Circadian regulation of lodopsin gene expression in embryonic photoreceptors in retinal cell culture

Neuron,

Vol. 10, 579-584,

April,

1993, Copyright

0 1993 by Cell Press

Circadian Regulation of lodopsin Gene Expression in Embryonic Photoreceptors in ‘Retinal Cell Cult&e Mary E. Pierce,*+ Hooshmand Sheshberadaran,* Zhe Zhang,* Lyle E. Fox,* Meredithe 1. Applebury,* and Joseph S. Takahashi* *NSF Center for Biological Timing and Department of Neurobiology and Physiology Northwestern University Evanston, Illinois 60208 *The University of Chicago Visual Sciences Center and Committee on Neurobiology Chicago, Illinois 60637

A circadian clock regulates a number of diverse physiological functions in the vertebrate eye. In this study, we show that mRNA for the red-sensitive cone pigment, iodopsin, fluctuates with a circadian rhythm in chicken retina. Transcript levels increase in the late afternoon just prior to the time of cone disc shedding. Furthermore, iodopsin mRNA levels are regulated similarly by a circadian oscillator in primary cultures of dispersed embry onic chick retina. Nuclear run-on experiments show that the circadian regulation of iodopsin transcript abundance occurs at the level of gene transcription. Our re suits provide a demonstration of clock-regulated gene expression in a vertebrate preparation maintained in cell culture.

pathways into, and molecular components of, cellular circadian oscillators. The maintenance of outer segment structure and function in both rod and cone photoreceptors involves the assembly of new photopigment-containing membrane at the base of the outer segment and its displacement toward, and eventual shedding from, the tip of the cell (for reviews see Besharse et al., 1988; Young, 1978). Data from a number of species indicate that light and a circadian clock interact to regulate these events (Besharse et al., 1988). However, rod and cone photoreceptors shed their outer segments at different times of the day. In rod photoreceptors, rhodopsin mRNA levels rise in the early morning before light onset just prior to the time of rod disc shedding (Korenbrot and Fernald, 1989). Since in chickens, the peak of cone photoreceptor disc shedding occurs soon after light offset (Young, 1978), we tested the hypotheses that mRNA levels of the predominant chicken cone pigment, iodopsin, would be elevated in the late afternoon and that transcript levels are regulated by a circadian clock. In this paper, we demonstrate that a circadian clock regulates the expression of iodopsin mRNA both in vivo and in primary cultures of embryonic retina and provide evidence that the regulation of iodopsin mRNA occurs at the level of gene transcription. Results and Discussion

Introduction A circadian clock regulates many aspects of retinal physiology, from gene expression to modulation of visual sensitivity (Besharse et al., 1988; Cahill et al., 1991; Reme et al., 1991). However, the cellular and molecular mechanisms that generate and control retinal circadianoscillationsarelargelyunknown.Anumber of observations indirectly support the hypothesis that photoreceptors may be self-sustaining oscillators (for review see Cahill et al., 1991). Recently, in a series of tissue reduction experiments on Xenopus laevis retinas, Cahill and Besharse (1993) definitively localized a circadian oscillator regulating melatonin synthesis to photoreceptors. Circadian rhythms of rhodopsin mRNA have been measured in toad and fish retinas in vivo (Korenbrot and Fernald, 1989). In addition, mRNA levels of several photoreceptor transduction proteins, including rhodopsin (Bowes et al., 1988; Korenbrot and Fernald, 1989), transducin (Brann and Cohen, 1987; Bowes et al., 1988), and S-antigen (Craft et al., 1990), vary diurnally. The study of clock-regulated gene expression in photoreceptors may identify

kurrent address: ment of Anatomy

University and Cell

of Kansas Medical School, DepartBiology, Kansas City, Kansas 66160.

