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Published March 1, 1977 Hereditary Retinal Degeneration in Drosophila melanogaster A Mutant Defect Associated with the Phototransduction Process WILLIAM A. HARRIS and WILLIAM S. STARK From the Division of Biology, California Institute of Technology, Pasadena, California 91125 and the Department of Psychology, The Johns Hopkins University, Baltimore, Maryland Downloaded from jgp.rupress.org on May 6, 2011 21218. Dr. Harris's present address is the Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115. AB S T R AC T Two genes in Drosophila, rdgA and rdgB, which when defective cause retinal degeneration, were discovered by Hotta and Benzer (Hotta, Y., and S. Benzer. 1970. Proc. Natl. Acad. Sci. U. S. A. 67:1156-1163). These mutants have photoreceptor cells that are histologically normal upon eclosion but subsequently degenerate. The defects in the rdgA and rdgB mutants were localized by the study of genetic mosaics to the photoreceptor cells. In rdgB mutants retinal degeneration is light induced. It can be prevented by rearing the flies in the dark or by blocking the receptor potential with a no-receptor-potential mutation, norpA. Vitamin A depri- vation and genetic elimination of the lysosomal enzyme acid phosphatase also protect the photoreceptors of rdgB flies against light-induced damage. The photo- pigment kinetics of dark-reared rdgB flies appear normal in vitro by spectrophoto- metric measurements, and in vivo by measurements of the M potential. In normal Drosophila, a 1-s exposure to intense 470-nm light produces a prolonged depolariz- ing afterpotential (PDA) which can last for several hours. In dark-reared rdgB mutants the PDA lasts less than 2 min; it appears to initiate the degeneration process, since the photoreceptors become permanently unresponsive after a single such exposure. Another mutant was isolated which prevents degeneration in rdgB flies but which has a normal receptor potential. This suppressor of degeneration is an allele of norpA. It is proposed that the normal norpA gene codes for a product which, when activated, leads to the receptor potential, and which is inactivated by the product of the normal rdgB gene. INTRODUCTION By screening chemically m u t a g e n i z e d Drosophila melanogaster for deficits in visual behavior, H o t t a and Benzer (1970), Pak et al. (1969), Pak et al. (1970), a n d H e i s e n b e r g (1971) isolated m a n y X - c h r o m o s o m a l mutants with altered electrore- tinograms (ERGs). Histological examination revealed that some o f these mutants suffer f r o m severe retinal d e g e n e r a t i o n (Hotta a n d Benzer, 1970; Heisenberg, 1971). All these retinal d e g e n e r a t i o n mutants fall into two c o m p l e m e n t a t i o n T H E J O U R N A L OF GENERAL PHYSIOLOGY ' VOLUME 6 9 , 1977 " pages 261-291 261 Published March 1, 1977 262 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69 ' 1977 groups, rdgA and rdgB. Hotta and Benzer (1970) showed that in mosaic flies with some parts genetically normal and some genetically mutant, only the eye tissue is relevant for the expression of retinal degeneration, i.e. the rdgA and rdgB defects are autonomous to the eye. In this paper, we have extended Hotta and Benzer's (1970) mosaic analysis to show that the photoreceptor cells themselves are primarily responsible for these mutant defects. Conditions that accelerate, decelerate, or prevent hereditary retinal degenera- tion can offer clues to the mechanism. Dowling and Sidman (1962) found that if pink-eyed retinal degeneration mutant rats were reared in the dark, the time course of hereditary retinal degeneration was slowed. Yates et al. (1974) and LaVail and Battelle (1975) found that black eye pigmentation mimicked the dark-rearing effect. The action of light on the disease suggests that rhodopsin metabolism may be involved. Vitamin A deprivation causes retinal degeneration in mammals (Dowling and Wald, 1960) and this effect is prevented in rats by Downloaded from jgp.rupress.org on May 6, 2011 raising them in the dark (Noell et al., 1971). Furthermore, vitamin A deprivation protects against the rat retinal degeneration that is caused by strong light (Noell and Albrecht, 1971). In this paper we examine conditions, including dark rearing and vitamin A deprivation, which are protective in Drosophila retinal degeneration mutants. This enables one to control precisely the onset of the degeneration process so that early physiological defects can be studied. Secondary mutations are also described which prevent retinal degeneration in rdgB flies. A brief introduction to some of the anatomy, physiology, and photochemistry of the Drosophila retina should aid in the interpretation of the experiments and results presented here. The Drosophila compound eye consists of approximately 800 ommatidia each of which contains eight photoreceptor cells of three mor- phologically and physiologically distinct classes. The six peripheral photorecep- tors, R1-6, in each ommatidium are blue and UV sensitive, (see Fig. 7), and contain a rhodopsin which absorbs maximally at about 470 nm with a secondary maximum in the UV, and which interconverts with a metarhodopsin absorbing maximally at about 570 nm (Pak and Liddington, 1974; Ostroy et al., 1974; Stark, 1975; Harris et al., 1976). The rhabdomeres of the central two photoreceptors, R7 and R8, are stacked on top of one another, and are, respectively, UV sensitive and blue sensitive (see Fig. 7); (Harris et al., 1976). R7, the distal central photoreceptor, contains a rhodopsin which absorbs maximally at about 370 nm and which interconverts with a metarhodopsin absorbing maximally at about 470 nm. R8, the proximal central photoreceptor, has a third photopigment (Harris et al., 1976). Maximal rhodopsin to metarhodopsin conversion (caused in R1-6 for example by bright 470 nm adaptation, and in R7 by 370 nm adaptation) produces a long-lived depolarization and inactivation in these cells which contin- ues even after the termination o f the stimulus (Minke et al., 1975a; Stark, 1975; Harris et al., 1976; Stark et al., 1976). The long-lived depolarization in inverte- brate photoreceptors, first discovered in Limulus median ocellus (Nolte et al., 1968) and well characterized in the barnacle (Hochstein et al., 1973) has been called the prolonged depolarizing afterpotential (PDA) (Minke et al., 1973). In the dark, metarhodopsin reconverts slowly to rhodopsin in flies (Stavenga et al., 1973; Pak and Liddington, 1974), allowing PDA decay and resensitization (Minke Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 263 et al., 1975a); d a r k r e c o n v e r s i o n m a y not o c c u r in all invertebrates (Minke et al., 1973). A m u c h m o r e r a p i d t e r m i n a t i o n o f the PDA a n d resensitization is accom- plished by p h o t o c o n v e r s i o n o f m e t a r h o d o p s i n to r h o d o p s i n (caused in R1-6, for e x a m p l e , by 570 n m a d a p t a t i o n ) ( H o c h s t e i n , et al., 1973; Pak a n d L i d d i n g t o n , 1974; Minke et al., 1975a). I n Drosophila R1-6 cells, s y n c h r o n o u s p h o t o c o n v e r - sion o f substantial a m o u n t s o f m e t a r h o d o p s i n to r h o d o p s i n is a c c o m p a n i e d by s o m e fast electrical potentials, collectively called the M potential, which can be r e c o r d e d in the E R G (Pak a n d L i d d i n g t o n , 1974). T h e basic m e c h a n i s m o f excitation in p h o t o r e c e p t o r cells is incompletely u n d e r s t o o d . Between p h o t o n c a p t u r e by r h o d o p s i n a n d g e n e r a t i o n o f the r e c e p t o r potential t h e r e m a y be m a n y i n t e r m e d i a t e steps (Fuortes a n d H o d g k i n , 1964; Baylor et al., 1974). Drosophila m u t a n t s in which the r e c e p t o r potential is blocked or altered m a y h a v e defects in these i n t e r m e d i a t e steps (Minke et al., 1975b; Pak, 1975). Downloaded from jgp.rupress.org on May 6, 2011 For instance, m u t a n t s o f the n o - r e c e p t o r - p o t e n t i a l A (norpA) g e n e are defi- cient in an excitation step s u b s e q u e n t to q u a n t u m catch (Alawi et al., 1972; Pak a n d L i d d i n g t o n , 1974; Ostroy et al., 1974). T h e e x p e r i m e n t s p r e s e n t e d h e r e indicate that the rdgB defect is associated with a step in the p h o t o t r a n s d u c t i o n process s u b s e q u e n t to p h o t o p i g m e n t action a n d yet not c o n s e q u e n t to the r e c e p t o r potential. F r o m these studies with m u t a n t s , we suggest bow the n o r m a l rdgB a n d norpA g e n e p r o d u c t s m a y be involved as i n t e r m e d i a t e s in the photo- t r a n s d u c t i o n process. MATERIALS AND METHODS Stocks Normal flies were from the wild-type Canton-S strain. The rdgA, rdgB, and norpA ~E5 mutants, the multiply marked y cho cv sn 3 X chromsosome, and the unstable ring-X In(1)wvc were from the collection of Seymour Benzer at the California Institute of Technology. The ora :Ks4 andJK910 mutants were from John Merriam at the University of California, Los Angeles. The acid phosphatase null mutant, Acph-1 "n, was from Ross MacIntyre at Cornell University. sd~ g w a s f r o m P. T. Ires at Amherst College. w, cn bw, and Df(1)g I were from Ed Lewis at the California Institute of Technology. Df(1)KA14 and Df(1)RA2 were from George Lefevre, California State University at Northridge. Several of these mutants were combined to study their interaction or to eliminate screening pigments from the eye. The following stocks were constructed by standard genetic techniques: (a) y w rdgAeC47; (b). w sn 8 rdgAnSt2; (c) w rdgBXS~2; (d) y cho rdgBXSm; ( e ) y cho rdgB xs~22,Acph-1,11; ( f ) rdgB xs~22 , ora :x~; ( g ) w norpA ~Es; ( h ) norpA xes rdgB t~s222, cn bw; (i) norpA BE5 rdgBt~°4s; 0") rdgBXS222; JK910. The single mutation w (white) and the double mutation cn bw (cinnabar brown) are equally effective at eliminating screening pigments from the eye while not interfering with the functioning of the photoreceptor cells (Alawi et al., 1972). Since cn and bw are located on the second chromosome while w and most of the visual mutants are on the first, it was often easier to use cn bw than w in the construction of white-eyed multiple mutants. Flies were raised at 25°C on standard yellow cornmeal medium (Lewis, 1960) in a 12 h: 12 h light-dark cycle unless otherwise stated. Isolation o f Suppressor M u t a t i o n s To find X-linked suppressors of degeneration rdgB xs222 and rdgA K°I4 males were muta- Published March 1, 1977 264 THE JOURNAL OF G E N E R A L P H Y S I O L O G Y • VOLUME 69 • 1977 genized with ethyl methane sulfonate according to the protocol of Lewis and Bacher (1968) and mated to virgin females having attached X chromosomes marked with yellow and forked (X~, y f ) . 