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Many invertebrates carry out a daily cycle of shedding and rebuilding of the photoreceptor's photosensitive rhabdomeric membranes. The mosquitoAedes aegyptishows a robust response, losing nearly all Aaop1 rhodopsin from the rhabdomeric membranes during the shedding process at dawn. Here, we made use of Aaop1 antibodies capable of distinguishing newly synthesized, glycosylated rhodopsin from mature nonglycosylated rhodopsin to characterize the fate of Aaop1 during the shedding and rebuilding processes. The rhabdomeric rhodopsin is moved into large cytoplasmic vesicles at dawn and is subsequently degraded during the standard 12 h daytime period. The endocytosed rhodopsin is trafficked back to the photosensitive membranes if animals are shifted back to dark conditions during the morning hours. During the daytime period, small vesicles containing newly synthesized and glycosylated Aaop1 rhodopsin accumulate within the cytoplasm. At dusk, these vesicles are lost as the newly synthesized Aaop1 is converted to the nonglycosylated form and deposited in the rhabdomeres. We demonstrate that light acts though a novel signaling pathway to block rhodopsin maturation, thus inhibiting the deglycosylation and rhabdomeric targeting of newly synthesized Aaop1 rhodopsin. Therefore, light controls two cellular processes responsible for the daily renewal of rhodopsin: rhodopsin endocytosis at dawn and inhibition of rhodopsin maturation until dusk.SIGNIFICANCE STATEMENTOrganisms use multiple strategies to maximize visual capabilities in different light conditions. Many invertebrates show a daily cycle of shedding the photoreceptor's rhabdomeric membranes at dawn and rebuilding these during the following night. We show here that theAedes aegyptimosquito possesses two distinct light-driven cellular signaling processes for modulating rhodopsin content during this cycle. One of these, endocytosis of rhabdomeric rhodopsin, has been described previously. The second, a light-activated cellular pathway acting to inhibit the anterograde movement of newly synthesized rhodopsin, is revealed here for the first time. The discovery of this cellular signaling pathway controlling a G-protein-coupled receptor is of broad interest due to the prominent role of this receptor family across all areas of neuroscience.

Wallenda (WND) is the Drosophila member of a conserved family of dual leucine-zipper kinases (DLK) active in both neuronal regeneration and degeneration. We examined the role of WND over-expression on sensory neuron morphology by driving WND in multiple subtypes of Drosophila photoreceptors. WND overexpression under control of the pan-retinal GAL4 driver GMR causes multiple photoreceptor defects including cell death, rhabdomere degeneration, and axonal sprouting. Individual photoreceptor subtypes were assayed using GAL4 drivers specific for each photoreceptor class. Many R7 and R8 cells exhibit axonal sprouting while some show cell degeneration. Delaying the onset of WND overexpression until 20 days of age showed that older adult R7 cells retain the ability to initiate new axon growth. R1–6 photoreceptor cells degenerate in response to WND expression and exhibit rhodopsin loss and rhabdomere degeneration. RNAi knockdown of the MAPK signaling components Kayak (KAY) and Hemipterous (HEP) attenuates the WND-induced loss of Rh1 rhodopsin. UAS-induced HEP expression is similar to WND expression, causing degeneration in R1–6 photoreceptors and axonal sprouting in R7 photoreceptors. These results demonstrate that WND in adult Drosophila photoreceptor cells acts through MAPK signaling activity with both regenerative and degenerative responses. These photoreceptors provide a tractable experimental model to reveal cellular mechanisms driving contradictory WND signaling responses.

Visual perception of the environment plays an important role in many mosquito behaviors. Characterization of the cellular and molecular components of mosquito vision will provide a basis for understanding these behaviors. A unique feature of the R7 photoreceptors in Aedes aegypti and Anopheles gambiae is the extreme apical projection of their rhabdomeric membrane. We show here that the compound eye of both mosquitoes is divided into specific regions based on nonoverlapping expression of specific rhodopsins in these R7 cells. The R7 cells of the upper dorsal region of both mosquitoes express a long wavelength op2 rhodopsin family member. The lower dorsal hemisphere and upper ventral hemisphere of both mosquitoes express the UV-sensitive op8 rhodopsin. At the lower boundary of this second region, the R7 cells again express the op2 family rhodopsin. In Ae. aegypti, this third region is a horizontal stripe of one to three rows of ommatidia, and op8 is expressed in a fourth region in the lower ventral hemisphere. However, in An. gambiae the op2 family member expression is expanded throughout the lower region in the ventral hemisphere. The overall conserved ommatidial organization and R7 retinal patterning show these two species retain similar visual capabilities. However, the differences within the ventral domain may facilitate species-specific visual behaviors.

