Rod Photoreceptor Regeneration Research Articles

Photoreceptor regeneration occurs normally in microglia-deficient irf8 mutant zebrafish following acute retinal damage

Microglia are resident immune cells in the central nervous system, including the retina that surveil the environment for damage and infection. Following retinal damage, microglia undergo morphological changes, migrate to the site of damage, and express and secrete pro-inflammatory signals. In the zebrafish retina, inflammation induces the reprogramming and proliferation of Müller glia and the regeneration of neurons following damage or injury. Immunosuppression or pharmacological ablation of microglia reduce or abolish Müller glia proliferation. We evaluated the retinal architecture and retinal regeneration in adult zebrafish irf8 mutants, which have significantly depleted numbers of microglia. We show that irf8 mutants have normal retinal structure at 3 months post fertilization (mpf) and 6 mpf but fewer cone photoreceptors by 10 mpf. Surprisingly, light-induced photoreceptor ablation induced Müller glia proliferation in irf8 mutants and cone and rod photoreceptor regeneration. Light-damaged retinas from both wild-type and irf8 mutants show upregulated expression of mmp-9, il8, and tnfβ pro-inflammatory cytokines. Our data demonstrate that adult zebrafish irf8 mutants can regenerate normally following acute retinal injury. These findings suggest that microglia may not be essential for retinal regeneration in zebrafish and that other mechanisms can compensate for the reduction in microglia numbers.

A Zebrafish Model of Retinitis Pigmentosa Shows Continuous Degeneration and Regeneration of Rod Photoreceptors.

More than 1.5 million people suffer from Retinitis Pigmentosa, with many experiencing partial to complete vision loss. Regenerative therapies offer some hope, but their development is challenged by the limited regenerative capacity of mammalian model systems. As a step toward investigating regenerative therapies, we developed a zebrafish model of Retinitis Pigmentosa that displays ongoing regeneration. We used Tol2 transgenesis to express mouse rhodopsin carrying the P23H mutation and an epitope tag in zebrafish rod photoreceptors. Adult and juvenile fish were examined by immunofluorescence, TUNEL and BrdU incorporation assays. P23H transgenic fish expressed the transgene in rods from 3 days post fertilization onward. Rods expressing the mutant rhodopsin formed very small or no outer segments and the mutant protein was delocalized over the entire cell. Adult fish displayed thinning of the outer nuclear layer (ONL) and loss of rod outer segments, but retained a single, sparse row of rods. Adult fish displayed ongoing apoptotic cell death in the ONL and an abundance of proliferating cells, predominantly in the ONL. There was a modest remodeling of bipolar and Müller glial cells. This transgenic fish will provide a useful model system to study rod photoreceptor regeneration and integration.

Midkine-a protein localization in the developing and adult retina of the zebrafish and its function during photoreceptor regeneration.

Midkine is a heparin binding growth factor with important functions in neuronal development and survival, but little is known about its function in the retina. Previous studies show that in the developing zebrafish, Midkine-a (Mdka) regulates cell cycle kinetics in retinal progenitors, and following injury to the adult zebrafish retina, mdka is strongly upregulated in Müller glia and the injury-induced photoreceptor progenitors. Here we provide the first data describing Mdka protein localization during different stages of retinal development and during the regeneration of photoreceptors in adults. We also experimentally test the role of Mdka during photoreceptor regeneration. The immuno-localization of Mdka reflects the complex spatiotemporal pattern of gene expression and also reveals the apparent secretion and extracellular trafficking of this protein. During embryonic retinal development the Mdka antibodies label all mitotically active cells, but at the onset of neuronal differentiation, immunostaining is also localized to the nascent inner plexiform layer. Starting at five days post fertilization through the juvenile stage, Mdka immunostaining labels the cytoplasm of horizontal cells and the overlying somata of rod photoreceptors. Double immunolabeling shows that in adult horizontal cells, Mdka co-localizes with markers of the Golgi complex. Together, these data are interpreted to show that Mdka is synthesized in horizontal cells and secreted into the outer nuclear layer. In adults, Mdka is also present in the end feet of Müller glia. Similar to mdka gene expression, Mdka in horizontal cells is regulated by circadian rhythms. After the light-induced death of photoreceptors, Mdka immuonolabeling is localized to Müller glia, the intrinsic stem cells of the zebrafish retina, and proliferating photoreceptor progenitors. Knockdown of Mdka during photoreceptor regeneration results in less proliferation and diminished regeneration of rod photoreceptors. These data suggest that during photoreceptor regeneration Mdka regulates aspects of injury-induced cell proliferation.

Xenopus laevisTadpoles Can Regenerate Neural Retina Lost after Physical Excision but Cannot Regenerate Photoreceptors Lost through Targeted Ablation

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). 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). 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. 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.

