Fig. 9. SU5402 treatment induces abnormal RGC axon trajectories but does not affect retinal lamination. (A-C,E-G) diI/diO-labeled RGC axons in SU5402-treated (A-C,E,F) and wild-type (G) embryos at 2 dpf (C) or 4 dpf (A,B,E-G). (A,B) Dorsal view showing ipsilateral projections (A) and mixed ipsi- and contralateral projections (B) (10 µM SU5402, 8-96 hpf). (C) Frontal view showing RGC axons extending ipsilaterally before reaching the optic chiasm (10µM SU5402, 8-48 hpf). (D) slit3 expression in SU5402-treated 30 hpf embryos. The distance between the anterior commissure and retinal axons is narrow (arrows/lines). White asterisks indicate ectopic slit3 expression at positions where retinal axons enter the midline area (compare with Fig. 6K). (E) Dorsal view showing retinal axons which fail to enter target areas in the tectum (white asterisks) (10 µM SU5402, 30-96 hpf). (F,G) Lateral views. Wild-type retinal axons project and arborize in the optic tectum, but SU5402-treated axon fails to enter the tectum (white asterisk). (H) SU5402-treated 60 hpf retinae (10 µM, soaked from 8 hpf) labeled with anti-phosphorylated-histone H3 antibody. The locations of dividing cells (arrowheads) resemble wild-type (compare with Fig. 4G). (I) SU5402-treated 3 dpf retinae (10 µM, soaked from 8 hpf) labeled with anti-Pax6 antibody. (J) SU5402-treated 60 hpf retinae (10 µM, soaked from 8 hpf) labeled with BODIPY-ceramide. Arrowheads indicate the IPL (compare with Fig. 2M). (K) SU5402-treated 60 hpf retinae (10 µM, soaked from 30 hpf) labeled with BODIPY-ceramide. (L) Lateral view of a 48 hpf living SU5402-treated eye (10 µM SU5402, 8-48 hpf). (M) Plastic section through the closed choroid fissure (red arrowheads) of a 4 dpf SU5402-treated retina. (N) Plastic section of 4 dpf SU5402-treated retina (10 µM, soaked from 8 hpf). Retinal lamination is globally normal, but some dying cells are observed as dark-stained nuclei (red arrowheads). (O) SU5402-treated 60 hpf retinae labeled with BODIPY-ceramide. Embryos were soaked with 1mM SU5402 from 30 hpf. No patches of IPL neuropil are observed, but instead large areas of cell degeneration are evident (red asterisks). Such severe cell death was not observed with treatment of 100 µM SU5402 (data not shown).

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