To studythe regulation of iodopsin gene expression, a cDNA library (ZAP II, Stratagene) was prepared from embryonic day 20 chick retinas and screened with a cDNA probe to human red cone opsin (clone hs7; from J. Nathans et al., 1986). We isolated and sequenced a number of partial clones that were homologous to a published cDNA clone for chicken iodopsin (data not shown; Kuwata et al., 1990). The iodopsin cDNA probe used in these experiments came from clone R7, which contains a 1.2 kb insert spanning all of the coding sequence from exon 2 to the poly(A) tail. Circadian Regulation of lodopsin mRNA In Vivo Northern blot analysis of total mRNA prepared at 6 hr intervals from chickens maintained on a 12 hr light: 12 hr dark cycle (12L:12D) shows a diurnal rhythm in iodopsin mRNA levels (Figure 1). The primary band recognized on blots is a 1.3 kb transcript; however, with longer exposures, two other bands appear at -2.5 and 5 kb. These latter bands fluctuate coordinately with the 1.3 kb band. In the early morning, iodopsin mRNA levels are low but increase E to IO-fold in thelateafternoon, several hours before light offset. The increase in iodopsin mRNA can be detected 6-7 hr after light onset (1500 Central Standard

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ous rhythm persisted through at least 2 days of constant dark treatment (Figure 2), indicating that a circadian clock regulates iodopsin expression. The rhythm of iodopsin mRNAabundance was similarto that seen in animals exposed to a light cycle; however, the peak of iodopsin mRNA appeared to be narrower in the second day of constant darkness, with iodopsin levels beginning to decline at 2300 CST (ZT 15). Although we have not excluded the possibility that light may influence iodopsin message levels, the light cycle is not necessary for the generation of the rhythm of mRNA in vivo.

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A Circadian Oscillator in Retinal Cell Culture Cultures of embryonic chick retinal cells have been used extensively in studies of retinal development (for review see Adler, 1986a). Under appropriate culture conditions, polarized cone-like photoreceptors are seen inculture(Adler, 1986b),and immunocytochemical data indicate that some of these photoreceptor-like cells synthesize iodopsin (Araki et al., 1990). We first determined whether a diurnal rhythm of io-

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Northern blot of retinal RNA prepared from chickens killed at 6 hr intervals beginning 3 hr after light onset (lights on = 0800 CST or ZT 0; lights off = 2ooO CST or ZT 12). In each lane, 10 pg of total RNA from a single eye was loaded. The time of day (11 = 1100 CST, ZT 3; 17 = 1700 CST, ZT 9; 23 = 2300 CST, ZT 15; 05 = 0500 CST, ZT 21) and light condition (thick diagonal lines indicate nighttime)when RNAwas prepared are indicated under the blot. The iodopsinlhistone band density ratios for each lane in this experiment were lane 1, 0.6; lane 2, 3.6; lane 3, 3.5; lane 4,2.3; lane 5,0.3; lane 6,2.9. The bar graph summarizes data from four experiments. Data presented in the graph are calculated by dividing the iodopsin/histone ratio at each time point by the iodopsin/histone ratio at 1100 CST (ZT 3) in a given experiment. Results are reported as the average f SEM for each time tested.

Time [CST’J = Zeitgeber Time [ZT] 7; data not shown) and peaks about 2 hr later (1700 CST = ZT 9). After light offset, iodopsin mRNA remains elevated in the early evening (2000-2300 CST = ZT 12-15), but falls off thereafter. lodopsin mRNA levels 30 min prior to light onset in the early morning are not significantly different from those seen 3 hr after light onset (1100 CST = ZT 3). When Northern blots were stripped and reprobed using a full-length chicken rhodopsin cDNA, bands were evident at 1.3 and 2.3 kb; however, no difference was seen in the abundance of either transcript at any of the times tested (data not shown). The observed diurnal rhythm of iodopsin mRNA might be modulated by the light cycle, a circadian clock, or an interaction between the two. To determine whether a circadian oscillator regulates iodopsin mRNA, total mRNA was prepared at 6 hr intervals from animals housed in constant darkness. An obvi-

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Chickens were placed in constant darkness at light offset the night before the experiment. Peak levels of iodopsin mRNA occur in late subjective afternoon and evening (subjective daytime is indicated by the thin diagonal lines). The ratios of iodopsinl histone band density for each lane (IO pg of total RNA per lane) in this Northern blot were lane 1, 0.30; lane 2, 1.5; lane 3, 1.4; lane 4, 1.1; lane 5, 0.7; lane 6, 1.6; lane 7, 0.8; lane 8, 0.23. The bar graph shows relative iodopsin mRNA levels in repeat animals (N = 4). Data presented in the graph are calculated by dividing the iodopsinlhistone ratio at each time point by the iodopsin/ histone ratio at 1100 CST (ZT 3) in a given experiment. Results are reported as the average f SEM for each time tested.

