Fig. 4. Retroviral expression of Zic3 caused abnormality in retinal axon projection toward the optic disc. (A) Retroviral constructs. LTR, long terminal repeat; gag, gene encoding the viral capsid proteins; env, gene encoding viral envelop protein; pol, gene encoding viral reverse transcriptase. Colored boxes indicate the inserted GFP (green) and Zic3 (pink) cDNAs. (B-G) Infected at HH stage 10-11, the retinas were analyzed at E12 by co-staining with an anti-neurofilament antibody (B-F) and an anti-viral GAG antibody (G). The axons were viewed from the optic fiber side of the flat-mount retinas. White arrows indicate the direction towards the optic disc. (B) In control virus RCAS-GFP-injected retinas, axons appeared normal, projecting straight toward the center of the retina. In the RCAS-Zic3-injected retinas, however, some axons showed abnormal trajectories (C-G). Although the infected area identified by anti-viral GAG antibody (P27) was much larger than the area with axonal phenotype, the area with phenotype appeared to be more intensely stained by anti-p27 (* in G). Note the whole area shown in G is infected. Images in F and G are from the same field. Scale bar: 50 µm.