In developing Drosophila photoreceptors, rhodopsin is trafficked to the rhabdomere, a specialized domain within the apical membrane surface. Rab11, a small GTPase implicated in membrane traffic, immunolocalizes to the trans-Golgi network, cytoplasmic vesicles and tubules, and the base of rhabdomeres. One hour after release from the endoplasmic reticulum, rhodopsin colocalizes with Rab11 in vesicles at the base of the rhabdomere. When Rab11 activity is reduced by three different genetic procedures, rhabdomere morphogenesis is inhibited and rhodopsin-bearing vesicles proliferate within the cytosol. Rab11 activity is also essential for development of MVB endosomal compartments; this is probably a secondary consequence of impaired rhabdomere development. Furthermore, Rab11 is required for transport of TRP, another rhabdomeric protein, and for development of specialized membrane structures within Garland cells. These results establish a role for Rab11 in the post-Golgi transport of rhodopsin and of other proteins to the rhabdomeric membranes of photoreceptors, and in analogous transport processes in other cells.

During the larval stages, the visual system of the mosquito Aedes aegypti contains five stemmata, often referred to as larval ocelli, positioned laterally on each side of the larval head. Here we show that stemmata contain two photoreceptor types, distinguished by the expression of different rhodopsins. The rhodopsin Aaop3 (GPROP3) is expressed in the majority of the larval photoreceptors. There are two small clusters of photoreceptors located within the satellite and central stemmata that express the rhodopsin Aaop7 (GPROP7) instead of Aaop3. Electroretinogram analysis of transgenic Aaop7 Drosophila indicates that Aaop3 and Aaop7, both classified as long-wavelength rhodopsins, possess similar but not identical spectral properties. Light triggers an extensive translocation of Aaop3 from the photosensitive rhabdoms to the cytoplasmic compartment, whereas light-driven translocation of Aaop7 is limited. The results suggest that these photoreceptor cell types play distinct roles in larval vision. An additional component of the larval visual system is the adult compound eye, which starts to develop at the anterior face of the larval stemmata during the 1st instar stage. The photoreceptors of the developing compound eye show rhodopsin expression during the 4th larval instar stage, consistent with indications from previous reports that the adult compound eye contributes to larval and pupal visual capabilities.

The tropical disease vector mosquito Anopheles gambiae possesses 11 rhodopsin genes. Three of these, GPROP1, GPROP3, and GPROP4, encode rhodopsins with >99% sequence identity. We created antisera against these rhodopsins and used immunohistology to show that one or more of these rhodopsins are expressed in the major R1-6 photoreceptor class of the adult Anopheles gambiae eye. Under dark conditions, rhodopsin accumulates within the light-sensitive rhabdomere of the photoreceptor. Light treatment, however, causes extensive movement of rhodopsin to the cytoplasmic compartment. Protein electrophoresis showed that the rhodopsin is present in two different forms. The larger form is an immature species that is deglycosylated during the posttranslational maturation process to generate the smaller, mature form. The immature form is maintained at a constant level regardless of lighting conditions. These results indicate that rhodopsin biosynthesis and movement into the rhabdomere occurs at a constant rate. In contrast, the mature form increases in abundance when animals are placed in dark conditions. Light-triggered internalization and protein degradation counteracts this rhodopsin increase and keeps rhabdomeric rhodopsin levels low in light conditions. The interplay of the constant maturation rate with light-triggered degradation causes rhodopsin to accumulate within the rhabdomere only in dark conditions. Thus, Anopheles photoreceptors possess a mechanism for adjusting light sensitivity through light-dependent control of rhodopsin levels and cellular location.

