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The visual cycle is an enzymatic pathway employed in the vertebrate retina to regenerate the chromophore following its release from light-activated rhodopsin. However, a visual cycle is thought to be absent in invertebrates such as the fruit fly, Drosophila melanogaster.
We demonstrate that an enzymatic visual cycle exists in flies for chromophore regeneration, and requires a retinol dehydrogenase, PDH, in retinal pigment cells. Absence of PDH resulted in progressive light-dependent loss of rhodopsin and retinal degeneration. These defects are suppressed by introduction of a mammalian dehydrogenase, RDH12, which is required in humans to prevent retinal degeneration. We demonstrate that a visual cycle is required in flies to sustain a visual response under nutrient deprivation conditions that preclude de novo production of the chromophore.
Our results demonstrate that an enzymatic visual cycle exists and is required in flies for maintaining rhodopsin levels. These findings establish Drosophila as an animal model for studying the visual cycle and retinal diseases associated with chromophore regeneration.
All animals that are endowed with the ability to detect light, do so through photoisomerization of vitamin A derivatives, such as 11-cis-retinal [1, 2]. Light-induced isomerization of 11-cis-retinal to all-trans-retinal causes a subsequent conformational change in the protein (opsin) subunit in rhodopsin, thereby initiating phototransduction . In vertebrates, the all-trans-retinal is released from the light-activated rhodopsin, and the chromophore is regenerated through an enzymatic cycle, referred to as the visual cycle or retinoid cycle . In the outer segments of rod photoreceptor cells, the all-trans-retinal is first converted to all-trans-retinol by a retinol dehydrogenase (RDH). All-trans-retinol is then transported to the closely associated retinal pigment epithelium (RPE) where it is converted into 11-cis-retinal . Mutations that disrupt proteins critical for regeneration of 11-cis-retinal, such as retinol dehydrogenases (RDHs), cause photoreceptor degeneration and progressive vision loss .
In contrast to vertebrate rods and cones, it is thought that photoreceptor cells in invertebrates such as Drosophila do not employ enzymes for chromophore regeneration. Rather, all-trans-3-hydroxy-retinal remains bound to the opsin and the photopigment remains stably activated. The stable conversion back to the inactive 11-cis-3-hydroxy-retinal is accomplished exclusively by exposure of the rhodopsin to a second photon of light . Interestingly, a number of studies indicate that a bistable photopigment exists in a third class of photoreceptor cell in mammals, the intrinsically photoreceptive retinal ganglion cells (ipRGCs) [6-8], which functions in circadian rhythm, pupillary constriction and sleep homeostasis [9-11]. Fly visual pigments and phototransduction has emerged as a model for the ipRGCs, as these latter cells appear to function through a cascade remarkably similar to Drosophila phototransduction [9, 10, 12].
Since the chromophore remains bound to bistable photopigments, it has been assumed that photoreceptor cells with bistable visual pigments do not employ a visual cycle. In fact, a visual cycle in Drosophila has not been described. However, flies have been used to characterize de novo synthesis of the chromophore, which is generated from dietary precursors. Indeed, multiple genes and proteins have been identified that are required for synthesis of the chromophore [13-17]. Moreover, de novo production of rhodopsin required a retinoid-binding protein in the retinal pigment cells [RPCs; 13], which lie adjacent to the photoreceptor cells. In flies, defects in chromophore production prevent normal expression and stability of the opsin [18, 19]. Thus, in the absence of the chromophore in the photoreceptor cells, the opsin is virtually eliminated.
In the current study, we generated a mutation in a gene encoding a pigment-cell-enriched dehydrogenase, PDH. However, the light responses and rhodopsin levels were normal in young pdh knockout flies, or in old mutant flies maintained in the dark. Thus, PDH did not function in de novo production of the chromophore. Rather, PDH was required for a previously unrecognized visual cycle in flies. Under a light/dark cycle, pdh1 flies underwent progressive loss of rhodopsin and light-dependent retinal degeneration. PDH was required in RPCs for the conversion of 3-OH-all-trans-retinal to 3-OH-all-trans-retinol. Ectopic expression of a human retinol dehydrogenase in RPCs partially rescued the loss of rhodopsin and the retinal degeneration in pdh1. These results indicate that a retinoid cycle exists in Drosophila, and that PDH and the RPCs play key roles in this cycle. Since the chromophore does not release from the opsin, the question arises as to the function of a visual cycle in flies. In flies, some rhodopsin is internalized and degraded in a light-dependent manner [20-25]. Thus, continued opsin synthesis is required to maintain rhodopsin levels in the presence of light. We found that the visual cycle allows light-exposed animals to regenerate the chromophore and sustain rhodopsin levels under nutrient deprivation conditions that prevent de novo synthesis of the chromophore, and would otherwise lead to blindness.
