Otd/Crx,a dual regulator for the specification of ommatidia subtypes in the Drosophila retina

Comparison between the inputs of photoreceptors with different spectral sensitivities is required for color vision. In Drosophila, this is achieved in each ommatidium by the inner photoreceptors R7 and R8. Two classes of ommatidia are distributed stochastically in the retina: 30% contain UV-Rh3 in R7 and blue-Rh5 in R8, while the remaining 70% contain UV-Rh4 in R7 and green-Rh6 in R8. We show here that the distinction between the rhodopsins expressed in the two classes of ommatidia depends on a series of highly conserved homeodomain binding sites present in the rhodopsin promoters. The homeoprotein Orthodenticle acts through these sites to activate rh3 and rh5 in their specific ommatidial subclass and through the same sites to prevent rh6 expression in outer photoreceptors. Therefore, Otd is a key player in the terminal differentiation of subtypes of photoreceptors by regulating rhodopsin expression, a function reminiscent of the role of one of its mammalian homologs, Crx, in eye development.     

Localization of Na/K-ATPase in developing and adult Drosophila melanogaster photoreceptors

Drosophila melanogaster photoreceptors are highly polarized cells and their plasma membrane is organized into distinct domains. Zonula adherens junctions separate a smooth peripheral surface, the equivalent of the basolateral surface in other epithelial cells, from the central surface (approximately equal to apical surface). The latter consists of the microvillar rhabdomere and the juxtarhabdomeric domain, a nonmicrovillar area between the rhabdomere and the zonulae adherens. The distribution of Na/K-ATPase over these domains was examined by immunocytochemical, developmental, and genetic approaches. Immunofluorescence and immunogold labeling of adult compound eyes reveal that the distribution of Na/K-ATPase is concentrated at the peripheral surface in the photoreceptors R1-R6, but extends over the juxtarhabdomeric domain to the rhabdomere in the photoreceptors R7/R8. Developmental analysis demonstrates further that Na/K-ATPase is localized over the entire plasma membrane in all photoreceptors in early pupal eyes. Redistribution of Na/K-ATPase in R1-R6 occurs at about 78% of pupal life, coinciding with the onset of Rh1-rhodopsin expression on the central surface of these cells. Despite the essential role of Rh1 in structural development and intracellular trafficking, Rh1 mutations do not affect the distribution of Na/K-ATPase. These results suggest that Na/K-ATPase and rhodopsin are involved in distinct intracellular localization mechanisms, which are maintained independent of each other.     

Characterization of Drosophila melanogaster rhodopsin

A polypeptide present in Drosophila eye homogenates was identified as opsin. This polypeptide pI 7.8, with Mr 39,000 is a retina-specific protein. It has the spectral characteristics of rhodopsin contained in the R1-6 photoreceptors and decreases in amount with vitamin A deprivation. It contains a chromophore derived from vitamin A and linked to the protein moiety by a Schiff base. Moreover, the polypeptide identified corresponds to a retina-specific polypeptide that was shown previously to undergo light-dependent phosphorylation in living flies. These results indicate that many properties of Drosophila rhodopsin do not differ significantly from those reported for rhodopsins of other organisms. However, the isoelectric point of Drosophila opsin is considerably more basic than those reported for vertebrate rhodopsins.     

Iroquois complex genes induce co-expression of rhodopsins in Drosophila

The Drosophila eye is a mosaic that results from the stochastic distribution of two ommatidial subtypes. Pale and yellow ommatidia can be distinguished by the expression of distinct rhodopsins and other pigments in their inner photoreceptors (R7 and R8), which are implicated in color vision. The pale subtype contains ultraviolet (UV)-absorbing Rh3 in R7 and blue-absorbing Rh5 in R8. The yellow subtype contains UV-absorbing Rh4 in R7 and green-absorbing Rh6 in R8. The exclusive expression of one rhodopsin per photoreceptor is a widespread phenomenon, although exceptions exist. The mechanisms leading to the exclusive expression or to co-expression of sensory receptors are currently not known. We describe a new class of ommatidia that co-express rh3 and rh4 in R7, but maintain normal exclusion between rh5 and rh6 in R8. These ommatidia, which are localized in the dorsal eye, result from the expansion of rh3 into the yellow-R7 subtype. Genes from the Iroquois Complex (Iro-C) are necessary and sufficient to induce co-expression in yR7. Iro-C genes allow photoreceptors to break the "one receptor-one neuron" rule, leading to a novel subtype of broad-spectrum UV- and green-sensitive ommatidia.     

