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First published online 18 May 2005
doi: 10.1242/dev.01856

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Wnt2b inhibits differentiation of retinal progenitor cells in the absence of Notch activity by downregulating the expression of proneural genes

Fumi Kubo^1,2,*, Masatoshi Takeichi^1,2 and Shinichi Nakagawa^1,*,

¹ RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minamimachi, Chuo-ku, Kobe 650-0047, Japan
² Department of Cell and Developmental Biology, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan

View larger version (63K): [in a new window]   Fig. 1. Wnt2b induces prolonged proliferation of retinal progenitor cells. (A,B) Exogenous gene expression by replication-competent retroviruses. The optic vesicles were electroporated with a GFP-expressing RCASBP provirus vector and fixed after 24 hours (A) or 72 hours (B). Transverse sections of neural retinas were observed using confocal microscopy. Nuclei were counterstained with propidium iodide and pseudocolored in purple. (C) Whole-mount view of E5 retina. The purple box shows the region from which retinal explants were prepared. (D-F) Whole-mount view of the retinal explants taken from control (D,G), Wnt2b-expressing (E,H), and Delta1-expressing (F,I) E6 retina immediately after the dissection (D-F) or after 7 days in culture (G-I). The small insets in G-I show the explants after the dissection at the same magnification. Note that Wnt2b-expressing explants increased in size enormously, making folded sheets (H), whereas control or Delta1-expressing explants made a much smaller sphere (G,I). (J) The change in cell number in the explants during the culture period (shown on the x axis). The cell number is shown in a logarithmic vertical scale. Note the exponential growth in the Wnt2b-expressing explants. Scale bars: 20 µm (A,B); 250 µm (D-I).

View larger version (73K): [in a new window]   Fig. 2. Wnt2b inhibits the differentiation of retinal progenitor cells. Retinal explants were prepared from E6 retinas electroporated with control (A,D,G,J), Wnt2b-expressing (B,E,H,K), or Delta1-expressing (C,F,I,L) provirus plasmids. They were cultured for 2 days, and sections were prepared and stained for molecular markers shown in the left column: visinin (green) and Hu (purple) in A-C, Pax6 (green) and Chx10 (purple) in D-F, glutamine synthetase (green) in G-I, and BrdU (green) and phosphorylated histone H3 (purple) in J-L. Note the decrease in the number of visinin- or Hu-expressing cells (B,C) and uniform co-expression of progenitor markers Pax6 and Chx10 (E,F) in Wnt2b- or Delta1-expressing retinal explants compared with the marker levels in the control explants. A number of cells incorporated BrdU in Wnt2b-expressing retinal explants (K), whereas few cells were positive for the proliferation markers in control (J) or Delta1-expressing (L) explants. (M) Percentage of BrdU+ cells in the explants expressing each construct (n=4). (N) Thickness of the retinal explants expressing each construct (n=4). **P<0.01, *P<0.05. Scale bars: 30 µm.

View larger version (77K): [in a new window]   Fig. 3. Wnt2b suppresses premature neurogenesis induced by an inactivation of Notch signaling. (A-L) Effects of DAPT and Wnt2b on progenitor cell differentiation. The retinal explants were prepared from E5 retina electroporated with control (A,B,E,F,I,J) or Wnt2b-expressing (C,D,G,H,K,L) provirus vectors, and were cultured for 2 days in the absence (A,E,I,C,G,K) or presence (B,F,J,D,H,L) of DAPT, a -secretase inhibitor that blocks Notch signaling. The sections were stained for visinin (green), and Hu (purple) in A-D, for Pax6 (green) and Chx10 (purple) in E-H, and for BrdU (green) and phosphorylated histone H3 (purple) in I-L. In the control explants, the DAPT treatment induced premature neuronal differentiation, as revealed by the increase of neuronal markers (B) and a decrease of retinal progenitor cell markers (F). In the Wnt2b-expressing retinal explants, however, a large population of the cells still co-expressed the progenitor markers (H) and incorporated BrdU (L). (M-P) Effects of a dominant negative Delta1 and Wnt2b on retinal progenitor cell differentiation. The optic vesicles were electroporated with provirus vectors encoding control RCASBP (A,B) in M, RCASBP (A) encoding a dominant-negative form of Delta1 and RCASBP (B) in N, RCASBP (A) and RCASBP (B) encoding Wnt2b in O, or RCASBP (A) encoding a dominant-negative form of Delta1 and RCASBP (B) encoding Wnt-2b in P. The retinas were fixed at E5, and sections were stained for visinin (green) and Hu (purple). Note that Wnt2b completely suppressed the effect of the dominant-negative Delta in promoting the neurogenesis. (Q-T) Effect of Delta1 and Wnt2b on Müller cell differentiation. Note that Wnt2b suppressed the effect of Delta1 in inducing Müller glia differentiation when co-expressed in the same explants. (U) Percentage of BrdU+ cells in the explants expressing each construct (n=4). Scale bars: 30 µm.

