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The mechanisms for directing axons to their targets in developing limbs remain largely unknown though recent studies in mice have demonstrated the importance of neurotrophins in this process. We now report that in co-cultures of larval Xenopus laevis limb buds with spinal cords and dorsal root ganglia of Xenopus and axolotl (Ambystoma mexicanum) axons grow directly to the limb buds over distances of up to 800 microm and in particular to sheets of epidermal cells which migrate away from the limb buds and also tail segments in culture. This directed axonal growth persists in the presence of trk-IgG chimeras, which sequester neurotrophins, and k252a, which blocks their actions mediated via trk receptors. These findings indicate that developing limb buds in Xenopus release diffusible factors other than neurotrophins, able to attract growth of sensory and motor axons over long distances.
Fig. 1. Spontaneous, but random axonal outgrowth occurs from larval Xenopus DRGs (arrows) in the absence of added growth factors (A). In co-cultures, axons show directed growth from the DRGs (arrows) towards sheets of migrating cells (arrow heads) surrounding the limb bud viewed at high (B) and lower (C) magnification. Phase-contrast (D1) and immunofluorescence (D2) images of migrating cells showing some nuclei (arrows) labelled by antibody to phosphorylated histone H3, a mitotic marker. Directed axonal growth towards limb buds also occurs from the DRGs (arrows) of metamorphosing (approximately stage 60) larva (E) and juvenile Xenopus (F). Scale bar = 100 μm (A, B, E, F); 200 μm (C) and 50 μm (D).
Fig. 2. RT-PCR analysis of migratory cells from Xenopus limb buds (lanes 2–5) and stage 28 embryo controls (lanes 6–9). Lane 1, markers; lanes 2 and 6 (BMP-2); lanes 3 and 7 (XIRG); lanes 4 and 8 (XLK); lanes 5 and 9 (XSLUG). The larval epidermal phenotype of the migratory cells is shown by the expression of XLK and X-IRG. X-IRG, but not BMP-2 and XSLUG (markers of mesoderm and neural crest, respectively).
Fig. 3. Axons (arrows) from the spinal roots of Xenopus (A) and Ambystoma (B) spinal cords growing towards sheets of cells (arrow heads) migrating from limb buds. Scale bar = 200 μm (A), 100 μm (B).
Fig. 4. Axonal outgrowth from Ambystoma PN-DRGs in collagen. In a control preparations (A) some spontaneous axonal outgrowth occurs (arrows) but is greatly enhanced in media conditioned by tail segments cultured in matrigel (B) or free-floating tail segments (C). Scale bar = 100 μm.
Fig. 5. Axonal outgrowth from Xenopus PN-DRGs in Matrigel. Control (A); NGF (B, C); BDNF (D, E); NT-3 (F, G); NT-4 (H, I). In the absence of neurotrophic factors, some spontaneous axonal outgrowth (arrows) is observed (A) but the numbers are markedly increased in the presence of the neurotrophins (50 ng/ml). However, in the presence of k252a, the stimulatory effects of the neurotrophins are abolished (C, E, G, I). Scale bar = 100 μm.
Fig. 6. Axonal outgrowth from Xenopus PN-DRGs in collagen. Control (A); NGF (B, C); BDNF (D, E); NT-3 (F, G); NT-4 (H, I). In the absence of neurotrophic factors, very little spontaneous axonal outgrowth (arrows) is observed (A). Axon numbers are markedly increased in the presence of the neurotrophins (B, D, F and H) but their stimulatory effects are abolished by k252a (C, E, G and I) where the mean numbers of outgrowing axons per preparation (six for each condition) in NGF (5.1 ± 0.9); BDNF (4.5 ± 1.4), NT-3 (3.8 ± 1.3) and NT-4 (5.8 ± 1.2) were all significantly less (P < 0.001) than in control preparations (11.8 ± 0.8, n = 12). A similar abolition of the effects of the neurotrophins on axonal growth was also observed in the presence of the trk-IgG fusion proteins. The mean numbers of outgrowing axons per preparation (6 for each condition) in the presence of NGF+ trkA-IgG (12 ± 5.9); NT-3 + trkC-IgG (11.6 ± 2.0) and NT-4 + trkB-IgG (12.5 ± 3.1) were similar to controls (11.8 ± 0.8, n = 12) whilst in the presence of BDNF+ trkB-IgG the mean number (5 ± 1.3) was significantly less (P < 0.001) than in control preparations. Scale bar = 100 μm.
Fig. 7. Directed axonal outgrowth from Xenopus PN-DRGs towards sheets of migrating cells (arrow heads) from limb buds persists in the presence of k252a (A) and trk-IgG chimeras (B) and from a larval Xenopus spinal cord in the presence of trk-IgG chimeras (C). Scale bar = 100 μm (A and B); 200 μm (C).
Fig. 8. RT-PCR analysis of neurotrophin expression in Xenopus CNS (lanes 2–6) and migrating epidermal cells (lanes 7–11). Lane 1, markers; lanes 2 and 7 (ODC); lane 3 and 8 (BDNF); lanes 4 and 9 (NGF); lanes 5 and 10 (NT-3); lanes 6 and 11 (NT-4). The neurotrophins are expressed in the CNS but not in the migrating epidermal cells.
