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Abstract
In contrast to mammals, the brain of adult non-mammalian vertebrates exhibits a higher proliferative and/or neurogenic activity. To provide new models on this issue, we have examined origin, distribution and fate of proliferating cells in the entire brain of juvenile and adult Xenopus laevis. Using immunohistochemistry for the Proliferation Cell Nuclear Antigen (PCNA), and/or the thymidine analog, 5-Bromo-2'' deoxyUridine (BrdU), the labeled cells are located in ventricular zones of the olfactory bulbs, cerebral hemispheres, preoptic region, ventralhypothalamus and cerebellum. Qualitatively, the highest level of proliferative cells was found in the telencephalic ventricles. By using in situ hybridization/immunocytochemistry double-labeling techniques, we demonstrate for the first time in post-metamorphic frog brain that the proliferative cells are localized in very close vivinity to the radial glial cells, progenitor cells that we have also identified in the ventricular layer using classical molecular markers (BLBP, Vimentin). In addition, after long post-BrdU administration survival times ranging between 14 and 28days, BrdU labeling combined with immunohistochemistry for markers of cell migration (DoubleCortin) or radial glial cells (BLBP), reveals that the proliferative cells are able to migrate from the ventricular zone into the brain parenchyma, most likely by migrating along the radial processes. Finally, at survival time of 28days and by using a combination of BrdU labeling and in situ hybridization for markers of differentiation states (Neuro-β-tubulin, Proteolipid Protein), we demonstrate that newborn cells can differentiate in large portion into either neurons or oligodendrocytes.
Fig. 1. Drawing of the transverse sections (A1–J1) and tissue transverse sections showing the distribution of proliferative zones (A2–J2, A3–J3 and A4–J4) in the brain of juvenile X. laevis (NF stage 66). The drawing at the top of the figure shows a dorsal view of the X. laevis brain. The letters correspond to the rostro-caudal location of sections as depicted in the whole brain drawing. Proliferating cell nuclear antigen (PCNA) immunohistochemistry (A2–J2 and A4–J4) and 5-bromo-2′ deoxyuridine (BrdU) labeling/detection (A3–J3) visualized by epifluorescence and counterstained with DAPI staining. (A4–J4) High magnifications of PCNA immunodetection images (A2–J2). Asteriks in A2–J2 images indicate the areas which are enlarge in A4–J4. The arrows in D2, D3, F3, I2 and I3 indicate labeling areas. The arrowheads in F3 indicate non-specific labeling due to vascular cells. (K) Proliferating cell nuclear antigen (PCNA) immunohistochemically visualized in Xenopus transverse larval stage 57. (L, M) Proliferating cell nuclear antigen (PCNA, L) and 5-bromo-2′ deoxyuridine (BrdU, M) double immunodetection on the same Xenopus telencephalon transverse section. Note the differences between the PCNA (L) and BrdU proliferation markers (M). Arrows in M indicate specific BrdU labeling. Abbreviations are defined in Table 1. The anatomical drawings, with the exceptions of G1 and J1, are from (Tuinhof et al., 1998), with modifications of basal ganglia subdivisions according to (Marin et al., 1998). Scale bar = 400 μm in A2, A3, F2 and F3 (applies to B2–E2, B3–E3, G2–J2, and G3–J3) and K; 100 μm in A4 and F4 (applies to B4–E4 and G4–J4); 40 μm in L–M.
Fig. 2. Drawing of the transverse sections (A1–J1) and tissue transverse sections showing the distribution of proliferative zones (A2–J2, A3–J3 and A4–J4) in the brain of juvenile X. laevis (NF stage 66). The drawing at the top of the figure shows a dorsal view of the X. laevis brain. The letters correspond to the rostro-caudal location of sections as depicted in the whole brain drawing. Proliferating cell nuclear antigen (PCNA) immunohistochemistry (A2–J2 and A4–J4) and 5-bromo-2′ deoxyuridine (BrdU) labeling/detection (A3–J3) visualized by epifluorescence and counterstained with DAPI staining. (A4–J4) High magnifications of PCNA immunodetection images (A2–J2). Asteriks in A2–J2 images indicate the areas which are enlarge in A4–J4. The arrows in D2, D3, F3, I2 and I3 indicate labeling areas. The arrowheads in F3 indicate non-specific labeling due to vascular cells. (K) Proliferating cell nuclear antigen (PCNA) immunohistochemically visualized in Xenopus transverse larval stage 57. (L, M) Proliferating cell nuclear antigen (PCNA, L) and 5-bromo-2′ deoxyuridine (BrdU, M) double immunodetection on the same Xenopus telencephalon transverse section. Note the differences between the PCNA (L) and BrdU proliferation markers (M). Arrows in M indicate specific BrdU labeling. Abbreviations are defined in Table 1. The anatomical drawings, with the exceptions of G1 and J1, are from (Tuinhof et al., 1998), with modifications of basal ganglia subdivisions according to (Marin et al., 1998). Scale bar = 400 μm in A2, A3, F2 and F3 (applies to B2–E2, B3–E3, G2–J2, and G3–J3) and K; 100 μm in A4 and F4 (applies to B4–E4 and G4–J4); 40 μm in L–M.
