Rhodopsin activation causes retinal degeneration in drosophila rdgC mutant

Neuron,

Vol. 4, 883-890,

June, 1990, Copyright

0 1990 by Cell Press

Rhodopsin Activation Causes Retinal Degeneration In Drkophila rdgC Mutant Fintan Steele* and Joseph E. O’Tousa Department of Biological Sciences University of Notre Dame Notre Dame, Indiana 46556

Summary Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgCinduced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the C protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation. Introduction Drosophila is a promising experimental system to investigate the molecular components responsible for photoreceptor function. The phototransduction pathway, in which rhodopsin activation leads to membrane depolarization, is known to require the phospholipase C activity coded by the norpA gene (Bloomquist et al., 1988). Other genes already identified by mutations affecting photoreceptor physiology (Pak, 1979) are currently being analyzed at the molecular level to determine the nature of the encoded gene product (e.g., Monte11 and Rubin, 1988; Sheih et al., 1989). Many additional photoreceptor genes have been identified by molecular techniques, including protein kinase C (Schaeffer et al., 1989) and arrestin (Smith et al., 1990). The isolation and study of mutants in these genes can define the in vivo role of these gene products in photoreceptor physiology. The genetic causes of retinal degeneration can also be investigated in Drosophila. Characterizing Drosophila retinal degeneration mutants has the potential to contribute to the description of basic photoreceptor metabolism as well as to provide a new approach toward understanding the inherited retinal degeneration diseases seen in human populations. Some mutants that show retinal degeneration have already been identified in Drosophila (Harris and Stark,

*Present address: LRCMB, tutes of Health, Bethesda,

National Maryland

Eye Institute, 20892.

National

Insti-

1977). The first of these, rdgA, shows aln abnormal photoreceptor morphology at or near rthe time of eclosion (Matsumoto et al., 1988). Rhodopsin activity is not required for the expression of the rdgA mutant phenotype (Harris and Stark, 1977; Stark and Carlson, 1985). In contrast, the rate of degeneration in mutants of a second gene, rdgB, is enhanced by rhodopsin activity, and genetic evidence suggests that the rdgB gene product interacts with the norpA-encoded phospholipase C (Harris and Stark, 1977; Stark let al., 1983). Retinal degeneration is seen in certain phospholipase C mutants (Meyertholen et al., 1987) and in mutants of other loci originally identified by phototransduction defects (Chen and Stark 1983; W. L. Pak, personal communication). Thus, genes identified by mutants exhibiting retinal degeneration may encode components of light-activated cascades. This report concerns the characterization of a novel Drosophila gene, rdgC, involved in retinal degeneration. We show that retinal degeneration in rdgC mutants is a consequence of light stimulation of rhodopsin, but does not require a functional phospholipase C gene. At young ages, prior to the onset of retinal degeneration, mutant photoreceptors show normal lightinduced photoreceptor responses. Thus, the rdgC+ function is required in a metabolic process that results from rhodopsin stimulation, but is not needed in the phospholipase C-activated transduction pathway.

Results Mutants Show Age- and light-dependent Degeneration Photoreceptors of rdgC mutants are phenotypically

rdgC

Retinal

wild type when young, but undergo age- and lightdependent degeneration. The time course of retinal degeneration is easily monitored by examining the deep pseudopupil. Figure 1 shows the deep pseudopupil structure and the corresponding histological phenotype of rdgC flies before and after degeneration. Retinal degeneration measured by the deep pseudopupil was largely complete by 5 days of age in

rdgC flies rdgC flies

reared reared

in constant light, and by 8 days in on a 12 hr light/l2 hr dark cycle. Photoreceptor degeneration did not occur in rdgC mutants reared in complete darkness (Figure IC). If mutant flies were reared in the dark for any length of time and then introduced to light, the same 5 or 8 day time course of degeneration was observed. rdgcflies maintained in constant light for up to 2 days or in 12 hr light/l2 hr dark for up to 4 days and then transferred to dark conditions failed to show degeneration. These results indicate a requirement of greater than 48 hr of light exposure to induce degeneration in rdgC mutants. A summary of an ultrastructural analysis of rdgC photoreceptor degeneration is shown in Figure 2. All

