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Purpose: Human Crb1 is implicated in some forms of retinal degeneration, suggesting a role in photoreceptor maintenance. Multiple Crumbs (Crb) polarity genes are expressed in vertebrate retina, although their functional roles are not well understood. To gain further insight into Crb and photoreceptor maintenance, we compared retinal cell densities between wild-type and Tg(RH2-2:Crb2b-sfEX/RH2-2:GFP)pt108b transgenic zebrafish, in which the extracellular domain of Crb2b-short form (Crb2b-sfEX) is expressed in the retina as a secreted protein, which disrupts the planar organization of RGB cones (red, green, and blue) by interfering with Crb2a/2b-based cone-cone adhesion., Methods: We used standard morphometric techniques to assess age-related changes in retinal cell densities in adult zebrafish (3 to 27 months old), and to assess effects of the Crb2b-sfEX transgene on retinal structure and photoreceptor densities. Linear cell densities were measured in all retinal layers in radial sections with JB4-Feulgen histology. Planar (surface) densities of cones were determined in retinal flat-mounts. Cell counts from wild-type and pt108b transgenic fish were compared with both a "photoreceptor maintenance index" and statistical analysis of cell counts., Results: Age-related changes in retinal cell linear densities and cone photoreceptor planar densities in wild-type adult zebrafish provided a baseline for analysis. Expression of Crb2b-sfEX caused progressive and selective degeneration of RGB cones, but had no effect on ultraviolet-sensitive (UV) cones, and increased numbers of rod photoreceptors., Conclusions: These differential responses of RGB cones, UV cones, and rods to sustained exposure to Crb2b-sfEX suggest that Crb-based photoreceptor maintenance mechanisms are highly selective.

Purpose: Zebrafish neurons regenerate from Müller glia following retinal lesions. Genes and signaling pathways important for retinal regeneration in zebrafish have been described, but our understanding of how Müller glial stem cell properties are regulated is incomplete. Mammalian Müller glia possess a latent neurogenic capacity that might be enhanced in regenerative therapies to treat degenerative retinal diseases., Methods: To identify transcriptional changes associated with stem cell properties in zebrafish Müller glia, we performed a comparative transcriptome analysis from isolated cells at 8 and 16 hours following an acute photic lesion, prior to the asymmetric division that produces retinal progenitors., Results: We report a rapid, dynamic response of zebrafish Müller glia, characterized by activation of pathways related to stress, nuclear factor-κB (NF-κB) signaling, cytokine signaling, immunity, prostaglandin metabolism, circadian rhythm, and pluripotency, and an initial repression of Wnt signaling. When we compared publicly available transcriptomes of isolated mouse Müller glia from two retinal degeneration models, we found that mouse Müller glia showed evidence of oxidative stress, variable responses associated with immune regulation, and repression of pathways associated with pluripotency, development, and proliferation., Conclusions: Categories of biological processes/pathways activated following photoreceptor loss in regeneration-competent zebrafish Müller glia, which distinguished them from mouse Müller glia in retinal degeneration models, included cytokine signaling (notably NF-κB), prostaglandin E2 synthesis, expression of core clock genes, and pathways/metabolic states associated with pluripotency. These regulatory mechanisms are relatively unexplored as potential mediators of stem cell properties likely to be important in Müller glial cells for successful retinal regeneration.

Purpose: To determine whether retinal progenitor cells in the inner nuclear layer give rise to regenerated cones after laser ablation of photoreceptors in adult goldfish retina., Methods: Using a technique developed previously in this laboratory, photoreceptors in the retina of adult goldfish were ablated with an argon laser. The mitotic marker, bromodeoxyuridine, was used to label proliferating and regenerated cells, which were identified with cell-specific markers., Results: Cells proliferating locally within lesion included microglia, Müller glia, and retinal progenitors in the inner nuclear layer (INL). The nuclei of both Müller glia and associated retinal progenitors migrated from the inner to the outer nuclear layer. The proliferating retinal progenitors, which express Notch-3 and N-cadherin, regenerated cone photoreceptors and then rod photoreceptors., Conclusions: Previous work has demonstrated that photoreceptors in the goldfish retina regenerate selectively after laser ablation, but the source of regenerated cones has not been identified. The results reported here provide support for the existence of retinal stem cells within the adult fish retina that are capable of regenerating cone photoreceptors. The data also support the involvement of Müller glia in the production of regenerated cones.