Fig. 3. (A–K) Transverse sections at the level of the lateral ventricles of a juvenile telencephalon, showing PCNA (H–I and K) or BrdU (E and F) immunoreactivity in radial glial cells. Radial glial cells were revealed by immunohistochemistry using BLBP antibodies (B, C, G, I, J and K), and/or by in situ hybridization using a Vimentin riboprobe (A, C, D, and F). Fast red (A and C) or NBT/BCIP (D and F) in situ hybridization revelation of vimentin transcripts. (A–B, C, J and K) confocal images. (D–H and I) bright field and/or epifluorescence images. Note the radial morphology of the radial glial cells in B and J (arrowheads). The arrows in A, B J and K indicate a ventricular region devoid of radial glial cells (absence of BLBP and Vimentin markers), and PCNA immunoreactivity. LV, lateral ventricles. Dorsal–Ventral (d–v) direction is indicated. Scale bar = 40 μm in A–K.
Fig. 4. (A–J) Single (A–D) or double immunofluorescence (E–J) for BrdU and DoubleCortin (DCX; E–G) or BLBP (H–J) at juvenile stages. Telencephalon transverse (A–B, H–J) or sagittal (E–G) sections. (C,D) Cerebellum transverse sections. BrdU labeling at 2-day survival (A, C) or 14-day survival (B, D, E–G, H–J) time. (E–G) Anterior is to the left. LV, lateralventricle; OV, olfactory ventricle. Epifluorescence (A–G) and confocal (H–J) images. For all images, dorsal is to the top. Rostral–caudal (r–c) direction is indicated when necessary. Scale bar = 300 μm in A–D; 40 μm in E–G and H–J.
Fig. 5. (A–H) Neurogenesis and gliogenesis in the juvenile Xenopus brain. Double in situ hybridization using NβT (A–B, C–E) and PLP (A–B, F–H) riboprobes combined with BrdU immunodetection (28-day survival time) on transverse sections. (A–B) Double in situ hybridization revelation of neurons (NβT) and oligodendrocytes (PLP) in the diencephalon (A) and cerebellum regions (B). (C–H) Colocalizations of NβT/BrdU (C–E) and PLP/BrdU (F–H) in the cerebellum. Arrows (C–H) indicate colocalizations. (E, H) DAPI staining to ascertain the presence of the nucleus. For all images, dorsal is to the top. Scale bar = 350 μm in A–B; 40 μm in C–H.
Fig. 6. (A–O). Proliferation mapping and neurogenesis/gliogenesis in the adult Xenopus brain. (B–G) PCNA immunodetection on transverse sections at the level of the olfactory bulbs (B), anterior (C) or middle (D, G) cerebral hemispheres, ventralhypothalamus (E) and cerebellum (F). (G–I) Confocal images at high magnification of a PCNA immunofluorescence assay (G, I) combined with DAPI staining (H, I). (C, D and G–I) Arrows indicate PCNA positive cells. (J–O) Double in situ hybridization using NβT (J–L) and PLP (M–O) riboprobes, combined with BrdU immunodetection (28-day survival time) on transverse sections. (J–L) Colocalization of NβT and BrdU. (M–O) Colocalization of PLP and BrdU. The arrows (J–O) indicate these colocalizations. (L, O) DAPI staining to ascertain the presence of the nucleus. (A) Brain size comparison between Xenopus juvenile and adult stages. For all images, excepted G–I, dorsal is to the top. Scale bar = 1000 μm in A; 40 μm in B–F, G–I, J–L and M–O.
