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BACKGROUND: Regeneration of the damaged central nervous system is one of the most interesting post-embryonic developmental phenomena. Two distinct cellular events have been implicated in supplying regenerative neurogenesis with cellular material - generation of new cells through cell proliferation and recruitment of already existing cells through cell migration. The relative contribution and importance of these two mechanisms is often unknown.
METHODS: Here, we use the regenerating radial nerve cord (RNC) of the echinoderm Holothuria glaberrima as a model of extensive post-traumatic neurogenesis in the deuterostome central nervous system. To uncouple the effects of cell proliferation from those of cell migration, we treated regenerating animals with aphidicolin, a specific inhibitor of S-phase DNA replication. To monitor the effect of aphidicolin on DNA synthesis, we used BrdU immunocytochemistry. The specific radial glial marker ERG1 was used to label the regenerating RNC. Cell migration was tracked with vital staining with the lipophilic dye DiI.
RESULTS: Aphidicolin treatment resulted in a significant 2.1-fold decrease in cell proliferation. In spite of this, the regenerating RNC in the treated animals did not differ in histological architecture, size and cell number from its counterpart in the control vehicle-treated animals. DiI labeling showed extensive cell migration in the RNC. Some cells migrated from as far as 2 mm away from the injury plane to contribute to the neural outgrowth.
CONCLUSIONS: We suggest that inhibition of cell division in the regenerating RNC of H. glaberrima is compensated for by recruitment of cells, which migrate into the RNC outgrowth from deeper regions of the neuroepithelium. Neural regeneration in echinoderms is thus a highly regulative developmental phenomenon, in which the size of the cell pool can be controlled either by cell proliferation or cell migration, and the latter can neutralize perturbations in the former.
Fig. 1. Effect of aphidicolin treatment on radial nerve cord regeneration, day 16 post-injury. a The aphidicolin treatment significantly reduced (by a factor of two) the number of BrdU +-cells in the regenerating RNC, but did not affect the cell density (b) in the rudiment, absolute cell number in the rudiment (c), nor the length of the regenerate. e and (f) show representative micrographs of the regenerating RNC in a control animal and in an animal injected with aphidicolin, respectively. The growing tip is on the right. These micrographs are merged images showing three different channels simultaneously: ERG1 (an antibody labeling echinoderm radial glia) in red, BrdU in green, and propidium iodide (PI) (nuclear stain) in blue. These channels are shown separately in Additional File 4. The dotted lines in (e) and (f) mark the area where cells were counted (the distal regenerate and 100 μm of the proximal stump tissue — see “Methodsâ€)
Fig. 2. Unilateral DiI labeling of the injured radial nerve cord, longitudinal sections. Immediately after bisection, the dye was applied to one side of the wound, while the opposite side was left unlabeled. The arrow shows the site of the original dye application. a On day 2 after the surgery, the dye remained on one side of the wound. (a’) Higher magnification of the boxed area in (a). b By day 25 after surgery, the wound gap is bridged and the two stumps of the radial nerve cord have reconnected across the wound gap. The dashed lines show the original wound margins. Note extensive migration of labeled cells into the new tissue bridging the wound gap. The dotted line marks the outline of the radial nerve cord
Fig. 3. DiI labeling at a distance of 2 mm from the cut on days 2 (a, a’), 16 (b, b’), and 25 (c) after labeling and injury. The site of dye application is indicated by an arrow. The radial nerve cord is outlined by dotted lines. Dashed lines show the original wound magins. (a’) and (b’) are higher magnification views of the boxed regions in a and b, respectively. d Uninjured radial nerve cord 25 days after labeling
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