FGF signaling regulates rod photoreceptor cell maintenance and regeneration in zebrafish

Fgf signaling is required for many biological processes involving the regulation of cell proliferation and maintenance, including embryonic patterning, tissue homeostasis, wound healing, and cancer progression. Although the function of Fgf signaling is suggested in several different regeneration models, including appendage regeneration in amphibians and fin and heart regeneration in zebrafish, it has not yet been studied during zebrafish photoreceptor cell regeneration. Here we demonstrate that intravitreal injections of FGF-2 induced rod precursor cell proliferation and photoreceptor cell neuroprotection during intense light damage. Using the dominant-negative Tg(hsp70:dn-fgfr1) transgenic line, we found that Fgf signaling was required for homeostasis of rod, but not cone, photoreceptors. Even though fgfr1 is expressed in both rod and cone photoreceptors, we found that Fgf signaling differentially affected the regeneration of cone and rod photoreceptors in the light-damaged retina, with the dominant-negative hsp70:dn-fgfr1 transgene significantly repressing rod photoreceptor regeneration without affecting cone photoreceptors. These data suggest that rod photoreceptor homeostasis and regeneration is Fgf-dependent and that rod and cone photoreceptors in adult zebrafish are regulated by different signaling pathways.

Microarray Analysis of XOPS-mCFP Zebrafish Retina Identifies Genes Associated with Rod Photoreceptor Degeneration and Regeneration

XOPS-mCFP transgenic zebrafish experience a continual cycle of rod photoreceptor development and degeneration throughout life, making them a useful model for investigating the molecular determinants of rod photoreceptor regeneration. The purpose of this study was to compare the gene expression profiles of wild-type and XOPS-mCFP retinas and identify genes that may contribute to the regeneration of the rods. Adult wild-type and XOPS-mCFP retinal mRNA was subjected to microarray analysis. Pathway analysis was used to identify biologically relevant processes that were significantly represented in the dataset. Expression changes were verified by RT-PCR. Selected genes were further examined during retinal development and in adult retinas by in situ hybridization and immunohistochemistry and in a transgenic fluorescent reporter line. More than 600 genes displayed significant expression changes in XOPS-mCFP retinas compared with expression in wild-type controls. Many of the downregulated genes were associated with phototransduction, whereas upregulated genes were associated with several biological functions, including cell cycle, DNA replication and repair, and cell development and death. RT-PCR analysis of a subset of these genes confirmed the microarray Three transcription factors (sox11b, insm1a, and c-myb), displaying increased expression in XOPS-mCFP retinas, were also expressed throughout retinal development and in the persistently neurogenic ciliary marginal zone. This study identified numerous gene expression changes in response to rod degeneration in zebrafish and further suggests a role for the transcriptional regulators sox11b, insm1a, and c-myb in both retinal development and rod photoreceptor regeneration.

Notch and Wnt Signaling Mediated Rod Photoreceptor Regeneration by Müller Cells in Adult Mammalian Retina

BackgroundEvidence emerging from a variety of approaches used in different species suggests that Müller cell function may extend beyond its role of maintaining retinal homeostasis to that of progenitors in the adult retina. Enriched Müller cells in vitro or those that re-enter cell cycle in response to neurotoxin-damage to retina in vivo display multipotential and self-renewing capacities, the cardinal features of stem cells.Methodology/Principal FindingsWe demonstrate that Notch and Wnt signaling activate Müller cells through their canonical pathways and that a rare subset of activated Müller cells differentiates along rod photoreceptor lineage in the outer nuclear layer. The differentiation of activated Müller cells along photoreceptor lineage is confirmed by multiple approaches that included Hoechst dye efflux analysis, genetic analysis using retina from Nrl-GFP mice, and lineage tracing using GS-GFP lentivirus in wild type and rd mice in vitro and S334ter rats in vivo. Examination of S334ter rats for head-neck tracking of visual stimuli, a behavioral measure of light perception, demonstrates a significant improvement in light perception in animals treated to activate Müller cells. The number of activated Müller cells with rod photoreceptor phenotype in treated animals correlates with the improvement in their light perception.Conclusion/SignificanceIn summary, our results provide a proof of principle for non-neurotoxin-mediated activation of Müller cells through Notch and Wnt signaling toward the regeneration of rod photoreceptors.

The Zebrafish Galectin Drgal1-L2 Is Expressed by Proliferating Müller Glia and Photoreceptor Progenitors and Regulates the Regeneration of Rod Photoreceptors

The purpose of this study was to identify secreted proteins in the retina of the adult zebrafish that are induced by the selective death of photoreceptors and to test experimentally the function of these proteins during the regeneration of photoreceptors. Induced selective death of photoreceptors in the retina of the adult zebrafish was combined with in situ hybridization and immunocytochemistry to identify the induced cellular expression of the secreted beta-galactoside binding protein Galectin 1-like 2 (Drgal1-L2). Electroporation of morpholino oligonucleotides was used to knock down protein synthesis, and regenerated photoreceptors were counted in control and experimental retinas after labeling with cell type-specific RNA probes. Expression analysis and immunocytochemistry showed that Drgal1-L2 is induced de novo by photoreceptor death and is synthesized by microglia and proliferating Müller glia and their mitotic progeny. Knockdown of Drgal1-L2 expression in Müller glia results in reduced regeneration of rod photoreceptors without affecting injury-induced proliferation or the regeneration of cone photoreceptors. Based on these data, the authors conclude that Drgal1-L2 is induced by photoreceptor cell death and secreted by stem cells and neuronal progenitors and that it regulates the regeneration of rod photoreceptors. Drgal1-L2 is the first secreted factor shown to regulate aspects of regenerative neurogenesis in the teleost retina.