Figure 3. SoxC TFs repress Notch-Hes5 signaling to promote contralateral RGC differentiation.

(A) Expression of Hes5 and Notch1 mRNAs in Ki67⁺ progenitor cells and Sox4 mRNA in Islet1/2⁺ RGCs at E15.5. (B) Luciferase assay in HEK293 cells transfected with combinations of expression vectors for the Notch intracellular domain (NICD) (1.2 μg) and/or SoxC (4, 11, 12) (2.4 μg) with a reporter vector including the mouse Hes5 promoter region (0.4 μg) and a Renilla luciferase construct (0.03 μg). Data are presented as fold change in relative luciferase activity normalized to the mean of empty vector (Vec). n = 4, two-way ANOVA. (C) Representative images of Islet1/2⁺ RGCs after overexpression of CAG-Hes5 plasmid into WT DT retina (arrows). (D, E) Quantification of Islet1/2⁺/GFP⁺ cells (%) (D) and Ki67⁺/GFP⁺ cells (%) (E) in WT or Sox4^−/−Sox11^−/−Sox12^−/− DT retina when Hes5 is overexpressed.(>360 of total GFP⁺ cells counted for each condition, n = 4, one-way ANOVA) (F) A representative image of Islet1/2⁺ RGCs in Sox4^−/−Sox11^−/−Sox12^−/− DT retinal cells with DAPT added to the medium (+ DAPT) (arrows). (G, H) Quantification of Islet1/2⁺/GFP⁺ cells (%) (G) and Ki67⁺/GFP⁺ cells (%) (H) in WT or Sox4^−/−Sox11^−/−Sox12^−/− DT retinal cells cultured with control or DAPT (>370 of total GFP⁺ cells counted for each condition, n = 4 – 5, one-way ANOVA). (I) qRT-PCR measurements of Hes5, Hes1, and Ccnd1 mRNAs of WT and Sox4^−/−Sox11^−/−Sox12^−/− GFP⁺ cells. n = 4, Student’s t test. See also Figures S3 and S4. Scale bars: 100 μm in B; 10 μm in C and F. * p<0.05, ** p<0.01, *** p<0.001, N.S. not significant.