PMC

Retinal explants were treated with clustered 0.5 µg/ml Fc or ephrin-B2 for 30 minutes, then fixed and immunostained for neurofilament (NF, green), with Cy3-conjugated phalloidin (Phal, red) used to visualize growth cones. Insets depict individual examples of growth cones for each condition. Prominent growth cones with lamellipodia and/or filopodia were visible on VT and DT axons 30 minutes after Fc treatment, as well as DT axons treated with ephrin-B2. Conversely, nearly all VT growth cones were collapsed 30 minutes after ephrin-B2 exposure. Scale bars = 20 µm.

(A, B) Graphs depicting the extension and retraction of RGC axons from VT explants exposed to either 0.5 µg/ml Fc or ephrin-B2 after 15 minutes (black arrows). Each individual axon is represented by a different colored line. Note that the trace of some VT axons flattens out after ephrin-B2 treatment, indicating that the axon retracted out of the field of view. (C) Traces from individual DT axons reveal significant variability in their response to ephrin-B2, with many axons pausing and then either extending or undergoing moderate levels of retraction. (D) Average trace of VT and DT axon extension and retraction in response to ephrin-B2 or Fc control. Fc treatment has no effect on either VT or DT axon extension. Ephrin-B2 induces strong and rapid retraction of VT axons, whereas DT axons display slower and weaker retraction upon ephrin-B2 exposure. Average extension/retraction ± standard error for each condition 30 minutes after ephrin-B2 or Fc treatment (t₄₅ − t₁₅), + signifies extension and − signifies retraction: VT + Fc = +48.97 ± 20.82 µm; DT + Fc = +56.70 ± 17.10 µm; VT + ephrin-B2 = −249.98 ± 35.63 µm; DT + ephrin-B2 = −50.10 ± 34.10 µm.

Still frames taken from DIC time-lapse movies of growth cones from VT or DT retinal explants exposed to 0.5 µg/ml ephrin-B2 or human-Fc at t₁₅ minutes (dashed grey arrow). Black and white arrows highlight representative growth cones in each frame, with their corresponding location at t₀ indicated with a black or white dot, respectively. (A) VT growth cones (and DT growth cones, not shown) continue to advance unhindered after application of human-Fc. (B) The majority of VT growth cones collapse and retract within several minutes after exposure to ephrin-B2, with some axons retracting out of the field of view (white arrow). (C) The response of DT RGC growth cones upon ephrin-B2 treatment is dampened and more variable, with some axons retracting (black arrow) and other axons continuing to advance (white arrow). Scale bars = 20 µm.

(A) Still frames taken from DIC time-lapse imaging of VT growth cones incubated in SFM + 10 µM Y-27632 for 1–2 hours (last 15 minutes of Y-27632 pretreatment is t₀–t₁₅). At t₁₅, medium was replaced with SFM + 10 µM Y-27632 + 0.5 µg/ml ephrin-B2 for 30 minutes. Red and blue arrows highlight representative growth cones that were located at the corresponding red and blue dots at t₀. (B) Graph displays the extension and retraction of VT axons (individual axons represented by a colored line) pre-exposed to Y-27632, with ephrin-B2 added after t₁₅ (black arrow). Note that several axons recovered and extended forward after ephrin-B2 treatment, which was rarely observed in control ephrin-B2-treated VT explants (). n = 23 VT axons, average retraction 30 minutes after treatment: VT + ephrin-B2 + Y-27632 = −29.72 ± 34.10 µm (C) Summary of extension and retraction behaviors of axons from VT and DT explants exposed to Fc, ephrin-B2, and ephrin-B2 + Y-27632. Note that ROCK inhibition causes weaker and delayed axon retraction from VT explants, comparable to DT explants in ephrin-B2. Scale bar = 20 µm.

(A) Traces depict average path of RGC axons from VT, DT and VN retinal explants exposed to varying concentrations (5.0 µg/ml to 5.0 ng/ml) of ephrin-B2 at t₁₅ minutes (black arrow). 5.0 ng/ml ephrin-B2 had little effect on all retinal regions. DT axons (blue traces) undergo very weak axon retraction even at the highest ephrin-B2 doses. Axons from both VT (red traces) and VN (green traces) explants rapidly retract at a similar distance and timescale when treated with 5.0 µg/ml or 0.5 µg/ml ephrin-B2, but a significant difference is observed at 50 ng/ml ephrin-B2 (black arrowheads). n ≥ 12 DT growth cones, n ≥ 17 VN growth cones, n ≥ 11 VT growth cones for each condition. (B) Dose-response curves of different ephrin-B2 concentrations (5 ng/ml = 0.70, 50 ng/ml = 1.70, etc.) on the rate of axon retraction in the first four minutes immediately following ephrin-B2 treatment (t₁₆–t₂₀). Although not statistically significant, VT axons show an increased retraction rate, and thus ephrin-B2 sensitivity, compared to VN axons, as demonstrated by the leftward shift of the VT trace. Both VT and VN are significantly more sensitive compared to DT axons at 50, 500 and 5000 ng/ml ephrin-B2 (*, p ≤ .005).