Figure 7.
RGC axon projection toward the optic disc was similarly disrupted by misexpression of Shh. A, Optic vesicles were injected with retroviruses expressing either Shh (B, C, E, F) or a control GFP protein (A, D) at HH stage 10-11. The infected retinas were harvested at E5.5-E6, flat mounted, and analyzed by staining with an anti-neurofilament antibody (A-F). As shown in A and D, the RGC axons appeared normal in the control RCAS-GFP-injected retinas, similar to uninjected wild-type retinas (data not shown). However, in the RCAS-Shh-injected samples, the axons appeared grossly disorganized and lacked central orientation (B, C). White arrows indicate the direction toward the optic disc. Another common phenotype caused by misexpression of Shh was the abnormal crossing of RGC axons, forming ectopic optic discs. Retinal axons normally converge only at the optic disc (arrowheads in D) in which they exit the retina, as shown in this control RCAS-GFP-infected sample (D). However, misexpression of Shh resulted in abnormal crossing of RGC axons at ectopic sites before reaching the optic disc (E, F, arrowheads). G-I, RCAS-Shh-injected samples were sectioned and labeled with an anti-neurofilament antibody (G) and an anti-viral GAG antibody (H). Despite the gross mistargeting of RGC axons in the RCAS-Shh-injected retina, the axons were mostly confined to the GCL and were not present in the deeper layers of the retina. Scale bars: A-C, F, 50 μm; D, E, 400 μm.