5-day old male progeny were checked for retinal degeneration by the pseudopupil technique (see below). Those that showed no degeneration were pair mated to X-'X, y f virgin females, and the male progeny tested by the same method. Suppressors were kept as stocks. One o f these, found to be allelic to norpA and designated norpA ~tI, was combined with various other mutations to produce the following stocks: (a) norp A sun rdgB XS~22; ( b ) nor# A *uu, cn bw ; ( c ) norp A ~n rdgB xs222 , cn bw ; ( d ) norp A suu rdg B K°45. Examination o f the Eye in L i v i n g Animals A technique devised by Kirschfeld and Franceschini (1968) allows analysis of the photore- ceptor optics in living flies. T h e pseudopupil is formed by the superposition of the images of the r h a b d o m e r e tips from several neighboring ommatidia. It was observed by placing the fly on a glass slide and illuminating the head from below with a narrow beam of intense light, while focusing just below the surface o f the eye with about ×20 magnifica- Downloaded from jgp.rupress.org on May 6, 2011 tion in a c o m p o u n d microscope. Alternatively, individual r h a b d o m e r e s were examined directly, without sectioning the eye, by the technique o f optical neutralization of the cornea (Franceschini and Kirschfeld, 1971). In this case, the head of the fly to be examined was cut off at the neck with a razor blade, m o u n t e d on a glass slide with clear nail polish, and examined u n d e r oil at about x400 magnification. Histology For light and electron microscopy, heads of flies were cut off, sliced midsagittaily, and fixed immediately by the techniques of Poodry and Schneiderman (1970). T h e y were then e m b e d d e d in Epon-Araldite mixture. 1.5-/~m sections for light microscopy were collected on a glass slide and stained with toluidine blue. Thin sections of about 1,200 /~ were picked up on copper grids, and stained with lead citrate (Reynolds, 1963). Production o f Mosaics T h e first method was to use males carrying the retinal degeneration mutation o f interest linked to recessive eye and body color mutations (y, yellow body color, and cho, chocolate eye color). These were mated to females heterozygous for the unstable ring-X chromo- some In(I)w ve which contains d o m i n a n t normal alleles o f the genes for retinal degenera- tion, body color, and eye color. Approximately 7% of the progeny of such crosses were haplo-X diplo-X g y n a n d r o m o r p h s in which the mutations were expressed in the hemizy- gous male tissue but not in the heterozygous female tissue (see Hotta and Benzer, 1970). T h e second method was to X-ray female first and second instar larvae heterozygous for the white eye color and retinal degeneration mutations to induce somatic crossing over (Stern, 1936). T h e dose used was 1,200 rad, 325 rad/min, 50 kV, 20 mA, 13 cm from two 1- m m A1 filters to target. In this way, small patches of homozygous mutant tissue were produced in a background of heterozygous normal tissue. Stimulation a n d Recording These methods were similar to Stark's (1975). Monochromatic stimuli were from a 150 W xenon arc (Hanovia 901C) with a Bausch & Lomb 500-mm m o n o c h r o m a t o r (Bausch & Lomb, Inc., Roche"~tser, N.Y.). Achromatic optics were used to focus the light onto the specimen, and the intensity was adjusted with Inconnel-on-glass neutral density filters (Bausch & Lomb 31-34-38 series). Energy calibrations at the locus of the preparation were made with a calibrated United Detector Technology PIN-10 photodiode (United Detector Technology Inc., Santa Monica, Calif.). Electroretinograms were recorded DC by use of Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 265 a Medistor (A-35) or ELSA-4 electrometer with saturated NaCl-filled microelectrodes inserted through the cornea. Responses were displayed on a Tektronix (5100 series) oscilloscope (Tektronix, Inc., Beverton, Ore.) and a Physiograph DMP-4B r e c o r d e r and p h o t o g r a p h e d on a Grass C4R camera (Grass I n s t r u m e n t Co., Quincy, Mass.). Spectral sensitivities were d e t e r m i n e d as in Harris et al. (1976). Intense flashes o f white light for generating the M potential were from a Vivitar (152) camera flash attachment. Intense 10- s adaptation conditioning flashes o f 10~7-10TM quanta/cm 2, unless otherwise stated, were followed by approximately 1 min o f d a r k before data were collected. Spectrophotometry Samples were obtained from d a r k - r e a r e d w and w rdgB~s222flies by placing approximately 100 flies in a small glass bottle which was then d i p p e d in liquid nitrogen for 1 min. Vigorous shaking o f the bottle decapitated the frozen flies. Nylon mesh filters were used to separate the heads from the bodies. T h e heads were then homogenized in - 0 . 6 ml of 0.1 M phosphate buffer, p H 7.2, and the homogenate was then placed in a cuvette for Downloaded from jgp.rupress.org on May 6, 2011 spectrophotometry at room t e m p e r a t u r e . A dual wavelength spectrophotometer con- structed by Dr. Edward Lipson at the California Institute of Technology and described in Harris et al. (1976) was used to measure light-induced absorption changes o f the Drosoph- ila photopigments. Vitamin A Deprivation Drosophila were vitamin A deprived by raising sterilized eggs aseptically on Sang's syn- thetic diet, m e d i u m C (Doane, 1967). For vitamin A-enriched m e d i u m , fl-carotene (Nutritional Biochemicals Corp. 101287) was a d d e d to a final concentration of 125 mg/100 ml. See Stark and Zitzmann (1976) for details. ATPase Assay 100 retinas each were dissected from cold-anesthetized w and w rdgBxs222 d a r k - r e a r e d flies, kept overnight at 4°C, then homogenized in 200 tzl o f reaction buffer. For total or ouabain-sensitive ATPase determination, 25 /zl of homogenates were a d d e d to 75 tzi of buffer at 30°C, and the reaction was started with 10 izl o f 25 mM s2P-T-ATP (New England Nuclear, Boston, Mass.). T h e reaction was terminated after 30 min by addition of 50 Izl of ice-cold 20% T C A . When ouabain was present, its final concentration was 2 × 10-4 M. Determination o f inorganic 3zp was by the method o f Fahn et al. (1968). Specific activity was d e t e r m i n e d after assaying for protein (Lowry et al., 1951). RESULTS Mutants MAPPING G e n e t i c m a p p i n g o f t h e rdgA g e n e p l a c e s it at p o s i t i o n 26.3 +-- 1.2 o n t h e X c h r o m o s o m e ; t h e r e c e s s i v e rdgA is u n c o v e r e d by t h e s m a l l d e l e t i o n Df(1)KA14, w h i c h s p a n s s a l i v a r y c h r o m o s o m e r e g i o n 7 F 1 . 2 - 8 C 6 , b u t is n o t u n c o v e r e d b y Df(1)RA2, w h i c h s p a n s 7 D 1 0 - 8 A 4 . 5 . T h e r e f o r e rdgA is w i t h i n t h e 8 A 4 . 5 - 8 C 6 r e g i o n . M a p p i n g ofrdgB b y r e c o m b i n a t i o n p l a c e d it at 42.7 -+ 0.7 o n t h e X c h r o m o s o m e , rdgB was u n c o v e r e d b y t h e d e l e t i o n Df(1)g 1 a n d is t h e r e f o r e in s a l i v a r y r e g i o n 1 2 A - 1 2 E . ANATOMICAL DEFECTS U p o n e c l o s i o n s all rdgA a n d r d g B m u t a n t s r a i s e d a n d k e p t as a d u l t s in s t a n d a r d c o n d i t i o n s (12 h r light: 12 h d a r k at 25°C) h a v e Published March 1, 1977 266 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69 • 1977 n o r m a l - l o o k i n g p h o t o r e c e p t o r s , as j u d g e d by electron microscopy a n d by pseu- d o p u p i l e x a m i n a t i o n . 7 days later, however, all m u t a n t s showed d e g e n e r a t i o n o f the o u t e r six r e c e p t o r cells, R1-6, o f every o m m a t i d i u m . T h e central two p h o t o r e c e p t o r s , R7 a n d R8 (see Figs. 1 a n d 2) were p r e s e r v e d in almost every Downloaded from jgp.rupress.org on May 6, 2011 FIGURE 1. Normal eye. (a) Pseudopupil (bar = 100/zm); (b) rhabdomeres viewed by optical neutralization of the cornea (bar = 10 ixm); (c) light microscopy of retina (bar = 10/zm); (d) electron micrograph of ommatidium (bar = 2 /xm). o m m a t i d i u m in rdgB Ks222 a n d rdgOK045, in a b o u t 60% o f the o m m a t i d i a in rdgBKsl°° a n d rdgAKs199, and in f e w e r than 10% o f the o m m a t i d i a in rdgBEEl7°, rdgAK°14, a n d rdgA ns12. T h e s e results suggest that R I - 6 are m o r e sensitive to the effects o f the rdgA a n d rdgB m u t a t i o n s , a n d that the alleles o f each retinal Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 267 d e g e n e r a t i o n gene can be o r d e r e d with respect to how m u c h R7 and R8 are affected in each m u t a n t . T h u s , for r d g B the o r d e r is: r d g B celT° > r d g B Ks1°° > rdgB Ksl6 ~-- rdgB rs2°° > rdgB K045 = r d g B 1~s222. For rdgA: rdgA nsl2 = rdgA r°14 > rdgA Ks199 > r d g A ec47. Downloaded from jgp.rupress.org on May 6, 2011 FIGURE 2. Degenerate rdgB Ks2e2 eye. (a, b, c, and d) as in Fig. 1. PHYSIOLOGICAL AND BEHAVIORAL DEFECTS E R G s o f 7-day old adult retinal d e g e n e r a t i o n mutants, raised in n o r m a l conditions, showed r e d u c e d receptor potentials a n d the absence o f on-transients (Benzer, 1971; also see Figs. 7, 10, 14 and 17). T h e alleles in which R7 and R8 were most affected gave the smallest r e c e p t o r potentials. In rdgB Ks222 a n d r d g B K°45 in which R7 a n d R8 are least affected, a r e c e p t o r potential o f u p to 5 m V was c o m m o n . This is o f the same Published March 1, 1977 268 T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y • V O L U M E 69 - 1977 o r d e r as the maximal response o f R7 and R8 (Minke et al., 1975a). In rdgA K°14 and rdgB EEl7° in which R7 and R8 are most affected, no receptor potential greater than 0.5 mV was found. These results suggest that the residual receptor potential in these mutants originates from R7 and R8. Indeed, Harris et al. (1976) have shown that the receptor potential in rdgBKs222 has a spectral sensitiv- ity corresponding to that o f R7 and R8 in normal eyes (Fig. 8). T h e on-transient is absent in all o f the mutants in which R1-6 have degenerated. This is consistent with the idea that this transient arises in the lamina (see Goldsmith and Bernard, 1974), since only the axons o f R1-6 have synapses in the lamina; the axons of R7 and R8 pass through the lamina and have their first synapses in the medulla (Trujillo-Cenoz and Melamed, 1966). Phototaxis, measured by counter-current distribution (Benzer, 1967), was strongest in those retinal degeneration mutants in which R7 and R8 were most preserved. Spectral analysis o f this behavior (Harris et al., 1976; Stark et al., Downloaded from jgp.rupress.org on May 6, 2011 1976) showed that R7 and R8 mediate the residual phototactic response in these mutants. TIME COURSE OF DEGENERATION T h e time course o f degeneration was measured for rdgB Ks222. l0 groups o f about 20 rdgBKs222 flies raised in normal conditions were collected within 1 h o f eclosion, kept in constant room light at 25°C, and examined at various intervals by the pseudopupil technique. Fig. 3 shows as a function o f time the percentage o f pseudopupils in which no defect was evident. Since the sharpness o f a normal pseudopupil is d e p e n d e n t upon the precise optical alignment o f the photoreceptors in about 20 ommatidia (Frances- chini 1972), this is a sensitive assay for anatomical signs o f photoreceptor degeneration. By 24 h degeneration was beginning in some flies, at 72 h degeneration was well underway in almost all rdgB Ks222 flies. T h e steepness of the decline in Fig. 3 does not necessarily indicate an abrupt change from a nondege- nerate to a degenerate state, but is more likely to reflect the threshold o f the technique used for revealing anatomical changes. A pseudopupil was j u d g e d to be normal whenever the trapezoidal pattern of seven dots (Fig. I a) was visible. Genetically normal flies in the same conditions showed no degeneration whatso- ever. Although anatomical signs o f degeneration do not occur until after emergence o f the adult, it is evident from the ERG that R1-6 are already functionally defective at eclosion in all rdgA and rdgB mutants. Since the photoreceptors of Drosophila are fully developed in late pupal life (Waddington and Perry, 1960), the initial degenerative process (i.e. the irreversible physiological malfunction o f the photoreceptors, as distinguished from their subsequent structural degenera- tion) probably begins before emergence. Localization of Defect TISSUE LOCALIZATION By mosaic analysis of ERG deficits Hotta and Ben- zer (1970) found that the eye was the focus of both the rdgA and rdgB defects. Pseudopupil examination o f 100 y cho rdgA rm4 and 100 y cho rdgB rs222 mosaics produced by ring loss (see Materials and Methods) confirmed their results. Even in mosaics in which all external landmarks were ~enticallv normal except for one Published March 1, 1977 HARRIS AND STARK RetinalDegeneration in Drosophila 269 eye, that eye showed retinal d e g e n e r a t i o n . F u r t h e r m o r e , a mosaic dividing line often (in about 20% o f the mosaics) passed t h r o u g h an eye. In these cases the genetically m u t a n t part o f the eye showed d e g e n e r a t i o n while the genetically n o r m a l part did not. This was true for both rdgA x°14 and rdgB Ks222. T h e most closely related internal tissue, in terms o f fate map position, is the first optic ganglion. T h e latter is very rarely (<2%) split by mosaic dividing lines and is, in 10% o f these mosaics, o f g e n o t y p e d i f f e r e n t f r o m the retina (Kankel and Hall, 1976). T h e r e f o r e , the d e g e n e r a t i o n defects must be a u t o n o m o u s to the retina. CELLULAR LOCALIZATION OF THE DEFECT Ready et al. (1976) have shown that the cells o f a Drosophila o m m a t i d i u m are not clonally related; a single om- matidium at a mosaic borderline may be c o m p o s e d o f both normal and m u t a n t cells. Examination by light and electron microscopy o f borderlines in the eyes o f Downloaded from jgp.rupress.org on May 6, 2011 I00 ..L 5 _~ 80 - 40 i i zo _J U- I I I I 12 24 36 48 60 72 t~" - ~ Age of rdgO KS222 flies (h) FIGURE 3. Time course of degeneration in rdgB xs22~. The percentage of flies with no retinal degeneration (as judged by observation of the pseudopupil) as a function of age after eclosion (see text for details). y cho rdgA K°I4 and y cho rdgB ~cs222 mosaics reveals that within a single o m m a t i d i u m some receptors may d e g e n e r a t e while others may not. This was also shown for a n o t h e r allele o f r d g B by Benzer (1971). T h e d e g e n e r a t i o n is not d e p e n d e n t on the g e n o t y p e o f n e i g h b o r i n g pigment cells since d e g e n e r a t e and n o n d e g e n e r a t e p h o t o r e c e p t o r s can be f o u n d next to the same pigment cells. By X-ray-induced somatic crossing over, small patches o f w rdgB ~cs222 m u t a n t tissue may be made. In these the p i g m e n t cells and the p h o t o r e c e p t o r s can be scored individually for the absence o f screening pigments (caused by the w mutation). T h e s e results also indicate that rdgB ~s222 is a u t o n o m o u s to the p h o t o r e c e p t o r s themselves. In the diagrammatic reconstruction o f part o f such a patch (Fig. 4) n o r m a l p h o t o r e c e p - tors are next to m u t a n t pigment cells and vice versa. F u r t h e r m o r e , the only p h o t o r e c e p t o r s which survive in spite o f being m u t a n t are the central ones, R7 and R8, as expected in rdgB Ks2~z. This means that the act o f p h o t o r e c e p t o r d e g e n e r a t i o n is c o n s e q u e n t only on the g e n o t y p e o f the individual p h o t o r e c e p - tor cell. Published March 1, 1977 270 THE JOURNAL OF GENERAL PFIYSIOLOGY " VOLUME 69 • 1977 SUBCELLULAR LOCALIZATION OF THE DEFECT In the m u t a n t o v a JK84 isolated by Koenig and Merriam (1975), the r h a b d o m e r e s o f the o u t e r p h o t o r e c e p t o r s R1-6 fail to develop. By combining this m u t a n t with the retinal d e g e n e r a t i o n m u t a n t r d g B ^'s222, doubly m u t a n t flies were obtained. T h e s e had R1-6 photore- ceptor cells, without r h a b d o m e r e s , carrying the r d g B Ks22~ mutation. W h e n these double mutants were raised and kept as adults at 18°C their p h o t o r e c e p t o r cells Downloaded from jgp.rupress.org on May 6, 2011 FIGURE 4. Diagram of an X-ray induced w rdgB Ks222 mosaic patch. Semicircular shapes represent primary pigment cells, ovoid shapes represent secondary pigment cells, large circles represent peripheral photoreceptor cells R1-6, small circles represent central photoreceptor R7. Black = nonmutant, uncolored = mutant. Photoreceptors which are absent have degenerated. did not d e g e n e r a t e even after 20 days in constant light. T h a t no d e g e n e r a t i o n occurs at 18°C in r d g B Kszzz ora sKs4 double mutants while considerable degenera- tion occurs in r d g B ~s22~ single mutants raised in identical conditions is explained by the light-deprivation effect (see below) because these p h o t o r e c e p t o r s with no r h a b d o m e r e s have no light response (Harris et al., 1976). H o w e v e r , when these double m u t a n t flies were kept as adults at h i g h e r t e m p e r a t u r e (25°C) they did d e g e n e r a t e after about 10 days i n d e p e n d e n t o f light condition (Fig. 5). T h u s , the cells are defective even in the absence o f r h a b d o m e r e s . While the ora a~s4 single mutants raised in given conditions do not d e g e n e r a t e , these double m u t a n t Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 271 p h o t o r e c e p t o r s do, even t h o u g h no r h a b d o m e r e s are p r e s e n t (Fig. 5). T h i s indicates that the rdgB Ks22z m u t a n t defect is not localized to the r h a b d o m e r e itself; s o m e o t h e r p a r t o f the cell m u s t be defective, (of course, the r h a b d o m e r e m a y be also). Altering the Time Course o f Degeneration TEMPERATURE T e m p e r a t u r e has an accelerating effect on retinal d e g e n e r - Downloaded from jgp.rupress.org on May 6, 2011 FmURE 5. Effect of the rdgB ~szz2 gene on oraJK84. (a) Electron micrograph of oraJus4 control kept for 15 days as an adult at 25°C in the dark (bar = 2 /xm). (b) Electron micrograph of the double rdgBKS222; oras~84 raised in identical conditions (bar = 2 bern). Receptor cells R1-6 degenerate due to the rdgB Ksz2z mutation, even though they have no rhabdomeres. ation. M u t a n t rdgB Ks222 flies were raised a n d k e p t as adults at 18°C, 25°C, or 30°C either in constant light (i.e. in glass f o o d bottles 0.5 m in f r o n t o f a GE 15 W cool white fluorescent l a m p G e n e r a l Electric Co., Cleveland, Ohio) or in darkness. In constant light at 18°C, p s e u d o p u p i l e x a m i n a t i o n showed that d e g e n e r a t i o n b e c a m e e v i d e n t in a b o u t 3 days p o s t e m e r g e n c e a n d a p p r o a c h e d c o m p l e t i o n in a b o u t 12 days. I n constant light at 25°C, d e g e n e r a t i o n was evident 1 day poste- m e r g e n c e a n d b e c a m e c o m p l e t e in a b o u t 7 days. I n constant light at 30°C, newly e m e r g e d flies already showed s o m e d e g e n e r a t i o n , which r e a c h e d c o m p l e t i o n in a b o u t 3 days. I n the d a r k , t e m p e r a t u r e also h a d a large effect. As will be discussed below, Published March 1, 1977 272 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 69 • 1977 rdgB ^'s222 flies raised and kept in darkness at 18°C showed little or no degenera- tion even up to 30 days postemergence. At 25°C, d e g e n e r a t i o n became evident by about 7 days. At 30°C, d e g e n e r a t i o n was evident within 2 days postemerg- ence. N o r m a l flies showed no retinal d e g e n e r a t i o n u n d e r any o f the above conditions. ACID PHOSPHATASE DEPRIVATION Lysosomal enzymes, including acid phosphatases, are involved in digesting cellular debris and d e g e n e r a t i n g tissue. Acid phosphatase activity changes markedly d u r i n g retinal d e g e n e r a t i o n in the mouse rd m u t a n t (Sanyall, 1970). T h e r e f o r e , it was o f interest to combine the Drosophila m u t a n t Acph-1"11, which lacks acid phosphatase activity (Bell and Mac- Intyre, 1973), with the retinal d e g e n e r a t i o n m u t a n t rdgB Ks~22. About 60 y cho rdgB Ks222 flies and about 60 y cho rdgBXS~22; Acph-1 "u flies were raised at 25°C in constant light, and adults were e x a m i n e d by the pseudopupil technique when Downloaded from jgp.rupress.org on May 6, 2011 24, 48, and 72 h old. At 24 h, d e g e n e r a t i o n was evident in 49% o f the flies carrying the Acph-1 + gene, while only 12% o f the flies carrying the null Acph-1 "11 gene showed d e g e n e r a t i o n . By 48 h, 98% ofAcph-1 + flies showed d e g e n e r a t i o n as c o m p a r e d to 55% for Acph-1 "~ flies. By 72 h, d e g e n e r a t i o n n e a r e d completion in the Acph- 1 "n flies. T h u s the absence o f acid phosphatase activity does not p r e v e n t h e r e d i t a r y retinal d e g e n e r a t i o n in Drosophila but does seem to delay it by about 24 h. Prevention of Degeneration by Light Deprivation BASIC EFFECT Flies o f each o f the rdgA alleles were raised at 18°C in the dark f r o m the egg until about 5 days postemergence. Controls were raised in constant light at the same t e m p e r a t u r e . All showed the same a m o u n t o f degen- eration in light o r dark. A d i f f e r e n t result was obtained with rdgB. In this case, flies o f all the rdgB alleles showed considerably m o r e d e g e n e r a t i o n when raised in the light. T h e effect was most p r o n o u n c e d in rdgB Ksz2~ and rdgB K°4~, which showed no signs o f d e g e n e r a t i o n in the dark, as j u d g e d by the pseudopupil m e t h o d or in histologi- cal sections. Fig. 6 shows an example o f 10-day old adult rdgB ~s222 flies f r o m the same parents, which had been separated as larvae into two groups. T h e dark- raised g r o u p showed very little d e g e n e r a t i o n after 10 days c o m p a r e d to the light- raised g r o u p . Even after 30 days in the dark at 18°C, most rdgB ~s2~2 flies showed little or no retinal d e g e n e r a t i o n . T h e ERGs o f white-eyed rdgB xs2~2 flies raised in the dark at 18°C were r e c o r d e d . I f these flies were p r e p a r e d for physiological examination u n d e r dim red light, the flash-elicited ERGs looked normal in all respects (Fig. 7). Spectral analysis o f the ERG (Fig. 8) showed the high sensitivity two-peaked curve shown by Harris et al. (1976) to be g e n e r a t e d by R1-6. After e x p o s u r e o f these dark-raised mutants to intense stimulation (see below) or b r i e f r o o m light, at 20°C, the ERG waveform was o f the R7-8 type (Fig. 7), and showed R7-8 spectral sensitivity (Fig. 8). 3 days later the first anatomical signs o f d e g e n e r a t i o n became evident by pseudopupil examination. Published March 1, 1977 HARRIS AND STARK Rf~Tta~Degeneration in Drosophila 273 rdgB KIm~ robed in light tO0 eo GO (t~ 40 LU i 20 It. 0 , ,I I 0 kd rdgB m u z raised in dark ~ I00 Z 80 l.J 0 n- eo o. Downloaded from jgp.rupress.org on May 6, 2011 4o so | I AMOUNT OF DEGENERATION FIGURE 6. Induction o f degeneration in r d g B xs~zz flies by exposure to light. Flies were kept for 10 days at 18°C in the d a r k or in the light then examined by the p s e u d o p u p i l technique. 50 flies each group. cn bw w rdgB K~'~" 5 7 0 nm dark-raised adapted i ~ L 47'0 nm ]zmv light-raised adapted I= FIGURE 7. Typical ERG waveforms for cn b w and w rdgB KB222. 470-nm flashes (traces below ERGs) were about 2 × 10" quanta/cm 2"s (top) and 5 × 1013 quanta/ cm 2. s (bottom). T h e obtainability o f ERG on and off transients in 570 n m - a d a p t e d cn b w and d a r k - r e a r e d w r d g B ~s222 but not in 470 n m - a d a p t e d cn b w and light-reared w r d g B xs2~2 , as well as the higher sensitivity in the f o r m e r cases is consistent with the idea that the ERGs in the top panels are d o m i n a t e d by photoreceptors R1-6, and those in the bottom panels by photoreceptors R7 and R8. Such waveforms at similar intensities were obtainable after about 1 min o f d a r k adaptation after 570 or 470 nm bright adaptation. Published March 1, 1977 274 T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E 6 9 " 1 9 7 7 LIGHT-SENSITIVE PERIOD Dark-raised and light-raised (both at 18°C) r d g B •s222 flies were shifted to the opposite lighting condition at various times d u r i n g d e v e l o p m e n t and a d u l t h o o d . Shifting f r o m light to d a r k was effective in p r e v e n t i n g d e g e n e r a t i o n p r o v i d e d it was d o n e b e f o r e the adult p h o t o r e c e p t o r s were f o r m e d in the late pupal stage. Shifting f r o m dark to light was always effective in inducing d e g e n e r a t i o n even when it was d o n e in a d u l t h o o d . This result indicates that it is the adult p h o t o r e c e p t o r which is sensitive to light- induced d e g e n e r a t i o n and not, for example, a p r e c u r s o r cell. DARK RECOVERY Reversibility o f light-induced d e g e n e r a t i o n was tested by raising r d g B xs~22 flies in the dark at 18°C until about 3 days posteclosion and then 10 ~,.~cn bw lC wrd~ t~ Downloaded from jgp.rupress.org on May 6, 2011 11 o \ i 12 "u ,~ 13- 13 ~e I-,. 14 14 I I I i I I I I I 400 ,500 600 4OO 50O eOO Wavolongth (rim) W~elength (nm) FIGURE 8. Spectral sensitivities of cn bw (n = 8) and w rdgB xs222 (n = 4). Dark- adapted cn bw and dark-reared w rdgB nsz2z (circles, solid lines) show R1-6 responsiv- ity; 470 nm-adapted cn bw and light-reared dark-adapted r d g B xs2~2 (triangles, dotted lines) show R7 plus R8 responsivity; 370 nm-adapted light-reared w rdgB xs22~ and cn bw (squared, dashed lines) show R8 responsivity. Standard errors computed between subjects normalized to mean sensitivity. (All curves except dark-reared w r d g B Ks222 are drawn from Harris et al., 1976.) exposing them to one o f three light regimes shown in Fig. 9 (series I): (a) 1 day (24 h) in light (in glass food vials 0.5 m f r o m a GE 15 W cool white fluorescent lamp) followed by 7 days in darkness; (b) 8 days in light; or (c) 8 days in darkness. T h e technique o f optical neutralization o f the cornea was used to count the n u m b e r o f normal R1-6 p h o t o r e c e p t o r s . Fig. 9 shows that 8 days in light caused severe degeneration. T h e n u m b e r o f R1-6 r h a b d o m e r e s r e m a i n i n g per omma- tidium was 0.9 - 0.2 (SEM) (n = 50 ommatidia e x a m i n e d - 1 0 each f r o m five flies). 8 days in darkness caused little if any d e g e n e r a t i o n (5.7 - 0.1 R1-6/ ommatidium). After 1 day in light followed by 7 in darkness there was mild d e g e n e r a t i o n (4.3 + 0.1 R1-6/ommatidium). Genetically normal flies after 8 days in the light or the dark showed no d e g e n e r a t i o n . In these flies, it is possible that the d e g e n e r a t i o n had p r o c e e d e d slightly in 1 Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 275 day of light and was halted by 7 days of dark, or that there was some recovery in the dark. To distinguish between these possibilities, 3-day old rdgB Ks222 adults, dark raised at 18°C, were transferred to one of the four light-dark schedules shown in Fig. 9 (series II). It is clear from these results that 4 consecutive days of light caused more retinal degeneration than 4 days of light separated by 3 days of dark. This recovery may occur only in photoreceptor cells that have not yet reached a critical stage in the degeneration process, since the gross histological retinal degeneration in those rdgB Ks~2~flies kept in constant light for 8 or 10 days was not reversed by putting the flies into the dark at 18°C. Properties of Mutant Photoreceptors on First Exposure to Light PHOTOPtGMENT CONVERSIONS The question arises of whether the light- overoge number of Downloaded from jgp.rupress.org on May 6, 2011 Light-Dark Sequence tntoct rhobdomeres at end of sequence Series I (RI-6 only) 4.~ tO.I I 0.9t02 5.1''0.1 Series n m m ] 3 . 4 t 0 s~ 1.2t02 III oJ~o., I o.oa'oJ I I I I I I II I I 2 3 4 5 6 79 8D Day number FIGURE 9. Dark recovery in rdgBKs~22. Horizontal line shows light-dark sequence. At right is the resultant average number of nondegenerate peripheral photorecep- tors per ommatidium (scoring only R1-6) + SEM. Each number represents meas- urements on 50 ommatidia, 10 each from five flies. induced degeneration in rdgB Ks222 flies is an invertebrate analog of light-sensitive degeneration in mammals deprived of vitamin A. Dowling and Wald (1960) showed that in mammals, vitamin A deficiency caused an inability to regenerate rhodopsin from opsin, and also that the opsin was a structurally less stable pro- tein than rhodopsin. Thus, rod outer segment membranes, which are normally composed mostly of rhodopsin, disintegrate under vitamin A-deprived condi- tions. In invertebrates, including Drosophila, rhodopsin is converted by light of one range of wavelengths into metarhodopsin which is stable at room tempera- ture and is converted back into rhodopsin by light of a second range of wave- lengths (Hamdorf et al., 1971; Pak and Liddington, 1974; Ostroy et al., 1974; Harris et al., 1976). In the squid, metarhodopsin is structurally less stable than rhodopsin (Hubbard and St. George, 1958), suggesting that the defect in rdgB t~s222 might be in the regeneration of rhodopsin from metarhodopsin. Published March 1, 1977 276 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69 " 1977 T o test this idea, a s p e c t r o p h o t o m e t r i c analysis was carried out on the visual pigment f r o m white-eyed rdgB ~s222 flies raised at 18°C in the dark. It showed that rdgB ~s~z contained as m u c h r h o d o p s i n as do normal flies (see Harris et al., 1976) and that the photointerconversion o f R1-6 r h o d o p s i n and m e t a r h o d o p s i n in dark-raised rdgB ~s222 was normal. T h a t the pigment regenerates p r o p e r l y in vitro does not m e a n it will do so in vivo, so a second e x p e r i m e n t was d o n e . Pak and Liddington (1974) and Grabowski and Pak (personal communication) have characterized two fast potentials in the Drosophila eye that are similar in some respects to the vertebrate early r e c e p t o r potentials (ERPs). In the Drosophila case, Pak and Liddington (1974) showed that these are g e n e r a t e d by the conversion o f dark reored ligM reared cn bw ~ / ~ W rdgBK'~'2 w rdge 1¢8~u~ 4"rOnm Downloaded from jgp.rupress.org on May 6, 2011 odopt,d 57Ohm adopted L._ \ I tom FIGURE 10. M Potentials. These responses were elicited by an intense white flash shown in stimulus monitor trace. 470 nm-adapted cn bw and dark-reared w rdgB nsz22 show the biphasic corneal negative then positive M potential. 570 nm-adapted cn bw and dark-reared w rdgB Ksz22 show much smaller M potential. M potentials were not obtainable from nonadapted dark-reared rdgB Ksz22. Such M potentials from 470 nm-adapted dark-reared w rdgB KS222 were obtainable from flies in which the periph- eral photoreceptors had recently been inactivated by 470 nm adaptation for several hours after the PDA decay. In the week-old, light-reared w rdgB xs~22 in which the photoreceptors have undergone morphological degeneration no M potential is seen. Voltage calibration equals 10 mV for cn bw 570 nm-adapted, 2 mV for all other traces. m e t a r h o d o p s i n back to r h o d o p s i n , since they have the spectral sensitivity o f Drosophila R1-6 m e t a r h o d o p s i n and are p r o p o r t i o n a l to the a m o u n t o f metarho- dopsin converted by the flash. For this reason, these potentials are collectively called the M potential (Pak and Liddington, 1974). Fig. 10 shows the M poten- tials in white-eyed control flies and rdgB xs~22 flies. In 18°C dark-raised w rdgB ~zs222 flies the M-potential properties were normal and r e m a i n e d so for several hours after the R1-6 r e c e p t o r potential had vanished, indicating that the rhodopsin- m e t a r h o d o p s i n interconversion was normal in vivo. Only after several days in light, when the R1-6 p h o t o r e c e p t o r s had completely d e g e n e r a t e d , was the M potential no longer obtainable (Fig. 10). Similar results have also been obtained by Grabowski and Pak (personal communication) in the same and a n o t h e r allele o f rdgB. T h e n o r m a l in vitro and in vivo interconversion o f the p h o t o p i g m e n t does not necessarily mean that everything about the p h o t o p i g m e n t is normal; Published March 1, 1977 HARRIS AND STARK RetinalDegenerationin Drosophila 277 these e x p e r i m e n t s do not rule out the possibility that some o t h e r aspect o f p h o t o p i g m e n t function may be defective in these mutants. PROLONGED DEPOLARIZING AFTERPOTENTIAL When intense 470-nm light, which converts R1-6 r h o d o p s i n to m e t