Summary Differential rhodopsin gene expression within specialized R7 photoreceptors cells divides the retinas of Aedes aegypti and Anopheles gambiae mosquitoes into distinct domains. Both mosquito species express the rhodopsin orthologs Aaop8 and Agop8, respectively, in a large subset of these R7 photoreceptors that function as ultraviolet receptors. We show here that a divergent subfamily of mosquito rhodopsins, Aaop10 and Agop10, are coexpressed in these R7 photoreceptors. The properties of the Ae. aegypti Aaop8 and Aaop10 rhodopsins were analyzed by creating transgenic Drosophila expressing these rhodopsins. Electroretinogram recordings, and spectral analysis of head extracts, obtained from the Aaop8 strain confirmed that Aaop8 is a ultraviolet-sensitive rhodopsin. Aaop10 was poorly expressed and capable of eliciting only small and slow light responses in Drosophila photoreceptors, and electroretinogram analysis suggested that it is a long wavelength rhodopsin with a maximal sensitivity near 500 nm. Thus, coexpression of Aaop10 rhodopsin with Aaop8 rhodopsin has the potential to modify the spectral properties of mosquito ultraviolet receptors. Retention of Op10 rhodopsin family members in the genomes of Drosophila species suggests this rhodopsin family may play a conserved role in insect vision.

Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites for N-linked glycosylation (N20I and N196I). We show that Asn(20) is the sole site for glycosylation. The Rh1(N20I) protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1(N20I); therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1(N20I)/NinaA interaction. We further show that Rh1(N20I) interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.

Rab6 is a GTP binding protein that regulates vesicular trafficking within the Golgi and post-Golgi compartments. We overexpressed wild-type, a GTPase defective (Q71L), and a guanine nucleotide binding defective (N125I) Rab6 protein in Drosophila photoreceptors to assess the in vivo role of Rab6 in the trafficking of rhodopsin and other proteins. Expression of Drab6(Q71L) greatly reduced the steady state levels of two rhodopsins, Rh1 and Rh3, whereas Drab6(wt) and Drab6(N125I) showed weaker effects. Analysis of a strain carrying Rh1 rhodopsin under a heat shock promoter showed that Drab6(Q71L), but not Drab6(wt) or Drab6(N125I), prevents the maturation of rhodopsin beyond an immature 40 kDa form. Drab6(Q71L) is a GTPase defective mutant, indicating that anterograde transport of rhodopsin requires Rab6 GTPase function. The three Drab6 strains had no effect on the expression of several other photoreceptor proteins. The Drab6(Q71L) photoreceptors show marked histological defects at young ages and degenerate over a two week time span. These results establish that rhodopsin is transported via a Rab6 regulated pathway and that defects in trafficking pathways lead to retinal degeneration.

ninaED1, a dominant allele of the major Drosophila rhodopsin gene, expresses a rhodopsin that is predominantly recovered in a 80-kD complex that likely represents rhodopsin dimers. By driving either ninaED1 or ninaE+ expression from a heat-shock promoter, we show that the 80-kD rhodopsin complex forms immediately after gene activation. In wild type, but not ninaED1, rhodopsin monomeric forms are detected at later times. The generation of monomeric forms of wild-type rhodopsin is suppressed in vitamin A-deprived flies or in flies heterozygous for the dominant rhodopsin mutation. We also show that ninaED1 expression does not affect the maturation of another Drosophila visual pigment, Rh3. These results are consistent with the view that the ninaED1 rhodopsin antagonizes an early posttranslation process that is specific for maturation of the ninaE-encoded rhodopsin.

The Drosophila genetic system has been exploited to identify and analyze many genes that are responsible for the specialized cellular processes and biochemical mechanisms of the photoreceptor cell. The results establish that disruption of many different cellular processes can trigger retinal degeneration. The rhodopsin gene family provides an example in which mutations are known to trigger retinal degeneration in both vertebrates and invertebrates. The Drosophila research has established that dominant rhodopsin mutations disrupt the protein maturation process. This review emphasizes the unique and powerful experimental approaches of Drosophila that are being applied towards understanding the many causes of retinal degeneration.

Dominant mutations of the Drosophila ninaE-encoded rhodopsin are described that reduce the expression of wild-type rhodopsin and cause a slow, age-dependent form of retinal degeneration. A posttranslational event subsequent to the requirement for the ninaA-encoded cyclophilin is disrupted by the dominant mutations. Most of these dominant mutations are missense mutations that affect the physical properties of one of the seven transmembrane domains; another affects the cysteine involved in a disulfide linkage. The results indicate that misfolded or unstable mutant rhodopsin can interfere with maturation of wild-type rhodopsin, and that these cellular conditions may trigger retinal degeneration. In addition, these dominant rhodopsin mutations suppress the rapid degeneration seen in rdgC and norpA flies, indicating that high levels of rhodopsin are required.

