Expression of UV-Sensitive Parapinopsin in the Iguana Parietal Eyes and Its Implication in UV-Sensitivity in Vertebrate Pineal-Related Organs

The pineal-related organs of lower vertebrates have the ability to discriminate different wavelengths of light. This wavelength discrimination is achieved through antagonistic light responses to UV or blue and visible light. Previously, we demonstrated that parapinopsin underlies the UV reception in the lamprey pineal organ and identified parapinopsin genes in teleosts and frogs of which the pineal-related organs were reported to discriminate light. In this study, we report the first identification of parapinopsin in the reptile lineage and show its expression in the parietal eye of the green iguana. Spectroscopic analysis revealed that iguana parapinopsin is a UV-sensitive pigment, similar to lamprey parapinopsin. Interestingly, immunohistochemical analyses using antibodies specific to parapinopsin and parietopsin, a parietal eye green-sensitive pigment, revealed that parapinopsin and parietopsin are colocalized in the outer segments of the parietal eye photoreceptor cells in iguanas. These results strongly suggest that parapinopsin underlies the wavelength discrimination involving UV reception in the iguana parietal eye. The current findings support the idea that parapinopsin is a common photopigment underlying the UV-sensitivity in wavelength discrimination of the pineal-related organs found from lampreys to reptiles

Developmental regulation of calcium-dependent feedback in Xenopus rods

The kinetics of activation and inactivation in the phototransduction pathway of developing Xenopus rods were studied. The gain of the activation steps in transduction (amplification) increased and photoresponses became more rapid as the rods matured from the larval to the adult stage. The time to peak was significantly shorter in adults (1.3 s) than tadpoles (2 s). Moreover, adult rods recovered twice as fast from saturating flashes than did larval rods without changes of the dominant time constant (2.5 s). Guanylate cyclase (GC) activity, determined using IBMX steps, increased in adult rods from ~1.1 s–1 to 3.7 s–1 5 s after a saturating flash delivering 6,000 photoisomerizations. In larval rods, it increased from 1.8 s–1 to 4.0 s–1 9 s after an equivalent flash. However, the ratio of amplification to the measured dark phosphodiesterase activity was constant. Guanylate cyclase–activating protein (GCAP1) levels and normalized Na+/Ca2+, K+ exchanger currents were increased in adults compared with tadpoles. Together, these results are consistent with the acceleration of the recovery phase in adult rods via developmental regulation of calcium homeostasis. Despite these large changes, the single photon response amplitude was ~0.6 pA throughout development. Reduction of calcium feedback with BAPTA increased adult single photon response amplitudes threefold and reduced its cutoff frequency to that observed with tadpole rods. Linear mathematical modeling suggests that calcium-dependent feedback can account for the observed differences in the power spectra of larval and adult rods. We conclude that larval Xenopus maximize sensitivity at the expense of slower response kinetics while adults maximize response kinetics at the expense of sensitivity.This work was supported by the National Institutes of Health grants EY-11256 and EY-12975 (B.E. Knox), EY-00667 (R.B. Barlow), and EY-13772 (G.A. Engbretson), Research to Prevent Blindness (unrestricted grant to SUNY UMU Department of Ophthalmology and Career Development Awards to E. Solessio and S.S. Mani) and Lions of CNY

Generation of functional eyes from pluripotent cells

Pluripotent cells such as embryonic stem (ES) and induced pluripotent stem (iPS) cells are the starting point from which to generate organ specific cell types. For example, converting pluripotent cells to retinal cells could provide an opportunity to treat retinal injuries and degenerations. In this study, we used an in vivo strategy to determine if functional retinas could be generated from a defined population of pluripotent Xenopus laevis cells. Animal pole cells isolated from blastula stage embryos are pluripotent. Untreated, these cells formed only epidermis, when transplanted to either the flank or eye field. In contrast, misexpression of seven transcription factors induced the formation of retinal cell types. Induced retinal cells were committed to a retinal lineage as they formed eyes when transplanted to the flanks of developing embryos. When the endogenous eye field was replaced with induced retinal cells, they formed eyes that were molecularly, anatomically, and electrophysiologically similar to normal eyes. Importantly, induced eyes could guide a vision-based behavior. These results suggest the fate of pluripotent cells may be purposely altered to generate multipotent retinal progenitor cells, whic

Ablation of the Proapoptotic Genes Chop or Ask1 Does Not Prevent or Delay Loss of Visual Function in a P23H Transgenic Mouse Model of Retinitis Pigmentosa

<div><p>The P23H mutation in rhodopsin (Rho^P23H) is a prevalent cause of autosomal dominant retinitis pigmentosa. We examined the role of the ER stress proteins, Chop and Ask1, in regulating the death of rod photoreceptors in a mouse line harboring the Rho^P23H rhodopsin transgene (<i>GHL⁺</i>). We used knockout mice models to determine whether Chop and Ask1 regulate rod survival or retinal degeneration. Electrophysiological recordings showed similar retinal responses and sensitivities for <i>GHL⁺</i>, <i>GHL⁺/Chop^−/−</i> and <i>GHL⁺/Ask1^−/−</i> animals between 4–28 weeks, by which time all three mouse lines exhibited severe loss of retinal function. Histologically, ablation of <i>Chop</i> and <i>Ask1</i> did not rescue photoreceptor loss in young animals. However, in older mice, a regional protective effect was observed in the central retina of <i>GHL⁺/Chop^−/−</i> and <i>GHL⁺/Ask1^−/−</i>, a region that was severely degenerated in <i>GHL⁺</i> mice. Our results show that in the presence of the Rho^P23H transgene, the rate of decline in retinal sensitivity is similar in <i>Chop</i> or <i>Ask1</i> ablated and wild-type retinas, suggesting that these proteins do not play a major role during the acute phase of photoreceptor loss in <i>GHL⁺</i> mice. Instead they may be involved in regulating secondary pathological responses such as inflammation that are upregulated during later stages of disease progression.</p></div

Regulation of rhodopsin-eGFP distribution in transgenic xenopus rod outer segments by light.

