Fig. 8

Effects of complete loss of function of Brn3a or Ret on RGCs. a Immunostaining of retina sections from Rax:Cre; Brn3a^CKOAP/WT (top row) and Rax:Cre; Brn3a^CKOAP/KO (bottom row) mice, using anti-Ret (green), anti-alkaline phosphatase (anti-AP, red) antibodies, and DAPI nuclear stain. Arrows indicate Ret-only-positive cells, arrowheads indicate Ret⁺AP⁺ double-labeled cells. b Pie-charts and (c) box-plots representing proportions of different cell categories according to the expression of Ret and AP in Rax:Cre; Brn3a^CKOAP/WT (left) and Rax:Cre; Brn3a^CKOAP/KO (right) animals. Total number of cells for each category are presented next to the pie-chart markers, and data spread for each category is shown in the boxplots, expressed as percent total DAPI positive cells. d RGCs (in red) and their respective retinas (in black) have matching genotypes in both Rax:Cre; Brn3a^CKOAP/WT (left) and Rax:Cre; Brn3a^CKOAP/KO (right) animals. e Immunostaining of sections through retinas from Rax:Cre; Ret^CKCFP/WT (top row, “Ret^KO/WT”) and Rax:Cre; Ret^CKCFP/CKCFP (bottom row, “Ret^KO/KO”) animals, using anti-GFP (staining CFP expressing cells, green) together with anti-Brn3a (red), anti-Brn3b (white) antibodies, and DAPI nuclear stain. Arrowheads indicate (CFP⁺Brn3a⁺) double-positive cells, and arrows indicate (CFP⁺Brn3a⁺Brn3b⁺) triple-labeled cells respectively. (f) Pie charts and (g) box-whiskers plots representing quantitation of immunostaining sections from Ret^KO/WT (left) and Ret^KO/KO (right) mice. Total number of cells for each category is presented next to the pie-chart markers. h CFP-positive RGCs (in green) and their respective retinas (in black) have matching genotypes in both Rax:Cre; Ret^CKCFP/WT (left) and Rax:Cre; Ret^CKCFP/CKCFP (right) animals. For each genotype in (a-c and e–g), sections from at least 3 different animals were stained and 9–20 images were quantified. For Box-Whisker plots, the red lines represent the median, the rectangles represent the interquartile interval, and the whiskers the full range of observations. Red crosses are outliers. Significance levels: *, p < 0.05; ** p < 0.01; *** p < 0.001; n.s. >  = 0.05. Total number of mice, images and cells in Supplementary Table 2. Scale bar in (a) and (e) is 25 μm