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First published online 25 February 2004
doi: 10.1242/dev.01041

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Disruption of gradient expression of Zic3 resulted in abnormal intra-retinal axon projection

Department of Medicine and Cell Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA

View larger version (15K): [in a new window]   Fig. 1. Intraretinal axon guidance. Ganglion axons at all positions inside the retina travel in the optic fiber layer (OFL) and project to the center of the retina, the optic disc (OD). At the optic disc, the ganglion axons exit the retina and join the optic nerve (ON). GCL, ganglion cell layer (where the cell bodies of the ganglion cell reside). PR, photoreceptor layer (where the photoreceptor cells are localized at the other side of the retina).

View larger version (71K): [in a new window]   Fig. 2. Expression of Zic3 mRNA in whole-mount chicken embryos (A-C) and flat-mount retinas (D-G) by in situ hybridization. (A) HH stage 10; (B) HH stage 12; (C) HH stage 17. Zic3 is expressed in early optic vesicle (arrows in A and B) and optic cup (arrow in C). (D) E5.5 retina; (E) E7 retina; (F) E9 retina; (G) high-magnification view of E7 retina. Zic3 is expressed in a periphery-to-center gradient with higher expression at the periphery. All the flat-mount retinas were shown with the ganglion cell side upwards, oriented with the anterior side (A) towards the left, the posterior side (P) towards the right, the dorsal side (D) upwards and the ventral side (V) downwards.

View larger version (114K): [in a new window]   Fig. 3. Expression of Zic3 mRNA on cross sections of embryonic chick eye. In situ hybridization experiments were performed on the cryosections of embryonic chick eye at various stages: (A) E4.5; (B) E6; (C,D) E9; (E,F) E16. Note that Zic3 mRNA expression is in a periphery-high, center-low gradient at E4.5 and E6 (A,B). At E9, most of Zic3 expression is confined to the retinal periphery (labeled `P' in C,D). Zic3 is also highly expressed in the ciliary margin area (C,E,F). At E16, Zic3 expression can be barely detected inside the retina while at a high level at the ciliary margin (E,F). C, center of the retina; P, periphery of the retina; R, retina; CM, ciliary margin. Arrowhead in C indicates the remaining undifferentiated cells located in the inner nuclear layer that are weakly expressing Zic3 mRNA at the retinal center. Scale bars: 100 µm in A,B,D-F; 400 µm in C.

View larger version (130K): [in a new window]   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.

View larger version (121K): [in a new window]   Fig. 5. The most common axonal phenotype of Zic3 misexpression is that the axons misrouted to the sub-retinal space at the photoreceptor side. The axons were photographed at the focal plane of the optic fiber layer (A,C,E). White arrows indicate the direction toward the optic disc. The same area in A,E was also photographed with the focal plane at the photoreceptor side to show that some axons were present at the sub-retinal space (B and F, respectively). The area shown in D at the photoreceptor side is a little distance away from the area shown in C. Note that the misrouted axons in the subretinal space were very disorganized without clear direction. Scale bar: 50 µm.

View larger version (118K): [in a new window]   Fig. 6. Misexpression of Zic3 did not alter the expression of islet 1 or induce the expression of a ciliary margin marker, collagen IX. RCAS-Zic3 virus-infected retinas were analyzed at E7. Samples were double stained with an anti-viral GAG antibody (B,F) and an anti-islet 1 antibody (A), or an anti-collagen IX antibody 2C2 (C,E). (D) A bright-field image is shown. The same areas are shown in A and B, C and D, and E and F, respectively. Note that there was no apparent difference in either the number or distribution of islet 1-expressing cells in the infected area (A,B) compared with the uninfected wild-type area. Collagen IX is highly expressed in the very periphery of the retina, the prospective ciliary margin region (arrow in C), but not inside the retina, identical to the patterns in the uninjected control retina (not shown). Scale bar: 50 µm.