Neuron 884

2C) and begin showing signs of degeneration in 5-dayold animals (Figure 2D). The time course of retinal degeneration can also be monitored by electrophysiologicaf recordings of w; rdgC mutants. Figures 3A-3C show electroretinogram tracings from rdgP6 flies at the same age time points as those in Figure 2. The young rdgC306 mutant had a normal electroretinogram response (Figure 3A). As the mutant ages, the degradation of the electroretinogram responses (Figures 3B and 3C) parallels the morphological degeneration seen by histological analysis. There is a near normal response at 3 days, but by S days, the only response is that expected from the R7 and R8 photoreceptors. A similar recording from a rdgC mutant reared in the dark for 2 weeks (Figure 3D) shows that photoreceptors protected from light damage retain normal physiological responses. Thus, both the physiological and the histological deficits seen in rdgC photoreceptors result from degeneration triggered by light.

Figure 1. Histological Mutants

and Deep

Pseudopupil

Phenotype

of rdgC

Eye structure is shown for rdgC mutants reared in constant light at the ages of 1 day (A) and 5 days (6) and for a IO-day-old rdgC mutant reared in darkness (C). Insets show the pseudopupil phenotype of flies at the same ages. Eye structure and trapezoidal image of the pseudopupil visible in young flies and darkreared flies are degenerated in 5-day-old flies reared in constant light. Magnifications: 1512x, insets 170x.

electron micrographs are from white-eyed rdgC306 flies maintained in constant light. The l-day-old r~IgC~~6 ommatidium has wild-type structure (Figure 2A, inset is wild type). By 3 days, RI-6 photoreceptors are in the process of degeneration (Figures 2B and 2C). By 5 days, the degeneration of RI-6 photoreceptors is essentially complete (Figure 2D and inset). In contrast, the R7 and R8 photoreceptors are normal at 3 days (the R7 photoreceptor is indicated in Figure 2B; the R8 in Figure

Genetic Analysis of r&C Meiotic recombination mapping located rdgC to 46.6 on the left arm of the third chromosome. Three gamma irradiation-induced alleles, Df(3L)rdgCCS5-4, Df(3LjrdgCco2, and T(2;3)rdgCco6, that localized the rdgC locus to the polytene chromosome band region 77Bi were recovered (Figure 4). The assignment of this cytclogical location is supported by the presence of P element sequences near this site in a rdgC allele, rdgCgx, recovered from hybrid dysgenic mutagenesis (data not shown). No previously described Drosophila mutation affecting vision maps to this location. The nature of the rdgP6 mutation was investigated by comparing the phenotypes exhibited by various genotypes. First, the rdgC3@ mutation is completely recessive to wild type because the photoreceptor morphology of heterozygous rdgP6 flies is indistinguishable, even at an advanced age, from that of wild type. Second, rdgC 3oG homozygotes showed a time course of degeneration similar to that of Df(3LJrdpYrdgC306 flies, indicating that rdgC 306 is a severe loss-of-function allele. Consistent with this interpretation, all existing mutant alleles of rdgC, including T(2;3)rdgP6 homozygotes, showed degeneration time courses similar to that of rdgC306. Rhodopsin Photoactivation Is Required for rdgC Expression Since rdgC mutant expression is light-dependent, experiments were carried out to determine whether light causes degeneration in rdgC flies as a consequence of rhodopsin stimulation. First, we examined whether normal levels of retinal, the chromophore of rhodopsin, are required for rdgC-induced degeneration. Flies raised on medium deficient in vitamin A (Nichols and Pak, 1985) have rhodopsin levels reduced to <3.0% of normal (Larrivee et al., 1981). Vitamin A-deprived rdgC flies showed no signs of degeneration, even after 4 weeks of constant light exposure. Supplementing

