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First published online June 8, 2006
doi: 10.1242/10.1242/dev.02412

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Growth cone navigation in substrate-bound ephrin gradients

¹ Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, 72076 Tuebingen, Germany.
² Universität Karlsruhe (TH), Zoologisches Institut I, Zell- und Neurobiologie, Haid-und-Neu-Strasse 9, 76131 Karlsruhe, Germany.
³ Institut für Mikro- und Nanotechnologie, Interstaatliche Hochschule für Technik Buchs NTB, Werdenbergstrasse 4, CH-9471 Buchs, Switzerland.
⁴ Labor für Mikro- und Nanotechnologie, Paul Scherrer Institut, CH-5232 Villigen-PSI, Switzerland.

View larger version (30K): [in a new window]   Fig. 1. Substrate-bound gradients fabricated by microcontact printing. (A) Schematic of a growth cone in a continuous gradient. (B,C) Growth cones in different discontinuous gradients fabricated by microcontact printing: (B) a steep gradient built by an array of stripes and (C) a shallow gradient built by dots. Antibody-stained ephrin is shown in red; phalloidin-stained actin in black. (D,E) The gradients shown in B and C in lower magnification extend over a distance of 250 µm. The relative slope 1 of the gradient in D is reduced to 1/3 in E by interrupting the ephrin stripes. (F) Averaged fluorescence intensities measured in a 20-µm-wide measuring field moved along the axis of the gradients in D (blue curve) and E (red curve). Scale bars: 15 µm in B,C; 50 µm in D,E.

View larger version (136K): [in a new window]   Fig. 2. Temporal, but not nasal, RGC axons form stop zones in ephrin gradients. (A) Nasal growth cones overgrow an ephrin gradient without being affected. (B) By contrast, temporal growth cones form a distinct stop zone in the ephrin gradient. As new axons continuously leave the explant, growth cones are observed in the area between the explant and the stop zone. Arrows mark growth cones that do not respond to the gradient. To the right, the ephrin gradient is shown in black with a red arrowhead marking the beginning of the gradient. Antibody-stained ephrin is shown in red; phalloidin-stained actin in black. Scale bar: 100 µm.

View larger version (156K): [in a new window]   Fig. 3. Growth cone dynamics within the stop zone of shallow and steep gradients. (A)Growth cones display an explorative equilibrium of forward and backward movements within the stop zone of a shallow gradient. A temporal growth cone approaches the gradient. After 15 minutes it pauses, rearranges its lamellipodia and starts to retract to the beginning of the gradient (30 minutes). Between 45 and 60 minutes the growth cone advances anew in the gradient and subsequently pauses (65 minutes). Small side branches appear along the axon shaft and a growth cone like structure builds at the beginning of the gradient (70 minutes). The axon shaft left deeper in the gradient retracts. Between 80 and 120 minutes the growth cone pauses under continued filopodial activity within the gradient. (B) Steep ephrin gradients can cause more pronounced reactions, including transient, partial growth cone collapse and change of growth cone morphology within the stop zone. A temporal growth cone entering a steep gradient starts withdrawing its lamellipodia (4 minutes), and retracts completely from the gradient (5 minutes). After 15 minutes the growth cone marked by an arrow recovers from the transient collapse and invades the gradient anew. A second growth cone (arrowhead) advances. Between 20 and 22 minutes the growth cone marked by an arrow undergoes a rapid change in morphology and realigns its filopodia along the printed ephrin pattern. The same happens to the growth cone marked by an arrowhead between 20 and 30 minutes. Afterwards, both growth cones remain stationary and undergo only small morphological changes, adhering partially to the lines of the gradient. Antibody-stained ephrin is shown in red; axons are shown in phase contrast. Scale bar: 20 µm.

View larger version (49K): [in a new window]   Fig. 4. The position of the stop zone depends on the slope of the gradient. (A) Temporal axons in three different gradients printed with 8 µg/ml ephrinA5. Antibody-stained ephrin is shown in red; axonal actin in black. In the steepest gradient (relative slope 1) the stop zone is located at the beginning of the gradient. In shallower gradients (relative slopes 2/3 and 1/3), stop zones are shifted deeper into the gradient. The position of the stop zone is marked with arrowheads and the overgrown distance within the gradient is indicated in µm. The axis to the right (in µm) corresponds to the x-axis in B. Scale bar: 100 µm. (B) Standardized fluorescence intensity curves of ephrin and the corresponding axonal fluorescence are depicted in blue (gradient slope 1), light blue (2/3) and red (1/3). The discontinuity of the gradient reflects in the sinuous course of the ephrin fluorescence intensity curve. The x-coordinate of the intersection of each axonal curve with the arithmetic mean (pink line) is defined as the position of the stop zone. The shallower the gradient, the further the stop zone is shifted in the direction of the positive x-axis.