To characterize further the chromophore de novo synthesis pathway, we focused on the pdh gene since it is expressed primarily in RPCs  and is homologous to known retinol dehydrogenases (RDHs). In addition, based on a microarray analysis that compared RNA expression in wild-type and eyeless heads, we found that the pdh mRNA displayed an eye-enrichment of >220-fold .
To produce pdh knockout flies, referred to as pdh1, we used ends-out homologous recombination to delete the initiation codon and the N-terminal 81 residues  (Figure 1A and Experimental Procedures). To confirm that the deletion eliminated expression of the PDH protein, we raised anti-PDH antibodies, which recognized PDH proteins of the predicted sizes of 28-30 kD in wild-type (Figure 1B). As previously reported, the anti-PDH signal was limited to the RPCs in wild-type (Figures 1C and 1D) . We did not detect PDH in the pdh1 flies either on Western blots or in whole-mount staining of compound eyes (Figures 1B and 1E).
Defects in multiple mammalian RDHs lead to retinal degeneration . To test whether pdh1 flies also underwent retinal degeneration, we examined retinal morphology. The fly compound eye consists of ~800 ommatidia, each of which contains seven photoreceptor cells in any tangential section. Each photoreceptor cell includes a microvillar structure, the rhabdomere, which is the functional equivalent of mammalian rod and cone outer segments (Figure 1C). We found that the pdh1 flies exposed to a light/dark cycle underwent a progressive loss of rhabdomeres (Figures 2A and 2B). The pdh1 flies started to lose rhabdomeres at about 10 days old, and virtually no rhabdomeres corresponding to the R1-R6 photoreceptor cells remained after 30 days. The cell bodies also showed an accumulation of prominent vacuoles as reported in other mutants displaying retinal degeneration [28, 29]. This was most notable in 40 day-old flies, consistent with age-dependent degeneration (Figure S1A). We did not detect retinal degeneration in pdh1 flies maintained in the dark for 30 days (Figures 2A and 2B). Thus, this phenotype was strictly light dependent.
To address whether the light-dependent retinal degeneration was reversible, we exposed newly-eclosed pdh1 flies to cyclic light for 10, 20 or 30 days, and then placed them in the dark. At 40 days of age, these flies all displayed severe retinal degeneration comparable to the 40-day-old pdh1 flies maintained continuously under a light/dark cycle (Figure S1). These data indicate that the light-dependent damage induced in pdh1 flies was not reversible.
In Drosophila, the chromophore is covalently linked to the opsin and required for stable production of rhodopsin [18, 19]. As a result, rhodopsin levels are reduced or eliminated by mutations that decrease or prevent generation of the chromophore. Since PDH is a predicted retinol dehydrogenase, which could potentially function in chromophore production, we monitored the levels of the major rhodopsin (Rh1) in the pdh knockout flies. The concentration of Rh1 was indistinguishable between young (1-day old) wild-type and pdh1 flies (Figure 3A), indicating that PDH was not required for de novo generation of chromophore.
To confirm that the Rh1 was functional in young pdh1 flies, we performed electroretinogram (ERG) recordings, which measure the summed retinal response to light . In wild-type flies, exposure to white or orange light results in a response coincident with the light stimulus (Figure 3B). Illumination with blue (480 nm) light causes stable activation of Rh1, resulting in a prolonged deactivation afterpotential (PDA) (Figure 3B). Termination of the PDA requires exposure of the light-activated metarhodopsin to orange or white light. The PDA is sensitive to the relative concentration of Rh1 to arrestin, which binds to metarhodopsin and arrests its activity . When Rh1 levels decrease to ≤30% wild-type levels, a PDA is not produced. Consistent with the Western blot data indicating that Rh1 levels were normal (Figure 3A), we found that young (1-day old) pdh1 flies displayed a PDA similar to wild-type (Figure 3B). These data indicated that the chromophore was produced in the absence of PDH and that PDH was not required for de novo synthesis of the chromophore.