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To determine whether there is a self-sustaining circadian oscillator regulating iodopsin gene expression in vitro, we repeated the above experiment using retinal cell cultures maintained in constant darkness beginning at light offset on day 5 in culture (Figure 4). As seen in vivo, iodopsin mRNA levels oscillate with a circadian rhythm in embryonic photoreceptors in culture. In every experiment (N = 3), there was a 2-to 3-fold increase in iodopsin message levels in the late subjective afternoon through 2 days in constant darkness. The shape of the iodopsin peak on the first day in constant darkness was similar to that measured in a light cycle (Figure 3). However, on the second day of constant dark treatment, the levels of iodopsin message were lower at all time points, and the peak of expression appeared to be narrower. A similar sharpening in the iodopsin mRNA peak was observed in vivo (Figure 2). Changes in a circadian rhythm’s waveformarecommonlyobservedduringthefirstfew days in constant environmental conditions (Pittendrigh, 1960). These data indicate that there is an endogenous clock in the chicken retina which is ex-

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Samples were collected from primary chick retinal cell cultures at 6 hr intervals beginning 9 hr after light onset on the fifth day in culture. Twenty micrograms of total RNA was loaded per lane. The iodopsinlhistone ratios for this Northern blot were lane 1, 0.2; lane 2, 1.12; lane 3, 0.8; lane 4, 0.5; lane 5, 1.5; lane 6, 1.1; lane 7,l.O. This experiment was repeated twice; in each experiment, four individual cultures were examined per time point. All replicate Northern blots showed rhythmicity. The bar graph shows relative iodopsin levels in retinal cell cultures from repeat experiments. Data presented in thegraph arecalculated bydividing the iodopsinlhistone ratio at each time point by the iodopsinl histone ratio at 1100 CST (ZT 3) in a given experiment. Results are reported as the average * SEM for each time tested.

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: dopsin mRNAis expressed byembryonic photoreceptorsinvitro. Beginningonday5ofculture,total mRNA was prepared at 6 hr intervals from chick retinal cultures that had been maintained on a 12Lz12D cycle. As seen in vivo, iodopsin mRNAabundance fluctuates rhythmically in vitro, with peak levels occurring in the late afternoon and early evening (Figure 3). A diurnal rhythm in iodopsin mRNA was clearly evident by day 6 in culture. On day 5, cultures were rhythmic, but showed more variability possibly as a result of photoreceptor development in vitro. Previous studies have shown that on the fifth day in cell culture, a polyclonal antiserum to opsin stains the entire embryonic photoreceptor cell; but byday7, immunoreactivity becomes polarized to a primitive outer segment-like structure (Adler, 1986b). Studies are underway to define and compare more accurately the development of the iodopsin mRNA rhythm in embryonic chick retina in vivo and in vitro.

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Figure 4. Chicken iodopsin mRNA a Circadian Oscillator In Vitro

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Cultures were placed into constant darkness at the time of normal light offset (2000 CST = ZT 12) the night before the experiment began. Samples were collected at 6 hr intervals beginning 3hraftersubjectivelightonsetonthesixthdayinculture.Twenty micrograms of RNA was loaded per lane. The ratios of iodopsin/ histone band density for this experiment were lane 1, 0.9; lane 2, 2.4; lane 3, 1.5; lane 4, 0.3, lane 5, 0.4; lane 6, 1.4; lane 7, 0.7 (histone band density was determined from a longer exposure for this blot). The bar graph plots the relative iodopsin levels in retinal cell cultures maintained in constant darkness from repeat experiments (N = 3); for each experiment, two cultures were examined pertime point.All replicate Northerns were rhythmic.