Retinal

Degeneration

Figure

2. Ultrastructure

885

in Drosophila

of rdgC

Photoreceptors

of Flies

Maintained

in Constant

Light

(A) Transmission electron micrograph of a cross section through a l-day-old rdgCommatidium that appears essentially wild type (insert shows wild-type ommatidium). (B) A distal cross section through a 3-day-old rdgC ommatidium shows that the RI-6 photoreceptors are degenerate; the R7 cell (closed arrow) is less affected. (C) A proximal cross section through a 3-day-old rdgC ommatidium is similar to (B), but shows that the R8 cell (open arrow) is also less affected than the RI-6 cells. The soma of R7 is visible at the top of this micrograph (open arrow). (D) A distal cross section through a 5-day-old rdgC ommatidium. RI-6 are highly degenerate, and R7 appears somewhat affected at this age. The inset shows an ommatidium from another fly of this age in which RI-6 are virtually undetectable. Bar, 1 urn (A and C); 1.5 urn (B and D).

vitamin A-deficient medium with p-carotene restored the degenerative phenotype of rdgC mutants (data not shown), indicating that light-triggered rdgC degeneration is mediated via a retinal-containing compound. To determine whether the retinal-containing intermediate is rhodopsin, flies were made double mutant for r&C and ninaE, the structural gene for the RI-6 opsin (OTousa et al., 1985; Zuker et al., 1985). Two different alleles of ninaE were used in this analysis: ninaE0’77 (an internal deletion of the ninaE gene [OTousa et al., 19851) and ninaEP332 (a missense muta-

tion in the third transmembrane domain [Washburn and OTTousa, 19891 that lowers functional rhodopsin levels to *O.l% of wild type uohnson and Pak, 19861). Figure 5A is an electron micrograph of the RI and R7 cells from a rdgC306 ninaE0177 fly maintained in constant light for 3 weeks. The disarray of membrane in the area of the rhabdomere’s normal location in the RI cell is characteristic of ninaE0177 (O’Tousa et al., 1989); there is no evidence of the RI cell degeneration expected from the rdgC mutation. ninaEP33.z is a milder allele that retains regular rhabdomeric membranes (OTousa et al., 1989). The rdgC306 ninaEP332 double

Neuron 886

Figure tants

A. 1 day rdgC

of

rdgC

MU-

Electroretinograms are shown for rdgCmutants maintained in constant light for 1 day (A), 3 days (B), and 5 days (C) and in the dark for 14 days (D). The response at I day is typical of wild type, but is gradually lost as the fly ages. A normal response is seen in Wday-old flies maintained in the dark. The electroretinogram protocol consisted of unattenuated ultraviolet, orange, and blue stimuli.

B. 3 day rdgC 1

3. Electrophysiology

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C. 5 day rdgC

D. 14 day rdgC

reared

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Dp (3:2)FM27 1-1

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1-1

Df(3L)rdgCCS5-4

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T(2:3)rdgCco6

A Figure

4. Genetic

and

Cytological

Mapping

of rdgC

(A) A summary of the mapping information on rdgC. The top panel shows the meiotic recombination (genetic) map position of rdgC relative to that of other third chromosome Drosophila genes used in mapping experiments. The middle panel shows the banding pattern of the proximal third of chromosome 3L seen in larval polytene chromosomes. Lines are drawn from the micrograph to a schematic diagram of the chromosomal region containing rdgC in the lower panel. This diagram summarizes the chromosomal rearrangements used to map rdgC and allows the cytological position of the rdgC gene to be assigned to 77Bl (arrows). (B) Polytene chromosome preparation of a T(2:3)rdgP heterozygote. The translocation break, and position of rdgC, is marked by an arrow. (C) Polytene chromosome preparation of a Dff3L)rdgC c0z heterozygote; an arrowhead marks the assigned position of rdgC. All micrographs are magnified 1350x.

