The sevenless signaling pathway: variations of a common theme

Many developmental processes are regulated by intercellular signaling mechanisms that employ the activation of receptor tyrosine kinases. One model system that has been particular useful in determining the role of receptor tyrosine kinase-mediated signaling processes in cell fate determination is the developing Drosophila eye. The specification of the R7 photoreceptor cell in each ommatidium of the developing Drosophila eye is dependent on activation of the Sevenless receptor tyrosine kinase. This review will focus on the genetic and biochemical approaches that have identified signaling molecules acting downstream of the Sevenless receptor tyrosine kinase which ultimately trigger differentiation of the R7 photoreceptor cell.     

Induction in the developing compound eye of Drosophila: multiple mechanisms restrict R7 induction to a single retinal precursor cell.

The development of the Drosophila R7 photoreceptor cell is determined by a specific inductive interaction between the R8 photoreceptor cell and a single neighboring precursor cell. This process is mediated by bride of sevenless (boss), a cell-surface bound ligand, and the sevenless (sev) tyrosine kinase receptor. The boss ligand is expressed specifically on the surface of the R8 cell, whereas the sev receptor is expressed on 5 cells contacting the developing R8 cell and other cells not in contact with R8. By altering the spatial and temporal expression of boss, we demonstrate that sev-expressing cells that do not contact R8 can assume an R7 cell fate. By contrast, the sev-expressing precursor cells to the R1-R6 photoreceptor cells that do contact R8 are nonresponsive to the inductive cue. Using the rough and Nspl mutations, we demonstrate that an early commitment to an R1-R6 cell fate blocks the pathway of sev activation in these cells.     

Photoconvertible pigment states and excitation in Calliphora; the induction and properties of the prolonged depolarising afterpotential

1. The proposed models of two independent groups, which relate the different states of the visual pigment to the excitation of the membrane in invertebrate photoreceptors (with particular reference to the prolonged depolarising afterpotential, the PDA) are compared and evaluated. 2. The validity of the late receptor potential (the "normal" receptor response) as an index of photoreceptor sensitivity, i.e., an index of the number of rhodopsin to metarhodopsin transitions, is verified by concurrent spectrophotometry. 3. Electrophysiological observations alone allow the calculation of 1.3 x 10(8) photopigment molecules in the rhabdom of an R1-6 photoreceptor of a vitamin A-bred Calliphora. 4. The PDA is shown to be quantifiable in terms of the number of rhodopsin to metarhodopsin conversions by the absorption of single light quanta. 5. The comparison of discrete membrane fluctuations (quantum bumps) during the PDA and during exposure to sustained light stimuli that mimic the PDA suggest that, the PDA, similar to the late receptor potential, may be due to the summation of quantum bumps.     

Photoreceptors for biosynthesis, energy storage and vision

Abstract Living organisms use light as a source of energy and as a means of obtaining information about their environment. Photoreactivating enzyme, provitamins D, retinal (rhodopsins and bacteriorhodopsin), porphyrins (chlorophyll, protochlorophyll and heme), photosynthetic accessory pigments (carotenoids and bilins), phytochrome and riboflavin: these are the molecules which life has settled upon to play the role of light receptor. For some of these photoreceptor molecules a great deal is now known about the chemistry which they perform upon absorbing light; for others virtually nothing is known. Riboflavin, the molecule believed to be functioning in a variety of organisms as the receptor for physiological responses to blue light, is an especially interesting case. Its widespread occurrence in cellular roles other than photoreception make it difficult to separate out the particular flavin which functions as the photoreceptor. It represents a case of a photoreceptor which is at once ubiquitous and elusive.     