Multiple mechanisms contribute to a photoreceptor's ability to adapt to ambient light conditions. The mosquitoAedes aegyptiexpresses the long-wavelength rhodopsin Aaop1 in all R1–R6 photoreceptors and most R8 photoreceptors. These photoreceptors alter the cellular location of Aaop1 and reorganize their photosensitive rhabdomeric membranes on a daily basis. During daylight periods, Aaop1 is excluded from the light-sensitive rhabdomeres and localized to multivesicular bodies (MVBs) within the photoreceptor cytoplasm. In the dark, Aaop1 accumulates in the rhabdomeres and no Aaop1-containing MVBs are present in the cytoplasm. Manipulation of light treatments shows the cellular movement of Aaop1 in and out of the rhabdomere is directly controlled by light. In a separate process, the photoreceptors reduce Aaop1 protein content during a time period spanning from late afternoon into the first 2 h of the dark period. Aaop1 levels then gradually increase through the dark period and remain high following movement of Aaop1 to the cytoplasm at dawn. These results demonstrate that mosquito photoreceptors control rhodopsin availability during the daily light-dark cycle by novel mechanisms not discerned from analysis of traditional invertebrate models. These mechanisms will maximize a photoreceptor's light sensitivity range and therefore may be common in organisms active in low light conditions.

The retina of the mosquito Aedes aegypti can be divided into four regions based on the non-overlapping expression of a UV sensitive Aaop8 rhodopsin and a long wavelength sensitive Aaop2 type rhodopsin in the R7 photoreceptors. We show here that another rhodopsin, Aaop9, is expressed in all R7 photoreceptors and a subset of R8 photoreceptors. In the dorsal region, Aaop9 is expressed in both the cell body and rhabdomere of R7 and R8 cells. In other retinal regions Aaop9 is expressed only in R7 cells, being localized to the R7 rhabdomere in the central and ventral regions and in both the cell body and rhabdomere within the ventral stripe. Within the dorsal-central transition area ommatidia do not show a strict pairing of R7-R8 cell types. Thus, Aaop9 is coexpressed in the two classes of R7 photoreceptors previously distinguished by the non-overlapping expression of Aaop8 and Aaop2 rhodopsins. Electroretinogram analysis of transgenic Drosophila shows that Aaop9 is a short wavelength rhodopsin with an optimal response to 400-450 nm light. The coexpressed Aaop2 rhodopsin has dual wavelength sensitivity of 500-550 nm and near 350 nm in the UV region. As predicted by the spectral properties of each rhodopsin, Drosophila photoreceptors expressing both Aaop9 and Aaop2 rhodopsins exhibit a uniform sensitivity across the broad 350-550 nm light range. We propose that rhodopsin coexpression is an adaptation within the R7 cells to improve visual function in the low-light environments in which Ae. aegypti is active.

In vitromutagenesis and germline transformation were used to create aDrosophilamutant, ΔAsn20lacking the N-linked glycosylation site near the amino terminus of the major rhodopsin (Asn20-Gly-Ser changed to Ile-Gly-Ser). Low opsin protein levels are detected in ΔAsn20photoreceptors. Electroretinogram responses of mutant flies show that the residual rhodopsin found in this mutant is capable of initiating phototransduction. The organization of rhabdomeres, the photoreceptor organelle containing nearly all of the rhodopsin, is aberrant in the ΔAsn20mutant and undergoes age-dependent deterioration. These results establish that an N-linked glycosylation site, and likely glycosylation itself, plays a critical role in the maturation ofDrosophilarhodopsin.

Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.

The ninaE-encoded Rh1 rhodopsin is the major light-sensitive pigment expressed in Drosophila R1-6 photoreceptor cells. Rh1 rhodopsin localizes to and is essential for the development and maintenance of the rhabdomere, the specialized membrane-rich organelle that serves as the site of phototransduction. We showed previously that the vertebrate bovine rhodopsin (Rho) is expressed and properly localized in Drosophila photoreceptor cells. Drosophila photoreceptors expressing only Rho have normal rhabdomere structure at young ages, but the rhabdomeres are not maintained and show extensive disorganization by 7-10 days of age. A series of Rho-Rh1 opsin chimeric rhodopsins were used to identify Rh1 domains required for maintenance of rhabdomeric structure. The results show that the Rh1 rhodopsin cytoplasmic tail domain, positioned to interact with cytoplasmic structural components, plays a major role in promoting rhabdomeric organization.