Expression of Drosophila rhodopsins during photoreceptor cell differentiation: insights into R7 and R8 cell subtype commitment

The R7 and R8 photoreceptor cells of the Drosophila retina are thought to mediate color discrimination and polarized light detection. This is based on the patterned expression of different visual pigments, rhodopsins, in different photoreceptor cells. In this report, we examined the developmental timing of retinal patterning. There is genetic evidence that over the majority of the eye, patterned expression of opsin genes is regulated by a signal from one subtype of R7 cells to adjacent R8 cells. We examined the onset of expression of the rhodopsin genes to determine the latest time point by which photoreceptor subtype commitment must have occurred. We found that the onset of rhodopsin expression in all photoreceptors of the compound eye occurs during a narrow window from 79% to 84% of pupal development (approximately 8 h), pupal stages P12-P14. Rhodopsin 1 has the earliest onset, followed by Rhodopsins 3, 4, and 5 at approximately the same time, and finally Rhodopsin 6. This sequence mimics the model for how R7 and R8 photoreceptor cells are specified, and defines the timing of photoreceptor cell fate decisions with respect to other events in eye development.     

Rab11 mediates post-Golgi trafficking of rhodopsin to the photosensitive apical membrane of Drosophila photoreceptors

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.     

Myosin V, Rab11, and dRip11 direct apical secretion and cellular morphogenesis in developing Drosophila photoreceptors

Sensory neuron terminal differentiation tasks apical secretory transport with delivery of abundant biosynthetic traffic to the growing sensory membrane. We recently showed Drosophila Rab11 is essential for rhodopsin transport in developing photoreceptors and asked here if myosin V and the Drosophila Rab11 interacting protein, dRip11, also participate in secretory transport. Reduction of either protein impaired rhodopsin transport, stunting rhabdomere growth and promoting accumulation of cytoplasmic rhodopsin. MyoV-reduced photoreceptors also developed ectopic rhabdomeres inappropriately located in basolateral membrane, indicating a role for MyoV in photoreceptor polarity. Binary yeast two hybrids and in vitro protein-protein interaction predict a ternary complex assembled by independent dRip11 and MyoV binding to Rab11. We propose this complex delivers morphogenic secretory traffic along polarized actin filaments of the subcortical terminal web to the exocytic plasma membrane target, the rhabdomere base. A protein trio conserved across eukaryotes thus mediates normal, in vivo sensory neuron morphogenesis.     

Angular and spectral sensitivity of fly photoreceptors. II. Dependence on facet lens F-number and rhabdomere type in<Emphasis Type="Italic"> Drosophila</Emphasis>

A wave-optical model for the integrated facet lens-rhabdomere system of fly eyes is used to calculate the effective light power in the rhabdomeres when the eye is illuminated with a point light source or with an extended source. Two rhabdomere types are considered: the slender rhabdomeres of R7,8 photoreceptors and the wider, but tapering R1-6 rhabdomeres. The angular sensitivities of the two rhabdomere types have been calculated as a function of F-number and wavelength by fitting Gaussian functions to the effective light power. For a given F-number, the angular sensitivity broadens with wavelength for the slender rhabdomeres, but it stays approximately constant for the wider rhabdomeres. The integrated effective light power increases with the rhabdomere diameter, but it is for both rhabdomere types nearly independent of the light wavelength and F-number. The results are used to interpret the small F-number of Drosophila facet lenses. Presumably the small head puts a limit to the size of the facet lens and favors a short focal length.     

Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster

A Drosophila mutant (ninaAP228) that is low in rhodopsin concentration but identical to the wild-type fly in photoreceptor morphology has been isolated. R1-6 photoreceptors of the mutant differ from those of wild type in that (a) the prolonged depolarizing afterpotential (PDA) is absent, (b) concentrations of rhodopsin and opsin are substantially reduced, and (c) intramembrane particle density in the membranes of the rhabdomeres is low. Each of these traits is mimicked by depriving wild- type flies of vitamin A. The ninaAP228 mutation differs from vitamin A deprivation in that in the mutant (a) the rhabdomeric membrane particle density is reduced only in the R1-6 photoreceptors and not in R7 or R8, (b) the PDA can be elicited from the R7 photoreceptors, and (c) photoconversion of R1-6 rhodopsin to metarhodopsin by ultraviolet (UV) light is considerably more efficient than in vitamin A-deprived flies. The absorption properties of the mutant rhodopsin in the R1-6 photoreceptors appear to be identical to those of wild type as judged from rhodopsin difference spectra. The results suggest that the mutation affects the opsin, rather than the chromophore, component of rhodopsin molecules in the R1-6 photoreceptors. The interaction between the chromophore and R1-6 opsin, however, appears to be normal.     

Characterization of the primary photointermediates of Drosophila rhodopsin

Invertebrate opsins are unique among the visual pigments because the light-activated conformation, metarhodopsin, is stable following exposure to light in vivo. Recovery of the light-activated pigment to the dark conformation (or resting state) occurs either thermally or photochemically. There is no evidence to suggest that the chromophore becomes detached from the protein during any stage in the formation or recovery processes. Biochemical and structural studies of invertebrate opsins have been limited by the inability to express and purify rhodopsins for structure-function studies. In this study, we used Drosophila to produce an epitope-tagged opsin, Rh1-1D4, in quantities suitable for spectroscopic and photochemical characterization. When expressed in Drosophila, Rh1-1D4 is localized to the rhabdomere membranes, has the same spectral properties in vivo as wild-type Rh1, and activates the phototransduction cascade in a normal manner. Purified Rh1-1D4 visual pigment has an absorption maximum of the dark-adapted state of 474 nm, while the metarhodopsin absorption maximum is 572 nm. However, the metarhodopsin state is not stable as purified in dodecyl maltoside but decays with kinetics that require a double-exponential fit having lifetimes of 280 and 2700 s. We investigated the primary properties of the pigment at low temperature. At 70 K, the pigment undergoes a temperature-induced red shift to 486 nm. Upon illumination with 435 nm light, a photostationary state mixture is formed consisting of bathorhodopsin (lambda(max) = 545 nm) and isorhodopsin (lambda(max) = 462 nm). We also compared the spectroscopic and photochemical properties of this pigment with other vertebrate pigments. We conclude that the binding site of Drosophila rhodopsin is similar to that of bovine rhodopsin and is characterized by a protonated Schiff base chromophore stabilized via a single negatively charged counterion.     

An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila.

We have used two techniques to isolate and characterize eye-specific genes from Drosophila melanogaster. First, we identified genes whose expression is limited to eyes, photoreceptor cells, or R7 photoreceptor cells by differential screening with [32P]cDNAs derived from the heads of mutant flies that have reduced amounts of these tissues and cells (Microcephalus, glass3, and sevenless, respectively). Secondly, we identified opsin genes by hybridization with synthetic [32P]oligonucleotides that encode domains that have been conserved between some opsin genes. We found seven clones that contain genes expressed only in the eye or optic lobes of Drosophila; three are expressed only in photoreceptor cells. One is expressed only in R7 photoreceptor cells and hybridizes to some of the previously mentioned oligonucleotides. The complete DNA sequence of the R7-specific opsin gene and its 5' and 3' flanking regions was determined. It is quite different from other known Drosophila opsin genes, in that it is not interrupted by introns and shares only 37-38% amino acid identity with the proteins encoded by these genes. The predicted protein structure contains many characteristics that are common to all rhodopsins, and the sequence differences help to identify four domains of the rhodopsin molecule that have been conserved in evolution.     