Rhodopsin Receptors of Phototaxis in Green Flagellate Algae

Green flagellate algae are capable of the active adjustment of their swimming path according to the light direction (phototaxis). This direction is detected by a special photoreceptor apparatus consisting of the photoreceptor membrane and eyespot. Receptor photoexcitation in green flagellates triggers a cascade of rapid electrical events in the cell membrane which plays a crucial role in the signal transduction chain of phototaxis and the photophobic response. The photoreceptor current is the earliest so far detectable process in this cascade. Measurement of the photoreceptor current is at present the most suitable approach to investigation of the photoreceptor pigment in green flagellate algae, since a low receptor concentration in the cell makes application of optical and biochemical methods so far impossible. A set of physiological evidences shows that the phototaxis receptor in green flagellate algae is a unique rhodopsin-type protein. It shares common chromophore properties with retinal proteins from archaea. However, the involvement of photoelectric processes in the signal transduction chain relates it to animal visual rhodopsins. The presence of some enzymatic components of the animal visual cascade in isolated eyespot preparations might also point to this relation. A retinal-binding protein has been identified in such preparations, the amino acid sequence of which shows a certain homology to sequences of animal visual rhodopsins. However, potential function of this protein as the phototaxis receptor has been questioned in recent time.     

Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes

The compound eye of D. melanogaster is a reiterative pattern of facets, each containing eight photoreceptor cells in a precise arrangement. This pattern is established in the eye imaginal disc during the third larval instar. A wave of morphogenesis sweeps from posterior to anterior across the disc, leaving in its wake organized clusters of photoreceptor cells. We have used monoclonal antibodies to highlight pattern elements that are not readily observable by other techniques. Monoclonal antibodies can be used to identify the molecules associated with particular patterns, providing links between observable structures and the genes. As an example, we present the purification and N-terminal sequence of a glycoprotein antigen specific to photoreceptor cells and their axons.     

Asymmetric localization of frizzled and the determination of notch-dependent cell fate in the Drosophila eye

BACKGROUND: During patterning of the Drosophila eye, a critical step is the Notch-mediated cell fate decision that determines the identities of the R3/R4 photoreceptor pair in each ommatidium. Depending on the decision taken, the ommatidium adopts either the dorsal or ventral chiral form. This decision is directed by the activity of the planar polarity genes, and, in particular, higher activity of the receptor Frizzled confers R3 fate. RESULTS: We present evidence that Frizzled does not modulate Notch activity via Rho GTPases and a JNK cascade as previously proposed. We find that the planar polarity proteins Frizzled, Dishevelled, Flamingo, and Strabismus adopt asymmetric protein localizations in the developing photoreceptors. These protein localizations correlate with the bias of Notch activity between R3/R4, suggesting that they are necessary to modulate Notch activity between these cells. Additional data support a mechanism for regulation of Notch activity that could involve direct interactions between Dishevelled and Notch at the cell cortex. CONCLUSIONS: In the light of our findings, we conclude that Rho GTPases/JNK cascades are not major effectors of planar polarity in the Drosophila eye. We propose a new model for the control of R3/R4 photoreceptor fate by Frizzled, whereby asymmetric protein localization is likely to be a critical step in modulation of Notch activity. This modulation may occur via direct interactions between Notch and Dishevelled.     

Small-angle neutron and X-ray scattering analysis of the supramolecular organization of rhodopsin in photoreceptor membrane

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering.     

semang affects the development of a subset of cells in the Drosophila compound eye

semang (sag), a mutation isolated as a suppressor of Drosophila Src42A, has previously been shown to affect some receptor tyrosine kinase mediated embryonic processes. Here we show that sag specifically affects the development of R1, R6 and R7 photoreceptor cells in a cell-autonomous manner. These cells are absent in the mutant at the time when they normally appear in the ommatidial pre-clusters. Genetic analyses suggest that sag functions downstream of, or parallel to, Mapk and Yan in the photoreceptor differentiation pathway. The autonomous requirement of sag for R1/R6/R7 development could be explained by a selective impairment of the late, but not early, rounds of Egfr-induced precursor cell assembly by the sag mutations. Egfr signaling is highly regulated by autocrine or paracrine mechanisms in different cells. Knowing that the photoreceptor cluster formation is a complex process involving dynamic changes in cell-cell contact, our hypothesis is that the sag alleles affected certain special aspects of Egfr-signaling that are unique for the recruitment of R1/R6/R7 cells.     