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Figure 5. Circadian Regulation of lodopsin Transcription In Vitro Nuclear run-on transcriptional analysiswas performed on nuclei prepared from retinal cell cultures beginning 3 hr after subjective light onset on the second day of constant dark treatment (iodopsin, closed circle; histone, open circle) or from cultures maintained in a diurnal light cycle (experiment 1, closed square; experiment 2, closed triangle). For all experiments, measurements were made on days 6-7 of culture. Transcription of iodop sin mRNA peaked between 1500 and 1700 CST (ZT 7-9) and is regulated by a circadian clock in vitro. The slot blots shown are from the constant dark experiment. Additionally, blots from run-on reactions (1700 CST, ZT 9) containing 0.5 or 1.0 &ml a-amanitin are shown.

pressed early in embryonic development when photoreceptors are differentiating oping organized outer segments.

at a time and devel-

Clock-Regulated lodopsin Gene Transcription In Vitro The circadian clock could generate the rhythm of iodopsin mRNA abundance by regulating transcriptional or posttranscriptional events. Nuclear run-on transcriptional assays measure the relative level of RNA polymerase activity associated with a specific gene as a function of cell state (Darnell, 1982; Loros and Dunlap, 1991). To determine whether there is an increase in iodopsin transcription, nuclear run-on analysis was performed using nuclei prepared from retinal cell cultures maintained in constant darkness for 2 days (Figure 5, closed circles). There is a circadian rhythm in iodopsin transcription in chick retinal cell cultures. Transcriptional rate was lowest nearthe time of expected light onset (0800 CST = ZT 0) and peaked 6-9 hr later (1400-1700 CST = ZT 6-9). A similar rise in iodopsin transcriptional rate was observed at the same time of day in cultures maintained on a light cycle (Figure 5, triangles and squares). There was no significant difference in transcription of histone 4 at

any time (Figure 5, open circles) or of total counts incorporated into RNA, indicating that there was not a general increase in transcription as a function of time of day. The addition of 0.5 &ml a-amanitin to nuclei prepared at 1700 CST (ZT 9) inhibited transcription 75%, whereas 1 @ml a-amanitin completely inhibited run-on transcription, suggesting that the increase in iodopsin transcription is likely the result of an increase in RNA polymerase II utilization (Figure 5). Our data indicate that a circadian clock regulates the transcription of the cone photopigment iodopsin, such that in the late afternoon, transcription of new iodopsin mRNA precedes the turnover of photopigment-containing outer segment membrane, which occurs after light offset. It appears that both cone and rod photoreceptors coordinate at least some of the physiological processes required for the efficient maintenance of outer segment function in a temporal manner; however, rod and cone renewal processes are out of phase. Since rhodopsin mRNA did not fluctuate at the time points examined in our experiments, the question remains as to whether rod and cone oscillations are out of phase in a single retina. In adult retinas, the circadian clock regulates the synthesis and turnoverof pigment-containing membrane in the photoreceptor outer segment (for review see Besharse et al., 1988). A circadian oscillator regulating iodopsin RNA is present in retinal cell culture at the time of photoreceptor outer segment development (Adler, 198615); however, it remains to be studied whether the circadian clock influences photoreceptor development. Cahill and Besharse (1993) have shown that inner retina neurons are not necessary for the generation of sustained circadian oscillations in Xenopus retinas maintained in vitro and have localized circadian pacemaker function to photoreceptors. Although we cannot unequivocally rule out that other retinal cells in culture may regulate the iodopsin rhythm, our results are consistent with the hypothesis that photoreceptor cells in chick retinal cells contain circadian oscillators that regulate iodopsin gene expression. Studies of circadian-regulated gene expression in diverse organisms are beginning to identify pathways into, and molecular components of, cellular circadian oscillators (for reviews see Takahashi, 1991; Dunlap, 1990; Hall, 1990; Morse et al., 1990; Kay and Millar, 1992). The circadian clock has been shown to regulate transcription of the ccgl and ccg2 genes in Neurospora (Loros and Dunlap, 1991) and the cab gene family in plants (Nagy et al., 1988; Giuliano et al., 1988; Millar and Kay, 1991). In vertebrates, circadian regulation of gene transcription has been reported previously for only one other gene, that encoding D-binding protein (DBP), the liver-enriched transcriptional activator (Wuarin and Schibler, 1990). Our results provide a demonstration of clock-regulated gene transcription in avertebrate preparation maintained in cell culture. This

