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.     

Coexpression of spectrally distinct rhodopsins in Aedes aegypti R7 photoreceptors

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.     

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.     

Ultraviolet polarisation sensitivity in the stomatopod crustacean <Emphasis Type="Italic">Odontodactylus scyllarus</Emphasis>

The ommatidia of crustacean eyes typically contain two classes of photoreceptors with orthogonally oriented microvilli. These receptors provide the basis for two-channel polarisation vision in the blue–green spectrum. The retinae of gonodactyloid stomatopod crustaceans possess a great variety of structural specialisations for elaborate polarisation vision. One type of specialisation is found in the small, distally placed R8 cells within the two most ventral rows of the mid-band. These ultraviolet-sensitive photoreceptors produce parallel microvilli, a feature suggestive for polarisation-sensitive photoreceptors. Here, we show by means of intracellular recordings combined with dye-injections that in the gonodactyloid species Odontodactylus scyllarus, the R8 cells of mid-band rows 5 and 6 are sensitive to linear polarised ultraviolet light. We show that mid-band row 5 R8 cells respond maximally to light with an e-vector oriented parallel to the mid-band, whereas mid-band row 6 R8 cells respond maximally to light with an e-vector oriented perpendicular to the mid-band. This orthogonal arrangement of ultraviolet-sensitive receptor cells could support ultraviolet polarisation vision. R8 cells of rows 5 and 6 are known to act as quarter-wave retarders around 500 nm and thus are the first photoreceptor type described with a potential dual role in polarisation vision.     

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.     

Freeze-fracture study of the Drosophila photoreceptor membrane: mutations affecting membrane particle density

The photoreceptor membrane of Drosophila melanogaster (wild type, vitamin A-deprived wild type, and the mutants ninaAP228, ninaBP315, and oraJK84) was studied by freeze-fracture electron microscopy. The three mutations caused a decrease in the number of particles on the protoplasmic face of the rhabdomeric membrane. The ninaAP228 mutation affected only the peripheral photoreceptors (R1-6), while the ninaBP315 mutation affected both the peripheral (R1-6) and the central photoreceptors (R7). The oraJK84 mutation, which essentially eliminates R1-6 rhabdomeres, was found to drastically deplete the membrane particles in the vestigial R1-6 rhabdomeres but not in the normal rhabdomeres of R7 photoreceptors, suggesting that the failure of the oraJK84 mutant to form normal R1-6 rhabdomeres may be due to a defect in a major R1-6 photoreceptor-specific protein in the mutant. In all cases in which both the rhabdomeric particle density and rhodopsin content were studied, the mutations or vitamin A deprivation was found to reduce both these quantities, supporting the idea that at least the majority of the rhabdomeric membrane particles are closely associated with rhodopsin. Vitamin A deprivation and the mutations also reduced the number of particles in the plasma membrane as in the rhabdomeric membrane, suggesting that both classes of membrane contain rhodopsin.     

Blue and double-peaked green receptors depend on ommatidial type in the eye of the Japanese yellow swallowtail Papilio xuthus

The compound eye of the butterfly Papilio xuthus is composed of three spectrally distinct types of ommatidia. We investigated the blue and double-peaked green receptors that are encountered distally in type I and III ommatidia, by means of intracellular recordings, in vivo fluorescence microscopy, and histology. The blue receptors are R1 and/or R2 photoreceptors; they contain the same mRNA encoding the opsin of the blue-absorbing visual pigment. However, here we found that the sensitivity in the UV wavelength region strongly depends on the ommatidial type; the blue receptors in type I ommatidia have a distinctly depressed UV sensitivity, which is attributed to lateral filtering in the fused rhabdom. In the main, fronto-ventral part of the eye, the R3 and R4 photoreceptors of all ommatidia contain the same set of two mRNAs encoding the opsins of green-absorbing visual pigments, PxL1 and PxL2. The spectral sensitivities are double-peaked, but the UV sensitivity of the R3 and R4 photoreceptors in type I ommatidia appears to be reduced, similar to that of the co-localized blue receptors.     

Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.     

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.     

Microvillar orientation in the photoreceptors of the ant Cataglyphis bicolor

Polarization sensitivity in arthropod photoreceptors is crucially dependent on the arrangement of the microvilli within the rhabdom. Here, we present an electron-microscopical study in which the degree of microvillar alignment and changes in the cross-sectional areas of the rhabdoms along their length were studied in the compound eye of the desert ant, Cataglyphis bicolor. Serial cross-sections through the retina were taken and the orientation of the microvilli was determined in the photoreceptors of individually identified ommatidia. The reconstructions of microvillar alignment were made in the three anatomically and functionally distinct regions of the Cataglyphis compound eye: the dorsal rim area (DRA), the dorsal area (DA), and the ventral area (VA). The following morphological findings are consistent with polarization sensitivities measured previously by intracellular recordings. (1) The microvilli of the DRA photoreceptors are aligned in parallel along the entire length of the cell from the distal tip of the rhabdom down to its proximal end, near the basement membrane. The microvilli of the retinular cells R1 and R5 are always parallel to each other and perfectly perpendicular, with only minor deviation, to the microvillar orientation of the remaining receptor cells. (2) In the DA and VA regions of the eye, the microvillar tufts of the small receptors R1, R3, R5, R7, and R9 change their direction repetitively every 1-4 7m for up to 90°. In contrast, the large receptor cells R2, R4, R6, and R8 maintain their microvillar orientation rigidly. (3) In the DRA ommatidia, the cross-sectional areas of the rhabdomeres do not change along the length of the rhabdom, but substantial changes occur in the DA and VA ommatidia.     

Role of the EGFR/Ras/Raf pathway in specification of photoreceptor cells in the Drosophila retina

The Drosophila EGF receptor is required for differentiation of many cell types during eye development. We have used mosaic analysis with definitive null mutations to analyze the effects of complete absence of EGFR, Ras or Raf proteins during eye development. The Egfr, ras and raf genes are each found to be essential for recruitment of R1-R7 cells. In addition Egfr is autonomously required for MAP kinase activation. EGFR is not essential for R8 cell specification, either alone or redundantly with any other receptor that acts through Ras or Raf, or by activating MAP kinase. As with Egfr, loss of ras or raf perturbs the spacing and arrangement of R8 precursor cells. R8 cell spacing is not affected by loss of argos in posteriorly juxtaposed cells, which rules out a model in which EGFR acts through argos expression to position R8 specification in register between adjacent columns of ommatidia. The R8 spacing role of the EGFR was partially affected by simultaneous deletion of spitz and vein, two ligand genes, but the data suggest that EGFR activation independent of spitz and vein is also involved. The results prove that R8 photoreceptors are specified and positioned by distinct mechanisms from photoreceptors R1-R7.     

Dissection and Immunohistochemistry of Larval,Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 ². The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 ^{3 4}. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes⁵: In the ''p'' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the ''y'' subtype, Rh4 in yR7s is associated with Rh6 in yR8s ^{6 7 8}.Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc⁹ and initiates the assembly of undifferentiated cells into ommatidia^10-11. The ''founder cell'' R8 is specified first and recruits R1-6 and then R7 ^12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes ¹⁵, including rhabdomere formation, synaptogenesis and eventually rh expression.In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).     

The Components of the Visual System of a Dragonfly

In this study of the electroretinograms of dragonflies (adults and nymphs) the objectives were to determine the number of classes of photoreceptors present in the visual system and to allocate these to particular morphological regions. There are probably five classes of photoreceptors present with peak sensitivities near 550, 530, 518, 420, and < 380 mµ. The dorsal ocelli contain two classes (518 mµ and < 380 mµ). The ventral (anterior) ommatidia of the adult compound eye contain at least two classes (near 518 mµ and < 380 mµ) and probably a third class (near 550 mµ). The dorsal ommatidia of the adult compound eye contain one class (420 mµ) and possibly another class (< 380 mµ). The compound eye of the nymph contains one class (530 mµ) and possibly another class (420 mµ).     

