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Figure 1. Organization of the uninjured RNC. (A – D) Microscopic anatomy of the RNC shown on transverse (A, B) and longitudinal (C, D) paraffin sections, Giemsa staining. (B) and (D) show higher magnification views of the RNC in (A) and (C), respectively. (E – I) Representative micrographs showing double immunolabeling of the RNC with the echinoderm radial glia-specific antibody ERG1 [22] and other glial and neuronal markers; cryosections. (E) ERG1 and AFRU (a rabbit polyclonal antiserum recognizing Reissner’s substance [30]), transverse section. (F) ERG1 and anti-GABA antibodies, longitudinal section. (G) ERG1 and anti-GFSKLYFamide [31] antibodies, longitudinal section. (I, H) ERG1 and anti-Nurr1 antibodies in the hyponeural (H) and ectoneural (I) neuroepithelia, longitudinal sections. Note a short bridge connecting the ectoneural and hyponeural neuroepithelia marked by an arrowhead in (H). bw, body wall connective tissue; c, coelom; ec, epineural canal; en, ectoneural neuroepithelium; h, hemal lacuna; hc, hyponeural canal; hn, hyponeural neuroepithelium; lmb, longitudinal muscle band; p, thin connective tissue partition separating the ectoneural and hyponeural neuroepithelia; re, roof epithelium; rnc, radial nerve cord; wvc, water-vascular canal.
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Figure 2. The surgical procedure. (A) The eviscerated animals were anesthetized to the point when they stopped responding to touch. (B, C) The inner layer of the body wall was exposed through the cloaca using a glass rod. (D, E) Using a razor blade, the mid-ventral radial organ complex, including the longitudinal muscle, water-vascular canal, and radial nerve cord, was cut at about the mid-body level without damaging the outer connective tissue layer and the epidermis.
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Figure 3. Organization of the injured RNC during the early post-injury phase (days 1 – 2). (A) Lesioned radial organ complex as viewed from the coelomic side of the body wall. (B – F) Longitudinal sections through the RNC. (B) Low-magnification view of the wound region; hematoxylin and esosin staining. (C) In the vicinity of the wound, the radial glial cells (magenta) still retain RS-like immunoreactivity (green) in the apical region of the ectoneural neuroepithelium. The dashed line shows the outlines of the cut end of the RNC. (D) Double labeling with the glial marker ERG1 (magenta) and the neuronal marker anti-GFSKLYFamide antiserum (green). (D’) and (D”) show these two types of labeling in separate channels. (E) Fragmentation (arrowheads) of glial processes (magenta) in the vicinity of the wound. (F) Neuronal processes (green) in injury area exhibit bulbous terminal swelling (arrow). bw, body wall connective tissue; c, coelom; ec, epineural canal; en, ectoneural neuroepithelium; hn, hyponeural neuroepithelium; rnc, radial nerve cord; roc, radial organ complex; wg, wound gap; wvc, water-vascular canal. Asterisk in (C, D, D’) indicates the zone of dedifferentiation in the ectoneural neuroepithelium with the radial glial cells preserving epithelial organization.
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Figure 4. Organization of the regenerating RNC during the late post-injury phase (days 6 – 8). (A) Regenerating radial organ complex as viewed from the coelomic side of the body wall. (B – E) Longitudinal sections through the RNC. (B) Low-magnification view of the wound region; hematoxylin and eosin staining. The inset shows a detailed view of the distal tip of the RNC. (C) Double labeling with the glial marker ERG1 (magenta) and the neuronal marker anti-GFSKLYFamide antiserum (green). (C’) and (C”) show these two types of labeling in separate channels. Note the extended zone of glial dedifferentiation and a terminal swelling of the epineural canal (asterisk). (D, E) Double immunolabeling with the ERG1 (magenta) and anti-RS AFRU (green) antibodies. Note that the dedifferentiated radial glial cells at the distal tip of the RNC (E) produce less RS-like material, than do the glial cells in the more proximal regions (D). c, coelom; ce, coelomic epithelium; ec, epineural canal; hc, hyponeural canal; rnc, radial nerve cord; roc, radial organ complex; wg, wound gap; wvc, water-vascular canal.
