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Pinopsin mRNA levels are significantly elevated in the pineal glands of chickens carrying a null mutation in guanylate cyclase-1
Molecular Brain Research 97 (2001) 51–58 www.elsevier.com / locate / bres
Research report
Pinopsin mRNA levels are significantly elevated in the pineal glands of chickens carrying a null mutation in guanylate cyclase-1 Susan L. Semple-Rowland*, Miguel Tepedino, Jason E. Coleman University of Florida McKnight Brain Institute, Department of Neuroscience, 100 S. Newell Drive, Bldg. 59, Rm L1 -100, Gainesville, FL 32610 -0255, USA Accepted 16 October 2001
Abstract The purpose of this study was to determine if the absence of guanylate cyclase-1 (RetGC1, GC1), a key visual phototransduction cascade enzyme that is expressed in both retinal photoreceptors and pinealocytes, disrupts light regulation of pinopsin mRNA levels in the chicken pineal gland. In this series of experiments, we compared levels of pinopsin and tryptophan 5-hydroxylase mRNA in the pineal glands of GUCY1*B (*B) and normal chickens housed under either cyclic light or constant dark conditions. The *B chicken carries a null mutation in the gene encoding guanylate cyclase-1 that results in blindness in these animals at hatching. The results of our experiments show (1) that the amount of pinopsin mRNA in *B pineal is significantly higher than the amount in normal pineal in both light and dark conditions, (2) that light induces an increase in pinopsin mRNA levels in *B pineal, (3) that the relative magnitude of the light-induced increase in pinopsin mRNA in *B pineal is not significantly different from that observed in normal pineal, and (4) that the changes in the regulation of pinopsin mRNA levels in *B pineal gland are not accompanied by changes in the circadian expression of tryptophan 5-hydroxylase mRNA. These results show that the absence of guanylate cyclase-1 expression in the *B pineal gland leads to a significant increase in basal levels of pinopsin mRNA in this gland but does not alter the magnitude of the increase in pinopsin mRNA levels that is observed as a result of light stimulation. 2001 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Retina and photoreceptors Keywords: Circadian; Transcription; Light-induced; Guanylate cyclase-1; GUCY1*B chicken
1. Introduction Light plays an important role in regulating gene transcription, enzyme activity and the function of circadian oscillators in chicken pinealocytes. Studies of light regulation of melatonin production in avian pineal gland suggest that at least two light transduction cascades are present in this organ, the first suppresses melatonin release and involves a pertussis toxin-sensitive G-protein and the second messenger, cAMP, and the second entrains the pineal oscillators that drive the melatonin rhythm and does not appear to involve either of these cascade components
*Corresponding author. Tel.: 11-352-392-3598; fax: 11-352-3928347. E-mail address: [email protected] (S.L. Semple-Rowland).
[27–29]. Prompted by the structural similarities exhibited by pinealocytes and retinal photoreceptors, several studies have been conducted to determine if pinealocytes possess a light transduction cascade similar to that present in retinal photoreceptors, the results of which suggest that such a cascade is present in avian pinealocytes [5] (for reviews see Ref. [16]). The primary pigment molecule expressed in avian pineal is pinopsin (P-opsin), a member of the opsin protein family [10,14,17]. Pinopsin forms a light-sensitive pigment with an absorption maximum of |470 nm when bound to 11-cis retinal that is capable of activating rod transducin in vitro [15]. Recent studies show that both the rod transducin alpha subunit and Gq / 11 are expressed in chicken pinealocytes [8,13], either of which could participate in light transduction cascades initiated by pinopsin. Several additional retinal phototransduction cascade proteins are
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00297-2
