Figure 6.

Overexpression of Sox4 and Sox11 is sufficient to promote RGC differentiation from RPCs. A, B, Nestin⁺ (A, red) E14 RPCs were transfected with a GFP reporter plasmid (B, green, marking transfected cells) and candidate differentiation genes and counterstained with DAPI for nuclei (blue) and for the RGC-specific marker Brn3 (red), as marked. C, Following transfection with candidate genes as marked, RPCs were cultured in pro-RGC differentiation and survival conditions for 4 d, fixed, and immunostained. Brn3⁺ and GFP⁺ cells out of all GFP⁺ cells were quantified. Overexpression of Math5, Sox4, and Sox11 increased the total number of GFP⁺ and Brn3⁺ cells (mean ± SEM, *p < 0.01, N = 3, ANOVA with post hoc Dunnett's test, mean ± SEM). D, RPCs were differentiated in the presence of EdU (5 μm) for 5 d and immunostained for the RGC marker Brn3 and EdU. Sox4 and Sox11 overexpression with lentivirus increased RGC differentiation (Brn3⁺, GFP⁺, and EdU⁺ cells) of proliferative, transfected RPCs (GFP⁺ and EdU⁺ cells) compared with control GFP overexpression (*p < 0.02, paired t test; mean ± SEM; all samples differ significantly from each other). E, Embryonic retinal cells from Math5-Cre/Sox4^fl/fl mice were differentiated in the presence of control or Sox11 shRNA; Sox11 knockdown further decreased RGC differentiation of Math5-Cre/Sox4^fl/fl progenitors compared with control mice. F, Sox4 and Sox11 overexpression with lentivirus had no effect on the survival or total number of RGCs that were postmitotic in vivo and then placed in culture (Brn3⁺, GFP⁺, and EdU⁻ cells; *p < 0.02, paired t test; mean ± SEM). G, Overexpression of Sox11-SUMO-1, Sox11^K91R-SUMO-1, and SUMO-1 in HEK cells and probed with Sox11 for Western blot. H–J, RPCs were further infected with Sox11 mutant gene (Sox11^K91R) for immunofluorescence (H), or probed with βIII-tubulin (I), or probed with Brn3 and EdU (J). K, Overexpression of REST in WT E14 RPCs decreased RGC differentiation compared with controls as measured by immunostaining of differentiated RGCs (Brn3⁺, GFP⁺, EdU⁺/GFP⁺, and EdU⁺; *p < 0.05, **p < 0.01 paired t test; mean ± SEM; all samples differ significantly from each other). L, REST overexpression in E14 RPCs derived from retinal-specific Sox4 cKO mice elicited no changes in RGC fate specification. M, Integrated model for regulatory mechanisms of RGC fate specification. Scale bars, 30 μm.