Isolation and structure of a rhodopsin gene from D. melanogaster

Using a novel method for detecting cross-homologous nucleic acid sequences we have isolated the gene coding for the major rhodopsin of Drosophila melanogaster and mapped it to chromosomal region 92B8-11. Comparison of cDNA and genomic DNA sequences indicates that the gene is divided into five exons. The amino acid sequence deduced from the nucleotide sequence is 373 residues long, and the polypeptide chain contains seven hydrophobic segments that appear to correspond to the seven transmembrane segments characteristic of other rhodopsins. Three regions of Drosophila rhodopsin are highly conserved with the corresponding domains of bovine rhodopsin, suggesting an important role for these polypeptide regions.     

The color-vision circuit in the medulla of Drosophila

BACKGROUND: Color vision requires comparison between photoreceptors that are sensitive to different wavelengths of light. In Drosophila, this is achieved by the inner photoreceptors (R7 and R8) that contain different rhodopsins. Two types of comparisons can occur in fly color vision: between the R7 (UV sensitive) and R8 (blue- or green sensitive) photoreceptor cells within one ommatidium (unit eye) or between different ommatidia that contain spectrally distinct inner photoreceptors. Photoreceptors project to the optic lobes: R1-R6, which are involved in motion detection, project to the lamina, whereas R7 and R8 reach deeper in the medulla. This paper analyzes the neural network underlying color vision into the medulla. RESULTS: We reconstruct the neural network in the medulla, focusing on neurons likely to be involved in processing color vision. We identify the full complement of neurons in the medulla, including second-order neurons that contact both R7 and R8 from a single ommatidium, or contact R7 and/or R8 from different ommatidia. We also examine third-order neurons and local neurons that likely modulate information from second-order neurons. Finally, we present highly specific tools that will allow us to functionally manipulate the network and test both activity and behavior. CONCLUSIONS: This precise characterization of the medulla circuitry will allow us to understand how color vision is processed in the optic lobe of Drosophila, providing a paradigm for more complex systems in vertebrates.     

Substitution of Pro206 and Ser86 residues in the retinal binding pocket of Anabaena sensory rhodopsin is not sufficient for proton pumping function

Anabaena sensory rhodopsin is a seven transmembrane protein that uses all-trans/13-cis retinal as a chromophore. About 22 residues in the retinal-binding pocket of microbial rhodopsins are conserved and important to control the quality of absorbing light and the function of ion transport or sensory transduction. The absorption maximum is 550 nm in the presence of all-trans retinal at dark. Here, we mutated Pro206 to Glu or Asp, of which the residue is conserved as Asp among all other microbial rhodopsins, and the absorption maximum and pKa of the proton acceptor group were measured by absorption spectroscopy at various pHs. Anabaena rhodopsin was expressed best in Escherichia coli in the absence of extra leader sequence when exogenous all-trans retinal was added. The wild-type Anabaena rhodopsin showed small absorption maximum changes between pH 4 and 11. In addition, Pro206Asp showed 46 nm blue-shift at pH 7.0. Pro206Glu or Asp may change the contribution to the electron distribution of the retinal that is involved in the major role of color tuning for this pigment. The critical residue Ser86 (Asp 96 position in bacteriorhodopsin: proton donor) for the pumping activity was replaced with Asp, but it did not change the proton pumping activity of Anabaena rhodopsin.     