View larger version (86K): [in a new window]   Fig. 7. Misexpression of Zic3 did not grossly alter retinal lamination. Cryosections of E10 retinas infected with RCAS-Zic3 were stained with anti-neurofilament (A-C) and various cell type-specific antibodies including: anti-visinin (G), anti-islet 1 (H) and anti-Pax6 (I). (C-F) Same RCAS-Zic3-infected retinal section was shown stained with anti-neurofilament (C), DAPI (D), anti-viral GAG antibody (F) and in phase contrast (E). Note some axons in the RCAS-Zic3-infected retinas misrouted to the subretinal spaces at the photoreceptor side of the retina (arrowheads, A-C). However, the laminated structure of the retina was largely normal (D-I), except some dispersion of cells towards the photoreceptor side (arrowheads, H and I). The optic fiber layer was largely normal, except for some protrusions of the axons (arrows in A,B). Frequently, however, the photoreceptor layer was broken (arrowheads, D and G). Note that the anti-viral GAG antibody staining was much more intense at the sites of axon crossing-over than the nearby infected region (arrowhead in F). PR, photoreceptor layer; OFL, optic fiber layer; GCL, ganglion cell layer; INL, inner nuclear layer.

View larger version (76K): [in a new window]   Fig. 8. Apoptosis was increased at the sites of axon crossing-over. E10 retinas infected with RCAS-Zic3 were analyzed by TUNEL assay (A,D-F), followed by staining with anti-neurofilament (B) and anti-viral GAG (P27) antibodies (C). At the sites with axon crossing-over (arrows in B), there was an increase in the number of apoptotic cells (arrows in A) and intensity of anti-viral GAG staining (arrows in C). Note that the adjacent area was also completely infected (marked with *), and some of the fluorescent signals were blocked by the DAB precipitates from the TUNEL assay. In addition, the apoptotic cells tended to concentrate at the photoreceptor side, not the ganglion side (A,D-F). PR, photoreceptor layer; G, ganglion cell layer. Scale bar: 50 µm.

View larger version (76K): [in a new window]   Fig. 9. Stripe assays. The membrane fragments prepared from various retinal tissues were compared for their abilities to support axonal growth by stripe assays. Green fluorescent beads were mixed in to mark the second stripes. (A) The membrane fragments prepared from the central halves of the uninjected retinas were laid down both in the first and second stripes. (B) The membrane fragments prepared from the RCAS-Zic3-injected retinas were laid down in the first stripes, whereas those from the RCAS-GFP-injected retinas were filled in as the second stripes. (C) Membrane fragments prepared from the peripheral E8 retinas were laid down in the first stripes, whereas those from the central E8 retinas were filled in as the second stripes. Note that the membrane fragments prepared from the RCAS-GFP-injected retina were more preferable than those from the RCAS-Zic3-injected retina, and the membrane preps from the central retinas were more preferable than those from the peripheral retinas. (D) Quantification of the stripe assay results. Scale bar: 100 µm.

View larger version (144K): [in a new window]   Fig. 10. Zic3 misexpression did not alter the expression of netrin 1 and CSPGs. The RCAS-Zic3-infected samples were hybridized with the netrin 1 probe (A) and then immunostained with an anti-GAG antibody (p27) (B). Netrin 1 is highly expressed in the optic disc (OD, in A) and optic fissure, similar to wild-type uninjected retinas. The RCAS-Zic3 virus-infected retinas were also double stained with an anti-chondroitin sulfate antibody (CS-56) (C,E) and an anti-viral GAG antibody (P27) (D,F). CS-56 staining is intense at the periphery of the retina. However, a lower level of CS-56 staining is also visible in the center of the retina (E). Comparing the RCAS-Zic3 virus-infected area (arrowheads in D,F) with the adjacent uninfected areas, there is no difference in the level of CS-56 expression (C-F). Identical fields are shown in A and B, C and D, E and F, respectively. Scale bar: 200 µm.

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