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expressed in avian pineal including arrestin [24], guanylate cyclase-1 (RetGC1, GC1), guanylate cyclase activating protein-1 (GCAP1) and GCAP2 [19], cGMP-gated channel proteins [2–4] and rhodopsin kinase [30]. The observations that levels of cGMP exhibit a diurnal rhythm with levels peaking during the dark phase of the light / dark cycle [25] and that cGMP levels in chicken pineal are reduced by light [21] provide additional support for the hypothesis that a functional cGMP-mediated light transduction cascade similar to that found in retinal photoreceptors is present in avian pinealocytes. A role for cGMP in pineal physiology has not yet been established. Studies using the cGMP analogue, 8-bromo cGMP, suggest that the effects of light on melatonin production and circadian oscillator function in avian pineal are not dependent upon cGMP [28]. The recent observation that levels of pinopsin mRNA in the chicken pineal gland are regulated by light, independent of the pineal circadian system [22], is of interest in this regard, and raises the question of whether a cGMP-mediated light transduction cascade might not be involved in this process. The GUCY1*B (*B) chicken carries a null mutation in the gene encoding GC1, an enzyme that is normally expressed in retinal photoreceptors and pineal gland [18–20]. GC1 plays a critical role in the retinal phototransduction cascade and its absence severely compromises the ability of photoreceptors to transduce light for vision [20,26]. Interestingly, although GC1 is not expressed in the retinas and the pineal gland of the GUCY1*B chicken, only the retinas appears to degenerate [19]. In this series of experiments, we examined levels of pinopsin mRNA in *B and normal pineal under cyclic light and constant dark conditions to determine if the absence of GC1 alters light regulation of pinopsin mRNA levels. Levels of tryptophan 5-hydroxylase mRNA (TPH), a circadian clock-regulated transcript [6,7], were monitored as a control.
were approved by the Institutional Animal Care and Use Committee and followed all federal guidelines. For these experiments, normal and *B chicken eggs were incubated on a 12 h light:12 h dark (12L:12D) cycle in separate incubators that were located in light-proof boxes illuminated by 20 W cool white fluorescent bulbs (90 lux ambient). Lights were controlled by electronic timers and were turned on at 07:00 and off at 19:00 h, times designated as Zeitgeber time (ZT) 0 and ZT12, respectively. On day 1 post-hatch, the chickens were transferred from the incubators to separate noise- and light-resistant holding pens that were on the same light / dark cycle as the incubators. The ambient light level in the pens was 90 lux. Animal care was administered on a random schedule and food and water were available to the animals ad libitum. For the cyclic light experiments, normal and *B chickens were maintained on a 12L:12D cycle for the first 4 days after hatching. On day 5 post-hatch, normal and *B chickens were sacrificed at ZT0, ZT6, ZT12 and ZT18 and the pineal glands were quickly removed and frozen in liquid nitrogen. The samples at ZT0 were taken just after the lights came on and the samples at ZT12 were taken just after the lights went off. For the constant dark experiments, normal and *B chickens were entrained to a 12L:12D cycle for the first 4 days after hatching and on day 5 post-hatch, the lights were not turned on at the normal time. The animals in these experiments were sacrificed at circadian time (CT) 0, CT6, CT12 and CT18 during the first 24 h in constant darkness. The samples at CT0 were taken just after the lights would have come on and samples at CT12 were taken just after the lights would have gone off. A low intensity red safe light (15 W bulb with Kodak [2 filter) was used to facilitate all dissections that were carried out in darkness. Twelve chickens were sacrificed at each time point for the cyclic light and for the constant dark experiment.
