Effects of exogenous thyroid hormones on visual pigment composition in coho salmon (Oncorhynchus kisutch).

The role of exogenous thyroid hormone on visual pigment content of rod and cone photoreceptors was investigated in coho salmon (Oncorhynchus kisutch). Coho vary the ratio of vitamin A1- and A2-based visual pigments in their eyes. This variability potentially alters spectral sensitivity and thermal stability of the visual pigments. We tested whether the direction of shift in the vitamin A1/A2 ratio, resulting from application of exogenous thyroid hormone, varied in fish of different ages and held under different environmental conditions. Changes in the vitamin A1/A2 visual pigment ratio were estimated by measuring the change in maximum absorbance (lambda max) of rods using microspectrophotometry (MSP). Exogenous thyroid hormone resulted in a long-wavelength shift in rod, middle-wavelength-sensitive (MWS) and long-wavelength-sensitive (LWS) cone photoreceptors. Rod and LWS cone lambda max values increased, consistent with an increase in vitamin A2. MWS cone lambda max values increased more than predicted for a change in the vitamin A1/A2 ratio. To account for this shift, we tested for the expression of multiple RH2 opsin subtypes. We isolated and sequenced a novel RH2 opsin subtype, which had 48 amino acid differences from the previously sequenced coho RH2 opsin. A substitution of glutamate for glutamine at position 122 could partially account for the greater than predicted shift in MWS cone lambda max values. Our findings fit the hypothesis that a variable vitamin A1/A2 ratio provides seasonality in spectral tuning and/or improved thermal stability of visual pigments in the face of seasonal environmental changes, and that multiple RH2 opsin subtypes can provide flexibility in spectral tuning associated with migration-metamorphic events.

Degeneration and regeneration of ultraviolet cone photoreceptors during development in rainbow trout.

Ultraviolet-sensitive (UVS) cones disappear from the retina of salmonid fishes during a metamorphosis that prepares them for deeper/marine waters. UVS cones subsequently reappear in the retina near sexual maturation and the return migration to natal streams. Cellular mechanisms of this UVS cone ontogeny were investigated using electroretinograms, in situ hybridization, and immunohistochemistry against opsins during and after thyroid hormone (TH) treatments of rainbow trout (Oncorhynchus mykiss). Increasing TH levels led to UVS cone degeneration. Labeling demonstrated that UVS cone degeneration occurs via programmed cell death and caspase inhibitors can inhibit this death. After the cessation of TH treatment, UVS cones regenerated in the retina. Bromodeoxyuridine (BrdU) was applied after the termination of TH treatment and was detected in the nuclei of cells expressing UVS opsin. BrdU was found in UVS cones but not other cone types. The most parsimonious explanation for the data is that UVS cones degenerated and UVS cones were regenerated from intrinsic retinal progenitor cells. Regenerating UVS cones were functionally integrated such that they were able to elicit electrical responses from second-order neurons. This is the first report of cones regenerating during natural development. Both the death and regeneration of cones in retinae represent novel mechanisms for tuning visual systems to new visual tasks or environments.

Proteomic analysis of opsins and thyroid hormone-induced retinal development using isotope-coded affinity tags (ICAT) and mass spectrometry.

PURPOSE: Analyses that reveal the relative abundance of proteins are informative in elucidating mechanisms of retinal development and disease progression. However, popular high-throughput proteomic methods do not reliably detect opsin protein abundance, which serve as markers of photoreceptor differentiation. We utilized thyroid-hormone (TH) treatment of rainbow trout (Oncorhynchus mykiss) as a model of cone apoptosis and cone regeneration. We used this model to investigate if emerging proteomic technology allows effective analysis of retinal development and opsin protein abundance. We also sought to begin a characterization of proteomic changes in the retina occurring with TH treatment and address whether TH affects proliferation or photoreceptor differentiation. METHODS: Retinal homogenates were prepared from control and TH-treated fish. Peptides from control and treated homogenates were differentially labeled, using isotope-code affinity tags (ICAT) and analyzed using capillary liquid chromatography-electrospray ionization-tandem mass spectrometry (capLC-ESI-MS/MS). This method identifies proteins and quantifies their relative abundance between two samples. RESULTS: The relative abundance of many retinal proteins changed during TH treatment. These included proteins from every functional class. We detected 1,684 different peptides, and our quantification suggests that 94 increased and 146 decreased in abundance more than 50% during TH treatment. Cell-cycle proteins appear to be increased, consistent with TH-inducing cell proliferation, similar to its effect in Xenopus. Other proteins associated with retinal development, such as deltaA and tubulins, changed in abundance during TH treatment. Rod opsin and three cone opsins were identified and the relative abundance of each changed with TH treatment. CONCLUSIONS: ICAT and capLC-ESI-MS/MS are an effective complement to other molecular approaches that investigate the mechanisms of retinal development. Unlike other proteomic techniques, this approach does not require development of species- or tissue-specific methodology, such as characterizing two dimensional (2D) gels or antibodies, in order to be practical as a high-throughput approach. Importantly, this technology was able to assess the relative abundance of opsin proteins. These findings represent the first high-throughput proteomic analysis of the retina and demonstrate the technique's ability to provide useful information in retinal development.