Since 1-day-old pdh1 flies did not display a phenotype, we examined rhodopsin levels in older flies. We found that under a light/dark cycle, the concentration of Rh1 decreased gradually (Figures 3A and 3C). This defect was light-dependent since pdh1 retained normal Rh1 expression even after 20 days in the dark (Figure 3A). The decline in Rh1 was paralleled by a loss in the PDA. After 7 days under a light/dark cycle, the concentration of Rh1 declined to about 30% of wild-type levels and the PDA was absent (Figures 3A-3C). In contrast, a PDA was observed in the pdh1 flies maintained for 20 days in the dark (Figure 3B). The losses in Rh1 levels and the PDA in older pdh1 flies exposed to a light/dark cycle were due to the pdh knockout since these phenotypes were rescued by a pdh+ genomic transgene (Figures 3D and 3E).
In addition to Rh1, which is expressed in the six outer photoreceptor cells (R1-6), there are four rhodopsins (Rh3–6) expressed in non-overlapping subsets of minor photoreceptor cells in the compound eye [R7 and R8; 5, 32]. If PDH functioned in maintaining the chromophore, we reasoned that the levels of the minor rhodopsins would also be affected. Therefore, we examined the levels of Rh4, and found that the concentration also decreased in a light-dependent manner (Figure S2). The decline in rhodopsins was not due to a nonspecific effect on retinal proteins or retinal degeneration since the levels of other photoreceptor cell proteins such as INAD were unaffected in pdh1 flies before massive retinal degeneration took place (Figure S2). After 30 days under a light/dark cycle when all the rhabdomeres except for the R7 cell degenerated, the concentration of INAD was also reduced significantly (Figure S2).
The preceding data indicate that PDH was not required for de novo synthesis of the chromophore, but was required to maintain chromophore levels in flies exposed to a light/dark cycle for many days. Since a portion of the rhodopsin pool is internalized and degraded upon exposure to light, we wondered whether PDH might function in the regeneration of the released chromophore. As a first test as to whether the age- and light-dependent loss of rhodopsin in pdh1 flies was due to a defect in chromophore regeneration, we profiled the levels of retinoids by performing HPLC analysis of fly head extracts.
In the mammalian retina, the first enzyme-dependent step in the regeneration of the chromophore involves reduction of the all-trans-retinal to all-trans-retinol . This reduction is controlled by at least two RDHs, including RDH12 [33, 34]. If the fly PDH functioned in vivo in the corresponding reduction, then the levels of retinols should be decreased. We found that in young flies (1-day post-eclosion), the retinoid concentrations were similar in wild-type and in the pdh knockout flies (data not shown; retinal/retinol ratios: wild-type. 7.0±1.4; pdh1, 8.3±0.5). After 7 days under a 12-hour light/12-hour dark cycle, the 3-OH-11-cis- and 3-OH-all-trans-retinols levels decreased dramatically in pdh1 flies as compared to wild-type, and the overall retinal/retinol ratio was 21.8-fold higher in pdh1 than in wild-type flies (wild-type, 8.6±3.7; pdh1, 187.9±43.8; Figure 4A and Figure S3A). The dramatic increase in this ratio in pdh1 indicated that the generation of retinols was disrupted in pdh1 flies. In the absence of regeneration, the all-trans-retinal may be degraded, since the levels were reduced ~two-fold.
Blue light locks Rh1 in the active state and continuous exposure to blue light for several hours greatly accelerates degradation of Rh1 . Therefore, we examined the retinal/retinol ratios after exposing the flies to blue light for 12 hours. We used 1-day-old pdh1 for these experiments, since these young flies show normal retinal/retinol ratios and rhodopsin levels when they are kept for just one day under 12-h light/12-h dark cycle (Figure 3A and data not shown). Under blue light conditions, 1-day-old pdh1 flies showed a similar decrease in retinol as pdh1 flies maintained under a light/dark cycle for 7 days (Figure 4B and Figure S3B; retinal/retinol ratios: wild-type 2.8±0.1; pdh1 94.3±45.4). These results suggest that PDH activity is required in vivo for the reduction of all-trans-retinal to retinol during turnover of Rh1.
