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Additional Supporting Information may be found in the online version of this article.

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DVDY_22168_sm_SuppFig1.tif4932KSupp. Fig. S1. Axin2 (Ch.1) and Lef1 expression pattern. A, C: Analysis of Axin2(Ch.1) expression (purple) detected by WMISH at st. 19 (A) and st. 24 (C). B, D: Analysis of Lef1 expression (purple) detected by WMISH at st. 19 (B) and st. 24 (D). Arrowheads, regions where both genes are expressed. Arrows, regions where only Axin2(Ch.1) is expressed. Scale bars = 100 μm.
DVDY_22168_sm_SuppFig2.tif16326KSupp. Fig. S2. Endogenous canonical Wnt signaling in the medaka retina. A: β-Cat immunohistochemistry and DAPI staining on eye cryosections. Z-sections cross at a dot of strong β-Cat staining (a). Z-axis projections are shown to the right (y axis) and below (x axis). B: Magnification of the area around (a) and corresponding z-sections, which show that β-Cat staining is surrounded by DAPI staining in all three axis. C: Control immunohistochemistry experiment using only secondary antibody showing no signal. D: β-Cat immunohistochemistry detection in the CMZ (arrows) at st. 24. Scale bars = 10 μm (A); 5 μm (B); 20 μm (C,D).
DVDY_22168_sm_SuppFig3.tif9757KSupp. Fig. S3. Marker gene expression pattern in the medaka developing eye. Expression patterns of (A) Ath5, (B) Prox1, (C) Vsx1, and (D) Rhod genes during different stages of eye development (sts. 23, 25, 27, 30, 34, 36).
DVDY_22168_sm_SuppFig4.tif8175KSupp. Fig. S4. Pharmacological inhibition of GSK3β regulates Ath5 and Vsx1 expression, but not Rhodopsin expression. A, D: Scheme showing the stages when LiCl or indirubin were administered and the stages when embryos were fixed and gene expression analyzed (arrows). CAB embryos were incubated with LiCl or indirubin from st. 17 until fixation at st. 30 or 35 and Ath5, Vsx1, or Rhodopsin expression was analyzed. B, C: Pharmacological inhibition of GSK3β activated Ath5 expression at st. 30. Indirubin increased Ath5 mRNA expression in 71% of embryos tested, n = 20. E–G: Pharmacological inhibition of GSK3β activated Vsx1 expression at st. 35. LiCl increased the expression of Vsx1 in 90% of embryos, n = 20, and indirubin in 75% of the embryos, n = 20. H, I: Pharmacological inhibition of GSK3β did not affect Rhodopsin expression at st. 35, n = 25. Scale bars = 50 μm.
DVDY_22168_sm_SuppFig5.tif5231KSupp. Fig. S5. WMISH using sense probes. Control WMISH analysis using sense probes for Ath5 at st. 30, Prox1 at st. 32, and Vsx1 or Rhodopsin at st. 35. Scale bars = 50 μm.
DVDY_22168_sm_SuppFig6.tif1833KSupp. Fig. S6. Apoptosis of NRP cells is regulated by Wnt signaling. Bar graphs of the TUNEL analysis representing the absolute numbers of apoptotic cells scored in HS-Wnt8 and HS-DNWnt8 transgenic embryos after the described HS treatments. A: Embryos collected at st. 22 were HS treated at st. 17, embryos collected at st. 26 were HS treated at st. 17 and st. 22, and embryos collected at st. 30 were HS treated at st. 17, st. 22, and st. 26. B: Embryos were HS treated at st. 17 and collected at st. 22 and 30. C: Embryos were HS treated at st. 22 and collected at st. 26 and 30. D: Embryos were HS treated at st. 26 and collected at st. 30. In every group, n = 35. *P < 0.05; **P < 0.01; ***P < 0.001. Bar graphs represent the mean ± SD (error bars) from three independent experiments.
DVDY_22168_sm_SuppFig7.tif2288KSupp. Fig. S7. Proliferation of NRP cells is regulated by Wnt signaling. Bar graphs of the pH3 immunostaining analysis representing the absolute numbers of proliferating cells scored in HS-Wnt8 and HS-DNWnt8 transgenic embryos after the described HS treatments. A: CAB embryos were incubated with LiCl or indirubin between st. 17 until fixation at st. 22 or 26. B: Embryos were HS treated at st. 17 and collected at st. 22, HS treated at st. 17 and st. 22 and collected at st. 26, or HS treated at st. 17, st. 22, and st. 26 and collected at st. 30. C: Embryos were HS treated at st. 17 and collected at st. 22 and 30. D: Embryos were HS treated at st. 22 and collected at st. 26, 30, and 32. E: Embryos were HS treated at st. 26 and collected at st. 30. Proliferation was not affected at this stage by Wnt signaling. In every group n = 60. *P < 0.05; **P < 0.01; ***P < 0.001. Bar graphs represent the mean ± SD (error bars) from three independent experiments.
DVDY_22168_sm_SuppFig8.tif1877KSupp. Fig. S8. GFP-positive transgenic embryos. GFP expression of HS-Wnt8 and HS-DNWnt8 transgenic embryos at st.19. Scale bars = 100 μm.
DVDY_22168_sm_SuppTables.doc157KSupplementary table 1. Primers and PCR conditions used for qRT-PCR. Supplementary table 2. Data results from apoptosis analysis (TUNEL) in HS-treated transgenic embryos. The data represent the number of cells stained with TUNEL in the developing retina. n is the number of retinas counted for each condition. This data was used to calculate relative values using the control condition as 1. Supplementary table 3. Data results for proliferation using pH3 staining in treated embryos. The data represent the number of cells stained with pH3 in the developing retina. n is the number of retinas counted for each condition. This data was used to calculate relative values using the control condition as 1. Supplementary table 4. List of primers used in RT-PCR and qRT-PCR, and RT-PCR conditions.

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