Purpose: To isolate and characterize a zebrafish CRX: homologue. Mammalian CRX: genes are expressed specifically in photoreceptors and pinealocytes, regulate photoreceptor gene expression, are necessary for normal photoreceptor differentiation, and when mutated cause a variety of photoreceptor degenerations., Methods: A zebrafish retinal cDNA library was screened with a human CRX cDNA probe. Radiation hybrid mapping, Northern blot analysis, in situ hybridization, and transient transfection studies were performed using standard methods., Results: Based on amino acid sequence comparisons, zebrafish crx shows 50% identity with human CRX, and 85% identity in the homeodomain. A phylogenetic analysis indicates that zebrafish crx is most closely related to the mammalian Crx proteins, and more distantly related to the Otx proteins. Zebrafish crx maps between 49.6 and 54.5 cm from the top of linkage group LG05C, a map position consistent with the location of the mouse and human CRX genes. Northern blot analysis and in situ hybridization indicate that zebrafish crx is expressed in the retina and pineal gland. In adult zebrafish, crx is expressed by both rods and cones in the outer nuclear layer, and in cells in the outer zone of the inner nuclear layer, in the region occupied by bipolar cells. Similar to mammalian Crx, zebrafish crx interacts with neural retinal leucine zipper (Nrl) to activate, although weakly, rhodopsin promoter activity., Conclusions: Based on molecular phylogeny, chromosomal location, expression pattern, and ability to activate rhodopsin promoter activity in transient transfection assays, zebrafish crx appears to be an orthologue and functional homologue of mammalian CRX:

Purpose: To examine the expression of the major isoforms of cyclic guanosine monophosphate (cGMP)-dependent protein kinase (cGK) in mouse eye., Methods: Immunohistochemical localization of cGMP in mouse eye cryosections was performed using an anti-cGMP antibody, followed by visualization with indirect fluorescence microscopy. The presence of types Ialpha, Ibeta, and II cGK mRNAs in mouse eye extracts was determined initially by RNase protection analysis. Further localization of cGK I and II mRNAs on cryosections was accomplished by in situ hybridization using digoxigenin-labeled cRNA probes and an alkaline phosphatase-conjugated anti-digoxigenin antibody. Finally, cGK I protein was localized to subcellular areas within the retina using an anti-cGK I-specific primary antibody., Results: In initial immunohistochemical experiments cGMP was present in numerous regions and layers within the eye and retina. Subsequent RNase protection studies demonstrated that cGK Ialpha, Ibeta, and II mRNAs were present in mouse eye and that type Ibeta mRNA were 6.6 and 30 times more abundant than type Ialpha and type II, respectively. By in situ hybridization, cGK I mRNA was localized to photoreceptor inner segments and the ganglion cell and inner nuclear layers of the retina, and lesser amounts were found in the ciliary epithelium, lens, and cornea. The cGK II mRNA expression pattern was similar but not identical with that of cGK I. Finally, within the retina, cGK I protein was most abundant in the inner plexiform layer, with significant amounts in ganglion cells and photoreceptor inner segments as well., Conclusions: The presence of these cGK isoforms in discrete areas throughout the eye suggests multiple roles for the cGMP-dependent signal transduction system in the regulation of physiologic and pathologic ocular processes.

Purpose: To examine expression of the zebrafish ultraviolet cone opsin pigment in goldfish and zebrafish retinas., Methods: Digoxigenin-labeled cRNA probes were prepared by run-off transcription from plasmids containing cDNAs for zebrafish ultraviolet opsin, goldfish ultraviolet cone opsin, and goldfish rod opsin. Probes were hybridized to cryosections of retina and visualized with immunocytochemistry., Results: The zebrafish ultraviolet opsin probe hybridized selectively to rod photoreceptors, but not to ultraviolet cones or any other cone type, in both zebrafish and goldfish retinas, and the pattern of expression was identical to that of the goldfish rod opsin probe. The goldfish ultraviolet opsin, in contrast, hybridized to ultraviolet cone photoreceptors in both goldfish and zebrafish., Conclusions: The cDNA previously identified by Robinson et al as zebrafish ultraviolet opsin is not a cone opsin but is likely to be a rod opsin.