a r h o d o p s i n , is presented to a normal, d a r k - a d a p t e d white-eyed Drosophila, the R1-6 cells stay depolarized for up to 6 h (Minke et al., 1975a). This has been called the p r o l o n g e d depolarizing afterpo- tential (PDA) and is observed in the ERG as a corneal-negative afterpotential (Minke et al., 1975a). D u r i n g a maximal PDA, p h o t o r e c e p t o r cells R1-6 are not responsive to stimulus flashes o f light (Minke et al., 1975a). E x p o s u r e to intense 570-nm light immediately resensitizes and repolarizes these receptors, and the R1-6-dominated ERG can once again be observed (see Fig. 7 and Fig. 11 a, b). With white-eyed rdgB xs22~ (dark-raised at 18°C) an intense 470 n m flash caused a PDA which lasted only for 30 s to 2 min (Fig. 1 l f ) . T h e ERG is an extracellular Downloaded from jgp.rupress.org on May 6, 2011 measure o f c u r r e n t flow, so the final m e m b r a n e potential o f the r e c e p t o r cells is not known. I f an intense 570 nm light was p r e s e n t e d to a w rdgB xs222 eye b e f o r e the PDA c u r r e n t had r u n down, say after 10 s (see Fig. 11 d), then, after the ERG had r e t u r n e d to base line, R1-6 were still capable o f r e s p o n d i n g (see Fig. 11 c-f). I f the PDA c u r r e n t was allowed to r u n down without i n t e r r u p t i o n by 570-nm light, the R1-6 cells became completely unresponsive (Fig. 11 g). At this point, even intense 570-nm light was incapable o f reactivating them. Receptor Potential and Degeneration VITAMIN A DEPRIVATION T h e intensity o f 470 n m adaptation r e q u i r e d to p r o d u c e irreversible loss o f R1-6 sensitivity in w rdgB xs2~ is the same as for reversible loss (and PDA) in n o r m a l white-eyed flies (Fig. 12). This suggested that the PDA-generating mechanism might be defective in the m u t a n t flies. Since vitamin A deprivation has been f o u n d to block the PDA and R1-6 inactiva- tion in normal flies (Stark and Zitzmann, 1976) while decreasing sensitivity by about 2.0 log units ( Z i m m e r m a n and Goldsmith, 1971), w rdgB xs2~2 flies were vitamin A deprived. T h e s e deprived flies, raised at 18°C in the dark and kept for several days as adults b e f o r e testing, showed R1-6 activity which consistently survived intense stimulation including 24 h o f r o o m light (Fig. 14), conditions which eliminated R1-6 activity in vitamin A-enriched controls r e a r e d in exactly the same conditions (Fig. 14). T h e m u t a n t and normal vitamin A-deprived flies showed a nearly identical sensitivity decrease induced by 470 n m adaptation without a PDA (Fig. 13), but in this case the sensitivity loss in both m u t a n t and normal was reversible. This protection caused by vitamin A deprivation, how- ever, did not last indefinitely as j u d g e d by ERG recordings and pseudopupil examinations. RECEPTOR P O T E N T I A L DEPRIVATION Raising rdgB gs*22 flies in the dark, eliminating the r h a b d o m e r e s (by oraJKS4), and desensitizing the p h o t o r e c e p t o r s by vitamin A deprivation all protect against degeneration; also, the m u t a n t defect does not a p p e a r to be in the r h o d o p s i n - m e t a r h o d o p s i n photoconversions. It was t h e r e f o r e conjectured that the defect might be electrical, i.e. that depolar- ization was lethal to the m u t a n t p h o t o r e c e p t o r s . Mutations o f the norpA gene can completely block the r e c e p t o r potential Published March 1, 1977 a 15 lOmV / ,! d ~ ~ 1 1 1 ~ 1 ~ .... ? __is lOmV i i q li I Downloaded from jgp.rupress.org on May 6, 2011 f d g --<_....__-- FIGURE l l . Responses prolonged afterpotentials in cn bw (a, b) and w rdgB Ks~22 (c, d, e , f , g). (a) Response o f c n bw to intense (5.75 × 1016 quanta/cm2"s) 1-s flash of 470 nm light followed by an equally intense 570 nm 1-s flash. (b) Response to the 470-nm flash alone causing a PDA (see text) which in this case is not terminated by 570 n m light. (c) First response of a dark-reared w rdgB Ks~22 fly to a dim 3.25 × 1011 quanta/cm2.s 470 n m light. It is essentially normal (like dark-adapted cn bw; Fig. 7). (d) shows the response to a 470 n m followed by a 570-nm flash (same intens- ity as in a and b). The 470-nm flash elicits a large (though not as large as in cn bw) receptor potential with a slow repolarization after the 570-nm flash. This stimula- tion sequence does not inactivate R1-6 as assayed by the normal waveform in the subsequent response to 3.25 × 1011 quanta/cm2"s of 470 nm, shown in (e). (f) First response of dark-reared w rdgB Ks2z2 to a single 470 n m flash (same intensity as a, b, and d). T h e extraceilularly recorded PDA current decays to base line in about 30 s. After this stimulus, RI-6 responsivity is lost as j u d g e d by reduced sensitivity and loss of ERG transients, shown in (g) (here stimulus intensity was 2 log units greater than in c and e). I n at least 10 experiments such as these, 570-rim stimulation was never f o u n d to reactivate R1-6 if applied after about a 30-s delay, while it could after a 10-s delay. In these experiments, the PDA c u r r e n t decay took typically 30- 100 s. Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 279 11"I *L15 ~,~ ........... "~ III ':t 1 -x s.,,,*...-.*+..-- 15+"1'2 113 14 15 16 1+ _ i i i 0 mr log Intensity 2 10- "0 11- .*** .• ...... o 12- * 13- W I- 14- * Downloaded from jgp.rupress.org on May 6, 2011 log Intemlty FIGURE 12. Sensitivity and adaptation o f vitamin A-enriched cn bw and w rdgB tcsm. T h e curves plot the sensitivity of the ERG receptor component as inverse threshold (3.0 mV criterion) in log quantum flux of 470-nm flashes as a function of adaptation at 470 nm (log intensity). Typical adaptation curves for cn bw (top) and dark-reared w rdgB Ksm (bottom) are shown. For cn bw, PDA (1 min poststimulus) is also plotted (dotted line) against the right ordinate. The threshold change and afterpotential for cn bw are reversible by long-wavelength adaptation; the threshold change for w rdgB x s m is irreversible. The threshold data were obtained between 1 and 2 min subsequent to each bright adaptation conditioning flash. log I n t a m z l t y 11Zr ,_~ i,zo. w ~ \ \+\ log I n t e n l l t y FIGURE 13. Sensitivity and adaptation of vitamin A-deprived cn bw and w rdgB xs222. Typical threshold changes as a function of adaptation for vitamin A- deprived cn bw (top) and w rdgB Ksm (bottom) are shown. These threshold changes are considerably less than those of vitamin A-enriched flies due to the fact that in neither deprived case is RI-6 inactivated as assayed by the obtainability of ERG on transients. Furthermore, in both cases the threshold changes are reversible by long- wavelength stimulation. T h e threshold data were obtained between 1 and 2 min subsequent to each bright adaptation conditioning flash. Published March 1, 1977 280 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69 " 1977 (Hotta a n d Benzer, 1970; Alawi et al., 1972). This block occurs at a step in the transduction process after r h o d o p s i n conversion since p h o t o p i g m e n t levels in these m u t a n t s are large fractions o f n o r m a l levels (Ostroy et al., 1974) a n d p h o t o p i g m e n t p r o p e r t i e s a p p e a r identical to n o r m a l (Pak a n d L i d d i n g t o n , 1974). T o test w h e t h e r blocking the r e c e p t o r potential would inhibit d e g e n e r a - tion, the norpA EEs m u t a t i o n which completely blocks the r e c e p t o r potential (Fig. 15) was genetically c o m b i n e d with rdgBKSZ2L T h e double m u t a n t s were checked by backcrosses to assure that both mutations were present. As in norpA EES, the double m u t a n t s had n o r m a l M-potential p r o p e r t i e s but no r e c e p t o r potential (Fig. 15), indicating p h o t o i n d u c e d p i g m e n t conversions. P r e v e n t i n g the r e c e p t o r vit.A deprived -- L..._f'" Downloaded from jgp.rupress.org on May 6, 2011 i Is vit.A erdched ! M----iz t ~ Is FIGURE 14. ERG waveforms of vitamin A-deprived and enriched w 7dgB KS222 flies which had been raised and aged for 7 days at 18°C in the dark and exposed for 24 h to white light at room temperature immediately before running. Responses in both cases were elicited by 470 nm flashes of 3.2 × 1014quanta/cm 2" s. The transients and larger (top) receptor potential indicate that R1-6 are still functioning in the deprived mutant but not in the enriched. potential in this way also p r e v e n t e d m o r p h o l o g i c a l signs o f retinal d e g e n e r a t i o n (Fig. 16). T h e s e norpA EE5 rdgB xs222 flies had n o r m a l - l o o k i n g R1-6 p h o t o r e c e p - tors, as j u d g e d by electron microscopy, even after 20 days in constant light at 18°C. This result suggests that a r e c e p t o r potential m a y be necessary for photo- r e c e p t o r d e g e n e r a t i o n to occur. DEPRIVATION OF ON AND OFF TRANSIENTS T h e norpA e~5 m u t a t i o n elim- inates both the r e c e p t o r potential a n d the transient c o m p o n e n t s o f the ERG. T h e r e f o r e , rdgBKs222 was also c o m b i n e d with a m u t a t i o n that eliminates the on a n d o f f transients o f the ERG but not the r e c e p t o r potential c o m p o n e n t . T h e m u t a n t JK910 was used for this (Koenig a n d M e r r i a m , 1975). In the double m u t a n t rdgBXS222;JK910, retinal d e g e n e r a t i o n p r o c e e d e d j u s t as rapidly as in the single m u t a n t rdgB xs222. T h u s it a p p e a r s that the r e c e p t o r potential, not the transients, is i m p o r t a n t in the retinal d e g e n e r a t i o n process. Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 281 w norpA [E5 n o r p A E [ 5 rdgBlCS222; 570 nm . . ~ . ~ _ ~ .