Activation and molecular recognition of the GPCR rhodopsin - insights from time-resolved fluorescence depolarisation and single molecule experiments

The cytoplasmic surface of the G-protein coupled receptor (GPCR) rhodopsin is a key element in membrane receptor activation, molecular recognition by signalling molecules, and receptor deactivation. Understanding of the coupling between conformational changes in the intramembrane domain and the membrane-exposed surface of the photoreceptor rhodopsin is crucial for the elucidation of the molecular mechanism in GPCR activation. As little is known about protein dynamics, particularly the conformational dynamics of the cytoplasmic surface elements on the nanoseconds timescale, we utilised time-resolved fluorescence anisotropy experiments and site-directed fluorescence labelling to provide information on both, conformational space and motion. We summarise our recent advances in understanding rhodopsin dynamics and function using time-resolved fluorescence depolarisation and single molecule fluorescence experiments, with particular focus on the amphipathic helix 8, lying parallel to the cytoplasmic membrane surface and connecting transmembrane helix 7 with the long C-terminal tail.     

The presence of two populations of sensory-type cells in the pineal organ of the five-bearded rockling,Ciliata mustela L. (Teleostei)

The pineal organ of the five-bearded rockling, Ciliata mustela L., was examined by means of electron microscopy. Two categories of sensory cells are described: 1) Sensory cells 1 (or photoreceptor cells sensu stricto) showing the characteristic ultrastructure of photoreceptor cells with a well-developed receptor pole (outer segment) and a transmitter pole (ribbon-type synapse in the basal pedicle contacting dendritic processes), and a segmental organization of organelles. 2) Sensory cells 2 (or photoneuroendocrine cells) displaying no particular segmentation. The ultrastructure of the receptor pole (outer segment) is variable in shape (with either long or short disks) and in the number of disks; some outer segments are simple cilia of the 9 + 0 type. This second cell category is rich in smooth endoplasmic reticulum, beta-particles of glycogen, dense inclusions of variable size and content, and dense-core vesicles 130 nm in diameter. These cells have an extended contact area with the perivascular space. The functional significance of both cell categories is discussed in terms of the known physiological responses of the pineal organ. A possible confusion in identification of interstitial cells and neuroendocrine cells in some teleost species is discussed.     

Decoding vectorial information from a gradient: sequential roles of the receptors Frizzled and Notch in establishing planar polarity in the Drosophila eye

The Drosophila eye is composed of several hundred ommatidia that can exist in either of two chiral forms, depending on position: ommatidia in the dorsal half of the eye adopt one chiral form, whereas ommatidia in the ventral half adopt the other. Chirality appears to be specified by a polarizing signal with a high activity at the interface between the two halves (the 'equator'), which declines in opposite directions towards the dorsal and ventral poles. Here, using genetic mosaics, we show that this polarizing signal is decoded by the sequential use of two receptor systems. The first depends on the seven-transmembrane receptor Frizzled (Fz) and distinguishes between the two members of the R3/R4 pair of presumptive photoreceptor cells, predisposing the cell that is located closer to the equator and having higher Fz activity towards the R3 photoreceptor fate and the cell further away towards the R4 fate. This bias is then amplified by subsequent interactions between the two cells mediated by the receptor Notch (N) and its ligand Delta (Dl), ensuring that the equatorial cell becomes the R3 photoreceptor while the polar cell becomes the R4 photoreceptor. As a consequence of this reciprocal cell fate decision, the R4 cell moves asymmetrically relative to the R3 cell, initiating the appropriate chiral pattern of the remaining cells of the ommatidium.     

Localization of DER and the pattern of cell divisions in wild-type and Ellipse eye imaginal discs.

The compound eye of Drosophila develops from a uniform layer of epithelial cells in the eye imaginal disc. One intriguing aspect of eye development is the establishment of the correct number and spacing of the photoreceptor clusters which give rise to the mature ommatidia. Ellipse (Elp) has been implicated as playing a role in this process because the Elp dominant gain of function mutation dramatically reduces the number of photoreceptor clusters in the compound eye without affecting the morphology of individual clusters that are formed (Baker and Rubin, 1989). Since Elp represents an allele of the Drosophila EGF receptor (DER) locus, it encodes a protein which is structurally capable of mediating inductive cell-cell interactions. In an effort to better understand the role of the DER locus in ommatidial patterning, we compared the localization of DER protein in eye imaginal discs of wild-type and Elp larvae. The distribution of this receptor is consistent with the notion of its mediating interactions between cells at the initial stages of photoreceptor precluster positioning and differentiation. However, the basis of the Elp gain of function mutation is not ectopic or increased expression of the DER protein. Rather, expression of the Elp form of the EGF receptor homolog in the normal localization leads to changes in the proliferative pattern of cells dividing posterior to the morphogenetic furrow.     