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in the circadian regulation of gene expression and may provide insight concerning retinal circadian oscillators and the mechanisms underlying the temporal regulation of photoreceptor metabolism. Experimental

Procedures

Animals and Retinal Cell Cultures For in vivo experiments, chickens were maintained on a 121:12D cycle for 3-4 weeks prior to use. Lights were turned on at 0800 CST and off at 2000 CST, which are equivalent to ZT 0 and ZT 12 by convention. At each time point, the anterior segment of the eye was removed, and the retina-pigment epithelium-vitreous was isolated and frozen in liquid NZ until RNA was prepared. Samples from time points in darkness were collected under a red safelight (Kodak IA filter). Retinal cell cultures were prepared on embryonic day 6 from chicken eggs maintained on a 12U12D cycle using procedures previously described by Adler (1986b). Enzymatically dispersed retinal cells were plated at high density (1 x 107to 1.5 x IO7 per 10 cm dish) in Medium 199 (GIBCO) supplemented with 10% heat-inactivated fetal calf serum (Hyclone) and 108 U/ml penicillin-100 uglml streptomycin. Cultures were incubated (37OC, 5% COJ on a 12L:12D cycle for at least 5 days before collecting RNA. Samples from time points in darknesswerecollected under infrared light. RNA Isolation and Northern Blot Analysis For both invivoand in vitroexperiments, total RNAwasprepared using the method of Chirgwin et al. (1979). Cells in culture were rinsed 2 times with Hank’s balanced salt solution (GIBCO) and scraped into the guanidinium isothiocyanate-containing solution, and RNA was prepared. Total RNA was separated by size in a 1% agarose denaturing formaldehyde gel, transferred, and UV cross-linked to nylon membranes. Blots were prehybridized for a least 1 hr at 42OC in 50% formamide, 10% dextran sulfate, 1 M NaCI, 0.1 mglml denatured salmon sperm DNA. Blots were hybridized overnight at 42OC in the same solution containing random-primed “P-labeled cDNA probes (alO cpm/ml). Blots were washed 3 times for 30 min at 65OC in 0.2 x SSC (Figure 1; Figure 2) or 2.0 x SSC (Figure 3; Figure 4), and 0.1% SDS. Autoradiographs were exposed at -70°C with an intensifying screen and analyzed using the Quantity One (Version 2) densitometry program (PDI, Inc., Huntington Station, NY). The chicken histone cDNA probe was from a fragment containing the full-length histone4gene (derived from pCH3dR8; Sugarman et al., 1983). Nuclear Run-on Transcriptional Assay Transcriptional analysis of nuclei prepared from cultured retinal cells was performed essentially as described by Banerji et al. (1984). At each time point, nuclei were prepared in room light or under infrared illumination (dark) as follows: culture plates were placed on ice, and cells were rapidly rinsed, scraped, and pelleted in ice cold phosphate-buffered saline (0.1 M, pH 7.2). While gently vortexing the pellet, 1 ml (per 1 x l(Y cells) of ice cold lysis buffer containing 10 mM NaCI, 3 mM MgCh, 10 mM Tris (pH 7.4), and 0.5% Nonidet P-40 was added. Cells were incubated for 5 min on ice, and then nuclei were pelleted. The pellet was resuspended in 1 ml of glycerol storage buffer (40% glycerol, 50 mM Tris [pH 8],5 mM MgCh, 0.1 mM EDTA), and nuclei were pelleted. When observed microscopically, nuclei were relatively clear of cellular debris. Isolated nuclei were resuspended in glycerol storage buffer (7 x 106 nuclei per 100 ul) and stored at-80°C. For run-on assays, 7 x 10s nuclei were used in each reaction. Elongation of nascent RNA transcripts was carried out at room temperature for 30 min in a reaction mixture containing 25 mM HEPES (pH 7.4), 2.5 mM MgCh, 2.5 mM dithiothreitol, 5% glycerol, ATP, CTP, and GTP (each at 350 PM), 0.4 pM UTP, and 300 NCi of [“P]UTP (3000 Ci/mmol; Amersham). The final reaction volume was 200 ~1. To stop the reaction, RNAase-free DNAase was added, and the mix was incubated for 10 min at 37OC. Reac-