View larger version (19K): [in a new window]   Fig. 5. Quantification of average axonal stop zones in different gradients. (A,B) The two graphs show the averaged axonal growth curves for five gradients printed with 8 µg/ml (A) or 4 µg/ml (B). The relative slope of the gradients is indicated with the colour code given in A. In A, the averaged curve of Fc controls is shown in black, the curve of nasal controls in gray and the arithmetic mean of the axonal fluorescence in pink. For both ephrin concentrations axonal curves intersect the arithmetic average later the shallower the gradient is. Curves in B intersect the arithmetic mean later than curves in A. The axonal curves were averaged from n=7 (gradient slope 1/6), n=8 (1/3), n=9 (1/2), n=5 (2/3), n=6 (1), n=10 (Fc controls), n=5 (nasal controls) experiments in A, and from n=9 (1/6), n=9 (1/3), n=8 (1/2), n=13 (2/3), n=7 (1) experiments in B. Mean stop point positions are 64.9±4.2 µm (1/6), 58.3±3.9 µm (1/3), 51.2±3.1 µm (1/2), 40.2±3.6 µm (2/3), 24.4±2.2 µm (1) in A and 97.9±6.8 µm (1/6), 86.8±7.3 µm (1/3), 80.3±8.0 µm (1/2), 73.8±5.1 µm (2/3), 52.3±5.5 µm (1) in B. (C) Plotting the stop point positions determined in A and B and stop point positions in gradients printed with 2 µg/ml against the relative slope of the corresponding gradients shows that the two parameters correlate in a linear way. Error bars indicate the standard error. Mean stop positions in gradients printed with 2 µg/ml are: 178.8 µm (1/6, n=4), 169.6±8.5 µm (1/3, n=5), 152.0±10.2 µm (1/2, n=5), 145.5±12.4 µm (2/3, n=5), 110.3±17.5 µm (1, n=5). Due to the data examination method, no standard error could be calculated for the mean stop point position in the gradient with the slope 1/6 (marked with a star, for details see Materials and methods). P<0.002 by ANOVA for both of the two groups of 8 µg/ml and 4 µg/ml gradients and P<0.156 for 2 µg/ml gradients. Studens t-test was applied to compare the mean stop points in gradients with the same slope but printed with 8 µg/ml and 4 µg/ml: P<0.002 (gradient slope 1/6), P<0.006 (1/3), P<0.003 (1/2), P<0.001 (2/3), P<0.002 (1).

View larger version (54K): [in a new window]   Fig. 6. Temporal axons stop in non-graded striped ephrin patterns. (A) Stop zones of temporal axons in non-graded patterns printed with 8 µg/ml ephrinA5. Antibody-stained ephrin is shown in red; phalloidin-stained axonal actin in black. In the pattern consisting of 0.3 µm thick stripes, growth cones stop later than in the pattern of 1.8 µm stripes. Scale bar: 100 µm. (B) The position of the axonal stop points in three different stripe patterns is plotted against the stripe thickness for patterns printed with 8, 4 and 2 µg/ml. Mean stop points were 49.1±2.3 µm (stripe thickness 0.3 µm, n=5), 37.4±3.0 µm (0.6 µm, n=6), 28.3±1.7 µm (1.8 µm, n=6) for 8-µg/ml patterns, 83.7±7.8 µm (0.3 µm, n=6), 66.0±4.3 µm (0.6 µm, n=7), 48.8±4.2 µm (1.8 µm, n=10) for 4 µg/ml patterns and 122.4 µm (0.3 µm, n=2), 93.7 µm (0.6 µm, n=3), 82.6 µm (1.8 µm, n=3) for 2 µg/ml patterns. Error bars indicate standard error. Due to the data examination method, no standard error could be calculated for the mean stop point position in the patterns printed with 2 µg/ml (marked with a stars, for details see Materials and methods). P<0.001 by ANOVA for 8 µg/ml patterns and P<0.006 for 4 µg/ml patterns. Studens t-test for small sample sizes was applied to compare the mean stop points in patterns with the same stripe thickness printed with 8 and 4 µg/ml: P<0.001 (stripe thickness 0.3 µm), P<0.001 (stripe thickness 0.6 µm), P<0.003 (stripe thickness 1.8 µm).

View larger version (28K): [in a new window]   Fig. 7. A balance of summation of the total encountered ephrin and adjustment of sensitivity to the local ephrin concentration may lead to growth cone stop. (A) Schematic of the total amount of encountered ephrin in the gradient (red dots in the rectangle on the left) and local encountered ephrin (red dots in the rectangle on the right). (B) When the local ephrin concentration is plotted against the total encountered ephrin-covered area at the stop point for all patterns, a linear correlation becomes apparent. Blue data points are derived from patterns printed with 8 µg/ml, red data points from patterns printed with 4 µg/ml ephrin printing ink. The relative slope of the gradient (1, 2/3, 1/2, 1/3, 1/6) or the thickness of the stripes in the non-graded patterns (1.8, 0.6, 0.3 µm) is noted next to the data point. Data points for gradients with the relative slope 1 (marked with stars) deviate noticeable from the linear smoothing function. For explanation, see text. (C) The correlation shown in B can be explained by a model in which the repulsive ephrin signal is summed up over time by accumulation of a slowly degrading signaling molecule X, which positively affects growth cone stop, retraction and/or collapse. Temporally delayed (t), a second, rapidly degrading component Y leads to an adjustment of the sensitivity to the local ephrin concentration, counteracting the output of X. The activity of X is postulated to be proportional to the total encountered ephrin, whereas the activity of Y is proportional to the local ephrin concentration. The growth cone stops when both activities are proportionable.