Multiple Mechanisms of Photoreceptor Spectral Tuning in Heliconius Butterflies

The evolution of color vision is often studied through the lens of receptor gain relative to an ancestor with fewer spectral classes of photoreceptor. For instance, in Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. However, color vision evolution is not well understood in the context of loss. In Heliconius melpomene and Heliconius ismenius lineages, the UV2 receptor subtype has been lost, which limits female color vision in shorter wavelengths. Here, we compare the visual systems of butterflies that have either retained or lost the UV2 photoreceptor using intracellular recordings, ATAC-seq, and antibody staining. We identify several ways these butterflies modulate their color vision. In H. melpomene, chromatin reorganization has downregulated an otherwise intact UVRh2 gene, whereas in H. ismenius, pseudogenization has led to the truncation of UVRh2. In species that lack the UV2 receptor, the peak sensitivity of the remaining UV1 photoreceptor cell is shifted to longer wavelengths. Across Heliconius, we identify the widespread use of filtering pigments and co-expression of two opsins in the same photoreceptor cells. Multiple mechanisms of spectral tuning, including the molecular evolution of blue opsins, have led to the divergence of receptor sensitivities between species. The diversity of photoreceptor and ommatidial subtypes between species suggests that Heliconius visual systems are under varying selection pressures for color discrimination. Modulating the wavelengths of peak sensitivities of both the blue- and remaining UV-sensitive photoreceptor cells suggests that Heliconius species may have compensated for UV receptor loss.     

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.     

Atypical Granules in the Eyes of the White Mutant of Drosophila melanogaster Are Lysosome-Related Organelles

In the pigment cells of the white mutant of Drosophila melanogaster, as described earlier, two types of abnormal granules are found by conventional electron microscopy. However, both types of abnormal granules, in addition to those in pigment cell invaginations, are also present in the cytoplasm of the photoreceptor cells. Three enzymes (acid phosphatase, peroxidase, and tyrosinase) are localized within the eyes of wild type and white mutant Drosophila melanogaster by electron microscopy. Peroxidase activity is present in lamellar bodies close to the rhabdomeral microvilli of both fly types. However the organelles containing peroxidase activity are 6-fold more frequent in the wild type than in the mutant. Acid phosphatase is present in lamellar bodies between and at the bases of the rhabdomeral microvilli of the wild type, as well as in ommochrome granules of the photoreceptor cells. In the white mutant, however, acid phosphatase was located in electron lucent vacuoles in the cytoplasm of the receptor cells. These acid phosphatase-positive vacuoles also contained both types of abnormal granules. The latter result indicates that abnormal granules in the receptor cells originate from lysosomal degradation and that targeting of lysosomal enzymes is altered in the white mutant. Due to the tyrosinase activity in the hemolymph of flies, the extracellular spaces are electron dense after DOPA incubation. Since some abnormal granules within the photoreceptor cells are not surrounded by an extracellular space, they can be assumed to originate within the photoreceptor cells.     

Figure-ground discrimination by relative movement in the visual system of the fly

The visual system of the fly performs various computations on photoreceptor outputs. The detection and measurement of movement is based on simple nonlinear multiplication-like interactions between adjacent pairs and groups of photoreceptors. The position of a small contrasted object against a uniform background is measured, at least in part, by (formally) 1-input nonlinear flicker detectors. A fly can also detect and discriminate a figure that moves relative to a ground texture. This computation of relative movement relies on a more complex algorithm, one which detects discontinuities in the movement field. The experiments described in this paper indicate that the outputs of neighbouring movement detectors interact in a multiplication-like fashion and then in turn inhibit locally the flicker detectors. The following main characteristic properties (partly a direct consequence of the algorithm's structure) have been established experimentally: a) Coherent motion of figure and ground inhibit the position detectors whereas incoherent motion fails to produce inhibition near the edges of the moving figure (provided the textures of figure and ground are similar). b) The movement detectors underlying this particular computation are direction-insensitive at input frequencies (at the photoreceptor level) above 2.3 Hz. They become increasingly direction-sensitive for lower input frequencies. c) At higher input frequencies the fly cannot discriminate an object against a texture oscillating at the same frequency and amplitude at 0° and 180° phase, whereas 90° or 270° phase shift between figure and ground oscillations yields maximum discrimination. d) Under conditions of coherent movement, strong spatial incoherence is detected by the same mechanism. The algorithm underlying the relative movement computation is further discussed as an example of a coherence measuring process, operating on the outputs of an array of movement detectors. Possible neural correlates are also mentioned.     