The Drosophila genes ninaB and ninaD, encoding a beta-carotene oxygenase and a type B scavenger receptor respectively, are essential for the biosynthesis of the 3-hydroxyretinal chromophore of rhodopsin. We analyzed transgenic reporter strains and performed in situ hybridization to show that both ninaB and ninaD are expressed in the adult brain but not retinal tissues. Developmental RT-PCR and tissue expression studies showed that ninaB is only expressed in the adult brain, while ninaD is expressed in the adult brain, the adult body, and many larval tissues. The data support a model in which NinaD is required for uptake and storage of dietary carotenoids throughout the larval and adult stages of development. Beta-carotene is transported to the adult brain, where cellular uptake by NinaD allows cleavage by the NinaB enzyme to produce retinal. Retinal is then transported to the retina for rhodopsin biogenesis.

PURPOSE. Vertebrate and invertebrate visual pigments are similar in amino acid sequence, structural organization, spectral properties, and mechanism of action, but possess different chromophores and trigger phototransduction through distinct biochemical pathways. The bovine opsin gene (Rho) was expressed in Drosophila, to examine the properties of a vertebrate opsin within invertebrate photoreceptor cells. METHODS. Transgenic Drosophila expressing the bovine opsin gene (Rho) in photoreceptors were created. Protein expression and cellular location of bovine rhodopsin was assessed by protein blots and immunofluorescence. The glycosylation state was determined by mobility profiles in SDS-PAGE before and after treatment with endoglycosidase. The rhodopsin chromophore was determined by HPLC-mass spectroscopy (MS) and the spectral properties by spectroscopy. The ability of the bovine rhodopsin to couple to Drosophila phototransduction components was assessed by electroretinography and to couple to vertebrate transducin by light-mediated GTPS-binding assays. RESULTS. Rho showed stable expression even in the absence of endogenous Rh1 opsin and chromophore. It was correctly targeted to the rhabdomeric membranes. Rho remained glycosylated during the maturation process and possessed a distinct glycosylation pattern from that of native Rho. The Drosophilaexpressed Rho associated with the 3-hydroxyretinal chromophore but failed to evoke an electroretinogram response from fly photoreceptors. However, the Drosophila-expressed Rho activated transducin in a light-dependent manner. CONCLUSIONS. Drosophila photoreceptors express a vertebrate rhodopsin as a functional visual pigment, but the expression does not activate the Drosophila phototransduction pathway. The system allows the characterization and comparison of vertebrate and invertebrate visual pigment properties in a common cell type. (Invest Ophthalmol Vis Sci. 2006;47:3722‐3728) DOI:10.1167/iovs.06-0281

Invertebrate and vertebrate rhodopsins share a low degree of homology and are coupled to G-proteins from different families. Here we explore the utility of fly-expressed chimeras between Drosophila rhodopsin Rh1 and bovine rhodopsin (Rho) to probe the interactions between the invertebrate and vertebrate visual pigments and their cognate G-proteins. Chimeric Rh1 pigments carrying individual substitutions of the cytoplasmic loops C2 and C3 and the C-terminus with the corresponding regions of Rho retained the ability to stimulate phototranduction in Drosophila, but failed to activate transducin. Surprisingly, chimeric Rho containing the Rh1 C-terminus was fully capable of transducin activation, indicating that the C-terminal domain of vertebrate rhodopsins is not essential for the functional coupling to transducin.

