Microphthalmia is due to increased cell death in cdh4 knockdowns. A-D,F-M: Zebrafish embryos were injected with either standard control morpholino oligonucleotide (MO, A,F,H,J,L) or cdh4 MO (B-D,G,K,I,M). Mitotic cells were visualized in eyes of 48 hours postfertilization (hpf) embryos using histone-H3 immunostaining (control, n = 31; slight, n = 16; moderate, n = 15; severe n = 13). A-D: In both control (A) and in cdh4 knockdowns (B-D), dividing cells were confined to the peripheral lamina of the retina. E: Comparison of the number of H3-positive nuclei per μm2 showed no significant difference between controls and cdh4 knockdowns. Statistical comparisons of these data were made in two ways. A two-tailed unpaired Students t-test was used to compare controls and pooled data from all cdh4 morphant groups (P = 0.07). One-way analysis of variance was used to compare controls and unpooled cdh4 morphant groups (P = 0.08). E: Chart shows unpooled group data. Error bars = 1 SD. A-D: Representative confocal images are shown of control (A) and cdh4 knockdowns from the slightly affected (B), moderately affected (C), and severely affected (D) cdh4 morphant groups. Dying cells were visualized using acridine orange (AO) vital staining at 36 hpf (control, n = 9; cdh4 MO, n = 14) and at 50 hpf (control, n = 5; cdh4 MO, n = 6). F-M: Representative differential interference contrast images (F-I) and a corresponding fluorescent image (J-M) are shown for each group; control at 36 hpf (F,J), cdh4 MO injected at 36 hpf (G,K), control at 50 hpf (H,L), and cdh4 MO at 50 hpf (I,M). AO-labeled cells were counted. Cell death was increased in the retina of cdh4 knockdowns compared with controls at both 36 hpf (P < 0.01) and 50 hpf (P < 0.01). Randomly selected embryos were used for AO staining to minimize potential bias. Cell death was increased in the lens at 50 hpf (P < 0.05) but not at 36 hpf. Error bars = 1 SD.
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