2.2. Northern blot analyses 2. Materials and methods
2.1. Animals and tissue collection Breeding colonies of normal and *B Rhode Island Red chickens are maintained at the University of Florida. The *B and normal Rhode Island Red chickens used in this study were genetically related as a result of a series of conventional matings. Firstly, the *B chickens were outcrossed to normal Rhode Island Red chickens. The F1 heterozygotes were then backcrossed to the *B chickens. Finally, the resulting *B mutants from the backcross were bred and the progeny were used in these experiments. The normal chickens used in the study were the progeny of matings between members of the normal Rhode Island Red colony. All experimental protocols involving these animals
Total RNA was extracted from the pineal glands using an RNeasy kit (Qiagen, CA). Four pineal glands were pooled to obtain each RNA sample and three samples (n53) were analyzed at each time point. Northern blots were prepared (8 mg RNA / lane) and hybridized consecutively with radiolabeled cDNA probes specific for pinopsin, TPH and 18S rRNA as described previously [11]. The template for the pinopsin probe was a 1.36 kb EcoRI / HindIII fragment of the pinopsin cDNA clone, V2L1 (clone obtained from Dr. T. Okano, University of Tokyo). The cDNA templates for the TPH and 18S rRNA probes were obtained using PCR and the following primers: TPH, 59-AGG GAA CAA CTG AAT GAG AT and 59-GAC TGG GCG AAT GGT GAA AC; 18S rRNA, 59-GGT TGA TCC TGC CAG TAG CA and 59-CCC CCI GCC GTC CCT CTT A. Hybridization signals were quantita-
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tively evaluated using a BioRad Molecular Imager FX system. Analyses of pinopsin and TPH mRNA levels were carried out in two ways. Each blot was prepared so that it contained samples from both normal and *B pineal. Blots for the 12L:12D experiments and constant dark experiments contained samples that were taken at ZT0, 6, 12 and 18 and CT0, 6, 12 and 18, respectively. Running the samples in this manner made it possible to examine the relative changes in the levels of pinopsin and TPH transcripts that occurred within normal and *B pineal gland as a function of time, and to obtain normalized values for the amounts of each transcript in normal and *B pineal glands at each time point. Relative changes in pinopsin and TPH mRNA levels were calculated by dividing the values obtained for the pinopsin and TPH hybridization signals in each sample by the value obtained for the 18S rRNA in that same sample and expressing these values relative to the average pinopsin or TPH value obtained across time. To determine the normalized values for the amount of pinopsin and TPH mRNA present in each sample, the pinopsin and TPH hybridization signal values obtained for each sample were corrected for loading differences by multiplying the values by an18S rRNA correction factor that was determined by dividing each sample 18S rRNA hybridization signal value by the average of the 18S rRNA hybridization signal values obtained from all of the samples run on the blot. The resulting pinopsin and TPH values, corrected for differences in sample load, were expressed relative to the average of the pinopsin and TPH values obtained across time, respectively. Data were analyzed using one-way ANOVA of the ranked values (Kruskal–Wallis) and twoway ANOVA, balanced design (SigmaStat, Jandel, CA).
3. Results
3.1. Analyses of pinopsin mRNA levels in pineal glands of * B and normal chickens Light induces increases in the amount of pinopsin mRNA in the pineal gland through a mechanism independent of the circadian oscillators present in this gland [22]. In this series of experiments we sought to determine if the absence of GC1 alters light regulation of pinopsin mRNA levels in the pineal glands of *B chickens. To address this question, we compared the levels of pinopsin mRNA in the pineal glands of normal and *B chickens that were entrained to a 12L:12D cycle and were then either maintained on this cycle or were placed in constant dark conditions. In our first experiment, we analyzed pinopsin mRNA levels over the course of a single 12L:12D cycle taking samples at ZT0, 6, 12 and 18. Examination of the Northern