Retinal a87

Degeneration

in Drosophila

normal levels of their respective rhodopsins in ninaE mutants, failed to degenerate by 5 days in rdgC306 ninaEo117 or rdgC3@ ninaEf332 mutants. These results indicate that the rdgC mutant phenotype is strongly expressed only in the RI-6 photoreceptors; the histological evidence of degeneration of R7 and R8 cells seen in rdgC mutants (Figure 2D) is an indirect consequence of neighboring RI-6 cell degeneration. However, if the rdgC306 ninaEP332 flies were maintained for 3 weeks under constant light, some R7 cells showed a degenerated appearance (data not shown). Thus, although the RI-6 cells are most sensitive tom the loss of rdgC gene product, this gene product is probably required in all photoreceptor cells to prevent degeneration. loss

of Phospholipase

C Fails

to

Block

rdgC Degeneration

Figure 5. Photoreceptor Structure of norpA; rdgC Double Mutants Reared in Constant Light

and rdgC ninaf

(A) Transmission electron micrograph of the R7 (arrow) and RI photoreceptors of a IO-day-old rdgC% ni~@‘~ ommatidium. The disarray of the rhabdomeric membrane of RI is characteristic of this ninaE allele. Inset shows the normal rhabdomeric reninaE”” double mutant of the same age. (B) gion of a rd@” Transmission electron micrograph of several ommatidia of a mutant maintained in con5-day-old norpA”; rdgC 306 double stant light. The RI-6 cells are degenerate; the R7 cell is less affected. Bar, 1 urn (A); 3 urn (B).

failed to show retinal degeneration even after IO days of constant light exposure, as assayed by deep pseudopupil (data not shown). The inset in Figure 5A shows the intact rhabdomeres of a lo-day-old rdgC306 ninaP3z mutant. Therefore, the results from both ninaE alleles establish that rhodopsin is required to trigger degeneration in rdgC flies. The R7 and R8 photoreceptors, although containing

mutant

The results presented above suggest that,, in the absence of the rdgC gene product activity, excitation of rhodopsin initiates metabolic activities resulting in retinal degeneration. The only enzyme known to be activated by rhodopsin in Drosophila is the phospholipase C encoded by norpA (Bloomquist et al., 1988). To determine whether the norpA gene product activity is required for rdgC-induced degeneration, we constructed rdgC flies that were also mutant for norpA and reared these under standard light conditions. We confirmed that the norpApz4 mutant shows photoreceptor degeneration in a rdgC+ background (Meyertholen et al., 1987), and so it was not informative in these experiments. However, we were able to use the norpAtEs mutant because it entirely eliminates electrophysiological activity without showing retinal degeneration (Stark et al., 1989; Wilson and Ostroy, 1987). Figure 5B is an electron micrograph of a 5-dayold norpACEs; rdgC306 fly maintained in constant light. The RI-6 cells completely degenerate, and the R7 cell shows the expected intermediate state of degeneration. These experiments show that the double mutant phenotype is indistinguishable from the rdgC single mutant phenotype. Thus, the absence of phospholipase C activity fails to block rdgC mutant expression. To investigate whether rdgC exhibited nonallelic complementation or other genetic interactions with other known Drosophila retinal degeneration mutants, we constructed rdgAH4; rdgCJo6 and rdgBKS”*; rdgC306 strains and assayed each for retinal degeneration by deep pseudopupil analysis. No interaction was evident; these strains degenerated with the same fast time course as the respective rdgAH4 and rdgBKS2” single mutants.