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Figure 5. Organization of the regenerating RNC during the growth phase (days 8 – 12 post-injury). (A) Regenerating radial organ complex as viewed form the coelomic side of the body wall. The growing regenerates are labeled with arrowheads. Note that in this case the radial organs growing from the opposite sides of the wound are not aligned along the same axis. (B – F) Longitudinal sections through the regenerating RNC. (B) Low-magnification view of the regenerating radial organs; hematoxylin and eosin staining. (C) Detailed view of the growing tip of the RNC; hematoxylin and eosin staining. Note that the tip of the hyponeural cord grows slower than the ectoneural cord. (C’) shows higher magnification view of the boxed area in (C). (D) Growing ectoneural cord. Double labeling with the glial marker ERG1 (magenta) and the neuronal marker anti-GFSKLYFamide antiserum (green). Note that the leading tip (arrow) is made up of differentiated glial cells (magenta) and contains no neuronal elements (green). (E, E’, F) Double immunolabeling with the ERG1 (magenta) and anti-RS AFRU (green) antibodies. Note that the glial cells of the leading tip of the growing ectoneural cord do not produce RS-like material (E, E’), unlike the glial cells of the more proximal regions (F). (E’) shows a detailed view of the leading tip of the growing glial tube (boxed area in E). bw, connective tissue of the body wall; c, coelom; ce, coelimic epithelium; ec, epineural canal; en, ectoneural cord; hc, hyponeural canal; hn, hyponeural cord; rnc, radial nerve cord; roc, radial organ complex; wvc, water-vascular canal.
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Figure 6. Organization of the regenerating RNC during the late regenerate phase (21+ days after injury). (A, B) Regenerating radial organ complex as viewed form the coelomic side of the body wall on day 28 and day 42 post-injury. Arrowheads indicate the regenerated radial organ complex bridging the wound gap. The shape of the regenerated structure in (A) suggests that the growing regenerates were not aligned along the same axis, but, nevertheless, were able to meet and fuse. (C – H) Longitudinal sections through the regenerated RNC. (C) General morphology of the regenerated radial organs on day 21 post-injury; hematoxylin and eosin staining. (D) Double immunolabeling of the RNC on day 21 post-injury with the ERG1 (magenta) and anti-RS AFRU (green) antibodies. Note that the radial glial cells of the newly regenerated segment of the RNC fully restored their palisade-like morphology and fully resumed their ability to produce and secrete the RS-like material. (E – F’) Double labeling with the glial marker ERG1 (magenta) and the neuronal marker anti-GFSKLYFamide antiserum (green) of the newly regenerated segment of the RNC (E, E’) and the region not affected by the injury (F, F’) on day 21 post-injury. (E’) and (F’) show the labeling with the anti-GFSKLYFamide antiserum in a separate channel. Note that the neuropil on day 21 has not yet completely restored its normal organization (compare with Figure 7). (G, H) Double labeling of the newly regenerated segment of the RNC with the glial marker ERG1 (magenta) and the anti-GABA (G) or the anti-Nurr1 antisera (H) (green) on day 21 post injury. bw, body wall connective tissue; c, coelom; ce, coelomic epithelium; ec, epineural canal; en, ectoneural neuroepithelium; hc, hyponeural canal; hn, hyponeural neuroepithelium; re, roof epithelium; roc, radial organ complex; wvc, water-vascular canal.
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Figure 7. Day 145 post injury. Organization of the neuropil in the fully regenerated segment of the RNC (A, A’) and in the region of the RNC not affected by the injury (B, B’). Double labeling with the glial marker ERG1 (magenta) and the neuronal marker anti-GFSKLYFamide antiserum (green). (A’) and (B’) show the labeling with the anti-GFSKLYFamide antiserum in a separate channel.
