We report that in spite of its regenerative capacity, rod photoreceptor loss in X. laevis
results in secondary cellular changes similar to those observed in nonregenerative models. Cone cell degeneration and death are observed in patients with RP and in all nonregenerative RP animal models.2,35–43
Similarly, ablation of rod photoreceptors using the NTR-metronidazole enzyme-prodrug system resulted in outer segment degeneration and cone cell death in X. laevis
(). In contrast to these results, a recent study also using X. laevis
did not report cone loss after rod cell ablation.9
Activation of a modified caspase-9 (iCasp9) in rod photoreceptors resulted in rod cell death in both premetamorphic and postmetamorphic X. laevis
. Although cone death was not reported, cone function was compromised after 3 months in postmetamorphic animals. Interestingly, photopic ERGs recovered by 5 months, prompting the speculation that recovery resulted from either functional restoration or regeneration of cones. The differences observed in these two Xenopus
models might have been due to the enzyme-prodrug system used, the time at which it was activated, or both. For instance, XOPNTR tadpoles were continuously treated with metronidazole, whereas post-metamorphic frogs received periodic subcutaneous injections of the iCasp9 activator AP20187 (the effect of AP20187 on cone survival in tadpoles was not addressed). The discontinuous delivery method that must be used in older animals may not result in cone cell degeneration and death. Alternatively, the extent and rate of secondary degeneration may be age dependent. Frog cones may be more resistant to the effects of rod ablation than are the cones of the tadpole retina. Examining the fate of cones in metronidazole-treated XOPNTR frogs and AP20187-treated iCasp9 tadpoles should distinguish between these possibilities.
Leakage of the cytotoxic-form of the drug into neighboring cells could also explain the loss of cones in XOPNTR animals. Several lines of evidence, however, suggest that this mechanism is unlikely to be driving cone loss in rod-ablated Xenopus
retinas. First, cone cell loss was progressive, mimicking the temporal sequence of morphologic changes observed in other animal RP models in which outer segments degenerate first, followed by the loss of cone soma.28,29
Second, cones continue to die in the absence of rods, which suggests cone loss is independent of the NTR-Mtz system because rods are no longer present to convert Mtz to its cytotoxic form. Consistent with this interpretation, cones lacking outer segments were observed in the ONL nearly 3 weeks after the last rods had been ablated (C″). Third, metronidazole was specifically developed as a substrate for NTR to avoid the prodrug-related death of neighboring cells observed with previous substrates. In cell culture studies, the death of neighboring cells was minimal, even under conditions in which targeted and nontargeted cells share gap junctions.44
Fourth, a recent study investigating the regenerative response of the zebrafish retina to rod ablation found no evidence of cone cell death when using the NTR-Mtz system.13
In addition to cone loss, Müller glia hypertrophy was also observed in the retinas of Mtz-treated XOPNTR tadpoles. Expression of the Müller cell marker R5 () and the intermediate filament protein vimentin (not shown) were dramatically increased in Mtz-treated animals. Enlarged Müller processes extend throughout the retina, most notably into the subretinal space (G). These changes mimic those observed in mammalian retinal degenerations.3,45
Zebrafish Müller glia also respond to rod loss by upregulating the expression of intermediate filaments such as glial fibrillary acidic protein.46,47
In contrast to Xenopus
, however, extensive gliosis in the subretinal space has not been reported in fish. These results are intriguing given the distinct response of these two regenerative models to rod ablation. Cone loss is not observed in the rodless zebrafish retina.13,48
Rod ablation driven by misexpression of a membrane-targeted form of cyan fluorescent protein under the control of the Xenopus
rhodopsin promoter did not result in cone degeneration.48
Similarly, cone loss was not detected in Mtz-treated, rodless transgenic fish expressing NTR under the control of the zebrafish rod opsin promoter.13
In zebrafish, retinal damage results in the activation of Müller glia, which reenter the cell cycle to produce neuronal progenitors that differentiate into retinal neurons and heal the damaged region.46,49–54
In contrast to fish, retinectomy experiments in both premetamorphic and postmetamorphic Xenopus
indicate transdifferentiating RPE is the source of new retinal neurons.10,11
The correlation between the extent of Müller cell hypertrophy and cone cell death may point to a role for gliosis in cone cell degeneration.
, rod ablation also resulted in a reduction in the thickness of the outer plexiform layer (C″; asterisks) observed in other models of photoreceptor degeneration.55,56
In contrast to nonregenerative models, however, we observed no statistically significant change in the number of INL or GCL cells in Mtz-treated tadpoles. Previous studies indicate that near complete cone cell loss is necessary for extensive neuronal remodeling, including the death of INL and GCL cells.28
After 17 days of Mtz exposure, the number of cone cells was reduced to approximately 30% of wild-type levels, possibly explaining the lack of cell death in other retinal layers. In future experiments, it will be important to determine whether the late phases of degeneration (INL, GCL cell death, and neuronal remodeling) are observed in rodless and coneless XOPNTR tadpoles.
When given time to recover, the retinas of rod-ablated XOPNTR tadpoles generated new rod photoreceptors with outer segments; however, regeneration was not complete, possibly because of insufficient recovery time. Alternatively, secondary cellular or molecular changes in these regions might have permanently inhibited rod regeneration. Consistent with this hypothesis, rod regeneration appeared less robust in transgenic animals treated with Mtz for 17 days compared with 12-day treated animals (not shown). However, additional experiments will be necessary to distinguish between these two possibilities.
Two sources of new cells in the amphibian retina are the adult retinal stem cells of the CMZ and the retinal pigment epithelium (RPE). Additional experiments will be necessary to determine whether the CMZ, RPE, or an unidentified cell class is the source of the newly born rods. Recently, Müller glial cells have been speculated to contribute to the regeneration of the retina of higher order vertebrates, as occurs in teleost fish.52,57
However, our preliminary evidence shows no statistically significant difference in the number of mitotic or EdU-labeled Müller glial cells between Mtz-treated transgenic and control animals after 3, 5, or 10 days (data not shown). A more extensive study is necessary to conclusively determine whether Müller glial cells play a role in retinal regeneration.
During normal retinal development, cell classes are born in a stereotypical order. Retinal ganglion, horizontal, and cone cells are born early, followed by rods, bipolar, amacrine, and Müller cells. Are the mechanisms of rod regeneration distinct from those of retinal development? Are the early retinal cell fates skipped to directly generate rods in XOPNTR tadpoles? How rapidly is rod vision restored during regeneration? If retinal degeneration is allowed to progress, will regeneration no longer be possible? Or will rods, cones, INL, and RGCs all regenerate and reform the complex neural network necessary for functional vision? The rapid, synchronous degeneration of rods, their regeneration, and the ability to control the timing of these events, coupled with the behavioral assay, makes the XOPNTR model useful for studying the mechanisms regulating both retinal degeneration and regeneration.