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First published online 16 April 2008
doi: 10.1242/dev.020693

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Zic2 promotes axonal divergence at the optic chiasm midline by EphB1-dependent and -independent mechanisms

¹ Instituto de Neurociencias de Alicante (Consejo Superior de Investigaciones Científicas-Universidad Miguel Hernández, CSIC-UMH). Campus San Juan, Avd. Ramón y Cajal s/n, Alicante 03550, Spain.
² Departments of Pathology and Cell Biology, Department of Neuroscience, Columbia University, College of Physicians and Surgeons, 630 W. 168th Street, New York, NY 10032, USA.

View larger version (34K): [in this window] [in a new window]   Fig. 1. Ectopic expression of Zic2 in mouse RGCs by in utero electroporation. (A) Schematic summarizing electroporation procedures. Plasmids (blue) were microinjected into the eye cup at embryonic stages. Two or 3 days later, embryos were analyzed to visualize EGFP-targeted cells in retinal whole-mounts (right) and axons in the optic chiasm (left). (Ba) Retinal section of a E16.5 embryo co-electroporated at E13.5 with plasmids bearing the coding sequences of EGFP and Zic2. Green cells in the center of the retina are targeted cells after the electroporation process. Scale bar: 100 µm. (Bb-d) Higher magnification of the boxed area in Ba showing Zic2/EGFP-targeted cells. EGFP is visualized in green and ectopic Zic2 is visualized in red by anti-Zic2 labeling. In the merged image (d), all of the targeted cells are Zic2-positive. Scale bar: 30 µm. (C) Quantification of the proportion of cells that co-express EGFP, Zic2 or both, in embryonic retinas as compared with the total number of transfected cells after electroporation.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Zic2 is sufficient to change axonal behavior at the midline. Left panels show axonal trajectories of targeted RGCs at the optic chiasm. Middle panels show the location of the targeted RGCs (green) in retinal whole-mounts (gray). Right panels are cartoons summarizing the results for each particular case. (A) In mouse embryos electroporated only with plasmids bearing EGFP, many green axons originating from the central retina have reached or crossed the midline (dashed line). (B) In embryos ectopically expressing Zic2/EGFP in the central retina, many targeted axons have crossed the midline but in this case some growth cones are positioned at the midline and a few axons are also observed turning to the ipsilateral side. (C) In embryos electroporated only with plasmids bearing EGFP at E13.5 and sacrificed at E17.5, all green axons coming from the central retina cross the optic chiasm midline. (D) By contrast, embryos ectopically expressing Zic2 plus EGFP in the central retina show a significant increase in the number of axons that project ipsilaterally at the chiasm. (E) In embryos electroporated at E14.5 with plasmids bearing only EGFP and sacrificed at E17.5, all green axons coming from the central retina cross the midline at the optic chiasm. (F) The increase in the proportion of axons projecting ipsilaterally after Zic2 ectopic expression is modest when electroporation is performed at E14.5, as compared with electroporation at E13.5. Yellow arrowheads point to the ectopic ipsilateral projection. ON, optic nerve; cOT, contralateral optic tract; IOT, ipsilateral optic tract; d, dorsal; n, nasal; t, temporal; v, ventral. Scale bars: chiasm panels, 200 µm; retina panels, 500 µm.

View larger version (58K): [in this window] [in a new window]   Fig. 3. Zic2 and EphB1 are expressed in the same RGCs in the ventrotemporal (VT) crescent. (A) Spatiotemporal representation of Zic2 (red) and EphB1 (green) in mouse retina (gray). Zic2 expression is first seen in VT retina at E14.5. Expression peaks at E16.5 and then Zic2 is downregulated by E17.5-18.5. EphB1 expression in the VT retina commences at E14.5, lasting until E17.5-18.5. EphB1 expression then expands into the entire retina in retinal cells other than RGCs. EphB1 expression is also observed in the central retina from E13.5-15.5. (Ba-c') Immunostaining against Zic2 in E15.5 retinas (a,a', red) combined with in situ hybridization for EphB1 (b,b', green) in the same section, indicates that Zic2 is expressed in the most-peripheral (younger) cells, whereas older and more-central cells co-express Zic2 and EphB1. (a',b',c') Higher magnification of the boxed area in c. Red line in c' indicates cells positive only for Zic2, which are presumably younger cells. Yellow line indicates the Zic2 and EphB1-positive region, in which more-mature cells are located. Scale bars: in a, 100 µm for a,b,c; in a', 30 µm for a',b',c'.

View larger version (90K): [in this window] [in a new window]   Fig. 4. Zic2 is essential for EphB1 expression in the VT retina. (A) White dashed lines delineate retinal sections from wild-type (a-a''), heterozygous (b-b'') and homozygous (c-c'') E16.5 littermate embryos. Immunostaining shows that Zic2 (red) is expressed in VT retina (yellow arrows) in Zic2+/+ mice, but is not detected in Zic2kd/kd embryos. Some Zic2-positive cells are detected in Zic2+/kd embryos. (a',b',c') In situ hybridization for EphB1 (green) in the same retinal sections. (a'',b'',c'') Higher-magnification of the respective boxed areas. These images reveal that EphB1 is expressed in the same area as Zic2 in wild-type retinas. In Zic2+/kd retinas, EphB1 expression is reduced compared with the wild type in accordance with a downregulation of Zic2. In Zic2kd/kd retinas, in which Zic2 is not expressed, levels of EphB1 are undetectable. d, dorsal; v, ventral. Yellow arrowheads indicate high expression of Zic2 (a,b,c) and EphB1 (a',b',c'). Scale bar: 100 µm. (B) In situ hybridization for EphB1 in horizontal sections from E17.5 Zic2+/+ (a) and Zic2kd/kd (b) brains. EphB1 is extensively expressed in a very similar pattern in both genotypes. Lgp, lateral globus pallidus; Cx, cortex; hi, hippocampus; th, thalamus. Yellow arrows indicate high expression of EphB1.

View larger version (61K): [in this window] [in a new window]   Fig. 5. Zic2-induced turning of ipsilateral retinal axons away from the midline is mediated by both EphB1-dependent and -independent pathways. (A) Quantitative RT-PCR for EphB1 performed on non-electroporated or electroporated mouse retinas at E13.5 with CAG-EGFP or with CAG-Zic2/CAG-EGFP plasmids. EphB1 levels significantly increase after ectopic expression of Zic2 in the center of the retina by in utero electroporation, as compared with control retinas. More than ten retinas for each condition were pooled per experiment. Data from seven different experiments are shown. Error bars indicate s.e.m.; *P<0.005. (B) Left panels show axonal trajectories of targeted RGCs at the optic chiasm. Middle panels show the location of the targeted RGCs (green) in retinal whole-mounts (gray). Right panels are cartoons summarizing the results in each particular case. (a) In wild-type embryos electroporated at E13.5 with Zic2/EGFP, many of the green axons originating from the central retina turn at the midline. (b) In EphB1-/- embryos ectopically expressing only EGFP in the central retina, no ipsilateral axons are seen at the chiasm. (c) When Zic2/EGFP are ectopically expressed in the central retina of EphB1-null embryos at E13.5, the number of axons projecting ipsilaterally does not change as dramatically as when wild-type embryos are electroporated with Zic2/EGFP, but a significant proportion of axons that project ipsilaterally can be still detected. White arrows indicate ipsilateral projections. ON, optic nerve; cOT, contralateral optic tract; iOT, ipsilateral optic tract; d, dorsal; n, nasal; t, temporal; v, ventral. Scale bars: chiasm panels, 200 µm; retina panels, 500 µm.

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