Cell-fate determination in the developing Drosophila eye: role of the rough gene.

The homeobox-gene rough is required in photoreceptor cells R2 and R5 for normal ommatidial assembly in the developing Drosophila eye. We have used several cell-type-specific markers and double mutant combinations to analyze cell-fate determination in rough. We show that the cells that would normally become R2 and/or R5 express a marker, a lacZ insertion in the seven-up (svp) gene, which is indicative of the R1/3/4/6 cell fate. In addition, the analysis of mitotically induced svp,ro double mutant clones in the eye indicates that in rough all outer photoreceptors are under the genetic control of the svp gene. These results show that, in the absence of rough function, R2 and R5 fail to be correctly determined and appear to be transformed into cells of the R3/4/1/6 subtype. This transformation and the subsequent developmental defects do not preclude the recruitment of R7 cells. However, the presence of ommatidia containing more than one R7 and/or R8 cell in rough implies a complex network of cellular interactions underlying cell-fate determination in the Drosophila retina.     

Three distinct roles for notch in Drosophila R7 photoreceptor specification

Receptor tyrosine kinases (RTKs) and Notch (N) proteins are different types of transmembrane receptors that transduce extracellular signals and control cell fate. Here we examine cell fate specification in the Drosophila retina and ask how N acts together with the RTKs Sevenless (Sev) and the EGF receptor (DER) to specify the R7 photoreceptor. The retina is composed of many hundred ommatidia, each of which grows by recruiting surrounding, undifferentiated cells and directing them to particular fates. The R7 photoreceptor derives from a cohort of three cells that are incorporated together following specification of the R2-R5 and R8 photoreceptors. Two cells of the cohort are specified as the R1/6 photoreceptor type by DER activation. These cells then activate N in the third cell (the R7 precursor). By manipulation of N and RTK signaling in diverse combinations we establish three roles for N in specifying the R7 fate. The first role is to impose a block to photoreceptor differentiation; a block that DER activation cannot overcome. The second role, paradoxically, is to negate the first; Notch activation up-regulates Sev expression, enabling the presumptive R7 cell to receive an RTK signal from R8 that can override the block. The third role is to specify the cell as an R7 rather than an R1/6 once RTK signaling has specified the cells as a photoreceptor. We speculate why N acts both to block and to facilitate photoreceptor differentiation, and provide a model for how N and RTK signaling act combinatorially to specify the R1/6 and R7 photoreceptors as well as the surrounding non-neuronal cone cells.     

Time-resolved rhodopsin activation currents in a unicellular expression system.

The early receptor current (ERC) is the charge redistribution occurring in plasma membrane rhodopsin during light activation of photoreceptors. Both the molecular mechanism of the ERC and its relationship to rhodopsin conformational activation are unknown. To investigate whether the ERC could be a time-resolved assay of rhodopsin structure-function relationships, the distinct sensitivity of modern electrophysiological tools was employed to test for flash-activated ERC signals in cells stably expressing normal human rod opsin after regeneration with 11-cis-retinal. ERCs are similar in waveform and kinetics to those found in photoreceptors. The action spectrum of the major R(2) charge motion is consistent with a rhodopsin photopigment. The R(1) phase is not kinetically resolvable and the R(2) phase, which overlaps metarhodopsin-II formation, has a rapid risetime and complex multiexponential decay. These experiments demonstrate, for the first time, kinetically resolved electrical state transitions during activation of expressed visual pigment in a unicellular environment (single or fused giant cells) containing only 6 x 10(6)-8 x 10(7) molecules of rhodopsin. This method improves measurement sensitivity 7 to 8 orders of magnitude compared to other time-resolved techniques applied to rhodopsin to study the role particular amino acids play in conformational activation and the forces that govern those transitions.