The Drosophila visual cycle and de novo chromophore synthesis depends on rdhB

Abstract

In mammalian rods and cones, light activation of the visual pigments leads to release of the chromophore, which is then recycled through a multistep enzymatic pathway, referred to as the visual or retinoid cycle. In invertebrates such as Drosophila, a visual cycle was thought not to exist since the rhodopsins are bistable photopigments, which consist of a chromophore that normally stays bound to the opsin following light activation. Nevertheless, we recently described a visual cycle in Drosophila that serves to recycle the free chromophore that is released following light-induced internalization of rhodopsin, and a retinol dehydrogenase (RDH) that catalyzes the first step of the pathway. Here, we describe the identification of a putative RDH, referred to as RDHB (retinol dehydrogenase B), which functions in the visual cycle and in de novo synthesis of the chromophore. RDHB was expressed in the retinal pigment cells (RPCs), where it promoted the final enzymatic reaction necessary for the production of the chromophore. Mutation of rdhB caused moderate light-dependent degeneration of the phototransducing compartment of the photoreceptor cells-the rhabdomeres, reminiscent of the effects of mutations in some human RDH genes. Since the first and last steps in the visual cycle take place in the RPCs, it appears that these cells are the sites of action for this entire enzymatic pathway in Drosophila.

Figure 1.

Spatial and temporal expression of RDHB. A, Schematic diagram of a cross-sectional view of a single ommatidium from a Drosophila compound eye. B–D, Immunostaining of a 0.5 μm head section from an rdhB-GAL4;rdhB¹, UAS-rdhB::myc fly. B, Myc antibodies (red). C, Rhodopsin (Rh1) antibodies (green). D, Merged image. E, Developmental Western blots probed with antibodies to RDHB, Rh1, and tubulin (Tub). Samples were prepared from flies at the indicated pupal times and from adult flies of the indicated ages. F, G, Immunostaining of dissected pupae eyes (∼45 h after puparium formation) with rabbit anti-RDHB. F, wild-type. The arrows indicate labeling of tertiary pigment cells. G, rdhB¹.

Figure 2.

Generation of rdhB¹ and effects of mutation on chromophore and Rh1 production during the pupal period. A, Schematic illustration of the rdhB (CG7077) gene and generation of the rdhB knock-out (rdhB¹) by ends-out homologous recombination. B, Western blot containing head extracts from the indicated flies probed with anti-RDHB and reprobed with anti-Tubulin (Tub). C, Western blot of head extracts from ≤1-d-old wild-type and rdhB¹. The flies were raised from the second instar larval stage on retinoid-free medium supplemented with 5 mm β-carotene or 500 μm all-trans-retinal or all-trans-retinol. The flies were kept under a 12 h light/dark cycle. D, Quantification of Western blot results shown in C. n ≥ 3. E, Decreased chromophore levels in rdhB¹ flies fed all-trans-retinal or all-trans-retinol. The flies were ≤1 d old and raised as indicated in C. n = 3. The concentration of 3-OH-11-cis-retinal and 3-OH-all-trans-retinal were measured by HPLC. Error bars represent ±SEMs. *p < 0.05. Unpaired Student's t tests.

Figure 3.

Proposed pathways for de novo synthesis of the chromophore and for the visual cycle. The proposed function for RDHB is indicated.

Figure 4.

RDHB was required to maintain Rh1 levels in adult flies. A, Western blot of head extracts from 2- to 3-d-old wild-type and rdhB¹ flies. The flies were raised on retinoid-free medium during the larval stages. Newly eclosed flies were maintained on retinoid-free medium supplemented with 5 mm β-carotene, 500 μm all-trans-retinal or all-trans-retinol or. The flies were kept under a 12 h light/dark cycle for 48 h before performing the Western blots. B, Western blot of head extracts obtained from wild-type and rdhB¹ flies of the indicated ages, and under the indicated light conditions. The flies were raised on normal retinoid-containing fly food (corn meal and molasses) and kept under a 12 h light/dark cycle. C, D, Quantification of Western blot data shown in A and B. Error bars represent ±SEMs. *p < 0.05. Unpaired Student's t test. n ≥ 3.

Figure 5.

Testing for a PDA in rdhB¹ flies by performing ERG recordings. Flies of the indicated genotypes and ages were dark-adapted for 1 min and subsequently exposed to 5 s pulses of orange (580 nm) light (O) or blue (480 nm) light (B) interspersed by 7 s. Arrows indicate the PDAs induced by blue light. The PDAs were terminated by orange light. A, Wild-type (wt), 2 d old. B, rdhB¹, 2 d old. C, wt, 25 d old. D, rdhB¹, 25 d old. E, rdhB¹, 25 d old, expressing a rdhB⁺ transgene: rdhB-gal4;rdhB¹, UAS-rdhB::myc.

Figure 6.

Transmission EM images of cross-sections from adult retinas. The age in days and the light conditions are indicated. The light/dark cycles were 12 h each. A, wt, 30 d under light/dark cycles. B, rdhB¹, 30 d under a light/dark cycle. C, rdhB¹, 30 d under constant darkness. D, Quantification of the numbers of rhabdomeres per ommatidium based on analyses of thin EM sections after 30 d under a light/dark cycle, or in the dark. Sixty ommatidia from 3 flies were counted for each condition. Error bars represent ±SEMs. *p < 0.05. ANOVA.