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blots showed that light produces an increase in the levels of the pinopsin transcript (|1.6 kb) in both normal and *B pineal glands and suggested that the light-induced increase in pinopsin mRNA in the *B pineal glands is much greater than that observed in normal pineal glands (Fig. 1A). Formal analyses of the normalized pinopsin mRNA levels in *B and normal pineal confirmed these observations. Two-way ANOVA revealed that the increase in mean pinopsin levels over the course of the 12-h light period was significant (F56.08, df53, P,0.01) and that the mean pinopsin mRNA levels in *B pineal were significantly greater than those in normal pineal (F526.14, df51, P, 0.001) (Fig. 1B). Post-hoc tests (Student–Newman–Keuls) revealed that the pinopsin levels in *B pineal at ZT6 and ZT12, which were not significantly different from each other, were significantly greater (P,0.05) than those measured at all times in normal pineal and at ZT0 and ZT18 in *B pineal. Examination of the levels of pinopsin mRNA in *B and normal pineal during the 12-h dark period suggested that the basal or dark levels of pinopsin mRNA in *B pineal glands were higher than those in normal pineal glands (Fig. 1A and B, compare ZT0 and ZT18). A comparison of the combined pinopsin mRNA values obtained under dark conditions (ZT0 and at ZT18) for *B pineal (0.8660.12; mean6S.E.M.) and normal pineal (0.4460.10) showed that the mean dark level of pinopsin mRNA in *B pineal was significantly higher than that observed in normal pineal (t522.76, df510, P5 0.02). In addition to the comparison of normalized pinopsin mRNA in *B and normal pineal, we also examined the relative changes in the levels of pinopsin mRNA that occurred over the course of the 12L:12D cycle within normal and within *B pineal gland (Fig. 1C). Analyses of these data using two-way ANOVA revealed that the relative increases in pinopsin mRNA in *B and in normal pineal glands were not significantly different from each other (F50.0, df51, P51.0). In both *B and normal pineal there was a significant increase in the relative levels of pinopsin mRNA during the 12-h light period (F510.81, df53, P,0.001). Post-hoc tests (Student–Newman– Keuls) revealed that the relative amount of pinopsin mRNA measured in *B pineal at ZT6 was significantly higher (P,0.05) than the levels measured at ZT0 and ZT18. In normal pineal, the relative amount of pinopsin mRNA measured at ZT12 was significantly higher (P, 0.05) than that measured at ZT0. In our second experiment, we examined pinopsin mRNA levels in pineal glands of *B and normal chickens entrained to a 12L:12D cycle and then placed in continuous darkness. No significant changes were observed in the levels of pinopsin mRNA in *B and in normal pineal glands over the course of the constant dark period; however, a comparison of the mean dark levels of pinopsin mRNA measured in *B pineal (1.1060.11; mean6S.E.M.) and normal pineal (0.5660.04) (Fig. 1D) showed that the
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Fig. 1. Comparison of pinopsin mRNA levels in pineal glands of *B and normal chickens housed under cyclic light and constant dark conditions. (A) Representative Northern blot hybridized consecutively with pinopsin and 18S rRNA probes. Each sample (8 mg total RNA) contained RNA extracted from four pineal glands. The experiment was repeated three times with similar results. Normal and *B chickens entrained to and housed under a 12L:12D cycle were sacrificed at ZT0, ZT6, ZT12 and ZT18. (B) Normalized pinopsin mRNA levels in the pineal glands of *B (white) and in normal (black) chickens housed under 12L:12D conditions. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. The values obtained at ZT0 were replotted at ZT24. Mean levels of pinopsin mRNA in *B pineal at ZT6 and ZT12 were not significantly different from each other but were significantly (*P,0.05) higher than those measured at all times in normal pineal and at ZT0 and ZT18 in *B pineal. (C) Comparison of the changes in the relative levels of pinopsin mRNA within the pineal glands of *B chickens (white) and normal (black) chickens over the course of a 12L:12D period. The values obtained at ZT0 were replotted at ZT24. The relative amount of pinopsin mRNA measured in *B pineal was significantly higher (*P,0.05) at ZT6 than at ZT0 and ZT18. The relative amount of pinopsin mRNA measured in normal pineal was significantly higher (*P,0.05) at ZT12 than at ZT0. (D) Comparison of mean normalized pinopsin mRNA levels in pineal glands of normal and *B chickens maintained in constant darkness. Each bar represents the mean6S.E.M. of the pinopsin mRNA levels in 11 normal and 12 *B pineal samples (four pineal glands in each sample). Mean pinopsin mRNA in *B pineal were significantly higher (*P,0.05) than those measured in normal pineal. Pineal glands were procured at CT0, CT6, CT12 and CT18 from chickens that had been entrained to a 12L:12D cycle and then placed in constant darkness.
level of pinopsin mRNA in *B pineal was significantly higher than that observed in normal pineal (t524.57, df521, P,0.001). This result is consistent with our
comparison of the mean levels of pinopsin mRNA measured in *B and normal pineal during the 12-h dark period of the 12L:12D cycle.