tions were diluted with 3 volumes of buffer containing 2% SDS, 7 M urea, 0.35 M LiCI, 1 mM EDTA, and 10 mM Tris (pH 8). Proteinase K (500 @ml) and 100 ug of carrier tRNA were added, and tubes were incubated at 50°C for 1.5 hr. ZP-labeled RNA was precipitated using ice cold trichloroacetic acid (final IO%), followed by two ethanol precipitations. Air-dried pellets were resuspended in 100 ul of IO mM Tris, 1 mM EDTA. Filters with bound DNA were prehybridized (50% formamide, 6 x SSC, 10 x Denhardrs, 0.2% SDS) for at least 6 hr and then hybridized in the same solution containing SrP-labeled RNA for 36-72 hr at 50°C. The volume and counts per minute per hybridization reaction were equal (minimum 2 x 106 cpm/ml). Filters were washed in 2 x SSC, 0.5% SDS at 65°C and exposed to X-ray film. Under these conditions, no hybridization to vector sequences without insert was observed. Slot blot nitrocellulose filters were prepared using linearized plasmids containing iodopsin (see text), histone (Figure I), or Bluescript KS(+) vector (control) according to the manufacturer’s protocol (Schleicher and Schuell) using 5 ug of DNA per slot. To determine whether there was reinitiation of transcription, a time course of 32P incorporation into trichloroacetic acid-precipitable product was performed. Transcription was complete by 20 min, and no reinitiation was observed for at least 90 min. Acknowledgments The authors wish to thank Ms. D. Barker for technical assistance, Dr. J. C. Besharse for support while writing this manuscript, Drs. G. Cahill and C. Green for comments on this manuscript, Dr. D. Engel for pCH3dR8, used to generate histone probes, Dr. Tiansen Li for help in screening libraries, and Dr. J. Nathans for clone hs.7, used in the initial screening. This work was supported by National Eye Institute grants EYOEl67 to M. E. P., EY08467 to J. S. T., and EYO4801 to M. L. A. and by an NSF Center for Biological Timing grant to J. S. T. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”in accordance with 18 USC Section 1734 solely to indicate this fact. Received

October

29, 1992;

revised

December

18, 1992.

References Adler, R. (1986a). Trophic interactions in retinal development and in retinal degenerations. In vivo and in vitro studies. In The Retina, A Model for Cell Biology. Part I, R. Adler and D. Farber, eds. (Orlando, Florida: Academic Press), pp. 111-150. Adler, R. (1986b). Developmental tural and molecular polarization Biol. 117, 520-527.

predetermination of photoreceptor

of the struccells. Dev.

Araki, M., Fukada, Y., Shichida, Y., and Yoshizawa, T. (1990). Localization of iodopsin in the chick retina during in vivo and in vitro cone differentiation. Invest. Ophthalmol. Vis. Sci. 37,14661473. Banerji, S. S., Theodorakis, N. G., and Morimoto, R. I. (1984). Heat shock-induced translational control of HSP70 and globin synthesis in chicken reticulocytes. Mol. Cell. Biol. 4,2437-2448. Besharse, J. C., luvone, P. M., and Pierce, M. E. (1988). Regulation of rhythmic photoreceptor metabolism: a role for post-receptoral neurons. In Progress in Retinal Research, Volume 7, N. Osborne and C. Chader, eds. (New York: Pergamon Press), pp. 21-61. Bowes, C., van Veen, T., and Farber, D. (1988). Opsin, G-protein and 48-kd protein in normal and rd mouse retinas: developmental expression of mRNAs and proteins and light/dark cycling of mRNAs. Exp. Eye Res. 47, 369-390. Brann, M. R., and Cohen, L. V. (1987). Diurnal ducin mRNA and translocation of transducin Science 235, 585-587.

expression of transin rods of rat retina.