Discussion We have shown that rdgC mutants show photoreceptor degeneration when exposed to light as a result of rhodopsin photoactivation. Thus, it is not possible that the rdgC+ gene product is required to protect photoreceptors from free radicals or other com-

Neuron

888

pounds formed by ubiquitous photochemical reactions. Rather, the rdgC+ gene product is required to prevent photoreceptor degeneration that results from a specific metabolic activity initiated by rhodopsin stimulation. Normal levels of rhodopsin are required to induce degeneration in rdgC mutants. Degeneration can be prevented by depriving rdgC photoreceptors of either the retinal chromophore or the opsin protein. In either case, it is not necessary that rhodopsin activity be completely eliminated to prevent degeneration. Raising flies on vitamin A-deficient medium is estimated to reduce rhodopsin to less than 3.0% of normal levels (Larrivee et al., 1981). Similarly, the r~inaE~~~* mutant retains about 0.1% of active rhodopsin (Johnson and Pak, 1986). These levels of rhodopsin generate substantial transduction activity in the RI-6 photoreceptors at the light intensities used in our experiments. Therefore, the level of active rhodopsin needed to initiate the events requiring rdgC+ to prevent retinal degeneration is substantially greater than that needed to initiate the phototransduction pathway. Recent work has established some molecular details of the phototransduction cascade of invertebrates. Biochemical and physiological evidence suggests that rhodopsin stimulates a G protein, which in turn activates a phospholipase C (Baer and Saibil, 1988; Devary et al., 1987). Active phospholipase C hydrolyzes PIP2 to produce two potential secondary messenger molecules, IP3 and diacylglycerol, which are known in other systems to mediate, respectively, increases in calcium ion concentration and stimulation of protein kinase C (Nishizuka, 1988). It is not yet known how this metabolism eventually mediates the permeability change of the photoreceptor membrane, but roles for IP3, calcium, and cGMP are likely (Bacigalupo et al., 1990). The finding that norpA encodes a phospholipase C (Bloomquist et al., 1988) is strong evidence that activation of this enzyme is an essential event in the phototransduction pathway. We have presented data on norpA; rdgC mutants that suggest that the photoreceptor metabolism affected in rdgC mutants requires rhodopsin activity, but does not require the phospholipase C activity coded by norpA. It must be emphasized that this conclusion is based on our analysis of the norpAEE5 allele. This norpA allele behaves as a severe allele in physiological tests and is capable of blocking the lighttriggered degeneration seen in rdgf3 mutants (Harris and Stark, 1977; Stark et al., 1989). It is therefore a good choice for these studies. Because other severe norpA alleles show retinal degeneration in a rdgC+ background (see Results; see also Meyertholen et al., 1987; W. L. Pak, personal communication), we could not assess the effects of these alleles on rdgC-induced degeneration, leaving the possibility that the norpAEE5 result is somehow aberrant. However, other observations also suggest that rdgC degeneration is not coupled to norpA activity. In rdgC mutants reared on vitamin A-deficient medium and in rdgC; ninaEP332

mutants, the residual rhodopsin is estimated to be 3.0% and O.l%, respectively. When stimulated with bright light, the RI-6 photoreceptors give a maximal response and hence must be capable of substantial activation of the norpA enzyme. Yet, no rdgC-induced degeneration is exhibited by these flies. Thus, situations exist in which rdgC degeneration occurs when there is no norpA-encoded phospholipase C activity and in which rdgC-induced degeneration is prevented despite the activity of the norpA-encoded phospholipase C. These considerations make it iikely that the metabolism leading to degeneration in rdgC mutants requires high levels of activated rhodopsin, but not the phospholipase C enzyme known to be activated by rhodopsin via a G protein. In the following paragraphs, we discuss three classes of mechanisms capable of accounting for rdgC induced degeneration: -Light stimulation of rhodopsin initiates events that require rdgC+ but that do not involve a G protein intermediate. This group includes the possibility that rdgC+ is required for an aspect of rhodopsin cycling, such as conformational rearrangements, interactions with other proteins such as arrestin or C protein, or phosphorylationldephosphorylation. However, there is no evidence that the phototransduction pathway or rhodopsin cycling is abnormal in young rdgC mutants, With regard to rhodopsin cycling, stimuli triggering prolonged depolarizing afterpotential (see Pak, 1979) cause a net conversion of greater than 30% of the visual pigment to the stable photoproduct, metarhodopsin. Yet young rdgC flies, and older rdgC flies raised in the dark, show repeated prolonged depolarizing after potential responses, suggesting that rho-, dopsin-metarhodopsin transitions occur properly in this mutant. Also, the residual rhodopsin in ninaEP332 and vitamin A-deprived photoreceptors must undergo similar transitions, but does not trigger retinal degeneration. Other hypotheses in which rhodopsin stimulates a biochemical pathway requiring rdgC+ without using a G protein intermediate would also be included in this group.There is no precedent for rhodopsins or related receptors acting in this manner. - rdgC is required for proper functioning of the G protein involved in phospholipase C activation. Our results show that this G protein is able to trigger the transduction pathway in rdgG mutants. So, as in the case for a defect with rhodopsin, such a hypothesis needs to account for the finding that the function of the G protein in phototransduction is not impaired. - Rhodopsin stimulation activates two different bicchemical pathways via G proteins. One pathway, via phospholipase C, results in phototransduction. A second pathway has an unknown function, but does require rdgC+ to prevent retinal degeneration. This second pathway could be stimulated by the same G protein as that used in the phospholipase C pathway, by different subunits of the same G protein, or by a different G protein. There are reports of a single receptor triggering multiple pathways through the stim-

