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First published online 19 July 2006
doi: 10.1242/dev.02474

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Wnt2b/ß-catenin-mediated canonical Wnt signaling determines the peripheral fates of the chick eye

Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

View larger version (87K): [in a new window]   Fig. 1. Assay for retinal progenitor proliferation in explants and in ovo. (A) E5.5 retinal explants electroporated with pCAG:CA-ß-catenin or pCAG:GFP were cultured for 18 hours and exposed to [3H]thymidine for 6 hours. The percentage of [3H]thymidine+ cells were quantified following autoradiography. Bars represent the percentage of cells labeled with [3H]thymidine among the GFP+ population. Error bars represent the s.d. (B,C) An in vivo assay for retinal cell proliferation using BrdU (B) and pH3 (C) in an E7.5 retinas infected with RCAS:CA-ß-catenin or RCAS:GFP. (D-F) Representative images of an RCAS:GFP-infected retina at E7.5 showing BrdU incorporation (green) and pH3 (red) in the central retina (D), the peripheral retina including the CMZ (E) and in the ciliary/iris epithelia (F). (G) Relative position of sections shown in D,E and F and used for scoring in B and C. (H) Representative image of an E7.5 retinal section showing BrdU labeling (green) and pH3 (red) in a thin and folded region induced with RCAS:CA-ß-catenin from the central part of the retina. Le, lens. Scale bar: 75 µm.

View larger version (97K): [in a new window]   Fig. 2. Expression of Wnt signaling genes and Wnt reporter activity. (A-D) ISH for chick Wnt2b. Wnt2b signal was observed exclusively in the SE (arrowheads) at the OV stages (A,B), and in the RPE (arrow) and the peripheral tip of the retina (white arrowhead), in addition to SE (black arrowhead), at the OC stage (C,D). (E-H) ISH for Lef1 (E,G) and Chx10 (F,H). Stages are invaginating OV (E,F) and early OC (G,H). White lines in A,B,E,F,H outline the OV and OC. Arrowheads in E,F indicate the dorsal OV. Arrows in G,H indicate the peripheral OC. (I) Structure of the Wnt reporter SuperTopAP and the mutant reporter SuperFopAP. (J,K) AP staining of retinal sections electroporated with SuperTopAP and assayed at the OV (J) and early OC (K) stages. Note that the anterior OV (blue line), the anlage for the central retina, does not show Wnt activity, whereas the posterior/dorsal OV (red and green), the anlagen for RPE and ciliary/iris epithelia, respectively, show strong AP staining. Inset in K' shows a close-up of the central retinal cells showing weak Wnt activity in some of the cells adjacent to the RPE. (L) AP staining of a retinal section electroporated with the mutant Wnt reporter SuperFopAP. (M-O) Co-electroporation of pMIW III:GFP shows the areas of reporter DNA delivery in sections corresponding to panels L,J and K, respectively. Note that the areas with a high AP signal have reduced GFP signal due to quenching of the immunofluorescence. Di, diencephalons; Lv, lens vesicle; Lp, lens placode. Dorsal side is up. Scale bars: 150 µm.

View larger version (68K): [in a new window]   Fig. 3. Morphological phenotype of E7.5 retinas infected with RCAS:CA-ß-catenin. (A,B) Infection with RCAS:CA-ß-catenin produced abnormally protruding eyes (B) compared with RCAS:GFP-infected eyes (A). (C,D) Views from the vitreal surface of an uninfected retina (C) and a RCAS:CA-ß-catenin-infected retina (D) reveals numerous small retinal folds in D. (E,F) Hematoxylin and eosin (H&E) staining of sections from control RCAS-infected (E) and RCAS:CA-ß-catenin-infected (F) retinas. (G-J) Examples of an RCAS-infected control central retina (G) and ciliary/iris epithelia (H), and a RCAS:CA-ß-catenin-infected retina (I,J). G-I are enlargements of boxed areas in E and F; J is from an independent animal. Note the thin and folded epithelial characteristics of the RCAS:CA-ß-catenin-infected retina. Le, lens; ON, optic nerve exit. Scale bars: 1 mm.

View larger version (106K): [in a new window]   Fig. 4. Effects of Wnt signal activation on RPG expression and retinal differentiation. (A-D) ISH for retinal progenitor markers Chx10 (A,C) and Notch1 (B,D) at E5.5 following injection of RCAS:CA-ß-catenin (A,B) or RCAS:Wnt2b (C,D). Arrowheads indicate the areas with higher viral infection. (E-H) IHC of viral gag protein (p27) on the adjacent (A) or same sections (B-D) to show the areas of viral infection (red). (I-K) IHC of ß-Tubulin III (green), a marker of ganglion and amacrine cells, following the introduction of control RCAS (I), RCAS:CA-ß-catenin (J) and RCAS:Wnt2b (K) harvested at E7.5. Note the partial overlap of -ß-Tubulin III staining with viral infection (arrow, J), possibly due to axonal processes produced by uninfected neighboring ganglion cells. IHC of viral gag protein (p27) shows the area of viral infection (red). (L,M) IHC for Visinin, a marker for developing photoreceptor cells, in uninfected control (L) and RCAS:CA-ß-catenin-infected (M) retinas. (N,O) IHC of -Pax6 (green), a marker for horizontal (arrow) and amacrine (marked by the upper bar) cells, and cells in the ganglion cell layer (lower bar), in E7.5 wild-type retina (N) and RCAS:CA-ß-catenin-infected retina (O). Nuclear counter staining was done with DAPI (blue). Viral infection is visualized using IHC for viral gag (red). Scale bars: 75 µm.

