To induce rod photoreceptor apoptosis, we treated NF stage 47/48 (equivalent to 14 days postfertilization) iCasp9/GFP double transgenic tadpoles with AP20187 for 4 days. To test the inherent regenerative capacity of
X. laevis retina, drug treatment was withdrawn and tadpoles were allowed to recover for 4 weeks in normal rearing conditions. Retinal cryosections were prepared, immunolabeled, and examined by confocal microscopy. We confirmed that administration of AP20187 induced rapid rod cell death, with ablation of a majority of rod photoreceptors after the 4 days of drug treatment (
Fig. 1A). After the 4-week recovery period, the rod outer segment (ROS) layer remained absent in the central retina of the drug treated iCasp9 tadpoles (
Fig. 1C). There was no detectable GFP signal in the photoreceptor layer, namely, the outer nuclear layer (ONL), inner segment (IS), and outer segment (OS), indicating the absence of rod photoreceptors in the central retina. Only cone photoreceptors remained in the ONL of the central retina, as evidenced by immunolabeling with cone-specific calbindin antibodies (
Fig. 1C'). However, rod photoreceptors were observed adjacent to the ciliary marginal zone (CMZ) at the periphery of the growing tadpole retina, indicating that acute drug treatment did not prevent the generation of new rod photoreceptors. The CMZ is a zone of retinal stem cells at the anterior margin of the
X. laevis retina that facilitates the addition of new retina as the eye grows.
18 In contrast, there was no evidence of new rod cells repopulating sections of the retina where rods were lost due to drug-induced apoptosis. In the untreated retinas, rod photoreceptors could be detected by GFP in the photoreceptor layer (
Figs. 1B,
1D). This methodology is capable of detecting rod photoreceptors even if their morphology is significantly altered.
16 These observations indicate that rods lost by caspase 9–mediated apoptosis do not regenerate after the death-inducing signal is removed. Furthermore, new rods from the expanding CMZ do not migrate along the retina to repopulate the ablated region. Interestingly, we found that the morphology of the cone photoreceptors differed between rod-ablated retinas and untreated retinas (
Figs. 1C', 1D'), with the overall length of the cones in the rod-ablated retinas being shorter in length. We previously reported a similar result in drug-treated adult frogs.
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