Retinal

a89

Degeneration

in Drosophila

ulation of one or more G proteins, including activation of cGMP phosphodiesterase by the transducin a subunit (Fung et al., 1981; Stryer, 1986) and phospholi-

carried (Sigma

pase AZ by transducin /3r subunits (Jelsema and Axelrod, 1987) in vertebrate photoreceptors. Other studies demonstrate the coupling of receptors to multiple effectors (Ashkenaki et al., 1987; Magnaldo et al., 1988; Schnefel et al., 1988). In Drosophila, there is evidence that light stimulation of rhodopsin, via a mechanism not requiring the norpA-encoded phospholipase C activity, initiates rhabdomeric membrane renewal (Stark et al., 1988). The mechanisms by which membrane renewal is stimulated by rhodopsin is not known. Although it is possible that rdgC is needed in this membrane renewal pathway, our histological analyses have failed to detect alterations in these processes in young fdgC mutants. This report establishes that an unknown consequence of light stimulation of rhodopsin not directly involved in phototransduction requires the rdgC+ gene product to prevent photoreceptor degeneration. As such, the Drosophila rdgC mutant provides a new approach to the study of the molecular basis of retinal diseases. There are many genetic causes of the human degenerative diseases that are grouped under the clinical term of retinitis pigmentosa (Boughman et al., 1985), including one apparently caused by a point mutation in rhodopsin (Dryja et al., 1990). rdgC is the first Drosophila mutant that shows no conspicuous physiological defect in the photoreceptors prior to the onset of degeneration. To determine the nature of the rdgC gene product, the chromosomal rearrangements and the reported P element allele rdgCgx will be used to initiate a molecular characterization of the rcfgcgene. Molecular and biochemical analyses of the rcfgC gene and gene product will help define its role in Drosophila photoreceptors and provide a means to assess whether similar gene products play roles in vertebrate photoreceptors.

Genetic Analysis of r&C The original rdgC306 allele was recovered from the ethyl methanesulfonate-treated or@ third chromosome as described by OTousa et al. (1989) and separated from ort3& by recombination. Df(3L)rdgPZ and T(2:3/rdgCco6 were isolated by treating th st in ri pp males with 5 Krad of @‘cobalt irradiation, and Df(3L)rdgcCss-4 was isolated by treating ry*% males with 5 Krad of cesium irradiation. In both cases, irradiated males were crossed to virgin rdgC306 cu females and the Fl generation was maintained in constant light for 5 days and scored by deep pseudopupil analysis for retinal degeneration. Fl males showing retinal degeneration were used in genetic crosses designed to recover the new rdgC alleles. For each allele, polytene chromosomes were examined for chromosomal aberrations by standard techniques (Lefever, 1976). Double mutants were constructed for norpAEF5, norpAPZ4, rdgAH4, rdgBKSZZ2, ninaEP332, and ninaE0’77 by standard genetic crosses. All parental mutant stocks are maintained in our laboratory or kindly supplied by either W. L. Pak, Purdue University, or W. S. Stark, University of Missouri. In all cases, the genetic constitution of stocks constructed to be mutant at two loci was tested by appropriate outcrosses and subsequent analysis of the progeny.

Experimental

Procedures

Assaying the Mutant Phenotype Retinal degeneration was routinely assayed in flies by analyzing the structure of the deep pseudopupil (Franceschini, 1972). Electroretinograms were carried out on white-eyed flies using procedures described elsewhere (e.g., Larrivee et al., 1981). Histological analysis used the following fixation and embedding protocol. Heads of ether-anesthetized flies of desired genotype and age were bisected to allow easier infiltration of fixative and immersed in 2% paraformaldehyde/2% glutaraldehyde in Drosophila Ringers (Roberts, 1986) on ice for l-2 hr. The fixed tissue was processed as follows: four 15 min washes in Ringers, postfixation for 1 hr in 2% osmium tetroxide, four 15 min washes in Ringers, a standard ethanol dehydration series, I:1 Spurr’s low viscosity embedding medium:lOO% ethanol mixture for 30 min, 3:l Spurr’s:ethanol mixture for 30 min, and 100% Spurr’s. The material was placed at 18OC with gentle agitation, and the Spurr’s was changed every 12 hr for 5 days to allow for slow infiltration of the tissue. The tissue was then embedded and prepared for light or electron microscopy by standard procedures. Vitamin A deprivation of rd@” flies was carried out by growing flies for several generations on the medium described by Nichols and Pak (1985). Vitamin A replacement therapy was

out by supplementing the same Chemical Co., St. Louis, MO).

food

with

P-carotene

Acknowledgments We are grateful to Mr. William Archer for expert assistance with the ultrastructural analysis of rdgC. We also acknowledge the assistance of Ms. Robin Goldsmith, Mr. Reginald Ho, and Ms. Carol Schmidt at various stages of the work. This work was supported by grant NEI EY06808 from the National Institutes of Health to J. E. 0. Received

August

4, 1989;

revised

March

5, 1990.

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Bacigalupo, J., Johnson, E. C., Robinson, I?, and Lisman, J. (1990). Second messengers in invertebrate phototransduction. In Transduction in Biological Systems, C. Hidalgo, E. Jaimovich, J. Bacigalupo, and J. Vergara, eds. (New York: Plenum Publishing Corp.), in press. Bloomquist, B. T., Shortridge, R. D., Schneuwly, S., Perdew, M., Montell, C., Stellar, H., Rubin, G., and Pak, W. L. (1988). Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction. Cell 54, 723-733. Boughman, J. A., Holloran, S. L., and Cohen, M. M. (1985). Genetic aspects of retinitis pigmentosa. In Retinal Degeneration: Experimental and Clinical Studies, M. M. LaVail, J. C. Hollyfield, and R. E. Anderson, eds. (New York: Alan R. Liss), pp. 3-24. Chen, D.-M., and Stark, W. S. (1983). Sensitivity and1 adaptation in the Drosophila phototransduction and photoreceptor degeneration mutants trp and rdgB. J. Insect Physiol. 29, 133-140. Devary, O., Heichal, O., Blumenfeld, A., Cassel, IX, Suss, E., Barash, S., Rubinstein, C. T., Minke, B., and Selinger, Z. (1987). Coupling of photoexcited rhodopsin to inositol phospholipid hydrolysis in fly photoreceptors. Proc. Natl. Acad. Sci. USA. 84, 6939-6943. Dryja, T. P, McGee, T. L., Reichel, E., Hahn, Yandell, D. W., Sandberg, M. A., and Berson,

L. B., Cowley, G. S., E. L. (1990). A point

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