View larger version (75K): [in a new window]   Fig. 5. Infection with RCAS:CA-ß-catenin induces ectopic peripheral markers. (A-D) ISH for Collagen IX (A,B) and Bmp7 (C,D) at E5.5 and E6.5, respectively. An uninfected retina exhibits marker gene expression in the ciliary and iris epithelia (A,C), but not in the central retina (B,D). Arrow in D indicates optic nerve exit. (E-H) ISH for Collagen IX (E) and Bmp7 (G) in E5.5 retinas infected with RCAS:CA-ß-catenin. IHC for -gag marks the area of viral infection (red, F,H). Note that the strong AP signal quenches the fluorescence signal in some areas. (I-L) ISH for WFDC1 (I), Wnt2b (K) and Lef1 (L) in E7.5 retinas infected with RCAS:CA-ß-catenin. Note that some of the thin and/or folded regions show ectopic peripheral marker induction in I,K,L (arrows). IHC for -gag marks the area of viral infection (red, J). (M,N) IHC of -SMA at E7.5 in an RCAS:CA-ß-catenin-infected retina shows ectopic induction of SMA (red) in the abnormally thin and folded region (arrow). Arrowheads indicate the lack of SMA induction in the central retinal area. Viral infection was visualized with -gag antibody (green, N).

View larger version (112K): [in a new window]   Fig. 6. Infection with RCAS:Lef1-En causes a reduction in Collagen IX in the iris epithelium. (A,B) ISH for Collagen IX at E4.5 from eyes infected with RCAS:Lef1-En. Note only the area with higher infection [stronger -gag (3C2) staining at the peripheral tip of the retina in A' compared with B'] shows a reduction of Collagen IX expression (A); weaker infection of RCAS:Lef1-En does not affect expression (B). (C-H) Effects of sustained expression of Lef1-En on iris development. (C) A normal eye after removal of the cornea at E14 is shown at the top and an example of an eye infected with RCAS:Lef1-En is shown at the bottom. The arrow points to an area with a defect in the anterior structures, particularly the iris. (D-F) H&E staining on sections from an eye partially infected with RCAS:Lef1-En reveals a slightly disorganized pars plicata and a severe reduction in the iris on the left side (arrowhead, E), compared with the unaffected normal iris (arrow, F). (G,H) IHC of -SMA (red) in stroma of the affected (arrowhead, G) compared with the non-affected (arrow, H) areas of the iris. Nuclear counter staining was done with DAPI (blue). (I,J) Hypopigmentation of RPE induced by RCAS:Lef1-En at E14.5. Compared with a control RCAS:GFP-infected eye (left), an eye from a RCAS:Lef1-En-infected animal (right) shows patches with less pigmentation (arrows). H&E staining of the infected eye shows RPE areas with partial pigmentation defects (arrows). Le, lens. Scale bar: 3 mm.

View larger version (29K): [in a new window]   Fig. 7. Wnt2b/wg expression and function during peripheral eye development in vertebrates and invertebrates. (Left) In vertebrates, Wnt2b (blue) is expressed in, and functions in, the RPE and peripheral tip of the OC, where the ciliary body and iris are derived, but not in the retina (yellow; top). At later embryonic stages (below), Wnt2b is maintained in both pigmented and non-pigmented layers of the iris. Wnt2b/ß-catenin signaling is sufficient and necessary for the development of peripheral eye tissues, ciliary body and iris epithelia (green). (Right) In Drosophila, wg (blue) is expressed in the lateral margins, both anterior and posterior, of the eye imaginal disc of the third instar larvae, but not in the retina (yellow; top). In the adult eye (below), wg is restricted to the head capsule immediately adjacent to the pigmented rim (PR), and functions to pattern peripheral tissues, such as head capsule cuticle, pigment rim and dorsal rim ommatidia (green) in a dosage-dependent manner. wg/armadillo signaling in Drosophila is sufficient and necessary for the development of these tissues. The scheme showing a cross section of the adult fly eye is adapted from Tomlinson (Tomlinson, 2003). A, antenna disc; HC, head capsule cuticle; L, lens; PR, pigment rim; MF, morphogenetic furrow; RPE, retinal pigmented epithelium.