1. The localization of the holothurian radial nerve cords.The holothurian nervous system is characterized by an anterior nerve ring from which 5 radial nerve cords extend up to the anus. These can be divided into ventral and dorsal according to the localization of the tube feet (ventral). Samples were obtained from the ventro-lateral ambulacrum region (arrow) and dorso-lateral body wall (arrow head), which were divided into anterior, middle and posterior; and from the large intestine.
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Fig 2. The holothurian nervous system as seen from a transverse section.(A) Reconstruction of a transverse section of the nervous system of H. glaberrima in which the ventral region radial nerves (RNC) and the nervous components of the body wall (BW), longitudinal muscle (LM), and circular muscle (CM), can be appreciated by their immunoreactivity to the RN1 antibody. (B) The ventro-lateral ambulacrum region radial nerve is divided into the ectoneural component (ERNC) and the hyponeural component (HRNC) by a thin connective tissue layer (arrowheads), as shown by immunolabeling with the RN1 antibody. (C) Neuronal and glia somata in the ectoneural (arrow) and hyponeural (arrowhead) components as observed by DAPI staining. (D) For the purpose of this study, each component of the radial nerve was subdivided into central and lateral regions and these regions further subdivided into apical and basal. Scale bar = 300 μm in A; 80 μm in B and C. CC, coelomic cavity; ELA, ectoneural lateral apical region; ELB, ectoneural lateral basal region; EMA, ectoneural medial apical region; EMB, ectoneural medial basal region; HLA, hyponeural lateral apical region; HLB, hyponeural lateral basal region; HMA, hyponeural medial apical region; HMB, hyponeural medial basal region.
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Fig 3. Labeling by neuronal markers of transverse sections of the radial nerve cord is specific to each marker.(A, B) Anti-β-tubulin and the RN1 antibody labeled extensively both components of the radial nerve cord. (C) Anti-GABA immunoreactivity was preferentially distributed to the medial apical region of the ectoneural radial nerve cord (ERNC) and the central region of the hyponeural radial nerve cord (HRNC). (D) Anti-GFSKLYFamide immunolabeling was distributed throughout the RNC, except some central and lateral areas of the ectoneural component. (E) Anti-TH immunoreactivity was concentrated to the central region of the ERNC, and none was observed in the HRNC. (F) Anti-PH3 immunoreactivity was observed in the central and lateral basal regions of the ERNC, and in the lateral regions of the HRNC. (G) Anti-galanin immunoreactivity was uniform on all the ERNC, but not much was observed in the HRNC, except on the lateral areas where the hyponeural peripheral nerves form. (H) Anti-nurr1 immunoreactivity was observed within the basal region of the ERNC and uniformly through the HRNC. (I) Anti-pax6 labeling was very strong in two apical areas of the ERNC, but labeling was also observed in the basal region of the ERNC and uniformly on the HRNC. (J-L) Anti-calbindin, anti-parvalbumin, and anti-calretinin immunoreactivity on the radial nerve was very similar, being present in the apical region of the ERNC and in the medial region of the HRNC. Scale bar = 40 μm in A-L.
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Fig 4. Transverse sections of dorsal and ventral radial nerve cords from different regions of the anterior-posterior axis.(A-D) Immunoreactivity to anti-pax6 (A), anti-nurr1 (B), anti-parvalbumin (C), and anti-PH3 (D) showing the conservation of the distribution pattern (arrows) of the fibers identified by each marker along the length of the radial nerve cord and comparing dorsal versus ventral radial nerve cords. Scale bar = 40 μm in A-D. AD, anterior dorsal section; AV, anterior ventral section; MD, middle dorsal section; MV, middle ventral section; PD, posterior dorsal section; PV, posterior ventral section.
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Fig 5. Diversity of neurons identified in the radial nerve cords.Immunoreactive cells were observed in the ectoneural radial nerve cord (ERNC) and/or the hyponeural radial nerve cord (HRNC) as identified by each marker. Catecholaminergic cells were identified by anti-TH in the ERNC (A), and cells labeled by anti-calbindin were identified in the ERNC (B). Cells labeled by anti-nurr1 were identified in the HRNC (C), as well as labeled by anti-GFSKLYFamide (D), anti-galanin (E), and anti-pax-6 (F) were found in the ERNC. Scale bar = 8 μm in A-F.
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Fig 6. Labeling by neuronal markers of transverse sections of the radial nerve cord identify the peripheral nerves.In a transverse section, the ectoneural peripheral nerve extends from the ectoneural component of the RNC into the dermis (DE) to form the podial nerve (arrow). Immunoreactivity within the ectoneural peripheral nerves was observed with the RN1 antibody (A), anti-calbindin (B), and anti-pax6 (C). In a transverse section, the hyponeural peripheral nerve extends from the hyponeural component of the RNC into the longitudinal muscle (CM) (arrowhead) and longitudinal muscle (LM) (double arrowhead). Immunoreactivity within the hyponeural peripheral nerves was observed with the RN1 antibody (D), anti-calbindin (E), and anti-pax6 (F). Scale bar = 40 μm in C, and E, 150 μm in A, B, D, and F.
