Fig. 1: Effects of downregulation of Nmnat1 during retinal development lead to apoptosis.

a Transition of the expression of mRNAs of Nmnat family members during mouse retinal development was examined by RT-qPCR. Whole retinas at indicated developmental stages were isolated, and RT-qPCR was performed. Relative expression levels to Gapdh are shown. The values are average of 3 independent samples with standard deviation. b, c Plasmids encoding sh-Nmnat1 or scramble control in combination with EGFP expression plasmid were electroporated into mouse isolated retina at E17.5, and the retinas were cultured for 3 or 4 days as explants and frozen sectioned. Immunostaining was done by using anti-active Caspase 3 (AC3) antibody to detect apoptosis (b) or anti-Ki67 antibody to detect proliferating cells (c). Transfected cells were stained with anti-GFP antibody, and nuclei were visualized by staining with DAPI (b, c). d–f Population of AC3 + EGFP + (d), Ki67 + EGFP + (f) cells in total EGFP-positive cells are shown. The sections at day 3 were stained with anti-Chx10 and -AC3 antibodies (e), with anti-GFP antibody (Supplementary Fig. 3C). Populations of triple-positive cells in total EGFP-positive cells are shown in e. The values are average of three independent samples with standard deviation. g, h Retinas at E17.5 were transfected with sh-Nmnat1 or control with EGFP expression plasmid and cultured for 3 days in the presence of Z-VAD-FMK at 20 μM in the final concentration. Frozen sections were stained with anti-AC3 (g) or -Ki67 (h) antibody with anti-GFP antibody. Nuclei were visualized by DAPI staining. Statistical significance was calculated by Student’s T-test. ***p < 0.005. Scale bar = 50 μm