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First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02575

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Negative regulation of retinal-neurite extension by ß-catenin signaling pathway

Department of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan

View larger version (34K): [in a new window]   Fig. 1. Morphology of neural retina cells transfected with retrovirus containing EGFP, dnLEF-EGFP, caß-catenin-EGFP or caLEF-EGFP. (A) Retinal explants were prepared from E17.5 mouse embryos and infected with retrovirus encoding EGFP, dnLEF-IRES-EGFP, caß-catenin-IRES-EGFP or caLEF-IRES-EGFP. After 2 weeks in culture, the explants were fixed and frozen-sectioned. Immunohistochemistry used anti-GFP antibody, and DAPI was used to visualize nuclei. Bars, 10 µm (left panels); 50 µm (right panels). (B) Subretinal localization of EGFP-positive virus-infected cells was examined in several sections. More than 100 cells of each sample were counted and each set of experiments was conducted in triplicate. The percentage of the total EGFP-positive cell number at each location is shown. ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. (C) Neurite length distribution of the retinal cells expressing EGFP, dnLEF-EGFP, caß-catenin-EGFP or caLEF-EGFP. Retinal explants were prepared from E17.5 mouse embryos and infected with retrovirus encoding EGFP, dnLEF-IRES-EGFP, caß-catenin-IRES-EGFP or caLEF-IRES-EGFP. After 3 days of culture, cells were disaggregated, replated into chamber slides and cultured for a further 11 days. Cells were then stained with anti-GS and anti-GFP antibodies and the neurite length of more than 45 cells of GS-negative/GFP-positive neural cells of each samples was measured. (D) Average neurite length from Fig. 1C is shown in the left panel. Populations of cells with neurite length less than 10 µm (middle panel) and more than 30 µm (right panel) in Fig. 1C are compared. More than 50 cells of each sample were measured and each set of experiments was conducted in triplicate. Error bars represent s.e.m. *P<0.05; **P<0.01.

View larger version (58K): [in a new window]   Fig. 2. Proliferation and differentiation of retinal cells perturbed by the ß-catenin signaling pathway. (A,B) BrdU incorporation into retinal cells expressing EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP was examined. Retinal explants were infected with retrovirus and incubated with 5 µM BrdU at 2, 6 or 13 days after retrovirus infection and harvested 24 hours later. (A) Patterns of frozen sections immunostained with anti-BrdU and anti-GFP antibodies were visualized with secondary antibodies conjugated to Alexa 546 and Alexa 488 (Molecular Probes), respectively. (B) Percentage of BrdU-incorporated retinal cells in virus-infected cells. (C,D) Expression of rod photoreceptor and Muller glia markers was examined by immunohistochemistry. Retinal explants were infected with retrovirus and cultured for 2 weeks. Frozen sections were then made (C), or the explants were disaggregated with trypsin and replated into chamber slides (D). In D, the number of Rho4D2-positive cells were counted. More than 100 cells were measured for each sample and s.d. was calculated from three independent experiments. Both samples were subjected to immunohistochemistry for rod photoreceptor cells by using anti-Rho4D2 antibody and for Muller glia cells by using anti-GS antibody. These first antibodies were visualized by using secondary antibody conjugated to Alexa 546. Nuclei were stained with DAPI and infected cells were detected by anti-GFP antibody. Bars, 50 µm (A,C).

View larger version (42K): [in a new window]   Fig. 3. Effects of perturbation of the Wnt signaling pathway on neurite extension by PC12 cells. (A) Morphology of PC12 cells infected with EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP retroviruses. PC12 cells were infected with retrovirus encoding EGFP, caß-catnin-EGFP, caLEF-EGFP or dnLEF-EGFP; the cells were then incubated with 50 ng/ml NGF for 5 days to cause differentiation, and subsequently fixed. Cells were immunostained with anti-ßIII-tubulin and anti-GFP antibodies and visualized with Alexa 546 and Alexa 488, respectively. DAPI was used for staining nuclei. Bar, 100 µm. (B-E) Neurite length distribution of the PC12 cells expressing EGFP, dnLEF-EGFP, caß-catenin-EGFP or caLEF-EGFP. Neurite lengths of more than 75 cells of EGFP-positive and -negative cells were measured. Each experiment was conducted in triplicate. Error bars represent s.e.m. of three independent results. (F) Normalized neurite length of PC12 cells expressing EGFP, caß-catenin-EGFP, caLEF-EGFP or dnLEF-EGFP. The average neurite length of EGFP-positive transfected cells (n>75) were normalized by the average neurite length of EGFP-negative non-transfected cells (n>75). Each experiment was conducted in triplicate. Error bars represent s.e.m. *P<0.05; ***P<0.005 by the Student's t-test. (G) Effect of the ß-catenin-Lef-1 signaling pathway on MAPK-dependent transcription activity. PC12 cells were co-transfected with the c-fos promoter-luciferase plasmid (0.1 µg) together with caMEK or caLEF plasmids (0.1 µg), as described in Materials and Methods. After 10 hours of culture, cells were harvested and the luciferase activities were measured.

View larger version (56K): [in a new window]   Fig. 4. Activation of the MAPK pathway and its inhibition during neural retina development. (A,B) Phosphorylation of MAPK in neural retina. (A) Retinal explant cultures were frozen-sectioned at day 6 and 14, and immunostained using anti-phospho p44/42 MAPK antibody (-p-MAPK) or control IgG; the first antibody was visualized by using a horseradish peroxidase-labeled secondary antibody. (B) Retinal explant cultures in the presence or absence of PD98059 were harvested at day 1 or 3, and whole-cell lysates were subjected to western blotting using anti-MAPK antibody (-MAPK) or anti-phospho p44/42 MAP antibody (-pMAPK). Ba/F3 cells expressing human GM-CSF receptor were stimulated or not for 10 minutes in the presence or absence of PD98059 and used as a positive control. (C) Effects of PD98059 on neurite extension in retinal explant cultures. Retrovirus encoding EGFP was introduced into E17.5 retinal explant cultures at day 1, and the cultures were continued in the presence of 10 µM PD98059 from day 1-14 or day 7-14. The cultures were harvested and frozen-sectioned to examine EGFP-positive cells by immunostaining with anti-GFP antibody. (D) Retinal explants were prepared from E17.5 mouse embryos and infected with retrovirus encoding dnMEK-IRES-EGFP, dnRas-IRES-EGFP or EGFP. After 2 weeks in culture, the explants were fixed and frozen-sectioned. Immunohistochemistry used anti-GFP antibody, and DAPI was used to visualize nuclei. (E) Subretinal localization of EGFP-positive virus-infected cells was examined in several sections. More than 100 cells of each sample were counted and each set of experiments was conducted in triplicate. ONL, outer nuclear layer, INL, inner nuclear layer, GCL, ganglion cell layer. Bars, 50 µm (A,C,D). (F,G) Neurite length was measured in cells from dissociated retinal culture expressing EGFP, dnMEK-EGFP or dnRas-EGFP, as described in the legend to Fig. 1C. Neurite length distribution of the retinal cells (F) and the average neurite length±s.e.m. (G) are shown. More than 70 cells of each sample were measured and each set of experiments was conducted in triplicate.

View larger version (33K): [in a new window]   Fig. 5. The expression and activation of the relevant Wnts in the mouse retinas. The expression of Wnts in mammalian retina is schematically summarized from a previous report (Liu et al., 2003). Tcf-binding-site-dependent EGFP expression was examined in the explanted retina from E17 by using TCF-binding-site-EGFP plasmid as described in the Results section.

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