Spatio-temporal characterization of retinal opsin gene expression during thyroid hormone-induced and natural development of rainbow trout.

The abundance and spatial distribution of retinal cone photoreceptors change during thyroid hormone (TH)-induced and natural development of rainbow trout (Oncorhynchus mykiss). These changes are thought to allow the fish to adapt to different photic environments throughout its life history. To date, the ontogeny of rainbow trout cone photoreceptors has been examined using physiological and morphological approaches. In this study, we extended these observations by measuring opsin gene expression in retinal quadrants during natural and TH-induced development. Gene expression during natural development was investigated in retinae from fish at both parr and smolt stages. The role of TH in modulating opsin gene expression was determined in TH-treated parr and control fish sampled after two, nine, and 22 days of treatment. Total RNA was isolated from each retinal quadrant and steady-state opsin mRNA levels were measured using reverse transcriptase real-time quantitative polymerase chain reaction (QPCR) analysis. Expression of ultraviolet-sensitive opsin (SWS1), rod opsin (RH1), middle wavelength-sensitive opsin (RH2), and long wavelength-sensitive opsin (LWS) transcripts vary spatially in the parr retina. Smolts, compared to parr, had downregulated SWS1 expression in all quadrants, lower LWS expression dorsally, higher RH1 expression nasally, and higher RH2 expression dorsally. In TH-treated parr, SWS1 opsin expression was downregulated in the nasal quadrants by two days. SWS1 displayed the greatest degree of downregulation in all quadrants after nine days of treatment, with an increase in short wavelength-sensitive (SWS2) and RH2 opsin mRNA expression in the temporal quadrants. This study reveals that opsin genes display spatially significant differences within rainbow trout retina in their level of mRNA expression, and that regulation of opsin expression is a dynamic process that is influenced by TH. This is particularly evident for SWS1 gene expression in parr following TH-induced and natural development.

Chromatin immunoprecipitation assay on the rainbow trout opsin proximal promoters illustrates binding of NF-kappaB and c-jun to the SWS1 promoter in the retina.

Misexpression of opsins has been linked to apoptosis of photoreceptor cells in the vertebrate retina. Salmonid fish lose their ultraviolet-sensitive (UVS) cones through post-natal developmental apoptosis mediated by thyroid hormone (TH). In order to identify genetic mechanisms that may play a role in the loss of UVS cones, the transcriptional regulation of the SWS1 opsin in the rainbow trout (Oncorhynchus mykiss) was investigated. The Transfac database was interrogated with promoter sequence acquired by genome-walking PCR using MatInspector V2.2 to identify putative transcription factor (TF) binding sites. Putative binding sites for AP-1 (c-jun) and NF-kappaB were found in the SWS1 opsin promoter and were chosen for further investigation due to their high MatInspector scores, their established role in photoreceptor apoptosis, and their relative exclusion from other opsin promoters. NF-kappaB and c-jun proteins were visualized in rainbow trout retinal tissue with immunohistochemistry and c-jun was identified in rainbow trout retinal protein homogenate by immunoblot. A chromatin immunoprecipitation-polymerase chain reaction technique was employed to examine the in vivo interaction of c-jun and NF-kappaB proteins with their proposed binding sites in the opsin promoters. This analysis demonstrated that NF-kappaB and c-jun bind to the SWS1 opsin promoter, but not to the other rod and cone opsin promoters tested. Given the role of NF-kappaB and c-jun during photoreceptor apoptosis, the influence of their activity through TH and their selective binding to the SWS1 opsin promoter in rainbow trout, these TFs represent good candidates of mechanisms underlying UVS cone degeneration in salmonids.