Calcium-activated Myosin V closes the Drosophila pupil

Approximately 40 years ago, an elegant automatic-gain control was revealed in compound eye photoreceptors: In bright light, an assembly of small pigment granules migrates to the cytoplasmic face of the photosensitive membrane organelle, the rhabdomere, where they attenuate waveguide propagation along the rhabdomere. This migration results in a "longitudinal pupil" that reduces rhodopsin exposure by a factor of 0.8 log units. Light-induced elevation of cytosolic free Ca(2+) triggers the migration of pigment granules, and pigment granules fail to migrate in a mutant deficient in photoactivated TRP calcium channels. However, the mechanism that moves photoreceptor pigment granules remains elusive. Are the granules actively pulled toward the rhabdomere upon light, or are they instead actively pulled into the cytoplasm in the absence of light? Here we show that Ca(2+)-activated Myosin V (MyoV) pulls pigment granules to the rhabdomere. Thus, one of MyoV's several functions is also as a sensory-adaptation motor. In vitro, Ca(2+) both activates and inhibits MyoV motility; in vivo, its role is undetermined. This first demonstration of an in vivo role for Ca(2+) in MyoV activity shows that in Drosophila photoreceptors, Ca(2+) stimulates MyoV motility.     

Angular and spectral sensitivity of fly photoreceptors. III. Dependence on the pupil mechanism in the blowfly<Emphasis Type="Italic"> Calliphora</Emphasis>

A wave optics model for the facet lens-rhabdomere system of fly eyes is used to analyze the dependence of the angular and spectral sensitivity of R1–6 photoreceptors on the pupil mechanism. This assembly of light-absorbing pigment granules in the soma interacts with the waveguide modes propagating in the rhabdomere. A fly rhabdomere carries two modes in the middle wavelength range and four modes at short wavelengths, depending on the rhabdomere diameter and the angle of the incident light flux. The extension of the mode to outside the rhabdomere strongly depends on wavelength, and this dependence plays a determinant role in the light control function of the pupil. The absorbance spectrum of the pigment in the pupil granules is severely depressed at short wavelengths by waveguide effects, resulting in a distinct blue peak. Accordingly, pupil closure suppresses the photoreceptors spectral sensitivity much more in the blue-green than in the UV. The pupil only narrows the angular sensitivity at short wavelengths. The geometrical size of the rhabdomere governs the angular sensitivity of fly photoreceptors in the dark-adapted state, but diffraction takes over in the fully light-adapted state.     

Ectoplasm, ghost in the R cell machine?

Drosophila photoreceptors (R cells) are an extreme instance of sensory membrane amplification via apical microvilli, a widely deployed and deeply conserved operation of polarized epithelial cells. Developmental rotation of R cell apices aligns rhabdomere microvilli across the optical axis and enables enormous membrane expansion in a new, proximal distal dimension. R cell ectoplasm, the specialized cortical cytoplasm abutting the rhabdomere is likewise enormously amplified. Ectoplasm is dominated by the actin-rich terminal web, a conserved operational domain of the ancient vesicle-transport motor, Myosin V. R cells harness Myosin V to move two distinct cargoes, the biosynthetic traffic that builds the rhabdomere during development, and the migration of pigment granules that mediates the adaptive "longitudinal pupil" in adults, using two distinct Rab proteins. Ectoplasm further shapes a distinct cortical endosome compartment, the subrhabdomeral cisterna (SRC), vital to normal cell function. Reticulon, a protein that promotes endomembrane curvature, marks the SRC. R cell visual arrestin 2 (Arr2) is predominantly cytoplasmic in dark-adapted photoreceptors but on illumination it translocates to the rhabdomere, where it quenches ongoing photosignaling by binding to activated metarhodopsin. Arr2 translocation is "powered" by diffusion; a motor is not required to move Arr2 and ectoplasm does not obstruct its rapid diffusion to the rhabdomere.     

Regulation of R7 and R8 differentiation by the spalt genes

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt was previously proposed to promote R7 and R8 terminal differentiation. Here we show that spalt is required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta0.5, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. We show that spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. We demonstrate that spalt and senseless are part of a genetic network, which regulates rhodopsin 6 and rhodopsin 1. Taken together, our results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression.