R7 photoreceptor specification requires Notch activity

The eight photoreceptors in each ommatidium of the Drosophila eye are assembled by a process of recruitment [1,2]. First, the R8 cell is singled out, and then subsequent photoreceptors are added in pairs (R2 and R5, R3 and R4, R1 and R6) until the final R7 cell acquires a neuronal fate. R7 development requires the Sevenless receptor tyrosine kinase which is activated by a ligand from R8 [3]. Here, we report that the specification of R7 requires a second signal that activates Notch. We found that a Notch target gene is expressed in R7 shortly after recruitment. When Notch activity was reduced, the cell was misrouted to an R1/R6 fate. Conversely, when activated Notch was present in the R1/R6 cells, it caused them to adopt R7 fates or, occasionally, cone cell fates. In this context, Notch activity appears to act co-operatively, rather than antagonistically, with the receptor tyrosine kinase/Ras pathway in R7 photoreceptor specification. We propose two models: a ratchet model in which Notch would allow cells to remain competent to respond to sequential rounds of Ras signalling, and a combinatorial model in which Notch and Ras signalling would act together to regulate genes that determine cell fate.     

Sensitivity and photopigments of R1-6, a two-peaked photoreceptor,inDrosophila, Calliphora andMusca

Summary Low vitamin A rearing decreases sensitivity and eliminates the ultraviolet but not the blue sensitivity maximum in R1-6 inDrosophila, Calliphora andMusca (Figs. 2–4). Spectral adaptation functions for control and vitamin A deprived flies yielded derived stable metarhodopsin absorption spectra from spectral sensitivity. Metarhodopsin has a long wavelength maximum and also has an ultraviolet maximum especially in the normal vitamin A condition (Figs. 2–4). M-potentials (fast early-receptor-like potentials) were obtained (Fig. 1) from all three genera in normal vitamin A rearing and were used for spectral adaptation studies (Figs. 2–3); the latter data are approximate inverses of sensitivity based spectral adaptation data. Thus, sensitivity must reflect proportion of rhodopsin, with metarhodopsin being inert in receptor potential generation.Vitamin A effects on spectral functions were further investigated inDrosophila. Ultraviolet (370 nm) and visible (470 nm) sensitivities varied approximately linearly with dietary vitamin A dose (Fig. 5); 370 nm sensitivity decreased more than 470 nm sensitivity at lower doses. Increasing adaptation intensities of 370 and 470 nm caused parallel decreases in spectral sensitivity assayed at 370 and 470 nm in normal vitamin A flies (Fig. 6); the adapting intensities were sufficient to convert photopigment. These and previous results suggest that the two R1-6 spectral peaks are ultimately mediated by one rhodopsin. R1-6 rhabdomeres were structurally similar in high and low vitamin A flies but emitted a long wavelength fluorescence to ultraviolet excitation in high vitamin A flies only (Fig. 7). These results suggest some form of energy transfer; i.e., a carotenoid may capture ultraviolet quanta and transfer energy to rhodopsin via inductive resonance. Spectral adaptation data are consistent with a calculated high rhabdomeric optical density of ECL=0.26 (i.e., 45% of incident light is absorbed) derived from presently available data onDrosophila. Calculations show electro-retinographic sensitivity to be extremely high, perhaps measurable at less than one absorbed quantum per rhabdomere.Supported by NSF grants BMS-74-12817 and BNS-76-11921. We thank M. Chapin, K. Hu, D. Lakin, G. Pransky, D. Sawyer and W. Zitzmann for technical assistance. We are indebted to numerous colleagues especially W. Harris, for comments and suggestions.Chalky Calliphora were obtained from the laboratories of Dr. G. McCann at Caltech and Dr. L. Bishop at the University of Southern California.W-II Musca were from Dr. D. Wagoner at the U.S.D.A. in Fargo, North Dakota.     

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.     

Cell-autonomous and -nonautonomous functions of LAR in R7 photoreceptor axon targeting.

During Drosophila visual system development, photoreceptors R7 and R8 project axons to targets in distinct layers of the optic lobe. We show here that the LAR receptor tyrosine phosphatase is required in the eye for correct targeting of R7 axons. In LAR mutants, R7 axons initially project to their correct target layer, but then retract to the R8 target layer. This targeting defect can be fully rescued by transgenic expression of LAR in R7, and partially rescued by expression of LAR in R8. The phosphatase domains of LAR are required for its activity in R7, but not in R8. These data suggest that LAR can act both as a receptor in R7, and as a ligand provided by R8. Genetic interactions implicate both Enabled and Trio in LAR signal transduction.     

Signal transduction and the fate of the R7 photoreceptor in Drosophila.

The sevenless protein tyrosine kinase receptor plays a central role in the pathway of cell fate induction that determines the development of the R7 photoreceptor in the Drosophila eye. In the last year we have learned much about the probable ligand for sevenless and have begun to dissect the signal transduction pathway that relays the information from the sevenless kinase. Studies of the mechanisms governing the specificity of signal transmission and reception suggest that the sevenless signal directs a bipotential cell towards a neuronal rather than a cone cell fate.     

Sensitivity changes and facilitation in the photoreceptor of phalangium opilio due to light adaptation. Preliminary notes

These preliminary notes were made on sensitivity changes and facilitation in the photoreceptor of phalangium opilio, due to light adaptation. They show that facilitation is a case opposite to light adaptation. Other measurements are planned in the progress of this work.     

Postlarval chromatophores as an adaptation to ultraviolet radiation

It is now well established that ultraviolet radiation (UVR) may have detrimental, even lethal effects on zooplankters. Unlike copepods and other holoplankters, which may avoid UVR by undergoing diel vertical migration, larvae of many decapod crustaceans and fishes recruit to adult populations by remaining in near-surface waters during the daytime. Consequently, they are exposed to biologically damaging UVR. A possible adaptation in these larvae is chromatophores, which may absorb UVR by expanding in high light environments. The supposition is that expanded chromatophores more effectively absorb UVR, but there is some fitness cost to having expanded chromatophores in low light environments. Since the ratio of visible light to UVR in the water column changes as result of season, latitude, dissolved organic carbon, and a host of other factors, the benefits of chromatophores would be maximized if they responded specifically to UVR. The purpose of this study was to determine whether the chromatophores of crab postlarvae (megalopae) could expand in response to UVR. Megalopae of two species of crabs (Cancer oregonensis, Telmessus cheiragonus) were collected from large surface-swarms during mid-day as they recruited onshore in early May 1998 at Friday Harbor, Washington, USA. Dark-adapted megalopae (held in the dark for 8 h before experiments) were exposed to UVR (UVBR+UVAR, 280-400 nm), UVAR (320-400 nm), and light (400-1700 nm) in the laboratory. Chromatophores expanded after only minutes of exposure to UVR, UVAR, and light for both species. Two alternative hypotheses may explain why both harmful and comparatively benign wavelengths stimulated chromatophores to rapidly expand. First, larvae may not distinguish among different wavelengths, which, if true, would increase the vulnerability of these larvae to intensifying UVBR due to ozone depletion. Second, chromatophores have functions other than blocking UVR, such as crypsis and thermoregulation, and must respond to light for these other functions to operate.     

Signal transduction and the fate of the R7 photoreceptor in Drosophila

The sevenless protein tyrosine kinase receptor plays a central role in the pathway of cell fate induction that determines the development of the R7 photoreceptor in the Drosophila eye. In the last year we have learned much about the probable ligand for sevenless and have begun to dissect the signal transduction pathway that relays the information from the sevenless kinase. Studies of the mechanisms governing the specificity of signal transmission and reception suggest that the sevenless signal directs a bipotential cell towards a neuronal rather than a cone cell fate.     

Sildenafil,N-desmethyl-sildenafil and Zaprinast enhance photoreceptor response in the isolated rat retina

Here we report that the active component of Viagra, Sildenafil and the first metabolite, N-desmethyl-sildenafil (UK-103, 320) increased the amplitude of flash-evoked electroretinogram (ERG) of dark-adapted albino rat retina. Effects of Sildenafil and N-desmethyl-sildenafil were comparable to those of the known phosphodiesterase inhibitor, Zaprinast. The photoreceptor cell response was isolated by blocking the glial K(+) ion-buffering and the on-bipolar components of the ERG with the use of BaCl(2) (500 microM) and the specific type VI metabotropic glutamate receptor agonist, DL-2-amino-4-phosphonobutyric acid (25 microM), respectively. Zaprinast, Sildenafil and N-desmethyl-sildenafil (1 microM each) increased the amplitude of photoreceptor cell response either. Besides, Sildenafil was significantly more effective than N-desmethyl-sildenafil. These findings suggest an increased sensitivity of photoreceptor cells in the presence of Sildenafil and it is metabolite.     

An ultraviolet photoreceptor in a Dipteran compound eye

Summary Intracellular recordings of the retinula cell receptor potential to flashes of monochromatic light demonstrate the presence of ultraviolet photoreceptors in the retina of the honeybee-mimic dronefly (Diptera, Syrphidae,Eristalis). Photoreceptors for visible light were also found. Hence, the dronefly has the neural capability for color vision. This is consistent with behavioral studies. Color vision would aid this animal in its natural habit of forraging with the honeybee.Supported by grants AFOSR-71-2112 and NSF GB-30733.     

Transplantation of photoreceptor precursor cells into the retina of an adult Drosophila

Blindness caused by the disconnection between photoreceptor cells and the brain can be cured by restoring this connection through the transplantation of retinal precursor neurons. However, even after transplanting these cells, it is still unclear how to guide the axons over the long distance from the retina to the brain. To establish a method of guiding the axons of transplanted neurons, we used the Drosophila visual system. By testing different conditions, including the dissociation and preincubation length, we have successfully established a method to transplant photoreceptor precursor cells isolated from the developing eye discs of third‐instar larvae into the adult retina. Moreover, we overexpressed N‐cadherin (CadN) in the transplant, since it is known to be broadly expressed in the optic lobe well after developmental stages, continuing through adult stages. We found that promoting the cell adhesive properties using CadN enhances the axonal length of the grafted photoreceptor neurons and therefore is useful for future transplantation. We tested the overexpression of a CadN::Frazzled chimeric receptor and found that there was no difference in axonal length from our wild‐type transplants, suggesting that the intracellular domain of CadN is necessary for axonal elongation. Altogether, using the Drosophila visual system, we have established an excellent platform for exploring the molecules required for proper axon extension of transplanted neuronal cells. Future studies building from this platform will be useful for regenerative therapy of the human nervous system based on transplantation.     

Sensitivity and adaptation in the Drosophila phototransduction and photoreceptor degeneration mutants trp and rdgB

We studied rdgB, a retinal degeneration mutant, and trp, a phototransduction mutant, separately and in combination in Drosophila. First we showed that trp did not block degeneration in white-eyed rdgB mutants. Thus, rdgB was useful in determining the defects which trp caused in the compound eye receptors R7 and R8; this is because rdgB selectively eliminates R1-6 photoreceptors which would, if present, dominate the compound eye responses. R7 and R8 both express the trptransient receptor potential phenotype in trp mutants. The trp mutation does not change receptor spectral sensitivities, nor does it alter the dark stability of R1-6's and R7's metarhodopsins as judged by dark adaptation studies. The dark adaptation is not significantly affected by trp. However, trp slows the dark adaptation of R8 considerably and seems to make the blue-induced inactivation of R1-6 less stable.     

In situ cGMP phosphodiesterase and photoreceptor potential in gecko retina

The possible involvement of phosphodiesterase (PDE) activation in phototransduction was investigated in gecko photoreceptors by comparing the in situ PDE activity with the photoreceptor potential. In the dark, intracellular injection of cGMP into a gecko photoreceptor caused a long-lasting depolarization. An intense light flash given during the depolarization phase repolarized the cell with a short latency comparable to that of the light-evoked hyperpolarizing response, which indicates that the activation of PDE in situ is rapid enough to generate the photoreceptor potential. PDE activity in situ was estimated quantitatively from the duration of the cGMP-induced depolarization, since it was expected that the higher the PDE activity, the shorter the duration. Under steady illumination, the enzyme exhibited a constant activity. On exposure to a light flash, PDE became activated, but recovered in the dark with a time course that was dependent on the intensity of the preceding stimulus. When PDE activity and photoreceptor sensitivity to light were measured in the same cell after a light flash, both recovery processes showed similar kinetics. Theoretical analysis showed that the parallelism in the recovery time courses could be explained if cGMP is the transduction messenger. These results suggest that PDE activation is involved not only in the generation but also in the adaptation mechanisms of the photoreceptor potential.     

Visual pigment and photoreceptor sensitivity in the isolated skate retina

Photoreceptor potentials were recorded extracellularly from the aspartate-treated, isolated retina of the skate (Raja oscellata and R. erinacea), and the effects of externally applied retinal were studied both electrophysiologically and spectrophotometrically. In the absence of applied retinal, strong light adaptation leads to an irreversible depletion of rhodopsin and a sustained elevation of receptor threshold. For example, after the bleaching of 60% of the rhodopsin initially present in dark-adapted receptors, the threshold of the receptor response stabilizes at a level about 3 log units above the dark-adapted value. The application of 11-cis retinal to strongly light-adapted photoreceptors induces both a rapid, substantial lowering of receptor threshold and a shift of the entire intensity-response curve toward greater sensitivity. Exogenous 11-cis retinal also promotes the formation of rhodopsin in bleached photoreceptors with a time-course similar to that of the sensitization measured electrophysiologically. All-trans and 13-cis retinal, when applied to strongly light-adapted receptors, fail to promote either an increase in receptor sensitivity or the formation of significant amounts of light-sensitive pigment within the receptors. However, 9-cis retinal isin. These findings provide strong evidence that the regeneration of visual pigment in the photoreceptors directly regulates the process of photochemical dark adaptation.     