~-~-___ adapted \ '\ 12mY IOtas Downloaded from jgp.rupress.org on May 6, 2011 FIGURE 15. ERGs and M potentials of w norpA rE5 and norpA EE5 rdgB~Sm; cn bw. Stimulation and adaptation as in Fig. 10. In both cases the M potentials could be seen after 470 nm adaptation but were considerably reduced after 570 nm adapta- tion. The later receptor potential component of the ERG (seen in Fig. 10) is completely absent in these mutants. rdgB KmuB I00 80 60 c~ 4O LU --J 20 It. LI. 0 I I 0 bJ norpAEEs rdgB Ks222 C9 I00 < I-- Z 80 LLI ¢..) w eO Lt.I O. 40 20 none portiol complete AMOUNT OF DEGENERATION FIGURE 16. Prevention of degeneration by norpA EES. These results are plotted as in Fig. 6, except in this case both groups were exposed to constant light 18°C for 10 days. SODIUM POTASSIUM PUMP T h e ouabain-sensitive Na+-K + A T P a s e is in- volved in p h o t o r e c e p t o r r e p o l a r i z a t i o n at the cessation o f the light stimulus ( B r o w n a n d L i s m a n , 1972). O n e possible h y p o t h e s i s that m i g h t a c c o u n t f o r the c o r r e l a t i o n o f d e g e n e r a t i o n with the r e c e p t o r potential is t h a t this e n z y m e is Published March 1, 1977 282 THE JOURNAL OF GENERAL PHYSIOLOGY "VOLUME 6 9 - 1977 defective in the rdgB Ks222 mutant. Failure of this mechanism might also account for the slow repolarization at the 570 nm-induced termination o f the PDA (Fig. l l d ) . T h e ATPase level was measured directly by a biochemical assay. It was found that about 60% o f the ATPase activity in the retina of normal white-eyed flies was sensitive to 0.2 mM ouabain. Dark-reared white-eyed rdgB ~s~22 mutants showed essentially an identical (within 5%) amount of total and ouabain-sensitive ATPase activity. Suppressors of Degeneration SCREENING FOR SUPPRESSORS TO investigate further the relation between the receptor potential and retinal degeneration, mutants were sought that would prevent retinal degeneration in the presence o f the rdgBKs222 mutation. Since one mutant, norpA EES, which eliminates the receptor potential suppresses degenera- tion, one might expect other mutations which eliminated the receptor potential Downloaded from jgp.rupress.org on May 6, 2011 to be among the suppressors. If all suppressors o f degeneration in rdgB xsz22 flies were found to have no receptor potential, that would suggest that the receptor potential is both necessary and sufficient for causing degeneration in rdgBxs22~ flies. Suppressors on the X chromosome were sought by mutagenizing rdgBKs2~2and rdgA pc47 males and mating them to attached-X females. Male progeny o f this cross carry the X chromosome with the rdg mutation and any other mutations that the mutagen might have caused. Approximately 1,000 mutagenized rdgA Pc47 and 1,000 rdgB Xs2~2 flies were checked by pseudopupil examination for retinal degeneration. No suppressors o f the rdgA ec47 were found. T h r e e suppressors o f rdgB xs2~ were found, all o f which proved to be alleles o f the norpA gene and were named norpA suI etc. Two of these, norpA"ul and norpA ~In, gave very small receptor potentials. Like other norpA mutants these were recessive. Thus, norpA'UVrdgBX~22/+ rdgB x~22 did show a receptor potential and also retinal degeneration. Neither allele complemented norpA EES. T h u s norpA *ux rdgBXSZ22/norpA~e~ rdgB Ks222 had almost no receptor potential and did not degenerate. T h e suppression o f degeneration caused by these mutants can be understood as a mimicking o f norpA EES, i.e. preventing the receptor potential and hence preventing degeneration. SUPPRESSORII T h e notion o f the receptor potential's being both necessary and sufficient for degeneration was shattered by the third allele, norpA "uH, which suppressed degeneration yet permitted a normal receptor potential (Fig. 17). T h a t is, norpAsun rdgBxsz22 flies had a normal ERG yet showed no degeneration. This suppressor, like other norpA mutants, was recessive. Thus, norpA ''n rdgBKS222/+ rdgBKszzz degenerated. Mapping experiments done with norp^,un using the suppression o f degeneration as a character for scoring recombination placed it at 1-6.3 -+ 0.6. Previous maps o f other norpA mutants by using ERGs placed norpA at 1-6.5 -+ 0.7 (Pak, 1975). F u r t h e r m o r e , norpA vau did not comple- ment with norpA eES. Thus, norpA "uu rdgBKS22~/norpAE~ rdgBKs2~2 flies have an ERG but do not degenerate. T h e presence of an ERG in norpA "~n is dominant to its absence in norpA EES. From these results, it is clear that norpA '~n is an allele o f norpA. Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 283 T h e norpA *un m u t a t i o n does not s u p p r e s s d e g e n e r a t i o n by simply lowering the sensitivity o f the p h o t o r e c e p t o r s . T h i s was shown by genetically s e p a r a t i n g the norpA Sun m u t a n t f r o m rdgB xs~2, m a k i n g it white eyed, a n d testing the responsiv- ity. T h e intensity o f 470-nm light n e e d e d to elicit a 3.0 m V r e s p o n s e in norpA'un; cn bw (log q u a n t u m flux = 10.56 -+ 0.37 SD, n = 4) was identical to that for n o r m a l white-eyed flies (10.63 + 0.25, n = 8). F u r t h e r m o r e , the w a v e f o r m , the m a x i m a l flash-induced E R G r e c e p t o r waves (about 25 mV), the intensity-re- sponse functions, a n d the PDA p r o p e r t i e s were n o r m a l (Fig. 18). T h e s u p p r e s - sion o f d e g e n e r a t i o n was not perfect, however; 15 days' e x p o s u r e to r o o m light a n d t e m p e r a t u r e caused s o m e d e g e n e r a t i o n in a b o u t 15% o f 60 norpA ~n norpA~ rdgBKs~z Downloaded from jgp.rupress.org on May 6, 2011 [11 f | rdgBKs222 m"'% FIGURE 17. ERG waveforms of red-eyed norpA~u rdgB ~mz2 and rdgBxmzz. These flies were raised in room light and temperature. Flash intensities were 1.0 x 1014 (for norpA =n rdgB xn'Ya) and 3.2 x 1015 (for rdgB x~2) quanta/cmU.s of 570 nm. This wavelength was chosen because its leakage through the screening pigments favors the obtainability of ERG transients. The waveform and higher sensitivity of the norpA sul*rdgBKm** compared to rdgB xm*2 indicate that in the former, photoreceptors R1-6 are functioning, while in the latter they are not. rdgB ~s222 adults e x a m i n e d , while in rdgB ~s22~ control flies u n d e r identical condi- tions t h e r e was d e g e n e r a t i o n in 100% o f the flies. T h e existence o f a s u p p r e s s o r o f retinal d e g e n e r a t i o n with a n o r m a l r e c e p t o r potential shows that the r e c e p t o r potential, while p e r h a p s necessary, is certainly not sufficient for retinal d e g e n e r - ation to occur. THE INTERACTION OF norpA ANn rdgB W h e n a mutational c h a n g e in o n e protein is c o m p e n s a t e d with restoration o f function by a mutational alteration in a second, interaction between these two proteins can usually be i n f e r r e d (e.g., W o o d a n d Bishop, 1973). T h i s raises the possibility that the g e n e p r o d u c t o f the n o r m a l rdgB g e n e [call it gp(rdgB+)], interacts with the g e n e p r o d u c t o f the n o r m a l norpA g e n e [gp(norpA+)], a n d that the defect in gp(rdgB Ks~z~) is c o u n t e r - acted by the defect in gp(norpASuI*). I n o t h e r words, gp(norpA ~n) is specifically Published March 1, 1977 norpASul]; cn bw cn bw 20 mV 1C I 10 14" 10 14 Io9 Intensity i ! Downloaded from jgp.rupress.org on May 6, 2011 ,/ I '~ 11 .4 s FIGURE 18. Left side shows typical responsivity ofnorpj=un; cn bw; right side o f c n bw controls. T h e top figure shows intensity-response functions for the ERG nega- tive (receptor) potential elicited by 1-s 470-nm flashes from a 570-nm then dark- adapted condition. They are calculated for 0.5, 1,3, 6, 10, 15, and near-maximal 22 mV with standard errors between preparations shown (for norpA~u"; cn bw, n = 3; for cn bw n = 6). Typical 1-s flash-elicited ERGs are shown for both strains for a 1.5 mV receptor potential (elicited by almost 101° quanta/cm 2. s of 470-nm light, first pair of traces with stimulus monitor below and 4 mV positive calibration) and for a 7 mV receptor potential (elicited by about 1011 quanta/cm2.s, second pair of traces with 10 mV calibration). At the bottom are responses to intense (about 5.75 × 10~6 quanta/cm 2"s) 2-s stimuli in the sequence 470, 470, 570, 570 n m to show the afterpotential properties in the two strains. Within limits of experimental variabil- ity, responsivity in norpASU'; cn bw a n d cn bw are the same. 284 Published March 1, 1977 HARRIS AND STARK RetinalDegeneration in Drosophila 285 tailored to interact with gp(rdgBXS2~2). I f this were so then one might expect the norpA s~n mutation to be allele specific, i.e. it might not suppress the degeneration caused by other rdgB mutations. On the other hand, one would not expect norpA EEn suppression to be allele specific since there is no restoration o f function in norpA EE5rdgB xs22~ double mutants, i.e. since norpA ~5 completely prevents the receptor potential it should suppress the degeneration in all rdgB mutants. T h e allele rdgB x°45, though physiologically similar to rdgBxs222, was induced by a separate mutational event. T o test the action o f norpA sulI and norpA EE5 on this allele the appropriate double mutants were constructed and checked for degen- eration by pseudopupil examination, norpA ~H rdgB K°45 double mutants did show retinal degeneration which proceeded at the normal r a t e , while norpA EE5 rdgB x°45 double mutants did not. Thus, norpA s~II suppression is indeed allele specific while norpA eE5 is not. Downloaded from jgp.rupress.org on May 6, 2011 DISCUSSION Anatomical Localization of the Defect In o r d e r to understand the mechanism o f hereditary retinal degeneration it is important to identify the tissue primarily responsible for the defect. By making mosaic individuals, part normal and part mutant, it is possible to determine the primary focus o f the defect, i.e., the tissue that must be mutant in o r d e r for the mutant property to appear. Such mosaic analysis has been used in mouse hereditary retinal degeneration caused by the rd mutation to show that the photoreceptor cells themselves are probably responsible for the defect (LaVail and Mullen, 1974). In rat retinal degeneration, however, the pigment epithe- lium has been implicated ( H e r r o n et al., 1969; Bok and Hall, 1969, 1971) and shown by mosaic analysis to be the primary focus o f the defect (Mullen and LaVail, 1976). Genetic mosaics in humans caused by random inactivation o f the X chromosomes in females heterozygous for sex-linked mutations (Lyon, 1961) revealed that some cases o f hereditary retinal degeneration are autonomous to the retina (Goodman et al., 1965; Berson et al., 1969). Another type o f heredi- tary retinal degeneration in humans is caused by a defect in absorption o f vitamin A in the intestine (Gouras et al., 1971). In Drosophila various techniques are available for making mosaics (Hall et al., 1976). Hotta and Benzer (1970) used mosaics to show that the rdgA and rdgB defects are autonomous to the eye. In this study, histological examination o f mosaic retinas shows that it is the photoreceptors which are defective. Further- more, a mutant, orasKs4, which blocks the formation o f rhabdomeres in the outer photoreceptor cells but still allows retinal degeneration (at high temperature) in rdgB flies, shows that the defect is not restricted to the rhabdomeres and must be present in the cell body. Physiological Localization of the Defect Given that the photoreceptor cells are responsible for their own degeneration, what is wrong with them? T h e rdgB mutants are conditional in that retinal degeneration is light sensitive. By turning light on and o f f at various times in the life o f an rdgB mutant, it is possible to show that the photoreceptor is light Published March 1, 1977 286 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 69. 