To provide additional evidence that PDH was required in vivo for the regeneration of chromophore from 3-OH-all-trans-retinal, we asked whether pdh1 flies could utilize all-trans-retinal for Rh1 synthesis. Wild-type and pdh1 flies raised on vitamin A-free medium did not produce detectable Rh1 (Figure 4C). When we supplemented the food with all-trans-retinol, both wild-type and pdh1 flies produced Rh1 after 48 hours under a light/dark cycle (Figure 4C). In contrast, only wild-type but not pdh1 flies produced detectable Rh1, after they were provided all-trans-retinal (Figure 4C). Thus, pdh1 flies were capable of generating functional Rh1 from all-trans-retinol but not from all-trans-retinal. The combination of HPLC analyses and food supplementation data indicate that PDH is required in vivo for the conversion of 3-OH-all-trans-retinal to 3-OH-all-trans-retinol to regenerate the chromophore following degradation of Rh1.
The natural precursors for de novo synthesis of the chromophore in Drosophila are carotenoids such as β-carotene and zeaxanthin. When we supplemented the food with β-carotene at the adult stage, wild-type and pdh1 flies were both capable of producing Rh1 (Figure 4C). NINAB, which is the Drosophila carotenoid oxygenase, also has isomerase activity, and cleaves β-carotene to yield both 11-cis-retinal and all-trans-retinal [Figure 5; 15, 35, 36]. Since supplementation of all-trans-retinal is insufficient to promote rhodopsin synthesis in pdh1 flies, we tested whether the other NINAB-induced product, 11-cis-retinal, could promote chromophore synthesis in the absence of PDH. Indeed, when we supplied pdh1 flies with 11-cis-retinal, Rh1 was generated within 48 hours in the dark (Figure 4D). These data, combined with the finding that all-trans-retinol promoted Rh1 synthesis in the absence of PDH, suggested that the NINAB-independent isomerization requires the retinol form of the chromophore (Figure 5). Therefore, during the visual cycle, the reduction of 3-OH-all-trans-retinal to 3-OH-all-trans-retinol by PDH appears to be a critical step prior to the re-isomerization of the chromophore.
To examine further a requirement for the visual cycle, we asked whether regeneration of the chromophore is sufficient to maintain rhodopsin levels, in the absence of new chromophore synthesis. To conduct this analysis, we disrupted the de novo generation of chromophore by eliminating the dietary supply of β-carotene in adult flies. Under these conditions, wild-type flies were able to maintain normal Rh1 levels, even after 25 days under a light/dark cycle (Figure 6A). In contrast, supplementing normal carotenoid-containing food with additional β-carotene (5 mM) was insufficient to prevent the loss of Rh1 in the pdh knockout (Figure 6B). These results indicate that regeneration of the chromophore is the major pathway for maintaining rhodopsin levels in adult flies exposed to a light/dark cycle. This was the case either under conditions in which de novo chromophore synthesis was blocked or when β-carotene was supplied to permit de novo synthesis to take place.
If PDH acts as a RDH to reduce all-trans-retinal to all-trans-retinol, then expressing the mammalian enzyme with this activity in Drosophila RPCs might rescue the pdh1 phenotype. RDH12 is a human RDH that catalyzes the reduction of all-trans-retinal to all-trans-retinol during the regeneration of chromophore, but is not required for de novo synthesis of the chromophore [37-39]. Patients with mutations in RDH12 undergo a progressive, severe and early-onset retinal dystrophy. Unlike PDH, the RDH12 protein is primarily expressed in photoreceptor cells and is only 24% identical to PDH [4, 34]. We created a UAS-RDH12 transgene, which we expressed in pdh1 flies under control of a GAL4 line expressed in RPCs [CG7077-GAL4; 14]. We found that expression of human RDH12 suppressed the pdh1 phenotype. Rh1 was maintained and a PDA was generated even after 7 days under a 12-h light/12-h dark cycle, although they were reduced relative to wild-type (Figures 7A-7D). Expressing RDH12 in RPCs also diminished the severity of the retinal degeneration in pdh1 flies (Figures 7E-7H). In contrast, introduction of RDH12 exclusively in photoreceptor cells, using the rh1 promoter (ninaE-GAL4 and UAS-RDH12) did not suppress the pdh1 phenotype (Figure 7D). These results indicate further that PDH functions in vivo as an all-trans-retinol dehydrogenase in RPCs during the visual cycle.