Purpose: Cone photoreceptors in teleost fish retina are organized into a precise, crystalline mosaic in which the four spectral subtypes have a consistent position relative to each other. The objective of the current study was to describe the spatial and temporal progression of photoreceptor differentiation in the embryonic goldfish retina to understand how the retinal cone mosaic might be produced., Methods: To identify developing photoreceptors when they first begin to express a specific opsin, the authors used in situ hybridization with cRNA probes generated from cDNA for rod opsin and red, green, blue, and ultraviolet cone opsins from goldfish (Carassius auratus)., Results: In the retina, rod opsin was expressed first, and it was restricted to a small patch of regularly spaced, precocious rods located near the ventronasal edge of the retina, close to the choroid fissure. The patch enlarged by recruitment of additional rods in a circular path, moving from ventral to nasal to dorsal to temporal retina. Expression of cone opsins began approximately 10 hours after rod opsin was first expressed, and differentiation of cone photoreceptors followed the spatial pattern laid down by the early rods. The temporal order of onset of cone opsin expression was red, then green, then blue, then ultraviolet. When rod and red cone opsin probes were combined, the number of labeled cells was additive, suggesting that these two opsins are expressed in separate populations of photoreceptors., Conclusions: The onset of opsin expression in goldfish retina follows a highly ordered spatio-temporal pattern. Early differentiation and regular spacing of the precocious rods was unexpected and suggested that they may play a role in cone mosaic patterning. The order of subsequent cone opsin expression was related to the relative positions of cone subtype in the mosaic, suggesting the possibility that inductive interactions among developing photoreceptors may be responsible for patterning the cone mosaic array.

The retinas of 63 goldfish were examined after varying durations of exposure to one of three environmental lighting conditions beginning before hatching: constant light (340 lux), cyclic light (12 hr 320 lux, 12 hr dark) and constant dark. Up to 8 months, no effects of constant light or dark on photoreceptor numbers or structure were apparent. Densities of rod and cone nuclei were normal and all retinal layers appeared normal by light microscopy. Exposure to constant light for 12 months or longer resulted in a reduction in rod density by 37%. Cone numbers were unaffected by constant light, even with exposures of 3 yr, and rod and cone outer segments were normal in length at 11-20 months under all environmental conditions. Due to poor survival, only one animal was available for quantitative examination from the group reared in constant dark 12 months or longer. Photoreceptor size and number in this retina were similar to those in the constant light condition. The results suggest that the formation and maturation of rods and cones in goldfish retina is unaffected by rearing in constant light. However, long-term exposure (greater than or equal to 12 months) may disrupt maintenance of differentiated rods.

Goldfish (Carassius auratus) were reared from hatching in constant light (340 lux), cyclic light (12 hr 320 lux, 12 hr dark) or constant dark. Absolute visual threshold was determined psychophysically in animals that still responded to visual stimuli after 1-3 years of exposure, by means of a classically conditioned respiration suppression technique wherein animals were presented with different intensities of large diffuse flashes of monochromatic light. Fish reared in constant light and fish reared in cyclic light responded reliably to stimuli above threshold, but fish reared in constant light were on average 0.58 log unit less sensitive at 532 nm, near the peak of the rod action spectrum. Two of the four fish reared in darkness did not respond to the stimuli, and thus could not be conditioned, and another fish reared in darkness responded only occasionally; threshold could not be measured in these three fish. The one fish reared in darkness that responded consistently enough to be conditioned was more than 5 log units less sensitive than normally reared fish on the first day of testing, and became progressively less sensitive over the next 2 days. Rearing under constant dark or constant light had no obvious effect on spectral sensitivity at absolute threshold. The effect of rearing in constant light on absolute threshold correlates with morphological changes in rod density, but the effect of rearing in constant darkness does not.