1977 sensitive only when fully differentiated. This does not necessarily mean that the defect first appears only in the adult. The immature photoreceptor cell could, for instance, already be defective in the uptake of some substance that is necessary for the adult photoreceptor's response to light. Several studies with light deprivation and vitamin A deprivation in rodents have suggested that defective photopigment metabolism may be important in leading to degeneration (Dowling and Sidman, 1962; Herron et al., 1969; Bok and Hall, 1971; Noell et al., 1971; Noell and Albrecht, 1971; LaVail et al., 1972; Yates et al., 1974; LaVail and Battelle, 1975). Similar experiments, described here, on the rdgB mutants of Drosophila also suggest that the photopigment metabolism may be defective in these mutants. However, direct studies of the photointerconversion of rhodopsin and metarhodopsin showed that, both in vivo and in vitro, there are normal conversions of the photopigment. This suggests that the defect is expressed at a step in the transduction process Downloaded from jgp.rupress.org on May 6, 2011 subsequent to photopigment conversion. Conversion of a net amount of rhodopsin to metarhodopsin induces a pro- longed depolarizing afterpotential (PDA) (Hochstein et al., 1973; Minke et al., 1973), which lasts up to 6 h in normal Drosophila (Minke et al., 1975a). The duration is less than 2 min in rdgB mutants on their first exposure to 470-nm light, after which the photoreceptors become permanently inactive. Vitamin A deprivation prevents the PDA in normal Drosophila (Stark and Zitzman, 1976) and delays degeneration in rdgB mutants. The intensity of 470-nm light needed to cause a PDA approximates the intensity needed to produce long-term damage to rdgB photoreceptors. These results suggest that long-lasting depolarization of the photoreceptors is causally related to the degeneration in these mutants. This idea was confirmed by depriving the photoreceptors of depolarization by use of the norpA~ mutation. The norpA mutants have normal photopigment metabo- lism but are defective in the generating mechanism for the receptor potential (Pak, 1975). The norpA~5 mutation results in no receptor potential and prevents rdgB photoreceptors from degenerating. Thus, the rdgB defect was shown to act during or subsequent to the action of the norpA gene product. The finding of a suppressor of degeneration with normal receptor potential, norpA*un, demon- strated that the degeneration process is not consequent to the receptor potential. Thus, the rdgB defect is associated with a step in the phototransduction process of the adult photoreceptor which begins after the photopigment action, is after or during the norpA+gene product action, and is not consequent on the receptor potential. Model of Drosophila Photoreceptor Degeneration We propose the following scheme for degeneration in Drosophila rdgB mutants. Each absorbed photon converts one rhodopsin to metarhodopsin and, as a result, one or more molecules of gp(norpA+), the gene product of the normal norpA gene, is either directly or indirectly activated. This activated gp(norpA +) which may be an enzyme, an internal transmitter, a channel, etc., is somehow involved in the eventual generation of a receptor potential. In nonmutant flies circulating gp(rdgB +), the gene product of the normal rdgB gene, terminates the action of gp(norpA +) by direct interaction with it. In rdgB mutants, however, the Published March 1, 1977 HARRIS AND STARK RetinalDegenerationin Droso#hila 287 defective gp(rdgB ~s~22) is incapable o f t e r m i n a t i n g the action o f gp(norpA+), and this abnormal state o f affairs leads somehow to cell death. This model explains the results o f this p a p e r . In the dark, gp(norpA +) does not b e c o m e activated and thus does not have to be inactivated, so d e g e n e r a t i o n is p r e v e n t e d . Vitamin A deprivation in flies reduces the a m o u n t o f r h o d o p s i n (Razmjoo and H a m d o r f , 1976). This would lead to a r e d u c t i o n in the a m o u n t o f activated gp(norpA+). This should delay the onset o f d e g e n e r a t i o n , as observed. In mutants such as norpA EE5 there is no r e c e p t o r potential because gp(norpA EEs) is absent o r nonfunctional. T h e r e f o r e , it does not n e e d to be inactivated for the cell to be protected against d a m a g e . In the norpA ~H mutant, which was selected for suppression o f d e g e n e r a t i o n in the presence o f the rdgB xw222 mutation, a modified gp(norpA) molecule is p r o d u c e d so that it can act in the usual way to p r o d u c e a r e c e p t o r potential. T h e modified f o r m o f the molecule, however, is such that it can be inactivated by gp(rdgB xs222) so that there is no d e g e n e r a t i o n . Downloaded from jgp.rupress.org on May 6, 2011 T h e genetic evidence so far obtained can be c o n s i d e r e d in light o f this model. T h e rdgB defect is recessive; this would be e x p e c t e d if, in rdgB/+ heterozygotes, there is half the normal level o f gp(rdgB +) and that is still sufficient to inactivate all the gp(norpA+). T h e suppression o f d e g e n e r a t i o n in norpA ee5 and norpA suil is recessive since in norpAeEn/+ and norpA~/+ heterozygotes there is still half the normal level o f gp(norpA +) which c a n n o t be p r o p e r l y inactivated by any a m o u n t o f m u t a n t gp(rdgBXS222). T h e norpA ~II suppression o f d e g e n e r a t i o n is allele specific whereas the norpA ~5 suppression is not because gp(norpA ~a) has been specifically modified to be inactivated by gp(rdgB xs222) while gp(norpA xEs) is simply inactive; it c a n n o t p r o d u c e a r e c e p t o r potential and t h e r e f o r e does not have to be inactivated. T h e m o d e l predicts suppression o f d e g e n e r a t i o n in norpA ~It rdgBKS~22/norpA8uII rdgB x°4s heterozygotes since in this case while gp(rdgB ~°4~) c a n n o t inactivate any gp(norpA~al), gp(rdgB xs222) can inactivate it all. T h e model also predicts no suppression in norpA xes rdgBX°45/norpA su" rdgB s°45 heterozygotes because even t h o u g h gp(norpA EEs) is inactive, gp(norpA su~l) can generate a r e c e p t o r potential and its action c a n n o t be termi- nated by gp(rdgB~°45). T h e s e heterozygotic combinations were constructed and f o u n d to c o n f o r m to prediction. T w o o f the results p r e s e n t e d in this p a p e r may, at first, a p p e a r contradictory to the model p r o p o s e d . T h e first is that the time n e e d e d to irreversibly d a m a g e the p h o t o r e c e p t o r s o f the rdgB xs2~2 m u t a n t with a bright flash is on the o r d e r o f tens o f seconds (Fig. 1 l f, g), whereas the visual excitation process takes only a few milliseconds (see, for e x a m p l e , Fig. 10). T h u s , one might argue that this rdgB xs222 p h e n o m e n o n is m u c h too slow to be involved in the excitation mecha- nism. T h e role p r o p o s e d for gp(rdgB+), however, is one o f de-excitation r a t h e r than excitation. According to the model, the rdgB mutations should, t h e r e f o r e , have no effect on the initial rise time o f the r e c e p t o r potential. It may be fairer to propose, then, that gp(rdgB +) is involved in the adaptation r a t h e r than transduc- tion p h e n o m e n a in the broadly d e f i n e d processes o f excitation. T h e second a p p a r e n t l y troublesome result is that d e g e n e r a t i o n can proceed in the absence o f r h a b d o m e r e s , i.e. in the rdgBxS2e2; ora:xs4 double m u t a n t (Fig. 5). T h e r e is good evidence in flies that the r h a b d o m e r e contains the visual pigment (Langer and T h o r e l l , 1966; Stavenga et al., 1973) and that the p h o t o r e c e p t o r c u r r e n t in some Published March 1, 1977 288 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 6 9 " 1977 invertebrates flows t h r o u g h the r h a b d o m e r i c m e m b r a n e or closely associated m e m b r a n e s (Hagins et al., 1962; Lasansky a n d Fuortes, 1969). T h u s , it m i g h t seem u n e x p e c t e d that a defect in the t r a n s d u c t i o n process should be e x p r e s s e d in the absence o f so m u c h t r a n s d u c t i o n m a c h i n e r y . T h a t there can be d e g e n e r a - tion in rdgB rs222 m u t a n t s at high t e m p e r a t u r e without r h a b d o m e r e s a r g u e s that there m a y be s o m e cytoplasmically located i n t e r m e d i a t e s in the p h o t o t r a n s d u c - tion process such as p r o p o s e d by Cone (1973). For instance, if gp(norpA +) a n d gp(rdgB +) are cytoplasmic, a n d if gp(norpA +) can be thermally activated, one m i g h t expect to see d e g e n e r a t i o n at high t e m p e r a t u r e in the rdgBrS2z2; oraJrs4 double m u t a n t . It is i m p o r t a n t to recall, however, that the d e g e n e r a t i o n seen in these double m u t a n t s proceeds only slowly a n d only at high t e m p e r a t u r e . Eliminating the r h a b d o m e r e s does have a substantial saving effect on the photo- r e c e p t o r s , a p p r o x i m a t e l y equivalent to d a r k - r e a r i n g . This kind o f protection is just what the m o d e l predicts. Downloaded from jgp.rupress.org on May 6, 2011 Alawi et al. (1972), Pak a n d L i d d i n g t o n (1974), a n d Ostroy et al. (1974) have shown that the norpA m u t a n t s are defective in a step in the transduction process between q u a n t u m catch a n d r e c e p t o r depolarization. Minke et al. (1975b) have shown that trp, a n o t h e r Drosophila m u t a n t , which leads to a transient r e c e p t o r potential, is also defective in an i n t e r m e d i a t e step in p h o t o t r a n s d u c t i o n . In this study we have p r e s e n t e d evidence suggesting that the rdgB gene also codes for a step in the transduction process. T h e evidence for direct interaction between the p r o d u c t s o f the norpA and the rdgB genes, while based solely on genetic evi- dence, e n g e n d e r s the h o p e that the f u r t h e r study o f interactions a m o n g these a n d o t h e r Drosophila visual m u t a n t s at genetic, physiological, a n d biochemical levels will yield a c o m p l e t e stepwise description o f the p h o t o t r a n s d u c t i o n proc- ess. This work was supported in part by a Gordon Ross Medical Foundation Fellowship (to William A. Harris), National Science Foundation Grants BG 27228 (to Seymour Benzer), BMS 74-12817, and BNS 7~-11921, and Johns Hopkins National Institutes of Health Biomedical Sciences Support Grant (to William S. Stark). We thank B. Butler, M. Chapin, Y. Dudai, E. Eichenberger, J. Gorn, R. Greenberg, D. Lakin, E. Lipson, and G. Pransky for technical assistance. We are indebted to many for advice and criticism, notably Seymour Benzer. Receivedfor publication 11 June 1976. REFERENCES A~wx, A. A., V. JENN[NGS, J. GROSSrIELD, and W. L. PAX. 1972. Phototransduction mutants of Drosophila melanogaster. In The Visual System: Neurophysiology Biophysics and Their Clinical Applications. G. B. Arden, editor. Plenum Publishing Corp., New York. 1-21. BAYLOR, D., A. L. HODGKIN, and T. LAMB. 1974. Reconstruction of the electrical responses of turtle cones to flashes and steps of light. J. Physiol. 