Yoshino J and Tochinai S (2004), Successful reconstitution of the non-regenerati...

XB-ART-2572

Dev Growth Differ 2004 Dec 01;466:523-34. doi: 10.1111/j.1440-169x.2004.00767.x.

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Successful reconstitution of the non-regenerating adult telencephalon by cell transplantation in Xenopus laevis.

Yoshino J , Tochinai S .

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The South African clawed frog (Xenopus laevis) can regenerate the anterior half of the telencephalon only during larval life, but such regeneration is no longer possible after metamorphosis. In order to gain a better understanding of differences between larvae and adults that are potentially related to regeneration, several experiments were conducted on larvae and froglets after the partial removal of the telencephalon. As a result, it was found that the cells in the brain proliferated actively, even in non-regenerating froglets, just as was observed in regenerating larvae after the partial removal of the telencephalon. Moreover, it was shown that although the structure was usually imperfect, even isolated single cells derived from the frog brain were able to reconstitute the lost portion when the cells were transplanted to the partially truncated telencephalon. It is therefore likely to be critical for massive organ regeneration that ependymal layer cells promptly cover the cerebral lateral ventricles at an initial stage of wound healing, as is the case observed in larvae. However, in froglets, these cells strongly adhere to one another, and they are therefore unable to move to seal off the exposed ventricle, which in turn is likely to render the froglet brain non-regenerative.

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Species referenced: Xenopus laevis
Genes referenced: rbfox3
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	Fig. 1. Regeneration of larval telencephalon. The anterior half of the telencephalon was removed from stage 53 tadpoles (A,H), and was then observed macroscopically (A–G) and histologically on horizontally sectioned materials (H–N). Just after the tissue dissection, the ventricles were open at the anterior end (B,I). As early as 4 days after the operation, the ventricle was completely covered by cells that had apparently aggregated from the vicinity (D,K). The covered area then gradually increased in thickness with time, and the regenerated brain was extended anteriorly (L,M). After 30 days, the telencephalon returned to a normal structure with olfactory nerve bundles, which were connected to the olfactory organs. The arrowheads in (B) indicate the anterior end of the remaining telencephalon domain. The asterisk in (B) indicates the space created by the partial removal. The arrow in (G) indicates the connection between the olfactory nerve and the regenerated telencephalon. on, olfactory nerve; t, telencephalon; d, diencephalon; m, mesencephalon; lv, lateral ventricle; cp, choroid plexus. Bars, 2 mm (A–G); 300 μm (H–N).
	Fig. 2. No regeneration occurred in the adult telencephalon. The anterior half of the telencephalon was removed from froglets 10 days after metamorphosis (A,D), and the results were observed macroscopically (A–C), and histologically in horizontal sections (D–F). Just after the tissue dissection, the ventricles were open at the anterior end (B–E). As late as 30 days after the removal of tissue, the ventricles were still open at the anterior end (C,F). The arrowheads in (B,C) indicate the anterior end of the remaining telencephalon domain. The asterisks in (B,C) indicate the space created by partial removal. ob, olfactory bulb; cb, cerebrum; d, diencephalon; m, mesencephalon; lv, lateral ventricle. Bars, 2 mm (A–C); 300 μm D–F.
	Fig. 3. Mitotic changes before and after partial telencephalon removal. After removing the anterior half of the telencephalon, the number of mitotic figures strikingly increased (compare B,E with A,D), both in stage 53 larvae (A–C, red line in G) and in froglets 10 days after metamorphosis (D–F, blue line in G), as examined histochemically on crosssectioned materials (A–F). The mean mitotic indices shown in (G) were calculated on BrdUincorporating cells for all cells on three randomly selected histological sections from one animal. Three animals were used to illustrate each point. The vertical bars indicate the SD of nine samples (three sections from three animals). lv, lateral ventricle. Bar, 200 μm.
	Fig. 4. Antigenic characterization of mitotically active ependymal layer cells surrounding the lateral ventricle. Intact larvae (A–F) and froglets (G–L) were injected with BrdU, and the telencephalon was examined histochemically 8 h later (A–L). Anti-BrdU antibody indicates replicating cells (green); anti-glial fibrillary acidic protein (GFAP) indicates the radial projection of ependymal layer cells (red); anti- Musashi1 indicates undifferentiated neuroblasts (red). In (M–O), froglets were injected with BrdU and the telencephalon was examined 30 days later in order to determine whether mitotically active ependymal layer cells would give rise to differentiated neurons positive for NeuN antigens. The right-most panel shows merged figures showing yellow cells that are double-positive for cell proliferation and characteristic molecules for each cell type. Each arrowhead in (A–C, G–I) indicates one replicating cell having radial projection. The arrowhead in (M–O) indicates a cell replicated 30 days ago which did not differentiate into neuron. The arrows in (M–O) indicate the replicated cells 30 days later differentiated into neuron. lv, lateral ventricle. Bar, 200 μm.
	Fig. 5. Reconstitution of the froglet telencephalon by transplantation of cell suspensions. Dispersed cells obtained from larvae (A,D) and froglets (B,E) were introduced into the space created in the adult brain by partially removing the telencephalon. After 30 days, the reorganized brain structure consisted primarily of donor-origin cells (non-granular nuclei). The rectangles in (A,B) show the locations of (D,E), respectively. Control individuals (no transplantation) show no indication of regeneration (C). The dotted line indicates the host–graft boundary, as distinguished by the quinacrine stainability of the nuclei (D,E). The donor cell region is indicated by asterisks. The arrowheads in (D,E) indicates recipient cells found in the donor cell region. lv, lateral ventricle. Bars, 200 μm (A–C); 50 μm (D,E).