Xenopus laevis tadpoles can regenerate neural retina lost after physical excision but cannot regenerate photoreceptors lost through targeted ablation.

PURPOSE: To determine whether the Xenopus laevis retina is capable of regenerating photoreceptor cells lost through apoptotic cell death in an inducible transgenic X. laevis model of retinitis pigmentosa (RP). METHODS: Acute rod photoreceptor apoptosis was induced in transgenic X. laevis expressing drug-inducible caspase 9. We subsequently monitored the ability of the retina to regenerate lost photoreceptors in the absence of drug, and in combination with physical injury or ectopic supplementation of basic fibroblast growth factor (FGF2). RESULTS: Direct activation of caspase 9 in rod photoreceptors resulted in the initiation of apoptosis and complete removal of rod photoreceptors within 4 days. Photoreceptors lost by apoptosis were not replaced over a 4-week recovery time frame. In contrast, physical disruption of rod-ablated retina was repaired by the end of a 3-week time frame, but did not result in rod photoreceptor regeneration other than at the site of injury. Furthermore, ectopic supplementation of FGF2 did not stimulate regeneration of photoreceptors lost by apoptosis. However, FGF2 supplementation increased the rate of regeneration of retina (including rod photoreceptors) in eyes from which retinal tissue was surgically removed. CONCLUSIONS: In the X. laevis retina, rod photoreceptors that undergo drug-induced caspase-9-mediated apoptosis are permanently lost and do not regenerate. In contrast, the neural retina (including rod photoreceptors) can regenerate in injured or retinectomized eyes, and this regeneration is promoted by supplementation with FGF2. However, FGF2 does not promote regeneration of rod photoreceptors that are selectively lost by apoptosis.

Dysmorphic photoreceptors in a P23H mutant rhodopsin model of retinitis pigmentosa are metabolically active and capable of regenerating to reverse retinal degeneration.

This study evaluated the capacity of Xenopus laevis retina to regenerate photoreceptor cells after cyclic light-mediated acute rod photoreceptor degeneration in a transgenic P23H mutant rhodopsin model of retinits pigmentosa. After discontinuation of cyclic light exposure, we monitored histologic progression of retinal regeneration over a 3 week recovery period. To assess their metabolomic states, contralateral eyes were processed for computational molecular phenotyping. We found that retinal degeneration in the P23H rhodopsin mutation could be partially reversed, with regeneration of rod photoreceptors recovering normal morphology (including full-length rod outer segments) by the end of the 3 week recovery period. In contrast, retinal degeneration mediated by directly induced apoptosis did not recover in the 3 week recovery period. Dystrophic rod photoreceptors with truncated rod outer segments were identified as the likely source of rod photoreceptor regeneration in the P23H retinas. These dystrophic photoreceptors remain metabolically active despite having lost most of their outer segments.

Fourier domain optical coherence tomography as a noninvasive means for in vivo detection of retinal degeneration in Xenopus laevis tadpoles.

PURPOSE: To determine the efficacy of Fourier domain optical coherence tomography (FD-OCT) as a noninvasive, nonlethal method for detecting in vivo, pathologic signs of retinal degeneration in Xenopus laevis larvae. METHODS: A prototype OCT system using FD detection customized for tadpole imaging was used to noninvasively obtain retinal scans in two different transgenic X. laevis models of retinal degeneration. FD-OCT retinal scans were compared with laser scanning confocal micrographs of histologic sections of the same eye. Retinal thickness was measured in the histologic micrographs and compared with in vivo measurements acquired with FD-OCT. RESULTS: In vivo retinal images of X. laevis tadpoles were obtained that visualized the major retinal layers. FD-OCT successfully detected the ablation of rod outer segments (OS) in degenerating tadpole eyes. Measurements from FD-OCT and histology showed a decrease in retinal thickness in transgenic mutant tadpoles relative to the wild-type control. The accumulation of phagosomes from dying rod OS was also visualized in the retinal pigment epithelium (RPE) in a degenerating tadpole retina. CONCLUSIONS: This report demonstrates that FD-OCT is a viable technique for screening, diagnosing, and monitoring retinal degeneration in X. laevis tadpoles in vivo.

A-type nuclear lamins act as transcriptional repressors when targeted to promoters.

Regions of heterochromatin are often found at the periphery of the mammalian nucleus, juxtaposed to the nuclear lamina. Genes in these regions are likely maintained in a transcriptionally silent state, although other locations at the nuclear periphery associated with nuclear pores are sites of active transcription. As primary components of the nuclear lamina, A- and B-type nuclear lamins are intermediate filament proteins that interact with DNA, histones and known transcriptional repressors, leading to speculation that they may promote establishment of repressive domains. However, no direct evidence of a role for nuclear lamins in transcriptional repression has been reported. Here we find that human lamin A, when expressed in yeast and cultured human cells as a fusion protein to the Gal4 DNA-binding domain (DBD), can mediate robust transcriptional repression of promoters with Gal4 binding sites. Full repression by lamin A requires both the coiled-coil rod domain and the C-terminal tail domain. In human cells, other intermediate filament proteins such as lamin B and vimentin are unable to confer robust repression as Gal4-DBD fusions, indicating that this property is specific to A-type nuclear lamins. These findings indicate that A-type lamins can promote transcriptional repression when in proximity of a promoter.

G4 DNA unwinding by BLM and Sgs1p: substrate specificity and substrate-specific inhibition.

To understand the specific genetic instabilities associated with deficiencies in RecQ family helicases, we have studied the substrate preferences of two closely related members of this family, human BLM and Saccharomyces cerevisiae Sgs1p. Here we show that both BLM and Sgs1p preferentially unwind G4 DNA relative to Holliday junction substrates, and that substrate preference reflects binding affinity and maps to the conserved central helicase domain. We identify the porphyrin N-methyl mesoporphyrin IX (NMM) as a specific inhibitor of G4 DNA unwinding, and show that in the presence of NMM the helicase becomes trapped on the NMM-G4 DNA complex, consuming ATP but unable to unwind or dissociate. These results suggest that BLM and Sgs1p function proactively in replication to remove G4 DNA structures which would otherwise present obstacles to fork progression, rather than by promoting recombination to restart a fork that has stalled.