242:759-791. BELL, J., and R. MACINTYRE. 1973. Characterization of acid phosphatase-I gene in Drosophila melanogaster. Biochem. Genet. 10"39-55. BENZER, S. 1967. Behavioral mutants of Drosophila isolated by countercurrent distribu- tion. Proc. Natl. Acad. Sci. U. S. A. 58:1112-1119. B~NZER, S. 1971. From the gene to behavior.J. Am. Med. Assoc. 218:1015-1022. BERSON, E. L., P. GOURAS, R. D. GUNKEL,and N. C. MYRIANTMOPOULOS.1969. Rod and cone responses in sex-linked retinitis pigmentosa. Arch. Ophthalmol. 81:315-325. Published March 1, 1977 HARMS AND STARK Retinal Degeneration in Drosophila 289 BOK, D., and M. O. HALL. 1969. The etiology of retinal dystrophy in RCS rats. Invest: Ophthalmol. 8:649-650. BoK, D., and M. O. HALL. 1971. The role of the pigment epithelium in the etiology of inherited retinal dystrophy in the rat. J. Cell Biol. 49:664-682. BROWN, J. E., and J. E. LISMAN. 1972. An electrogenic sodium pump in Limulus ventral photoreceptor cells. J. Gen. Physiol. 59:720-733. CONE, R. 1973. The internal transmitter model for visual excitation: some quantitative implications. In Biochemistry and Physiology of Visual Pigments. H. Langer, editor. Springer-Verlag, New York. 275-284. DOANE, W. W. 1967. Drosophila. In Methods in Developmental Biology. F. H. Wilt, and N. K. Wessels, editors. Thomas Y. Crowell Co., New York. 219-244. DOWLING,J. E., and R. L. SIDMAN. 1962. Inherited retinal dystrophy in the rat. J. Cell Biol. 14:73-109. DOWUNG,J. E., and G. WALD. 1960. The biological activity of vitamin A acid. Proc. Natl. Downloaded from jgp.rupress.org on May 6, 2011 Acad. Sci. U. S. A. 46:587-608. FAHN, S., G. J. KOVAL,and R. W. ALBERS. 1968. Sodium-potassium-activated adenosine triphosphatase of Electrophorus electric organ. V. Phosphorylation by adenosine tri- phosphate-32P.J. Biol. Chem. 243:1993-2002. FRANCESCHINI,N. 1972. Pupil and pseudo-pupil in the compound eye of Drosophila. In Information Processing in the Visual Systems of Arthropods. R. Wehner, editor. Springer-Verlag, Berlin. 75-82. FRANCESCmNI, N., and K. KIRSCHFELD.1971. Etude optique in vivo des ~l~ments photo- recepteurs dans l'oeil compos~ de Drosophila. Kybernetik. 8:1-13. FUORTES, M. G. F., and A. L. HODGKIN. 1964. Changes in the time scale and sensitivity in the ommatidia of Limulus. J. Physiol. 172:239-263. GOLDSMITH, T. H., and G. D. BERNARD. 1974. The visual system of insects. In The Physiology of Insecta. 2. M. Rockstein, editor. Academic Press, Inc., New York. 165- 272. GOODMAN, G., H. l~PPS, and L. M. SIEGEL. 1965. Sex-linked ocular disorders: trait expressivity in males and carrier females. Arch. Ophthalmol. 73:387-398. GOURAS, P., R. E. CARR, and R. D. GUNKEL. 1971. Retinitis pigmentosa in abetalipopro- teinemia. Effects of vitamin A. Invest. Ophthatmot. 10:784-793. HAgINS, W. A., H. V. ZONANA,and R. G. ADAMS. 1962. Local membrane current in the outer segments of squid photoreceptors. Nature (Lond.). 194:844-847. HALL, J. C., W. M. GELBART,and D. R. KANKEL. 1976. Mosaic systems. In Genetics and Biology of Drosophila. la. M. Ashburner and E. Novitski, editors. Academic Press, Inc., London. 265-314. HAMDORF, K., J. SCHWEMER, and M. GOGALA. 1971. Insect visual pigment sensitive to ultraviolet light. Nature (Lond.). 231:458-459. HARRIS, W. A., W. S. STARK, and J. A. WALKER. 1976. Genetic dissection of the photoreceptor system in the compound eye of Drosophila melanogaster. J. Physiol. 256:415-439. HEISENBERG, M. 1971. Isolation of mutants lacking the optomotor response. Drosophila Information Service 46:68. HERRON, W. L., B. W. RIEGEL, O. E. MYERS, and M. L. RUBXN. 1969. Retinal dystrophy in the r a t - a pigment epithelial disease. Invest. Ophthalmol. 8:595-604. HOCHSTEIN, S., B. MINKE, and P. HXLLMAN.1973. Antagonistic components of the late receptor potential in the barnacle photoreceptor arising from different stages of the pigment process. J. Gen. Physiol. 62:105-128. Published March 1, 1977 290 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 69 • 1977 HOTTA, Y., and S. BENZER. 1970. Genetic dissection o f the Drosophila nervous system o f means o f mosaics. Proc. Natl. Acad. Sci. U. S. A. 67:1156-1163. HUBBARD, R., and R. C. C. ST. GEORGE. 1958. T h e rhodopsin system of the squid. J. Gen. Physiol. 41:501-528. KANKEL, D. R., and J. C. HALL. 1976. Fate m a p p i n g o f nervous system and other internal tissues in genetic mosaics of Drosophila melanogaster. Dev. Biol. 48:1-24. KIRSCHFELD, K., and N. FaANCESCHINI. 1968. Optische Eigenschaften d e r Ommatidien im Komplexauge von Musca. Kybernetik. 5:47-52. KOENIG, J., and J. E. MERRIAM. 1975. Autosomal ERG mutants. Drosophila Information Service. I n press." LANGER, H., and B. THORELL. 1966. Microspectrophotometric assay o f visual pigments in single r h a b d o m e r e s of the insect eye. In T h e Functional Organization o f the Com- p o u n d Eye. C. G. B e r n h a r d , editor. Pergamon Press, Oxford. 145-149. LASANSKY, A., and M. G. F. FUORTES. 1969. T h e site o f origin o f electrical responses in Downloaded from jgp.rupress.org on May 6, 2011 visual cells o f the leech, Hirudo medicinalis. J. Cell Biol. 42:241-252. LAVAIL, M. M., and B. A. BATTELLE. 1975. Influence o f eye/pigmentation and light deprivation on inherited retinal dystrophy in the rat. Exp. Eye Res. 21:167-192. LAVAIL, M. M., and R . J . MULLEN. 1974. Analysis o f the pigment epithelium in mice with inherited retinal degeneration using experimental chimeras. Association for Research in Vision and Ophthalmology Program. 61. (Abstr.). L A V A I L , M . M . , R . L . SIDMAN, and D. O'NEXL. 1972. Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. J. Cell Biol. 53:185-209. LEWIS, E. B. 1960. A new standard food medium. Drosophila Information Service. 34:117- 118. LEWIS, E. B., and R. BACHER. 1968. Method o f feeding ethyl methane sulfonate (EMS) to Drosophila males. Drosophila Information Service 43:193. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL. 1951. Protein m e a s u r e m e n t with Folin phenol reagent. J. Biol. Chem. 193:265-275. LYON, M. F. 1961. Gene action in the X-chromosome o f the mouse (Mus musculus L.) Nature (Lond.). 190:372-373. MINKE, B., S. HOCHSTEXN, and P. HILLMAN. 1973. Antagonistic process as source of visible light suppression o f afterpotential in Limulus UV photoreceptors.J. Gen. Physiol. 62:787-791. MINKE, B., C.-F. Wu, and W. L. PAK. 1975a. Isolation o f light-induced response o f the central refinula cells from the electroretinogram o f Drosophila ommatidia. J. Comp. Physiol. 98:345-355. MINKE, B., C.-F. Wu, and W. L. PAK. 1975b. Induction of photoreceptor voltage noise in the d a r k in Drosophila mutant. Nature (Lond.). 258:84-87. MULLEN, R. J., and M. M. LAVAIL. 1976. Inherited retinal dystrophy: primary defect in pigment epithelium d e t e r m i n e d with experimental rat chimeras. Science (Wash. D. C.). 192:799-801. NOELL, W. K., and R. ALBKECnT. 1971. Irreversible effects o f visible light on the retina: Role o f vitamin A. Science (Wash. D. C.). 172:76-79. NOELL, W. K., M. C. DELMELLE, and R. ALBKECHT. 1971. Vitamin A deficiency effect on retina: d e p e n d e n c e on light. Science (Wash. D. C.). 172:72-75. NOLTE, J., J. E. BROWN, and T. G. SMITH, JR. 1968. A hyperpolarizing c o m p o n e n t o f the receptor potential in the median ocellus of Limulus. Science (Wash. D. C.). 162:677-679. OSTROV, S. E., M. WILSON, and W. L. PAK. 1974. Drosophila rhodopsin" photochemistry, extraction and differences in the norpA P12 phototransduction mutant. Biochem. Biophys. Published March 1, 1977 HARRIS AND STARK Retinal Degeneration in Drosophila 291 Res. Commun. 59:960-966. PAK, W. L. 1975. Mutations affecting the vision of Drosophila melanogaster. In Handbook of Genetics. 3. R. C. King, editor. Plenum Publishing Corp., New York. 703-733. PAK, W. L., J. GROSSFIELD,and K. S. ARNOLD. 1970. Mutants of the visual pathway of Drosophila melanogaster. Nature (Lond. ). 227:518-520. PAK, W. L.,J. GROSS~ELD,and N. V. WroTE. 1969. Nonphototactic mutants in a study of vision of Drosophila. Nature (Lond.). 222:351-354. PAK, W. L., and K. L. LIDDINGTON.1974. Fast electrical potential from a long-lived, long- wavelength photoproduct of fly visual pigment. J. Gen. Physiol. 65:740-756. POODRY, C. A., and H. A. SCHNEIDERMAN.1970. The ultrastructure of the developing leg of Drosophila melanogaster. Wilhelm Roux's Arch. Dev. Biol. 166:1-44. RAZMJOO, S., and K. HAMDO~. 1976. Visual sensitivity and the variation of total photo- pigment content in the blowfly photoreceptor membrane. J. Comp. Physiol. 105:279- 286. Downloaded from jgp.rupress.org on May 6, 2011 READY, D. F., T. E. HAnSOn, and S. BENZER. 1976. Development of the Drosophila retina, a neurocrystalline lattice. J. Dev. Biol. 53:217-240. REYNOLDS, E. S., JR. 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy.J. Cell Biol. 17:208-212. SANYALL, S. 1970. Changes in lysosomal enzymes during hereditary degeneration and histogenesis of retina in mice. I. Acid phosphatase visualized by A20-dye and lead nitrate methods. Histochemie. 23:207-219. STARK, W. S. 1975. Spectral selectivity of visual response alterations mediated by inter- conversions of native and intermediate photopigments in Drosophila. J. Comp. Physiol. 96:343-356. STARK, W. S., A. M. IVANYSI~YN,and K. G. Hu. 1976. Spectral sensitivities and photopig- ments in adaptation of fly visual receptors. Naturwissen Schaften. In press. STARK, W. S., and W. G. ZITZMANN. 1976. Isolation of adaptation mechanisms and photopigment spectra by vitamin A deprivation in Drosophila. J. Comp. Physiol. 105:15- 27. STAVENGA, D. G., A. ZANTEMA,and J. W. KuIPER. 1973. Rhodopsin processes and the function of the pupil mechanism in flies. In Biochemistry and Physiology of Visual Pigment. H. Langer, editor. Springer-Verlag, New York. 175-179. STERN, C. 1936. Somatic crossing over and segregation in Drosophila melanogaster. Genetics. 21:625-730. TRUJILLO-CENoz, O., and J. MEmMED. 1966. Electron microscope observations of the peripheral and intermediate retinas of dipterans. In The Functional Organization of The Compound Eye. C. G. Berhard, editor. Pergamon Press, Oxford. 339-361. WADDtNGTON,C. H., and M. M. PERKY. 1960. The ultrastructure of the developing eye of Drosophila. Proc. R. Soc. Lond. Set. B Biol. Sei. 155:155-178. WooD, W. B., and R. J. BisHoP. 1973. Bacteriophage T4 tail fibers: structure and assembly of a viral organelle. In Virus Research. Second ICN-UCLA Symposium on Molecular Biology. C. F. Fox and W. S. Robinson, editors. Academic Press, Inc., New York. 303-326. YATES, C. M., A.J. DEWAR, H. WILSON,A. K. WINTrRBURN,and H. W. READING. 1974. Histological and biochemical studies on the retina of a new strain of dystrophic rat. Exp. Eye Res. 18:119-133. ZIMMERMAN, W. F., and T. H. GOI~DSMIa'H. 1971. Photosensitivity of the circadian rhythm and of the visual receptors in the carotenoid-depleted Drosophila. Science (Wash. D. C.). 171:1167-1169.
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