In vertebrate rods, light activation of rhodopsin leads to release of the trans form of the chromophore. The chromophore is regenerated through an enzyme-dependent visual cycle involving photoreceptor cells and cells in the RPE . In contrast, in invertebrates such as Drosophila, the chromophore is not released from the opsin, but is regenerated by exposure of rhodopsin to a second photon of light . Therefore, the need for a visual cycle in Drosophila has not been anticipated.
Similar to other G-protein-coupled receptors, in flies the light-activated rhodopsin is subject to internalization and degradation [20-24]. As a result, a proportion of the 3-OHall-trans-retinal is released from the opsin. Illumination might increase the release of 3-OH-all-trans-retinal through a second mechanism, since it is possible that the stability of the 3-OH-all-trans-retinal bound to the opsin is lower than the 3-OH-11-cis-retinal. However, the question as to the fate of the released chromophore has not been considered. Unexpectedly, our analysis of the pdh knockout revealed the existence of an enzymatic pathway involved in the regeneration of visual pigment in Drosophila.
The current work permits the formulation of a model for regeneration of the chromophore through an enzyme-mediated visual cycle (Figure 5). The 3-OH-all-trans-retinal, which is released from the degraded opsin, is reduced to 3-OH-all-trans-retinol through the activity of PDH. The 3-OH-all-trans-retinol is subsequently isomerized to 3-OH-11-cis-retinol, which is then oxidized to produce the chromophore necessary for generation of rhodopsin. However, the enzymes required for these latter steps remain to be determined. A light-dependent Drosophila retinoid isomerase activity was suggested many years ago, since 3-OH-all-trans-retinal and all-trans-retinol promoted Rh1 synthesis in the light, but not in the dark [19, 40]. The enzyme responsible for oxidation of the 11-cis-retinol is not known; although, candidates include several eye-enriched dehydrogenases, which remain to be investigated .
The proposed model is supported by multiple observations. These include the findings that PDH is not required for de novo synthesis since rhodopsin levels are normal in newly eclosed pdh1 flies, or in old pdh1 flies maintained in the dark, when very little rhodopsin internalized is degraded. Rather, PDH functions in RPCs, and only in the presence of light, to maintain a wild-type concentration of rhodopsin. PDH is required in vivo for conversion of 3-OH-all-trans-retinal to 3-OH-all-trans-retinol (Figure 5), since virtually no 3-OH-all-trans-retinol is present in pdh1, after the animals have been exposed to a light/dark cycle for seven days. If pdh1 flies are exposed to continuous blue light, which promotes rapid degradation of rhodopsin, the 3-OH-all-trans-retinol and 3-OH-11-cis-retinol are lost precipitously in just a few hours. In further support of the model, pdh1 flies are able to produce rhodopsin if they are supplied all-trans-retinol or 11-cis-retinal, but not if they are provided all-trans-retinal. Nevertheless, questions remain, including whether the oxidation of 11-cis-retinol takes place in the RPCs or in the photoreceptor cells, as is the case for mammalian cone cells [41, 42].
Mammalian RPE cells function in both de novo synthesis and in regeneration of the chromophore after it is released from rhodopsin . We found previously that a defect in a retinoid binding protein expressed in the RPCs prevents de novo synthesis of rhodopsin . Here, we demonstrate that RPCs also function in an enzymatic visual cycle necessary for chromophore regeneration. Thus the RPCs share both functions with the RPE.
