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

Repetitive induction responses in individual rods.

<p>(A) Schematic diagram of the Dex treatment paradigm. (B) Fluorescence (top) and merged with DIC (bottom) images of a live rod that received three Dex treatments. Labels I, II and III indicate fluorescence responses corresponding to the different inductions. Scale bar is 5 μm. (C) Relative fluorescence intensity profile of the rod in (B). For reference, the position of IS/OS junction was set as 0 μm. The maximum intensity (Peak) and minimum intensity (trough) between two induction responses are indicated. F₀ indicates the pre-induction background expression level. (D) Average normalized fluorescence intensity distribution of rods that received repetitive induction. Data were pooled from 112 inductions of 44 rods whose profiles were extracted from confocal images of 4 tadpoles ranging from St. 52-56. The fluorescence distribution for each rod was aligned at the position where fluorescence in the rising phase is 50% of maximum (designated as 0 μm, dotted line). The average relative fluorescence intensity for all responses is plotted (black line). The average lines of for induction I (red), II (green) and III (blue) are shown. Error bars represent 95% confidence. (E) Average peak and trough Rho-mCherry concentrations derived from the fluorescence intensity for the three different inductions are shown. The 'Ave' is the average concentration of all inductions. The 'Max' is the maximum response in each rod. Error bars represent standard deviation (n = 61, 66, 45, 172, 68 respectively).</p

b-wave threshold intensity (I_t) values.

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

Distribution of Rho-mCherry in live rods after repetitive 3-day induction.

<p>(A) Live rods with one to three responses in a retina chip are shown with the fluorescence merge with DIC. Scale bar is 10 μm. (B) Five individual rods with two (2,3) or one (4,5) responses are shown with fluorescence and merged with DIC . Scale bar is 5 μm. (C) Relative mCherry fluorescence intensity profiles of several different live rods, which received same treatment but exhibited different responses. Top scan is from the cell in A with three responses and the others from cells indicated in B. Scale bar on the x-axis represents 10 μm.</p