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Figure 1- EGCG impairs optic tract development. A: mical3 mRNA as assessed by in situ hybridization is expressed in the ganglion cell (gcl) layer and lens (ln) of the eye of a stage 35/36 X. laevis embryo. B: Schematic showing the brain exposure and the position of the leading RGC axons (arrow) at the time of exposure. C–F: Anterogradely HRPâ€labeled (brown) RGCaxon tracts in X. laevis brains exposed to control or EGCG solutions between stage 33/34 and stage 40. G: Graph of the percentage of embryos with optic tracts that reached the optic tectum (optic tectum), failed to navigate the turn in the diencephalon (diencephalon), or arrested in the ventralforebrain (v. forebrain) in control and EGCG exposed conditions. di, diencephalon; gcl, ganglion cell layer; ln, lens; oc, optic chiasm; pi, pineal gland; tec, optic tectum. The number of embryos examined under each condition is presented within the bars in G, and are pooled from at least three independent experiments.
Figure 2-EGCGâ€treatment in culture increases filopodial numbers. A–I: Representative images of phalloidinâ€labeled RGC axons exposed to control H2O (A–C), 25 μM EGCG (D–F), DMSO (G), 400 μM MCIâ€186 (H), and 200 μM apocynin (I) solutions. J–L: Graphs depicting the average number of growth cone filopodia and the percentage of collapsed growth cones for each treatment group. Conditions are indicated beneath each bar. The number of explants analyzed is presented within each bar, a minimum of two separate experiments were performed for each condition. Analysis: In J, a oneâ€way ANOVA indicated a main effect of treatment for number of filopodia (F(3,61) = 22.4; P < 0.01) and percentage collapse (F(3,61) = 22.3; P < 0.01). Post hoc analyses using the Dunnett test revealed a significant difference in number of filopodia in the 25 μM EGCG group (P < 0.01) and percentage collapse in the 10 μM (P < 0.05) and 25 μM (P < 0.01) EGCG groups when compared with controls. In K, a oneâ€way ANOVA indicated no effect of treatment for number of filopodia (F(2,19) = 1.76; P = 0.20) and percentage collapse (F(2,19) = 0.96; P = 0.40). In L, a oneâ€way ANOVA indicated no effect of treatment for number of filopodia (F(2,20) = 1.54, P = 0.24) and percentage collapse (F(2,20) = 2.19; P = 0.14). Data are mean ± SEM. ** P < 0.01, and * P < 0.05.
Figure 3-EGCG increases filopodial number and decreases rate of outgrowth of RGC axons in vivo. A: Schematic illustrating the approximate position of RGC axons during the recording period, as well as the mounting and imaging technique. Developing GFPâ€positive axons were exposed and embryos were anchored exposedâ€side down in a glass bottom dish for imaging with an inverted fluorescent microscope. B,C: The development of GFPâ€labeled RGC growth cones was monitored by time lapse image capture in vivo before and after the addition of EGCG to a final concentration of 25–50 μM. The time, in min, relative to the addition of EGCG is indicated in the lower right corner of each image. D–F: Graphs representing the number of filopodia per growth cone, the cumulative length of the filopodia on each growth cone, and the rate of outgrowth before and after the addition of EGCG. Number of axons tracked are presented within each bar. Analysis: A paired samples tâ€test was used for all comparisons. For number of filopodia, P < 0.01; for cumulative length of filpodia, P = 0.7; for outgrowth rate, P < 0.01. Scale bar = 10 μm in C. Data are mean ± SEM. **P < 0.01.
Figure 4- RGC axons fail to navigate the midâ€diencephalic turn in the presence of the actin stabilizer, jasplakinolide. A,B: X. laevis brains were exposed to control or jasplakinolide solutions from stage 33/34 to stage 40. C: Graph showing the percentage of embryos with optic tracts that reached the optic tectum (tectum), or failed to navigate the turn in the diencephalon (diencephalon). Number of brains analyzed for each condition indicated within the bars.
Figure 5-RGCaxon navigation at the optic chiasm and into the tectum is unaffected by EGCG. A: Schematic indicating the exposure technique for RGC axons at the optic chiasm. B,C: Developing RGC axons were exposed to control or 25 μM EGCG as they crossed the optic chiasm. Arrows in B illustrate chiasm width measurement. D: Graph comparing the width of the optic chiasm in control and EGCGâ€treated embryos. E: Schematic illustrating the position of leading RGC axons for the stage 37/38 exposure. F,G: X. laevis brains were exposed to control or 25 μM EGCG solutions during the period of RGC extension into the optic tectum. Arrows in F illustrate the width ratio (x/y) measurements. H: Graph comparing the width ratio for control and EGCGâ€treated embryos. For both D and H, the number of brains analyzed for each condition are indicated within the bars. A minimum of three experiments was performed for each condition. Data are mean ± SEM. Analysis: A Student's tâ€test was used to compare control and 25 μM EGCGâ€treated groups, and no significant difference was found for the chiasm width (P = 0.07) or the width ratio (P = 0.16). A, anterior; D, dorsal; di, diencephalon; oc, optic chiasm; P, posterior; rgc, retinal ganglion cell; tec, optic tectum; V, ventral. Dashed line in F indicates approximate anterior tectal boundary.