We previously reported (Sarfare, S., Ahmad, S. T., Joyce, M. V., Boggess, B., and O'Tousa, J. E. (2005) J. Biol. Chem. 280, 11895-11901) that the Drosophila ninaG gene encodes an oxidoreductase involved in the biosynthesis of the (3S)-3-hydroxyretinal serving as chromophore for Rh1 rhodopsin and that ninaG mutant flies expressing Rh4 as the major opsin accumulate large amounts of a different retinoid. Here, we show that this unknown retinoid is 11-cis-3-hydroxyretinol. Reversed phase high performance liquid chromatography coupled with a photodiode array UV-visible absorbance detector and mass spectrometer revealed a major product eluting at a retention time, t(r), of 3.5 min with a lambda(max) of approximately 324 nm and with a base peak in the mass spectrum at m/z 285. These observations are identical with those of the 3-hydroxyretinol standard. The base peak in the electrospray ionization mass spectrum arises from the loss of a water molecule from the protonated molecule at m/z 303 because of fragmentation in the ion source. These results suggest that 11-cis-3-hydroxyretinol is an intermediate required for chromophore biogenesis in Drosophila. We further show that ninaG mutants fed on retinal as the sole source of vitamin A are able to synthesize 3-hydroxyretinoids. Thus, the NinaG oxidoreductase is not responsible for the initial hydroxylation of the retinal ring but rather acts in a subsequent step in chromophore production. These data are used to review chromophore biosynthesis and propose that NinaG acts in the conversion of (3R)-3-hydroxyretinol to the 3S enantiomer.

The Drosophila ninaG mutant is characterized by low levels of Rh1 rhodopsin, because of the inability to transport this rhodopsin from the endoplasmic reticulum to the rhabdomere. ninaG mutants do not affect the biogenesis of the minor opsins Rh4 and Rh6. A genetic analysis placed the ninaG gene within the 86E4-86E6 chromosomal region. A sequence analysis of the 15 open reading frames within this region from the ninaG(P330) mutant allele identified a stop codon in the CG6728 gene. Germ-line transformation of the CG6728 genomic region rescued the ninaG mutant phenotypes, confirming that CG6728 corresponds to the ninaG gene. The NinaG protein belongs to the glucose-methanol-choline oxidoreductase family of flavin adenine dinucleotide-binding enzymes catalyzing hydroxylation and oxidation of a variety of small organic molecules. High performance liquid chromatography analysis of retinoids was used to gain insight into the in vivo role of the NinaG oxidoreductase. The results show that when Rh1 is expressed as the major rhodopsin, ninaG flies fail to accumulate 3-hydroxyretinal. Further, in transgenic flies expressing Rh4 as the major rhodopsin, 3-hydroxyretinal is the major retinoid in ninaG+, but a different retinoid profile is observed in ninaG(P330). These results indicate that the ninaG oxidoreductase acts in the biochemical pathway responsible for conversion of retinal to the rhodopsin chromophore, 3-hydroxyretinal.

The Drosophila ninaB gene encodes a beta,beta-carotene-15,15'-oxygenase responsible for the centric cleavage of beta-carotene that produces the retinal chromophore of rhodopsin. The ninaD gene encodes a membrane receptor required for efficient use of beta-carotene. Despite their importance to the synthesis of visual pigment, we show that these genes are not active in the retina. Mosaic analysis shows that ninaB and ninaD are not required in the retina, and exclusive retinal expression of either gene, or both genes simultaneously, does not support rhodopsin biogenesis. In contrast, neuron-specific expression of ninaB and ninaD allows for rhodopsin biogenesis. Additional directed expression studies failed to identify other tissues supporting ninaB activity in rhodopsin biogenesis. These results show that nonretinal sites of NinaB beta,beta-carotene-15,15'-oxygenase activity, likely neurons of the central nervous system, are essential for production of the visual chromophore. Retinal or another C(20) retinoid, not members of the beta-carotene family of C(40) carotenoids, are supplied to photoreceptors for rhodopsin biogenesis.

Drosophilavision research has benefited from simultaneous application of genetic, molecular, electrophysiological analyses. The work establishes an essential role of Ca2+in regulation of phototransduction. Many different proteins are the targets of Ca2+regulation and these proteins act at multiple steps of the process. These targets include proteins involved in the rhodopsin cycle, proteins responsible for intermediate steps of phototransduction, and the TRP and TRPL light-gated channels. The regulation of these phototransduction components by Ca2+occurs in three different ways. First, the presence of Ca2+/calmodulin-binding sites in phototransduction-mediating proteins places these proteins under Ca2+control. Second, a protein kinase C regulated by Ca2+phosphorylates proteins to modulate their activity. Finally, some proteins contain Ca2+-binding sites and apparently are directly regulated by Ca2+. Here I review the photoreceptor proteins regulated by Ca2+and summarize current views on the roles of these proteins in theDrosophilaphotoresponse.

