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doi: 10.1242/10.1242/dev.00386

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On the turning of Xenopus retinal axons induced by ephrin-A5

¹ Max-Planck-Institute for Developmental Biology, Spemannstrasse 35, 72076 Tübingen, Germany
² MRC Centre for Developmental Neurobiology, King's College London, New Hunt's House, Guy's Hospital Campus, London SE1 1UL, UK

View larger version (73K): [in a new window]   Fig. 1. Xenopus retinal axons express EphA receptors. Explants from 2 day Xenopus retinae were grown on matrigel for 48 hours. (A) Incubation with ephrin-A5-AP, which binds all EphA receptors, results in a dark labelling of retinal axons. (B) Incubation with AP alone leads to no staining. Scale bar:100 µm.

View larger version (137K): [in a new window]   Fig. 2. In vitro guidance of Xenopus retinal axons by ephrin-A5-Fc in the stripe assay. (A) Striped outgrowth of retinal axons is observed when they are given the choice of growing on either ephrin-A5-Fc containing (yellow) or Fc containing lanes (orange). Axons avoid the ephrin-A5 containing lanes. (B) Both stripes have been coated with Fc protein alone. Axons show no preference for either type of lanes. Laminin was used as an outgrowth promoting substratum.

View larger version (140K): [in a new window]   Fig. 3. Growth cone turning of retinal axons after contact with an ephrin-A5-coated bead. (Top, left) Xenopus retinal growth cone filopodia contact an ephrin-A5-Fc coated bead. The bead is located slightly to the right of the pathway of the growth cone. (Top, right) The growth cone has moved slowly forward and has made extensive filopodial and lamellipodial contact to the bead. (Bottom, left) The growth cone has started to detach from the bead, the central domain of the growth cone has moved away from the bead. (Bottom, right) The growth cone has almost detached from the bead. Part of the growth cone previously in contact with the bead appears to be collapsed. There are only very few filopodial contacts left with the bead. Its growth direction has changed by an angle of –41° to the left (away from the bead). New filopodia have been formed on the side opposite to the bead. In total, a mean turning angle of –41°±2.6° was determined for experiments with ephrin-A5-coated beads (n=8). In control experiments with non-coated or protein A-coated beads (n=10), growth cones did not show a change in growth direction (–6°±0.4°; n=10). As schematised in the picture (bottom, right), turning angles were measured by determining the angle between the axon shaft before bead contact and after passing the bead. Experiments were performed using laminin as an outgrowth promoting substratum. The pictures were taken using a Zeiss inverted phase contrast microscope with a magnification of 63. The beads were 4.5 µm diameter.

View larger version (13K): [in a new window]   Fig. 4. Summary of data from the growth cone turning assay using ephrin-A5-coated beads and laser tweezers. Ephrin-A5-coated or control beads were placed (using laser tweezers) in front of the retinal growth cones. After contact with ephrin-A5-coated beads, roughly half of the observed growth cones showed growth cone collapse (7 of 15; 47%), while the other half reacted with repulsion (7 of 15; 47%). One growth cone did not show any reaction. Control beads (uncoated or coated with protein A) did not elicit any change in growth cone behaviour in most cases (10 of 12; 83%). However, two growth cones showed a growth cone collapse. In all experiments laminin was used as an outgrowth promoting substratum.

View larger version (15K): [in a new window]   Fig. 5. Cumulative distribution plot of turning angles of retinal growth cones in response to gradients of various axon guidance molecules. On the x axis, turning angles with negative values indicate repulsion and positive values indicate attraction. The y axis gives the percentage of growth cones with turning angles greater than the corresponding angle on the abscissa. Both BDNF (50 µg/ml) and netrin 1 (5 µg/ml) induce repulsive reactions with growth cones turning away from the pipette (avoiding higher concentrations) (not shown here, see Fig. 6) (Mann-Whitney test: P0.012). In an ephrin-A5-Fc gradient, growth cones show substratum-dependent turning responses. On laminin, growth cones turn towards the pipette (attraction) with a mean turning angle of +20°, on fibronectin the turning behaviour is converted to repulsion with a mean turning angle of –25°. Controls consisted of axons growing on laminin or fibronectin without guidance molecules in the pipette. Here growth cones were neither repelled nor attracted. The turning responses are statistically significant (P<0.003, Mann-Whitney test).

View larger version (10K): [in a new window]   Fig. 6. Summary of the turning responses of retinal growth cones caused by different axon guidance molecules. Growth cones are repelled by netrin 1 (5 µg/ml) and BDNF (50 µg/ml). Ephrin-A5-Fc at 20 µg/ml induces a substratum-dependent turning on laminin leading to attraction, whereas on fibronectin it leads to repulsion. Shown are mean turning angles (see also Fig. 5). Error bars represent s.e.m.