Regulation of synaptic frequency: comparison of the effects of hypoinnervation with those of hyperinnervation in the fly's compound eye

At the anterior rim of the first optic neuropile, or lamina, of the housefly's (Musca domestica) compound eye, the terminals of photoreceptors (R) innervate postsynaptic neurons in variable numbers to provide a continuous range of natural hypo- and hyperinnervations. Frequencies of photoreceptor synapses have been measured from quantitative electron microscopy on single sections of the lamina's unit synaptic modules, called cartridges. These are normally innervated by six photoreceptor terminals (6R cartridges). At the lamina's edge hypoinnervated cartridges (2R-5R) are found, whereas hyperinnervated cartridges (7R, 8R) are located at the equator between dorsal and ventral eye halves. In 2R cartridges each presynaptic terminal forms up to 1.5 times the normal, 6R cartridge number of synapses, thereby offsetting the reduced number of terminals and partially conserving the input upon the postsynaptic neurons. Thus the terminals have a reserve synaptogenic capacity never normally revealed. By comparison, terminals in 8R cartridges form about the same numbers of synapses as in "normal" eye regions, so that their postsynaptic neurons have a synaptic input increased by the extra number of terminals. The number of synapses formed between input terminals and target neurons is therefore not fixed but changes as a function of the total receptor terminal complement. The size of a photoreceptor terminal covaries to a certain extent with the number of its presynaptic sites; the spacing density of presynaptic sites over the terminals' surface in a 2R cartridge compared with an 8R cartridge increases far less (only 17%) than the increase in the number of sites (43%). The pair of postsynaptic cell interneurons in each 2R cartridge also shows a decrease in axonal diameter compared with those in 8R cartridges. Thus both the pre- and postsynaptic cells show size changes correlated with changes in their synaptic engagement.     

Localization of the sevenless protein, a putative receptor for positional information, in the eye imaginal disc of Drosophila

The Drosophila gene sevenless encodes a putative trans-membrane receptor required for the formation of one particular cell, the R7 photoreceptor, in each ommatidium of the compound eye. Mutations in this gene result in the cell normally destined to form the R7 cell forming a non-neuronal cell type instead. These observations have led to the proposal that the sevenless protein receives at least part of the positional information required for the R7 developmental pathway. We have generated antibodies specific for sevenless and have examined expression of the protein by light and electron microscopy. sevenless protein is present transiently at high levels in at least 9 cells in each developing ommatidium and is detectable several hours before any overt differentiation of R7. The protein is mostly localized at the apices of the cells, in microvilli, but is also found deeper in the tissue where certain cells contact the R8 cell. This finding suggests that R8 expresses a ligand for the sevenless protein.     

Different structural organization of the encephalopsin gene in man and mouse

Encephalopsin, also called Panopsin, is a recently discovered extraretinal photoreceptor, which may play a role in non-visual photic processes such as the entrainment of circadian rhythm or the regulation of pineal melatonin production. Based on RT-PCR data and comparative genomic sequence analysis, we show that the human OPN3 gene consists of six exons and expresses various splice variants, while the murine homologue contains four exons and produces just one splice form. Furthermore, the human OPN3 gene overlaps with the neighboring KMO gene on a genomic as well as on an RNA level, whereas the corresponding genes in mouse lie close together but do not overlap. This finding is of particular interest, since differences in gene organization between man and mouse, that have been reported so far, occur within gene clusters, i.e. the number of genes within a certain cluster may differ between man and mouse. OPN3 provides an exception to this rule, since it is positionally uncoupled from other genes of the opsin family.