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Hereditary Retinal Degeneration

VIEWS: 9 PAGES: 31

									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




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                                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




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                          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).




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                               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-




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                          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




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                          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




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                                  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.




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                                 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.,




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                           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




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                                              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




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                                 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 -




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                                 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




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                          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.




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                                                                   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~




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                                                                                               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




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                                                 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




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                                   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




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                                                                                              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




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                          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




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                                                                       [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.




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                               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




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                          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 .




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                               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.




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                               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.
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