The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial variation of fluorescence intensity in cells expressing a fluorescently tagged rhodopsin transgene. There was a light synchronized fluctuation in intensity, with higher intensity in disks formed at night and lower intensity for those formed during the day. This fluctuation was absent in constant light or dark conditions. There was also a slow modulation of the overall expression level that was not synchronized with the lighting cycle or between cells in the same retina. The axial variations of other membrane-associated fluorescent proteins, eGFP-containing two geranylgeranyl acceptor sites and eGFP fused to the transmembrane domain of syntaxin, were greatly reduced or not detectable, respectively. In acutely light-adapted rods, an arrestin-eGFP fusion protein also exhibited axial variation. Both the light-sensitive Rho-eGFP and arrestin-eGFP banding were in phase with the previously characterized birefringence banding (Kaplan, Invest. Ophthalmol. Vis. Sci. 21, 395-402 1981). In contrast, endogenous rhodopsin did not exhibit such axial variation. Thus, there is an axial inhomogeneity in membrane composition or structure, detectable by the rhodopsin transgene density distribution and regulated by the light cycle, implying a light-regulated step for disk assembly in the OS. The impact of these results on the use of chimeric proteins with rhodopsin fused to fluorescent proteins at the carboxyl terminus is discussed

An Inducible Expression System to Measure Rhodopsin Transport in Transgenic <i>Xenopus</i> Rod Outer Segments

<div><p>We developed an inducible transgene expression system in <i>Xenopus</i> rod photoreceptors. Using a transgene containing mCherry fused to the carboxyl terminus of rhodopsin (Rho-mCherry), we characterized the displacement of rhodopsin (Rho) from the base to the tip of rod outer segment (OS) membranes. Quantitative confocal imaging of live rods showed very tight regulation of Rho-mCherry expression, with undetectable expression in the absence of dexamethasone (Dex) and an average of 16.5 µM of Rho-mCherry peak concentration after induction for several days (equivalent to >150-fold increase). Using repetitive inductions, we found the axial rate of disk displacement to be 1.0 µm/day for tadpoles at 20 °C in a 12 h dark /12 h light lighting cycle. The average distance to peak following Dex addition was 3.2 µm, which is equivalent to ~3 days. Rods treated for longer times showed more variable expression patterns, with most showing a reduction in Rho-mCherry concentration after 3 days. Using a simple model, we find that stochastic variation in transgene expression can account for the shape of the induction response. </p> </div

G3U inducible system in mammalian cell culture.

<p>(A) Diagram of G3U system using luciferase (pCMV:G3 and pUAS:Luciferase) or mCherry reporters (top, pCMV:G3 and pUAS:mCherry). A CMV promoter drives transcription of a chimeric transcription factor, G3, which encodes contains a GAL4 DNA binding domain, the VP16 transcription activation domain, a rat glucocorticoid receptor binding domain (GR) and eGFP. Synthesized G3 protein localizes to the cytosol. Dex treatment triggers the dimerization of G3, which translocates into the nucleus. Nuclear G3 activates a second construct containing five tandem repeats of the UAS sequence upstream of the hsp70 minimal promoter. The reporter gene (luciferase or mCherry) is under the control of this system. (B) Luciferase assay of G3U inducible system in cell culture. HEK293T cells transfected with pCMV:G3 and pUAS:Luciferase were lysed and luciferase activity measured at different concentrations (0-80 µM) and treatment durations (2-24 hr) of Dex. Relative luciferase activity is plotted as a function of duration and Dex concentration. (C) Live cell imaging of G3 translocation after induction. HEK293 cells were transfected with pCMV:G3 and pUAS:mCherry and induced with 10 μM Dex at 27°C and 37°C. Confocal images were taken before and 20 min after induction; G3 (eGFP), nucleus (Hoechst). Scale bar is 10 μm. (D) Nuclear translocation rate of G3 in HEK293T cells at different temperatures. Fluorescence intensity was measure in nuclear and cytoplasm of live 293T cells at 27°C and 37 °C. Dex (10 μM) was added and mixed into medium. Error bars represent standard deviation (n = 17 at 37 °C and n = 15 at 27 °C). (E) mCherry reporter expression after induction. HEK293T cells transfected with pCMV:G3-GFP and pUAS:mCherry were induced with 10 μM Dex and fixed at different times after induction. G3 (eGFP) and mCherry images show the movement of G3 and expression of mCherry. Scale bar is 10 μm.</p

b-wave threshold intensity (I_t) values.

<p>Threshold amplitude: 75 µV, I_t: Threshold intensity.</p