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Figure 8. Cell division dynamics in the normal and regenerating RNC. (A) BrdU labeling paradigm employed to quantify cell division. (B) Diagram showing the percentage of all BrdU-incorporating cells (irrespective of their phenotype) normalized to the total number of cells in the tissue. (C) Diagram showing how many of the BrdU-incorporating cells are ERG1-positive glial cells. (D) Diagram showing how many of the ERG1-positive glial cells incorporate BrdU. Results are represented as mean (percentage) ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001. ‡ indicates that the value is missing because BrdU-incorporating cells were absent in three of the four animals. The the ratio of the number of BrdU+ ERG1+ cells divided by the total number of BrdU+ cells is impossible to define in these animals (division of zero by zero). Therefore, neither mean value nor standard error were calculated. In the fourth animal, there were only two BrdU+ cells in the hyponeural neuroepithelium, one of them was BrdU+ ERG1+, whereas the other was BrdU+ ERG1-.
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Figure 9. Representative micrographs showing distribution of BrdU-incorporating cells (green) in the uninjured and regenerating RNC (single BrdU injection, 50 mg/kg, followed by a 4 h chase period). The radial glial cells (magenta) are visualized by immunostaining with the ERG1 monoclonal antibody. Nuclei are stained with propidium iodide (PI, blue). All micrographs are longitudinal sections with the plane of injury/regenerate to the left. (A) The RNC of an uninjured animal. The inset shows a high maginification view of a BrdU-incorporating radial glial cell. (B) Early post-injury phase (day 1). The inset shows a higher magnification view of the boxed area. (C, C’) Late post-injury stage (day 6 post-injury). Note numerous BrdU-incorporating cells among the dedifferentiating radial glia. (C’) shows higher magnification of the boxed area in (C) (dedifferentiating region of the RNC). (D) Growth phase (day 8 post-injury). Note abundant BrdU-positive cells in the growing tubular glial regenerate (arrowheads). (E) Late regenerate (day 21 post-injury). en, ectoneural neuroepithelium; hn, hyponeural neuroepithelium.
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Figure 10. Proliferating ERG1-positive glial cells give rise to neurons in the regenerating RNC. (A) BrdU labeling paradigm employed to label proliferating glial cells and trace their progeny. Multiple BrdU injections (50 mg/kg, every 12 hours) were given during the growth phase of regeneration (days 8 thru 12 post-injury). The tissues were fixed at two time points: 12 hours and 51 days after the last BrdU injection (on day 13 and day 64 post-injury, respectively). (B) Proportion of ERG1-positive glial cells among BrdU-positive cells. Note that shortly after BrdU administration during the growth phase, the vast majority of BrdU-incorporating cells show ERG-positive glial phenotype. This number significantly decreases on day 51 after the last BrdU injection, when ERG1-negative cells (neurons) represent almost half of the BrdU-positive progeny. (C, C’) Representative micrographs showing BrdU-incorporating ERG1-positive radial glial cells (arrowheads) 12 h after the last BrdU injection. (D, D’) Representative micrographs showing Nurr1+ BrdU+ neurons (arrows) on day 51 after the last BrdU injection (day 64 post-injury).
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Figure 11. Dynamics of programmed cell death as determined by TUNEL assay in the normal and regenerating RNC. (A) Relative abundance of all TUNEL-positive cells (irrespective of their phenotype) normalized to the total number of cells in the tissue. (B) Proportion of TUNEL-positive ERG1+ glial cells in relation to the total number of TUNEL-positive cells. (C) Proportion of TUNEL-positive ERG1+ glial cells in relation to the total number of ERG1+ glial cells. Results are represented as mean (percentage) ± standard error. *P < 0.05, **P < 0.01, ***P < 0.001.
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Figure 12. Representative micrographs showing distribution of TUNEL-positive cells (green) in the uninjured and regenerating RNC. The radial glial cells are stained with the ERG1 monoclonal antibody (magenta), and the nuclei are stained with DAPI (blue). All micrographs are longitudinal sections with the plane of injury or growing regenerate to the left. (A) Uninjured RNC. A’ and A” show a high magnification view of the TUNEL-positive ERG1-negative cell marked with an arrowhead. (B) Abundant TUNEL-positive cells in the vicinity of the injury plane at the early post-injury stage (day 1). (C) Late post-injury stage (day 8). (D) Growing glial tubular rudiment on day 10 post-injury. D’ and D” show a high magnification view of a TUNEL-positive ERG1-positive glial cell (marked with an arrow). (E) Late regenerate (day 21 post-injury).
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