Light exposure causes functional changes in the retina: increased photoreceptor cation channel permeability, photoreceptor apoptosis, and altered retinal metabolic function

Light exposure induces retinal photoreceptor degeneration and retinal remodeling in both the normal rat retina and in animal models of retinal degeneration. Although cation entry is one of the triggers leading to apoptosis, it is unclear if this event occurs in isolation, or whether a number of pathways lead to photoreceptor apoptosis following light exposure. Following light exposure, we investigated the characteristics of cation entry, apoptotic markers [using terminal deoxynucleotidyl transferase (EC 2.7.7.31) dUTP nick-end labeling (TUNEL) labeling] and metabolic properties of retina from Sprague-Dawley (SD) rats and a rat model of retinitis pigmentosa [proline-23-histidine (P23H) rat]. Assessment of cation channel permeability using agmatine (AGB) labeling showed that excessive cation gating accompanied the series of anomalies that occur prior to photoreceptor loss. Increased AGB labeling in photoreceptors was seen in parallel with the appearance of apoptotic photoreceptors detected by TUNEL labeling with only a smaller proportion of cells colocalizing both markers. However, SD and P23H retinal photoreceptors differed in the amounts and colocalization of AGB gating and TUNEL labeling as a function of light exposure. Finally, reduced retinal lactate dehydrogenase levels were found in SD and P23H rat retinas after a 24-h light exposure period. Short-term (2 h) exposure of the P23H rat retina caused an increase in lactate dehydrogenase activity suggesting increased metabolic demand. These results suggest that energy availability may be exacerbated during the early stages of light exposure in susceptible retinas. Also, the concomitant observation of increased ion gating and TUNEL labeling suggest the existence of at least two possible mechanisms in light-damaged retinas in both SD and the P23H rat retina.     

Dopaminergic interplexiform cells contact photoreceptor terminals in catfish retina

Summary Dopaminergic interplexiform cells in retinae of glass catfish were investigated using an antiserum against tyrosine hydroxylase and peroxidase-anti-peroxidase (PAP) visualization. In whole-mount preparations, we observed a homogeneous distribution of cell bodies throughout the retina without any indication of regional specializations. At the ultrastructural level, we studied the morphology of labelled telodendria within the outer plexiform layer. Apart from contacts with horizontal cells and bipolar cell dendrites, we report for the first time direct contacts with cone pepdicles and rod spherules. Quantitative evaluation of short series of sections showed that all cone pedicles, and a major part of the rod terminals, were approached in this way. The dopaminergic pathway terminating on photoreceptors is discussed in the context of pharmacological effects of this transmitter in the distal retina during light adaptation, i.e., cone contraction, spinule formation and horizontal cell coupling.     

The pigment system of the photoreceptor 7 yellow in the fly,a complex photoreceptor

Summary The 7y photoreceptor in the fly (Musca, Calliphora) retina harbours an unusually complex pigment system consisting of a bistable visual pigment (xanthopsin, X and metaxanthopsin, M), a blue-absorbing C₄₀-carotenoid (zeaxanthin and/or lutein) and a uv sensitizing pigment (3-OH retinol).The difference spectrum and photoequilibrium spectrum in single 7y rhabdomeres were determined microspectrophotometrically (Fig. 2).The extinction spectrum of the C₄₀-carotenoid has a pronounced vibrational structure, with peaks at 430, 450 and 480 nm (Fig. 3). The off-axis spectral sensitivity, determined electrophysiologically with 1 nm resolution shows no trace of this fine structure thus excluding the possibility that the C₄₀-carotenoid is a second sensitizing pigment (Fig. 4).The absorption spectra of X and M are derived by fitting nomogram spectra (based on fly R1–6 xanthopsin) to the difference spectrum. max for X is 425 nm, and for M 510 nm (Fig. 5). It is shown that the photoequilibrium spectrum and the difference spectrum can be used to derive the relative photosensitivity spectra of X and M using the analytical method developed by Stavenga (1975). The result (Fig. 6) shows a pronounced uv sensitivity for both, X and M, indicating that the uv sensitizing pigment transfers energy to both X and M. A value of 0.7 for, the relative efficiency of photoconversion for X and M, is obtained by fitting the analytically derived relative photosensitivity spectra to the absorption spectra at wavelengths beyond 420 nm.