Despite the presence of a visual cycle in Drosophila, it differs from that in the mammalian rod cells. In the case of the visual cycle that initiates in rod cells, the conversion of retinal to retinol, which is catalyzed by the PDH counterpart, RDH12, takes place in the outer segment of the photoreceptor cells. Another difference between the visual cycle in flies and mammals is that retinoid esterification may not play a role in the fly visual cycle, since we did not detect retinyl esters when we performed the HPLC analysis of fly head exacts (unpublished observations). Variations in visual cycles are not unprecedented as there are multiple differences in the cycles involving mammalian rods and cones, which couple to RPE and Müller cells respectively . In flies, the six outer photoreceptor cells (R1-6 cells) express one visual pigment, Rh1, and are high sensitivity photoreceptor cells, reminiscent of mammalian rods . The two, smaller, central R7 and R8 cells express multiple visual pigments and are the fly counterparts to mammalian cone cells, as they are less sensitive than the R1-6 cells and contribute to color vision . We propose that the R7 and R8 cells may function through the same visual cycle as R1-6 cells since Rh4, which is expressed in R7 cells , also depends on expression of PDH in the RPCs, during a normal light/dark cycle.
A key question concerns the function of a visual cycle, since the chromophore can be generated de novo from dietary sources, such as β-carotene. We found that the visual cycle is the major pathway contributing to the maintenance of rhodopsin levels in adult flies exposed to a light/dark cycle. When we disrupted de novo generation of chromophore by eliminating the dietary supply of retinoids in wild-type flies, they were able to maintain the rhodopsin levels for at least 25 days under a light/dark cycle. In contrast, providing very high levels of carotenoids did not prevent the loss of Rh1 in the pdh knockout flies. Since rhodopsin is degraded under light/dark conditions, the flies would become blind in environments with very limited supplies of dietary carotenoids if they were unable to employ a visual cycle. Thus, we suggest that the ability of the flies to recycle the chromophore provides a survival strategy that enables the flies to be independent of dietary carotenoids.
In conclusion, we described a visual cycle for chromophore regeneration in Drosophila and identified a key component of this pathway. This visual cycle exists despite the presence of a bistable pigment in fly photoreceptor cells. Several reports indicate that the visual pigment in the ipRGCs, referred to melanopsin, is also a bistable pigment [6, 7, 8]. Since we have shown that a bistable pigment and a visual cycle are not exclusive, our results suggest that mammalian photoreceptor cells that utilize bistable rhodopsin-like receptors also depend on enzymatic visual cycles.
Finally, defects in the mammalian visual cycle lead to multiple forms of retinal degeneration; however, treatment options are of limited value . Loss of PDH also results in retinal degeneration, possibly due to toxicity associated with the accumulation of retinaldehydes . The demonstration of a highly efficient visual cycle in Drosophila provides the opportunity to exploit this model organism to identify genetic and pharmacological suppressors of retinal dystrophies associated with a defective visual cycle.
We obtained the bw1;st1 and the y w;P[70FLP]11 P[70I-SceI]2B nocSco/CyO flies from the Bloomington Stock Center. Flies were raised at 25°C on standard cornmeal-yeast medium under a 12-h light/12-h dark cycle unless indicated otherwise. We used a retinoid replacement protocol similar to that described previously . Briefly, the retinoid-deficient medium consisted of: 240 ml H2O, 10 g dry yeast, 10 g glucose, 12 g rice powder, 2 g agar, 60 mg cholesterol, 3 ml 10% butyl p-hydroxybenzoate and 0.8 ml propionic acid. In some experiments the retinoid-deficient media was supplemented with either 5 mM β-carotene (Sigma), all-trans-retinal (Sigma), all-trans-retinol (Sigma) or 11-cis-retinal (a gift from Dr. R. Crouch).
The pdh knockout flies (pdh1) were generated by ends-out homologous recombination . The targeting construct deleted a 515-bp region encompassing the translation initiation site (Figure 1A; nucleotides −186 to +329; +1 is the predicted transcription initiation site). A 3.0 kb genomic fragment (extending from −3185 to −185) was inserted into the NotI site and another 3.0 kb genomic fragment (+330 to +3323) was inserted into the BamHI site of pw35 [Figure 1A; 27]. The two genomic fragments were in the same orientation. The targeting construct was injected into w1118 embryos, and transformants were selected on the basis of eye pigmentation. Flies carrying the targeting construct on the 2nd chromosome were crossed to y,w;P[70FLP]11 P[70ISceI]2B nocSco/CyO flies, and the progeny were screened for gene targeting by PCR.
To generate a genomic pdh rescue construct, we PCR-amplified a 1.5 kb DNA fragment (−515 to +1035; +1 is the transcription start site) and subcloned it between the NotI and XbaI sites of pCaSpeR4 . To express human RDH12 in flies, we subcloned the RDH12 cDNA (EST clone MGC-34762, ATCC) between the NotI and XbaI sites of pUAST . The constructs were introduced into w1118 flies by germline transformation.
