Barry E. Knox

Novel retinal photoreceptors

The rod and cone cells of the retina are thought to mediate all of the light responses of the vertebrate eye. But here we show that salmon vertebrate ancient (VA) opsin forms a functional photopigment in vitro, and is expressed in a subset of retinal horizontal and amacrine cells in vivo. These results indicate that retinal photoreception is not restricted to just rod and cone cells.

Transgene expression in Xenopus rods

The photoreceptors of the vertebrate retina express a large number of proteins that are involved in the process of light transduction. These genes appear to be coordinately regulated at the level of transcription, with rod‐ and cone‐specific isoforms (J. Hurley (1992) J. Bioenerg. Biomembr. 24, 219–226). The mechanisms that regulate gene expression in a rod/cone‐specific fashion have been difficult to address using traditional approaches and remain unknown. Regulation of the phototransduction proteins is medically important, since mutations in several of them cause retinal degeneration (P. Rosenfeld and T. Dryja (1995) in: Molecular Genetics of Ocular Disease (J.L. Wiggs, Ed.), pp. 99–126, Wiley‐Liss Inc.). An experimental system for rapidly producing retinas expressing a desired mutant would greatly facilitate investigations of retinal degeneration. We report here that transgenic frog embryos (K. Kroll and E. Amaya (1996) Development 122, 3173–3183) can be used to study cell‐specific expression in the retina. We have used a 5.5 kb 5′ upstream fragment from the Xenopus principal rod opsin gene (S. Batni et al. (1996) J. Biol. Chem. 271, 3179–3186) controlling a reporter gene, green fluorescent protein (GFP), to produce numerous independent transgenic Xenopus. We find that this construct drives expression only in the retina and pineal, which is apparent by 4 days post‐nuclear injection. These are the first results using transgenic Xenopus for retinal promoter analysis and the potential for the expression in rod photoreceptors of proteins with dominant phenotypes.

Characterization of the Xenopus rhodopsin gene.

The abundant Xenopus rhodopsin gene and cDNA have been cloned and characterized. The gene is composed of five exons spanning 3.5 kilobase pairs of genomic DNA and codes for a protein 82% identical to the bovine rhodopsin. The cDNA was expressed in COS1 cells and regenerated with 11-cis-retinal, forming a light-sensitive pigment with maximal absorbance at 500 nm. Both Southern blots and polymerase chain reaction amplification of intron 1 revealed multiple products, indicating more than one allele for the rhodopsin gene. Comparisons with other vertebrate rhodopsin 5′ upstream sequences showed significant nucleotide homologies in the 200 nucleotides proximal to the transcription initiation site. This homology included the TATA box region, Ret 1/PCE1 core sequence (CCAATTA), and surrounding nucleotides. To functionally characterize the rhodopsin promoter, transient embryo transfections were used to assay transcriptional control elements in the 5′ upstream region using a luciferase reporter. DNA sequences encompassing −5500 to +41 were able to direct luciferase expression in embryo heads. Reporter gene expression was also observed in embryos microinjected with reporter plasmids during early blastomere stages. These results locate transcriptional control elements upstream of the Xenopus rhodopsin gene and show the feasibility of embryo transfections for promoter analysis of rod-specific genes.

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which ∼40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

Rhodopsin from the fish, Astyanax: role of tyrosine 261 in the red shift.

PURPOSE To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on its spectral tuning. METHODS The rhodopsin cDNA was cloned using reverse transcription-polymerase chain reaction amplification and then sequenced. A mutant, Y261F, was generated by site-directed mutagenesis. Both wild type and mutant were transiently expressed in COS-1 cells, regenerated with 11-cis retinal, and purified by immunoaffinity chromatography. Ultraviolet-visible spectrophotometry was used to determine wavelength of maximum absorption. RESULTS A fasciatus rhodopsin cDNA exhibits 80% amino acid identity with bovine rhodopsin. In contrast to all known rhodopsins, this rhodopsin contains a tyrosine instead of a phenylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of the Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment. CONCLUSIONS A naturally occurring fish rhodopsin is red shifted about 8 nm due to one critical amino acid substitution.

Mouse cone arrestin gene characterization: promoter targets expression to cone photoreceptors

Cone arrestin (CAR) is a novel member of the arrestin superfamily expressed in retinal cone photoreceptors and the pineal gland. To understand the regulatory mechanisms controlling its cone‐ and pineal‐specific expression, and to facilitate further functional studies using gene knockout approaches, we characterized the genomic organization and the 5′‐flanking region of the mouse CAR (mCAR) gene. The mCAR gene is comprised of 17 exons and 16 introns, encoding five alternatively spliced transcripts. A 215‐bp proximal promoter fragment containing a TATA box, an Sp1 site and four cone‐rod homeobox‐binding sites is sufficient to direct expression in cultured retinoblastoma cells and in cone photoreceptors and the pineal gland in transgenic Xenopus laevis.

