Fig 2: Microarray analysis and screening of candidate genes regulated by KLF family members identifies Dusp14 as developmentally regulated axon growth suppressor. (A) Scatterplots of KLF-regulated gene targets undergoing significant change in RGCs. (B) Modified Venn diagram characterizing gene expression analysis of RGCs expressing different KLF family members. (C–E) Gene expression effects on neurite growth screened in P0 RGCs. KLF9 (pattern column) and Dusp14 (arrow) decreased average and maximum neurite length with no effect on neurite number (*P < 0.05, Tukey-Kramer HSD test). (F) Dusp14 expression in the retina, at different postnatal ages. (G) qRT-PCR for Dusp14 in RGCs after KLF9 or control vector transduction (**P < 0.01, paired t-test; n = 4). (H) AAV2-anti-luciferase-shRNA (control shRNA) or AAV2-anti-KLF9-shRNA were intravitreally injected in adult rats and expressed in RGCs in vivo (GFP reporter, green). At 2 weeks, KLF9 shRNA led to a decrease in Dusp14 expression (red) in RGCs (labeled with RBPMS, yellow). Scale bar: 25 µm in (F) and 50 µm in (H). Error bars: standard error of the mean (SEM).

Fig 3: Displaced amacrine cell density is unaffected in PItx2+/- mice. Comparison of Nonganglion cell densities (other cells) to ganglion cell densities in selected Pitx2+/+ versus Pitx2+/- retinas. Images (N = 13) from Pitx2+/- retinas exhibiting a =35% ganglion cell loss of were identified and the density of nonganglion cells “other cells” was deduced by determining the total number of DAPI+ nuclei and subtracting the number of RBPMS+ cells. “Other cells” densities from control images (N = 12) were determined in parallel. “Other cells” are presumed to represent displace amacrine cells, as they constitute the vast majority of nonganglion cells in the ganglion cell layer. Data are presented by dot plot with mean values ± SD. Significance: n.s., not significant; ** P = 0.01.

Fig 4: CDKL5 null mice show normal retinal morphology. (A) Western blot analysis on adult male CDKL5-/y and WT (CDKL5+/y) retinal homogenates showing CDKL5 expression. (B and C) Retinal sections stained for cone photoreceptors (cone arrestin, red) and rod outer segments (rhodopsin, green). The overall structural organization of photoreceptor cells is identical in the two experimental groups. (D and E) Representative images from retinal whole-mount preparations of CDKL5+/y (D) and CDKL5-/y (E) stained for cone arrestin as a cone marker. (F) Quantitation of the total number of cones showed no statistical significance between the two groups (two-tailed Student’s t-test P = 0.435). (G and H) Representative images from retinal whole-mount preparations of CDKL5+/y (G) and CDKL5-/y (H) stained for RBPMS as a RGCs marker. (I) Quantitation of the total number of cones (I) showed no statistical significance between the two groups (two-tailed Student’s t-test P = 0.098). Scale bars are equal to 20 µm. Error bars represent standard error of mean (SEM).

Fig 5: Characterization of Purified RGCs Isolated from Adult Mice. (a–d) Brightfield images (10x) were taken following each panning step during the isolation of adult RGCs from mice: (a) BSL-1 (fibroblasts, endothelial cells, macrophages and microglia), (b) CD11b/c (macrophages and microglia), (c) VC1.1 (amacrine cells), and (d) Thy1.2 (RGCs; scale bar = 250 µm). As expected, Thy1.2+ cells (inset, d) were larger than BSL-1+ (inset, a), CD11b/c+ (inset, b), and VC1.1+ (inset, c) cells (scale bar = 50 µm). (e) Bar graph showing the distribution of the diameter (µm) of the Thy1.2+ cells (n = 2,759 cells). The average cell diameter was 11.3 ± 1.8 µm. (f) 4-step immunopanning of RGCs average yield of 46,718 ± 2,176 cells per retina with an 86.7 ± 1.2% viability per isolation (n = 21). Immunostaining of the isolated cells against RGC Markers, TUJ1 (h) and RBPMS (i), as well as a nuclear stain, DAPI (g; scale bar = 50 µm). Merged image (j) identified 88.1 ± 4.2% of DAPI positive cells to be RBPMS positive. (k) Semi-quantitative q-PCR analysis identified a significant (p < 0.0001) increase in expression of Pou4f2 (RGC marker) and significant decrease (p < 0.0001) in the expression of Rhodopsin (Rho; Photoreceptor gene marker) in the purified RGCs samples compared to the whole retina.