DEV02474 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. (A,B) CA-β-catenin does not significantly affect RPC proliferation at E4.5. (A) A section showing an average region of the RCAS:CA-β-catenin-infected retina and the lack of change of BrdU uptake (green) compared with an uninfected area. Viral infection was visualized with α-gag antibody (red). (B) Rarely, CA-β-catenin causes a slight reduction in BrdU incorporation only in relatively large patches of infected cells. (C,D) Expression of CA-β-catenin reduces the level of cyclin D1 transcript at E5.5 (arrows). (E-H) ISH of Wnt2b at later embryonic stages, e.g. E5.5 (E), E8.5 (F), E10.5 (G) and E12.5 (H). Wnt2b is primarily expressed at the tip of the iris epithelium (arrowheads). (I,J) Lef1 is exclusively expressed at the periphery of the E6.5 retina (I), whereas Chx10 is expressed in the central retina (J). Le, lens.

• Supplemental Figure 2 -

  Fig. S2. (A) Co-electroporation of RCAS:CA-β-catenin with SuperTopAP elicits ectopic AP staining in the central retina at the OC stage. (B) Delivery of the reporter was detected by α-GFP staining of co-electroporated pMIW III:GFP. (C,D) Cells in a thin and folded region induced by CA-β-catenin show Pax6 expression and cuboidal morphology. IHC of Pax6 (green) and α-gag (red) from RCAS-infected (C) and RCAS:CA-β-catenin-infected (D) E7.5 retina. Normal ciliary/iris epithelia express Pax6 (C), whereas CA-β-catenin expresses Pax6 only in thin and folded epithelium (D). The lack of Pax6 induction in retinal cells is shown in Fig. 4O. (E,F) Magnified images from C and D show the cuboidal cell shape (arrowheads) of ciliary epithelium from RCAS-infected (E) and CA-β-catenin-infected thin (F) retina. (G,H) Effects of Wnt signal activation on Chx10 (G) at E7.5 following injection of RCAS:CA-β-catenin. (H) IHC of viral gag protein (p27) on the adjacent section shows the area of viral infection (red). Arrowheads indicate areas of higher viral infection. Dorsal side is up in A and B. Scale bar: 75 μm.

• Supplemental Figure 3 -

  Fig. S3. (A) Pax6 (red) is expressed in ciliary/iris epithelia in addition to horizontal, ganglion and amacrine cells at E7.5. Nuclear DAPI staining (blue) marks all cells. (B-D) Analysis of cell death (green) from RCAS-infected (B), RCAS:CA-β-catenin-infected (C) and RCAS:Wnt2b-infected (D) retinas, showing apoptotic cells in Wnt signal-activated retinas.

• Supplemental Figure 4 -

  Fig. S4. Collagen IX expression marks the development of the peripheral retinal tissues, ciliary body and iris. (A-E) IHC for Collagen IX (red) at E4 (A), E5 (B), E6 (C), E8 (D) and E18 (E). Arrowhead in E indicates the ora serrata, a region between the NR and ciliary body. Nuclear DAPI staining (blue) marks all cells. Le, lens.

• Supplemental Figure 5 -

  Fig. S5. The peripheral retina has lower mitotic activity and a lack of retinal ganglion cell differentiation. (A-E) IHC of α-pH3 (green), a marker for mitotic cells, and β-Tubulin III (red) at E4 (A), E5 (B), E6 (C), E8 (D) and E18 (E). Arrowhead in E indicates the ora serrata, a region between the NR and ciliary body, and shows sharp termination of ganglion cell differentiation at the retinal margin. Nuclear DAPI staining (blue) marks all cells.