Rhodopsin is essential for normal photoreceptor development inDrosophila(O'Tousaet al.,1989; Leonardet al.,1992; Kumar and Ready, 1995) and in mice (Humphrieset al.,1997). Here we report studies in which a rhodopsin transgene is expressed at restricted stages during the development ofDrosophilaphotoreceptors otherwise lacking rhodopsin. Substantial rescue of normal photoreceptor structure and physiology is effected by rhodopsin expression during the time of the normal onset of rhodopsin synthesis. Expression shortly before or after this critical period does not rescue these deficits. There is a critical developmental period in which rhodopsin plays its key role in photoreceptor morphogenesis.

Mutations within the human rod opsin gene are responsible for approximately 25% of the autosomal dominant forms of retinitis pigmentosa (ADRP) afflicting human populations (l).There are over 60 different mutations known in the rhodopsin gene that cause ADRP. To account for the mechanisms by which these mutations can cause retinal degeneration, three general types of models have been considered. First, improper folding mutant protein may prevent its proper maturation (2). A second model is that the mutated forms may produce rhodopsins that are constitutively active, leading to increased metabolic activity (3). A third possibility is that the mutant proteins show improper cellular localization (4). All three views are consistent with a scenario by which the dominant rhodopsin mutation reduces the health of the photoreceptor, eventually causing the cell to degenerate. Understanding the molecular basis of rhodopsin-based forms of ADRP represents a major advance, but it is likely that successful strategies for alleviating the disease will require additional knowledge on the mechanisms by which these rhodopsin mutants trigger retinal degeneration.

The Drosophila mutant, oraJK84, lacks rhabdomeres in the major (R1-6) class of photoreceptors because these rhabdomeres rapidly degenerate in young flies. Genetic analysis reveals that oraJK84 actually contains two mutations (a ninaE and an ort allele) that affect the visual process. The mutation in ort appears to have no effect on photoreceptor structure. The other mutation occurs within the ninaE gene, which encodes the species of rhodopsin found in the R1-6 class of photoreceptors. Our analysis shows that this mutation is responsible for R1-6 rhabdomere degeneration in oraJK84 mutants. We also examined a ninaE mutant, denoted ninaEo117, that produces no ninaE transcript. The morphological phenotype observed in ninaEo117 is similar to that seen in oraJK84 mutants. We conclude that rhodopsin plays a vital role in maintaining photoreceptor structure in Drosophila.

Mutations that decrease the amplitude of the prolonged depolarizing afterpotential (PDA) in Drosophila melanogaster have been shown to have reduced rhodopsin content in the rhabdomeres of photoreceptor cells. In the present study, a genetic analysis of a class of third chromosome PDA-defective mutants localized the ninaE locus to the salivary band region 92A-B. In flies with only one copy of this region instead of the normal two copies, and in ninaE heterozygotes, the rhodopsin content of the major class of photoreceptors is reduced. Three doses of this region increase the rhodopsin content of these photoreceptors. These characteristics of the ninaE locus are expected of the structural gene encoding the major species of opsin in the Drosophila compound eye.

We placed UAA, UAG and UGA nonsense mutations at two leucine codons, Leu205 and Leu309, in Drosophila's major rhodopsin gene, ninaE, by site-directed mutagenesis, and then created the corresponding mutants by P element-mediated transformation of a ninaE deficiency strain. In the absence of a genetic suppressor, flies harboring any of the nonsense mutations at the 309 site, but not the 205 site, show increased rhodopsin activity. Additionally, all flies with nonsense mutations at either site have better rhabdomere structure than does the ninaE deficiency strain. Construction and analysis of a 3'-deletion mutant of ninaE indicates that translational readthrough accounts for the extra photoreceptor activity of the ninaE309 alleles and that truncated opsins are responsible for the improved rhabdomere structure. The presence of leucine-inserting tRNA nonsense suppressors DtLa Su+ and DtLb Su+ in the mutant strains produced a small increase (less than 0.04%) in functional rhodopsin. The opal (UGA) suppressor derived from the DtLa tRNA gene is more efficient than the amber (UAG) or opal suppressor derived from the DtLb gene, and both DtLa and DtLb derived suppressors are more efficient at site 205 than 309.