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doi: 10.1242/10.1242/dev.00244

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Wnt2b controls retinal cell differentiation at the ciliary marginal zone

¹ Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa Oiwake-cho, Kyoto 606-8502
² RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami-Cho, Kobe 650-0047, Japan

View larger version (67K): [in a new window]   Fig. 1. Wnt signaling components are expressed in the marginal neural retina. (A-D) In situ hybridization of stage 22 (E4) neural retina probed with chicken Wnt2b (A), Fzd4 (B), Fzd5 (C) and LEF1 (D). le, lens. Scale bars: 100 µm. (E) Western blot of the immunoprecipitates from COS7 cells co-transfected with Fzds-CRD and Wnt2b-Fc. The cells were co-transfected with the construct described below, and the cell lysates were immunoprecipitated with protein A. The immunoprecipitates were detected with the antibodies shown at the top. The Wnt2b-Fc binds both mouse Fzd4-CRD and human Fzd5-CRD, whereas the control c-cad7-Fc did not. (F) Stabilization of cytosolic ß-catenin and upregulation of LEF1 mRNA by chicken Wnt2b. Retinal cultures were incubated for 6 hours in the absence or presence of chicken Wnt2b-conditioned medium (CM) or soluble mouse Fzd4 lysate described below. The membrane-associated and cytosolic ß-catenins were then analyzed by western blotting, and the level of LEF1 mRNA expression was analyzed by RT-PCR. mRNA expression of N-cadherin was used as a control for the RT-PCR analysis. Cytosolic ß-catenin and LEF1 mRNA expression were clearly upregulated in the presence of chicken Wnt2b, an effect completely blocked by the addition of mouse Fzd4-CRD. Molecular weight markers are 200 kDa, 116 kDa, 98 kDa, 66 kDa, 45 kDa and 31 kDa. (G) Relative ratio of cytoplasmic ß-catenin to membrane-bound ß-catenin and of LEF1 mRNA to N-cadherin mRNA. The ratio in the control culture medium was normalized to 1.

View larger version (74K): [in a new window]   Fig. 2. Subdivision of the marginal retina characterized by expression of a combination of markers. The expression patterns of various molecular markers were examined by using adjacent sections of the marginal region of the E5 retina. The S-shape of the retina is the artifact produced during the fixation (see Fig. 1D). In these figures, the marginal region is towards the left and the central region is towards the right. (A,B,D-H) In situ hybridization or (C,I) immunohistochemistry of E5 adjacent sections stained for chicken Wnt2b (A), Pax6 (B), collagen type IX (C), Ptmb4 (D), LEF1 (E), Chx10 (F), Rx1 (G), Notch1 (H) and NF-M (I). The small arrowheads, arrows and large arrowheads indicate the retinal regions that presumably give rise to the iris/ciliary epithelium, the CMZ and the neural retina, respectively (shown at higher magnification in the righthand panels). For Wnt2b, the marginal tip of the retina is shown at higher magnification instead of the presumptive iris region. Note that the LEF1-expressing CMZ is characterized by weak expression of collagen type IX and Ptmb4; co-expression of Pax6, Chx10 and Rx1; and the absence of Notch1 and NF-M (J). Scale bars: 50 µm.

View larger version (143K): [in a new window]   Fig. 3. Time course of Wnt2b overexpression and induction of LEF1 expression by in ovo electroporation. (A-D) In situ hybridization of normal E3 retina probed with chicken Wnt2b (A), Fzd4 (B), Fzd5 (C) and LEF1 (D). (E-J) In situ hybridization of the electroporated embryos probed with the overexpressed mouse Wnt2b (E-G) and LEF1 (H-J). The embryos were fixed 12 hours (E, H), 24 hours (F, I) and 48 hours (G,J) after the electroporation. The upregulation of LEF1 mRNA was detected 12 hours after the initiation of exogenous mouse Wnt2b expression, which continued for at least 24 hours. di, diencephalon; eb, eye bud. Scale bars: 100 µm.