Hypoglycemia leads to age-related loss of vision

The retina is among the most metabolically active tissues in the body, requiring a constant supply of blood glucose to sustain function. We assessed the impact of low blood glucose on the vision of C57BL/6J mice rendered hypoglycemic by a null mutation of the glucagon receptor gene, Gcgr. Metabolic stress from moderate hypoglycemia led to late-onset loss of retinal function in Gcgr−/− mice, loss of visual acuity, and eventual death of retinal cells. Retinal function measured by the electroretinogram b-wave threshold declined >100-fold from age 9 to 13 months, whereas decreases in photoreceptor function measured by the ERG a-wave were delayed by 3 months. At 10 months of age Gcgr−/− mice began to lose visual acuity and exhibit changes in retinal anatomy, including an increase in cell death that was initially more pronounced in the inner retina. Decreases in retinal function and visual acuity correlated directly with the degree of hypoglycemia. This work demonstrates a metabolic-stress-induced loss of vision in mammals, which has not been described previously. Linkage between low blood glucose and loss of vision in mice may highlight the importance for glycemic control in diabetics and retinal diseases related to metabolic stress as macular degeneration.

Activation of Transducin by a Xenopus Short Wavelength Visual Pigment

Phototransduction in cones differs significantly from that in rods in sensitivity, kinetics, and recovery following exposure to light. The contribution that the visual pigment makes in determining the cone response was investigated biochemically by expressing a Xenopus violet cone opsin (VCOP) cDNA in COS1 cells and assaying the light-dependent activation of transducin. Light-exposed VCOP stimulated [35S]guanosine 5′-(γ-thio)triphosphate nucleotide exchange on bovine rod transducin in a time-dependent manner with a half-time for activation of 0.75 min, similar to that of bovine rhodopsin. In exhaustive binding assays, VCOP and rhodopsin activity showed similar concentration dependence with half-maximal activation occurring at 0.02 mol of pigment/mol of transducin. Although VCOP was able to activate as many as 12 transducins per photoisomerization, rhodopsin catalyzed significantly more. When assays were performed with λ > 420 nm illumination, VCOP exhibited rapid regeneration and high affinity for the photoregenerated 11-cis-retinal. Recycling of the chromophore and reactivation of the pigment resulted in multiple activations of transducin, whereas a maximum of 1 transducin per VCOP was activated under brief illumination. The decay of the active species formed following photobleaching was complete in <5 min, ∼10-fold faster than that of rhodopsin. In vitro, VCOP activated rod transducin with kinetics and affinity similar to those of rhodopsin, but the active conformation decayed more rapidly and the apoprotein regenerated more efficiently with VCOP than with rhodopsin. These properties of the violet pigment may account for much of the difference in response kinetics between rods and cones.

Rhodopsin mutant P23H destabilizes rod photoreceptor disk membranes.

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and RhoP23H-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. RhoP23H-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with RhoP23H-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The RhoP23H-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing RhoP23H, suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss.

Immediate upstream sequence of arrestin directs rod-specific expression in Xenopus.

Arrestins are a family of proteins that modulate G protein-coupled receptor responses with distinct arrestin genes expressed in rods and cones. To understand the regulatory mechanisms controlling rod-specific expression, the abundant Xenopusrod arrestin cDNA and a partial genomic clone, containing the immediate upstream region and amino terminus of the polypeptide, have been characterized. The deduced polypeptide has ∼69% identity to other vertebrate rod arrestins. Southern blot analysis and polymerase chain reaction of intronic sequences demonstrated multiple alleles for rod arrestin. DNase I footprinting with retinal proteins revealed four major DNA binding sites in the proximal promoter, coinciding with consensus sequences reported in mammalian promoters. Purified bovineCrx homeodomain and mouse Nrl proteins protected a number of these sites. A dual approach of transient embryo transfections and transgenesis was used to locate transcriptional control sequences essential for rod-specific expression inXenopus. Constructs containing −1287/+113 of 5′ upstream sequence with or without intron 1 directed high level expression, specifically in rods. A construct containing only −287/+113 directed expression of green fluorescent protein solely in rod cells. These results suggest that the Crx and Nrl binding sites in the proximal promoter are the primary cis-acting sequences regulating arrestin gene expression in rods.