Requirement for an enzymatic visual cycle in Drosophila

Abstract

Background: The visual cycle is an enzymatic pathway employed in the vertebrate retina to regenerate the chromophore after its release from light-activated rhodopsin. However, a visual cycle is thought to be absent in invertebrates such as the fruit fly Drosophila melanogaster.

Results: 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.

Conclusions: 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.

Figure 1. Generation of pdh Knockout Flies

(A) Scheme for generating the pdh knock-out (pdh¹) by ends-out homologous recombination. (B) Western blot of wild-type and pdh¹ head extracts probed with anti-PDH antibodies and reprobed with anti-Tubulin antibodies. Molecular weight markers (kDa) are indicated to the left. (C) Schematic diagrams of longitudinal (left) and cross-sectional (right) views of a single ommatidium from a Drosophila compound eye [adapted from 5]. (D) Immunostaining of whole dissected adult retinas with rabbit anti-PDH antibodies. The images include approximately 25 ommatidia each. The box demarcates a single ommatidium, which corresponds to the indicated cartoon in panel C.

Figure 2. Light-dependent Retinal Degeneration in pdh¹ Flies

(A) Time course of retinal degeneration in pdh¹ flies. The numbers of rhabdomeres were based on analysis of transmission EMs of retinal cross-sections (n=20 from 5 flies each). The error bars represent standard errors. *p<0.01, unpaired Student t-test. (B) Transmission EM images of cross-sections from wild-type and pdh¹ retinas. The age in days and whether the flies were maintained in the dark or under a 12-h light/12-h dark cycle are indicated. See also Figure S1.

Figure 3. Light-dependent Loss of Rh1 in pdh¹ Flies

(A) Western blot of head extracts from wild-type and pdh¹ flies probed with anti-Rh1, anti-Tubulin and anti-PDH antibodies. (B) ERG recordings in wild-type and pdh¹ flies. Flies were dark-adapted for 1 min and subsequently exposed to 5 s pulses of orange light (O) or blue light (B) interspersed by 7 sec as indicated. A PDA was induced in wild type by blue light and terminated by orange light (arrows). (C) Quantification of Rh1 levels in wild-type and pdh¹ flies of the indicated ages, which were maintained under a light/dark cycle. The percentages were normalized to the wild-type day 1 samples. We performed Student t-tests to compare the differences between wild-type and pdh¹ (*p<0.01). (D) ERG recording in pdh¹ flies containing the pdh⁺ genomic rescue transgene. (E) Western blot of head extracts probed with anti-Rh1, anti-Tubulin and anti-PDH antibodies. The extracts were prepared from wild-type, pdh¹, and the knockout flies with the pdh⁺ genomic transgene. See also Figure S2.

Figure 4. PDH is required for the Conversion of Retinal to Retinol

(A and B) Retinoid composition in wild-type and pdh¹ fly heads measured by HPLC analysis. Before performing the HPLC analysis, 1 day-old-flies were collected and maintained under: (A) 12-h light/12-h dark cycle for 7 days; or (B) 12 h under blue light. The data represent the means ±SEMs (n=3). 3-hydroxy-11-cis-retinal (3-OH-11-cis-RAL); 3-hydroxy-all-trans-retinal (3-OH-all-trans-RAL); 3-hydroxy-11-cis-retinol (3-OH-11-cis-ROL); 3-hydroxy-all-trans-retinol (3-OH-all-trans-ROL). (C) The pdh¹ flies failed to utilize all-trans-retinal to synthesize Rh1. Newly eclosed wild-type and pdh¹ flies reared on retinoid-deficient food were fed retinoid-deficient medium only, or this same medium supplemented with 5 mM β-carotene, all-trans-retinal (all-trans-RAL), all-trans-retinol (all-trans-ROL) or 11-cis-retinal (11-cis-RAL) for 48-72 hours under a light/dark cycle before performing the Western blots. (D) Supplementation with 11-cis-retinal is sufficient to produce Rh1. The flies were treated as in (C) except that they were fed 11-cis-retinal and then maintained in the dark for 48 hours. See also Figure S3.

Figure 5

Model of the Drosophila visual cycle. The visual cycle is indicated by the dashed square. The “?” indicates that the molecular identities of the isomerase and 11-cis-RDH are not known. See text for other details.

Figure 6

The retinoid cycle is essential for maintaining Rh1 in adult flies exposed to light. (A) Rh1 levels in wild-type and pdh¹ flies maintained for 25 days under a light/dark cycle in the presence or absence of β-carotene. Larvae were raised on normal food. Following eclosion, the adult flies were fed normal food (+) or vitamin A-free food (−) and maintained for 25 days under a 12-h light/12-h dark cycle before performing the Western blot. (B) Rh1 levels in wild-type and pdh¹ flies maintained for 7 days in the presence of very high levels of β-carotene. Larvae were reared on normal food and the adults were fed normal food supplemented with either 5 mM β-carotene (++) or normal food only (+) and maintained for 7 days under a 12-h light/12-h dark cycle before performing the Western blot.

Figure 7

Partial rescue of pdh¹ phenotype by expressing human RDH12 in RPCs. (AC) ERG recordings of 7-day-old flies. (A) wild-type. (B) pdh¹. (C) 7077-Gal4;UASRDH12;pdh¹ flies. (D) Western blot of wild-type, pdh¹, and 7077-Gal4;UASRDH12;pdh¹ head extracts probed with anti-Rh1 and anti-Tubulin antibodies. The flies were maintained under a 12-h light/12-h dark cycle for 7 days before conducting the experiments. (E) Mean number of rhabdomeres per ommatidium in 20-day-old flies of the indicated genotypes. Each mean was based on analyses of retinal cross-sections of 20 ommatidia in five different flies using transmission EM. The error bars represent standard errors. We determined statistical significances using the unpaired Student's t-test (*p<0.01). (F-H) EM images of retinal cross-sections from 20-day-old flies held under a 12-h light/12-h dark cycle. (F) wild-type. (G) pdh¹. (H) 7077-Gal4;UASRDH12;pdh¹.