(A) Double labeling of wild-type (upper) and banprw337 mutant (lower) retinas with TUNEL (red) and anti-pH3 antibody (blue). Nucleus was counterstained with Sytoxgreen (green). (B) Only TUNEL and nuclear fluorescent channel of images shown in (A). TUNEL and nuclear staining are indicated in magenta and green, respectively. In banprw337 mutants, apoptotic cells start to be observed in neural retinas at 53 hpf, and increase in the apical region of retinas (white arrowheads) and at the interface between the CMZ and the central retina (yellow open arrowheads) at 77 and 101 hpf. (B’) Only pH3 and nuclear fluorescent channel of images shown in (A). pH3 signals and nuclear staining are indicated in magenta and green, respectively. Mitotic cells are accumulated at the apical surface of banprw337 mutant retinas at 53 hpf (white open arrowheads) and restricted in the apical region of the CMZ (yellow open arrow) at 77 and 101 hpf. (C) The number of TUNEL-positive cells per retina. Apoptosis is significantly increased in banprw337 mutants after 53 hpf. Unpaired t-test, two-tailed, Mean ± SD. [n=3, p**<0.01] (D) The number of pH3-positive cells per retina. The number of mitotic cells is significantly increased in banprw337 mutants at 53 hpf. The number is decreased, but still significantly higher in banprw337 mutants than in wild-type siblings after 77 hpf. Unpaired t-test, two-tailed, Mean ± SD. [n=3, p**<0.01] (E) Schematic diagram of wild-type retinas. Retinal progenitor cells undergo cell-cycle progression in which the nuclei move along the apico-basal axis depending on cell-cycle phase (the most basal location in S phase and the most apical location in M phase), called interkinetic nuclear migration. Mitosis occurs in the apical surface of the neural retina. Daughter cells reenter the cell cycle as retinal progenitor cells or start to differentiate into postmitotic neurons, which migrate into the basal region and become mature neurons. (F) Schematic diagram of banprw337 mutant retinas. A fraction of interphase progenitor cells (G1, S, or G2 phase cells) undergo apoptosis after 48 hpf. Mitotic cells are accumulated at the apical surface, which may be caused by prolonged mitotic duration. A fraction of immature differentiating neurons also undergo apoptosis. Thus, banprw337 mutant retinal cells show multiple defects in cell-cycle progression of retinal progenitor cells and retinal neurogenesis. Scale bars: 20 μm for (A, B, B’).