View larger version (54K): [in a new window]   Fig. 4. Wnt2b downregulates the expression of multiple proneural genes. (A-F) mRNA expression pattern of Notch1, Cath5, NeuroM, and Delta1 in the central region of control (A,C,E,G) or Wnt2b-expressing (B,D,F,H) E5.5 retina. These genes were robustly expressed in the control retina, but their expressions were reduced in the Wnt2b-expressing retina, except for Delta1, expression of which was unaffected by exogenous Wnt2b. (I) RT-PCR analysis of proneural gene expressions. The expression of all of the genes, Notch1, Cath5, NeuroM, Cash1, Neurogenin 1(Ngn1), and Neurogenin 2 (Ngn2) was downregulated in the Wnt2b-expressing retina (right lane) compared to the control retina (left lane), whereas the expression level of Delta1 and Gapdh was unchanged. The expression of Lef1 was upregulated in the Wnt2b-expressing retina. (J) Percentage expression of each proneural gene in Wnt2b-expressing retina to that in the control retina. Intensity of PCR products corresponding to each proneural gene was measured and normalized to that of Gapdh. Relative signal intensity of each gene in the control experiment was represented as 1. Scale bar: 30 µm.

View larger version (81K): [in a new window]   Fig. 5. Introduction of exogenous Cath5 suppresses the inhibitory effect of Wnt2b on the differentiation of retinal ganglion cells. Expression pattern of neurofilament (A-D) and islet1 (E-H) in the retina electroporated with plasmids expressing control (A,E), Cath5 (B,F), Wnt2b (C,G), and Cath5 and Wnt2b (D,H). The marker expressions are shown in purple, and co-electroporated GFP signals, in green. Arrowheads indicate the cells expressing co-electroporated GFP and the retinal ganglion cell markers. (I) The number of islet1-expressing cells per retina (n=4). The cells were counted on serial sections of the retina electroporated with each construct as indicated. Note that the exogenous expression of Cath5 increased the number of islet1-positive cells (B,F,I), whereas no islet1-positive cells were observed in the Wnt2b-expressing retina (C,G,I). When Cath5 was introduced together with Wnt2b, inhibition of the ganglion cell differentiation by Wnt2b was suppressed (D,H,I). Scale bar: 30 µm.

View larger version (105K): [in a new window]   Fig. 6. Blockade of cell-cycle progression by a CDK inhibitor, Ink4D (p19), does not affect the inhibitory effect of Wnt2b on ganglion cell differentiation. (A-D) Effect of Ink4D and Wnt2b on cellular proliferation. Optic vesicles of E1.5 embryos were electroporated with vectors, as indicated above. After 12 hours, the sections were stained for BrdU (purple) and for co-electroporated nlsGFP (green). Note that Ink4D-electroporated cells did not incorporate BrdU in B and D. (E-L) Expression patterns of neurofilament (purple in E-H) and islet1 (purple in I-L) in the retina 48 hours after the electroporation. Arrowheads indicate the cells expressing co-electroporated GFP and the retinal ganglion cell markers. Wnt2b inhibited ganglion cell differentiation in both the presence and absence of co-electroporated Ink4D (G,H,K,L). (M) Percentage of BrdU+ cells in retina (n=4) expressing each construct. (N) Ratio of islet1+ cells/co-electroporated nlsGFP in the retina (n=4). Scale bars: 20 µm (A-D); 30 µm (E-L).

View larger version (20K): [in a new window]   Fig. 7. Proposed model for the action of Wnt2b in the marginal retina. (A) Gene cascade leading to the inhibition of cellular differentiation by Wnt signaling. See the text for details. (B) During early retinal development, Wnt2b sends a signal to the peripheral region of the optic vesicles to keep the progenitor cells undifferentiated. Centrally located progenitor cells do not receive the Wnt signals and express proneural genes as well as Notch, which triggers the differentiation cascades. (C) At later stages, the marginal retina differentiates into iris and ciliary epithelium, induced by factors coming from surrounding tissues such as the lens or cornea.

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