View larger version (68K): [in a new window]   Fig. 4. Induction of the CMZ markers and inhibition of neural retinal markers by Wnt2b overexpression in ovo. (A-H) The expression pattern of NF-M (A,B), Notch1 (C,D), Pax6 (E,F), Chx10 (G,H), Rx1 (I,J), and collagen type IX (K,L) in the embryos electroporated with control (A,C,E,G,I,K) or chicken Wnt2b (B,D,F,H,J,L)-expressing plasmids. The embryos were fixed at stage 21, and adjacent transverse sections were stained for co-electroporated GFP (shown in left panels) and each molecular marker (shown in right panels). In the Wnt2b-electroporated embryos, the inner layer of the optic cup formed folded retinas that did not express neural retina-specific markers such as NF-M or Notch1 (B,D). The folded retinas expressed progenitor marker genes, including Pax6 (F), Chx10 (H) and Rx1 (J). Note that whole retina was affected by relatively uneven introduction of the electroporated chicken Wnt2b. Arrowheads indicate the central neural retina. di, diencephalon. Scale bar: 100 µm.

View larger version (97K): [in a new window]   Fig. 5. Precocious differentiation of the retinal stem cells by inhibition of the Wnt canonical pathway with LEF1. (A-D) Transverse sections of stage 21 embryo electroporated with control (A,B) or LEF1 (C,D) stained with DAPI (A,C) and the co-electroporated GFP (B,D). Note that the cell clump in the dorsal retinal margin strongly expressed GFP (arrow). (E-H) The incorporation of BrdU (red) in the control (F) and LEF1-electroporated (H) retina. The co-electroporated GFP is shown in E,G. (F,H) Pseudo-colored DAPI signals are shown in green. (I-P) The expression of co-electroporated GFP (I,K,M,O), NF-M (J,L), Islet1 (N,P) in the cell clump of LEF1-electroporated retina adjacent to the lens (le). Arrowheads indicate the ectopic NF-M expression. Scale bars: 100 µm.

View larger version (30K): [in a new window]   Fig. 6. CMZ progenitor cells produce a larger number of progenies compared with central progenitor cells. Histograms of the number of progenies derived from single progenitor cells prepared from the central retina (A,C) or the CMZ (B,D) either in the absence (A,B) or presence (C,D) of chicken Wnt2b CM. The number of progenies within a clone was counted and placed into the bins indicated below. Each value represents the number of clones in each bin divided by the total number of clones. Note that the CMZ progenitor clones contained a larger number of progenies compared with the central progenitor cells.

View larger version (133K): [in a new window]   Fig. 7. Clonal analysis of multipotential retinal progenitor cells using reaggregation culture. (A,E,I,M,Q) Low-magnification image of the clones visualized with anti-quail nucleus antibody, QCPN. The singly dissociated retinal progenitor cells, prepared from the central part of the E4.5 quail neural retina (A,I) or CMZ (E,M,Q), were mixed with feeder cells from E5 chicken retina, and cultured on a filter for 7 days either in the control (A,E) or chicken Wnt2b (I,M,Q)-conditioned medium. The CMZ progenitor cells produced larger clones than the central progenitor cells (A,E), and proliferation of both progenitor cells was promoted by the addition of chicken Wnt2b-conditioned medium (I,M). The CMZ progenitor cells produced secondary clones when they had been cultured in the presence of chicken Wnt2b (Q). Confocal images of progenitor clones visualized with QCPN (red), which were triple-labeled for Müller cell marker glutamine synthetase (Gln-Syn) and photoreceptor cell marker visinin (B,F,J,N,R), ganglion/amacrine cell marker Hu and visinin (C,G,K,O,S), or Hu and glutamine synthetase (D,H,L,P,T). Scale bar: 10 µm. Arrowheads indicate quail cells expressing those differentiation markers.