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Fig 7. Muscle nervous system components as revealed by immunoreactivity to the RN1 antibody.(A) Diagram of a transverse section of the body wall showing the location of the nervous system as identified by immunoreactivity of the RN1 antibody (dashed lines) in relation to the muscle components. The ectoneural peripheral nerve is shown extending from the ERNC to the dermis to form the podial nerve; while the hyponeural peripheral nerve is shown extending from the HRNC to the CM and LM. (B) A high magnification view of the LM, showing the hyponeural peripheral nerve within the coelomic epithelia and between the muscle fiber bundles of the LM. (C) The RN1 antibody immunoreactivity in fibers and cells of the connective tissue stalk (arrow) that connect the body wall with the LM. (D, E) A large subpopulation of fibers in coelomic epithelia and inside the LM was identified by immunoreactivity to RN1 (D) while smaller subpopulations were identified by other markers, such as anti-pax6 (E). (F) Multipolar cells immunoreactive to the RN1 antibody were present inside the LM. (G, H) Anti-GABA immunoreactive cells were present in the coelomic epithelia (G) and inside the LM (H). Scale bar = 40 μm in A, C, D, and E, 20 μm in B, and 10 μm in F-H. CC, coelomic cavity; CM, circular muscle; DE, dermis; ERNC, ectoneural radial nerve cord; HRNC, hyponeural radial nerve cord; LM, longitudinal muscle; WVS, water vascular system.
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Fig 8. Nervous system components distribution in the muscles and intestinal tissues as revealed by immunoreactivity to the RN1 antibody.(A) In a transverse section, many RN1 immunopositive fibers are observed in the epithelia and within the muscle bundles. Higher magnification of the LM shows the presence of RN1 antibody immunopositive cells (arrowhead, insert) and fibers surrounding the muscle fibers. (B, C) The RN1 antibody immunopositive cells (arrowhead) localized within the LM coelomic epithelium (B) and surrounding the muscle fibers (arrowhead, insert) (C). (D) RN1 antibody immunopositive fibers bundles (arrowhead) are present in the intestine, associated with the muscle component. (E) RN1 antibody immunopositive cells (arrowhead) were also observed to be present in the muscular layer of the intestine. Green = phalloidin labeling of muscle, red = RN1 antibody labeling of neural fibers and cells, blue = DAPI nuclear labeling. Scale bar = 20 μm in A, and 10 μm in B-E. CC, connective tissue cavity; LM, longitudinal muscle.
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Fig 9. The diversity of the enteric nervous system in H. glaberrima.(A) Diagrammatic transverse section of part of the wall of the large intestine detailing the composition of the main layers of the large intestine. (B, C) The main components of the enteric nervous system were identified by most markers, such as the RN1 antibody (B); while others, such as anti-PH3 (C), identified a small subpopulation of fibers. (D-F) Whole mounts of the serosa showing nerves (arrow) and individual fibers within the serosal plexus as shown by their immunoreactivity to the RN1 antibody (D), anti-GFSKLYFamide (E) and anti-galanin (F). (G) These nerves correspond to the nerve bundles (arrows) identified in transverse sections of the intestine immunolabeled with the RN1 antibody. (H-L) Unipolar and bipolar cells in the serosal plexus were identified by anti-galanin (H), anti-GFSKLYFamide (I), anti-calbindin (J), anti-pax6 (K), anti-nurr1 (L). (M-O) Multipolar and bipolar cells in the connective tissue plexus were identified by the RN1 antibody (M), anti-calbindin (N), and anti-GABA (O). (P-S) Bipolar cells in the luminal epithelium plexus were identified by anti-nurr1 (P), anti-pax6 (Q), anti-GFSKLYFamide (R), and anti-calbindin (S). (T, U) Anti-GABA immunoreactivity identified a novel plexus in the luminal epithelium (T), which was not immunoreactive to the RN1 antibody (U) or any of the other markers. (V) Anti-GABA immunopositive round and unipolar cells were observed in this plexus. Scale bar = 80 μm in B, 40 μm in D-F, 20 μm in C, G-I, S, 8 μm in J-Q, R, T-V. CE, coelomic epithelium; CM, circular muscle; CT, connective tissue; CTP, connective tissue plexus; LE, luminal epithelium; LEP, luminal epithelium plexus; LM, longitudinal muscle; SP, serosal plexus.
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Fig 10. The subdivisions of the radial nerve cord (RNC) in H. glaberrima.The RNC shows specific areas that can be identified by their specific immunoreactivity to a series of markers. Anti-pax6 (purple) identified two specific regions of large concentrations of immunopositive fibers in the ectoneural radial nerve cord (ERNC). Anti-calbindin (red) identified a subpopulation of fibers in the lateral apical regions of the ERNC, while anti-PH3 (brown) identified a subpopulation of fibers in the lateral basal regions of the ERNC. Anti-GABA and anti-GFSKLYFamide (blue) identified a subpopulation in the medial apical region of the ERNC. Anti-TH and anti-PH3 (dark green) identified a subpopulation in the central region of the ERNC. Anti-GFSKLYFamide, anti-galanin, and anti-nurr1 (orange) identified a subpopulation in the medial basal region of the ERNC. In the hyponeural radial nerve cord (HRNC), anti-GFSKLYFamide, anti-pax6, and anti-galanin (light blue) identified a subpopulation in the lateral region, while anti-GABA and anti-calbindin (yellow) identified a subpopulation in the apical region. Anti-TH (light green) and anti-calbindin (red) identified an extensive group of the cells in the RNC. Most of the anti-GFSKLYFamide (gray) immunoreactive cells are localized to the lateral apical region of the ERNC, while most of the anti-nurr1 (pink) immunoreactive cells are localized in the medial basal region of the HRNC.
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