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Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues

Departments of Ophthalmology and Physiology, University of California, San Francisco, San Francisco, CA 94143, USA

View larger version (37K): [in a new window]   Fig. 1. Characterization of recombinant proteins consisting of EphB receptor extracellular domains fused to the immunoglobulin Fc domain. (A) Polyacrylamide gel electrophoresis and silver staining of protein A column-purified EphB1•ECD-Fc (lane 1), EphB2•ECD-Fc (lane 2), EphB3•ECD-Fc (lane 3), and Fc control protein (lane 4). (B-E) Binding of EphB1•ECD-Fc (B), EphB2•ECD-Fc (C), EphB3•ECD-Fc (D) and Fc control (E) onto E14 embryonic retinal axons and growth cones in culture, visualized with Cy3-conjugated anti-Fc antibody. No binding of the Fc control protein (E) was detected. Scale bar, 10 µm.

View larger version (143K): [in a new window]   Fig. 2. Retinal axon responses to EphB•ECD-Fc proteins in substratum choice assays. (A,C) Retinal axons, visualized with TR-phalloidin, grow freely into regions containing Fc control protein. (B,D) Reduced retinal axon growth on EphB2-ECD substratum region. Note tendency of individual axons to stop or turn at the border. C,D are higher magnification views of A,B at substratum border. Asterisks in A,B indicate test substratum region. Scale bars, 200 µm (A and B are same magnification, as are C and D).

View larger version (21K): [in a new window]   Fig. 3. Quantitation of substratum choice assay. The response rate expressed as the percentage of axons turning or stopping at the border of EphB-ECD or Fc control containing substratum. ‘None’ shows the results of quantitation of a virtual border drawn on a uniform laminin substratum. Results from dorsal and ventral retinal explants were quantified separately. (A) Response rate (percentage either stopping or turning at border) from pooling all individual axon encounters of a given condition. (B) Mean percentage of axons stopping or turning at the border for each explant. Error bars show s.e.m.

View larger version (145K): [in a new window]   Fig. 4. Time-lapse microscopy of retinal growth cone responses to soluble EphB-ECDs or Fc protein applied by micropipette. Time in minutes relative to beginning of reagent application (0) is indicated in top left of panels. (A) Retinal axon elongation and growth cone behavior before and after application of soluble Fc protein. The growth cone was unaffected and continued towards the micropipette tip (outline in upper right). The 4th panel shows the fluorescent marker gradient after expulsion of reagent from the pipette. (B) Growth cone collapse triggered by EphB1-ECD. The micropipette tip was located at the top approx. 180 µm away from the growth cone and the direction of EphB1-ECD dispersal is indicated by the arrow. (C) Example of cessation of growth cone motile activity after application of EphB2-ECD. Black arrow indicates direction of protein dispersal from the micropipette tip located 70 µm away. Within 5 minutes of application, the growth cone ceased filopodial and lamellipodial movements (black arrowheads) and remained in this ‘frozen’ state without retraction. Cellular material (white arrowhead) accumulated within the growth cone body, indicating continuing axonal transport. Scale bars, 10 µm.

View larger version (15K): [in a new window]   Fig. 5. Graph quantifying the percentage of retinal growth cones inhibited by micropipette application of specific EphB-ECD or Fc control reagents. ‘Heat treated’ shows the responses to heat-inactivated EphB2-ECD. Responses include growth cone collapse, cessation of growth cone motility, cessation or significant reduction in axon elongation rate, or growth cone turning (see Table 1).

View larger version (24K): [in a new window]   Fig. 6. Model of EphB proteins acting as inhibitory axon guidance cues governing dorsal retinal axon pathfinding to the optic disc. In wild-type retina (left), the high-ventral to low-dorsal gradient of EphB protein expression (based primarily on EphB2 protein expression) is depicted in purple. Dorsal RGC axons growing ventrally towards the optic disc encounter an increasing gradient of EphB protein extracellular domains serving as inhibitory axon guidance cues. It is proposed that this inhibition leads to tight axon fasciculation as RGC axons approach the optic disc and that the high EphB protein expression in the ventral retina prevents inappropriate dorsal axon growth into the opposite half of the retina. Both these constraints are removed in the EphB2 EphB3 null mutant mice (right) resulting in the observed dorsal RGC axon defasciculation defects around the optic disc and the aberrant axon growth into the ventral half of the retina.

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