To generate anti-PDH antibodies, a pdh cDNA fragment encoding the N-terminal 120 residues was subcloned into the pGEX5X-1 vector (GE Healthcare). The glutathione Stransferase fusion protein was expressed in Escherichia coli BL21 codon-plus (Stratagene), purified using glutathione agarose beads (GE Healthcare) and introduced into rabbits (Covance) to create the anti-PDH antibodies.
We prepared fly head extracts by homogenizing the heads directly in 1×SDS sample buffer with a pellet pestle (Kimble-Kontes). The extracts were fractionated by 12% SDS-PAGE and transferred to Immobilon-P transfer membranes (Millipore) in Trisglycine buffer. We probed the blots with mouse anti-Tubulin primary antibodies (1:2000 dilution, Developmental Studies Hybridoma Bank), mouse anti-Rh1 antibodies (1:2000 dilution, Developmental Studies Hybridoma Bank), or rabbit anti-PDH antibody (1:1000 dilution) and subsequently with peroxidase conjugated anti-mouse or rabbit IgG secondary antibody (Sigma). The signals were detected using ECL reagents (GE Healthscience). For quantification, we used IRDye 680 goat anti-Rabbit IgG (LI-COR) or IRDye 800 Donkey anti-mouse IgG (LI-COR) as the secondary antibodies, and detected the signals using the Odyssey infrared imaging system (LI-COR).
Immunofluorescence stainings of whole adult fly retinas were performed as described previously . The primary antibodies used were rabbit anti-PDH antibodies (1:50 dilution). The secondary antibodies were anti-rabbit IgG conjugated with Alexa Fluor 568 (1:200 dilution, Molecular Probes). The samples were examined at room temperature using a laser scanning microscope (Zeiss LSM510-Zeta, Carl Zeiss MicroImagining Inc.) with plan-apochromat 20× objectives. The images were acquired using a Carl Zeiss LSM imaging system and transferred into Adobe Photoshop 7.0 for analysis.
ERG recordings were performed as described previously . Two glass microelectrodes filled with Ringer's solution were inserted into small drops of electrode cream placed on the surfaces of the compound eye and the thorax. A Newport light projector (model 765) was used for stimulation. The ERG signals were amplified with a Warner electrometer IE-210 and recorded with a MacLab/4s A/D converter and the Chart v3.4/s program (A/D Instruments). We used five pulses of orange (580) or blue (480) light in the following order: orange, blue, blue, orange, orange. Each pulse was 5 sec separated by 7-sec intervals. All recordings were carried out at room temperature.
Dissected fly heads were homogenized in a glass homogenizer in 200 μl 2 M NH2OH (pH 6.8) and 600 μl methanol. After 10 minutes, we added, 600 μl acetone, 250 μl diethyl ether and 250 μl petroleum benzene. To extract retinoids, the samples were vortexed three times for 10 sec, centrifuged (2000×g, 25°C, 15 sec) and the organic phases were collected. The extraction was repeated two times with 400 μl petroleum benzene and the collected organic phases were dried under a nitrogen stream. Lipophilic compounds were dissolved in 100 μl HPLC-solvent and subjected to quantitative HPLC analysis as described previously . All steps were carried out under a dim red safety light.
Dissected heads from flies reared under a 12-h light/12-h dark cycle or in constant darkness were fixed in glutaraldehyde and embedded in LR White resin as described . Thin sections (50 nm) prepared at a depth of 30 μm were examined using a Zeiss (Oberkochen) FEI Tecnai 12 transmission electron microscope. The images were acquired using a Gatan (Pleasanton) camera (model 794) and Gatan Digital Micrograph software and converted into tiff files.
We thank Dr. A. Mariani and Dr. R. Crouch for the gift of 11-cis-retinal, the Bloomington Stock Center for fly stocks and the Developmental Studies Hybridoma Bank for anti-Rh1 and anti-Tubulin antibodies. We thank M. Sepanski and M. Delannoy for preparing EM sections and Dr. D. McClellan for comments on the manuscript. This work was supported by a grant to C.M. from the NEI (EY08117).
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