Cahill, C. M., and Besharse, J. C. (1993). Circadian clock functions localized in Xenopus retinal photoreceptors. Neuron, this issue.

Neuron 584

Cahill, G. M., Grace, M. S., and Besharse, J. C. (1991). Rhythmic regulation of retinal melatonin: metabolic pathways, neurochemical mechanisms and the ocular circadian clock. Cell. Mol. Neurobiol. 77, 529-560. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 78, 5294-5299. Craft, C. M., Whitmore, D. H., and Donoso, L. A. (1990). Differential expression of mRNAand protein encoding retinal and pineal S-antigen during light/dark cycle. J. Neurochem. 55,1461-1473. Darnell, J. E. (1982). Variety in the level of gene otic cells. Nature 297, 365-371. Dunlap, J. C. (1990). Closely watched of circadian rhythms in Neurospora Cenet. 65, 159-165.

control

in eukary-

clocks: molecular and Drosophila.

analysis Trends

Ciuliano, C., Hoffman, N. E., Kenton, K., Scolnik, P. A., and Cashmore, A. R. (1988). A light-entrained circadian clock controls transcription of several plant genes. EMBO J. 7, 3635-3642. Hall, J. C. (1990). Genet. 24, 659-97.

Genetics

of circadian

rhythms.

Annu.

Rev.

Kay, S. A., and Millar, A. J. (1992). Circadian-regulated cab gene transcription in higher plants. In Molecular Genetics of Biological Rhythms, M. Young, ed. (New York: Marcel Dekker), 73-90. Korenbrot, J. I., and Fernald, R. D. (1989). Circadian light regulate opsin mRNA in rod photoreceptors. 454-457.

rhythm Nature

and 337,

Kuwata, O., Imamoto, Y., Okano, T., Kokame, K., Kojima, D., Matsumoto, H., Morodome,A., Fukada, Y., Shichida,Y.,Yasuda, K., Shimura, Y., and Yoshizawa, T. (1990). The primary structure of iodopsin, a red-sensitive cone pigment. FEBS Lett. 272, 128132. Loros, J. J., and Dunlap, J. C. (1991). Neurospora crassa controlled genes are regulated at the level of transcription. Cell. Biol. 17, 558-563. Millar, A. J., and Kay, S. A. (1991). Circadian control transcription and mRNAaccumulation in Arabidopsis. 3, 541-550. Morse, D. S., Fritz, L.,and Hastings,J. Translational regulation of circadian Biochem. Sci. 15, 262-265.

clockMol.

of cab gene Plant Cell

W. (1990). What istheclock? bioluminescence. Trends

Nagy, F., Kay, S. A., and Chua, N. (1988). A circadian lates transcription of the wheat cab-7 gene. Genes 382.

clock reguDev. 2, 376-

Nathans, J., Thomas, D., and Hogness, D. S. (1986). Molecular genetics of human color vision: the genes encoding blue, green and red pigments. Science 232, 193-202. Pittendrigh, C. S. (1960). Circadian rhythmsand zation of living systems. Cold Spring Harbor 25,159-W%

circadian organiSymp. Quant. Biol.

Reme, C. E., Wirz-Justice, A., and Terman, M. (1991). The visual input stage of the mammalian circadian pacemaker system: I. Is there a clock in the mammalian eye? J. Biol. Rhythms 6,5-29. Sugarman, B. J., Dodgson, J. B., and Engel, D. (1983). Genomic organization, DNA sequence and expression of chicken embryonic histone genes. J. Biol. Chem. 258, 9005-9016. Takahashi, J. S. (1991). Circadian rhythms: from gene expression to behavior. Curr. Opin. Neurobiol. 7, 556-561. Wuarin, J., and Schibler, U. (1990). Expression of the liver-enriched transcriptional activator protein DBP follows a stringent circadian rhythm. Cell 63, 1257-1266. Young, R. W. (1978). The daily coneouter segment membranes thalmol. Vis. Sci. 77, 105-116.

rhythm of shedding in thechick retina.

of rod and Invest. Oph-