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3.2. Analyses of TPH mRNA levels in pineal glands of * B and normal chickens In view of the changes observed in the regulation of pinopsin mRNA levels in *B pineal, we felt it was important to include in our analyses a gene whose expression in pineal is regulated by the pineal circadian clock. Transcription of these genes would not be expected to change in *B pineal since light entrainment of the pineal circadian oscillator has been shown to occur independent of changes in cGMP levels [28]. Previous studies have shown that the amount of TPH mRNA in chicken pineal is controlled by the pineal circadian clock, the levels of TPH mRNA increasing over the course of the day, reaching peak levels in early night and returning to low levels by the beginning of the next day [6,7]. Based on these results, we chose to measure transcript levels of the TPH gene in the pineal glands of *B and normal chickens that were entrained to a 12L:12D cycle and were then either kept on this cycle or were placed in continuous dark conditions. In our first experiment, we examined TPH mRNA levels in *B and normal pineal glands over the course of a single 12L:12D cycle taking samples at ZT0, 6, 12 and 18 (Fig. 2A). Comparisons of the normalized (Fig. 2B) and relative (Fig. 2C) TPH transcript profiles in *B and in normal pineal using two-way ANOVA revealed that the profiles in *B and normal pineal glands were not significantly different from each other. In both *B and normal pineal gland, the levels of TPH mRNA increased over the course of the 12-h light period, reaching maximum levels |6 h into the 12-h dark period. Post-hoc tests (Student–Newman–Keuls) revealed that the relative levels of TPH mRNA in both *B and normal pineal at ZT18 were significantly higher (P, 0.05) than those measured at ZT0 (Fig. 2C). In our second experiment, we analyzed TPH mRNA levels in the pineal glands of *B and normal chickens entrained to a 12L:12D cycle and then placed in continuous darkness. The results of these analyses showed that the TPH mRNA rhythms observed under cyclic light conditions were maintained in *B and in normal pineal glands in constant darkness (Fig. 2D and E). Comparisons of the normalized (Fig. 2D) and relative (Fig. 2E) TPH transcript profiles in *B and normal pineal using two-way ANOVA revealed that the TPH profiles in *B and normal pineal in constant dark conditions were not significantly different from each other. TPH levels in *B and in normal pineal did vary significantly as a function of time (F513.08, df53, P,0.001), the TPH mRNA levels reaching maximum levels at CT12.
4. Discussion The purpose of this study was to determine if light regulation of pinopsin mRNA levels in chicken pineal gland is dependent on GC1, a key enzyme in the visual
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phototransduction cascade. The results of our study show (1) that light is able to induce an increase in pinopsin mRNA in the pineal gland in the absence of GC1, (2) that the levels of pinopsin mRNA in *B pineal in dark conditions (basal levels) are significantly higher than those observed in normal pineal, (3) that the relative magnitudes of the light-induced increases in pinopsin mRNA are not statistically different in *B and normal pineal, and (4) that the changes observed in the regulation of basal levels of pinopsin mRNA in *B pineal gland are not accompanied by changes in the circadian expression of TPH mRNA. The results of our study show that the absence of GC1 alters basal levels of pinopsin mRNA in pineal without affecting the ability of light to produce increases in pinopsin mRNA. These data suggest that the mechanisms in pineal that regulate basal levels of pinopsin mRNA and mediate the light-dependent increases in pinopsin mRNA levels function independently of each other, and that the signal transduction cascade that mediates the light-driven increases in pinopsin mRNA in pineal is not dependent on GC1. One possible mechanism by which the absence of GC1 could alter regulation of basal levels of pinopsin mRNA in pineal is suggested by our studies of *B retinal photoreceptors. In *B retina, the absence of GC1 produces a significant and permanent reduction in the amount of cGMP in the photoreceptor cells, a condition which prevents these cells from transducing light for vision [20]. We have proposed that reduced cGMP levels in these cells leads to chronic closure of the cGMP-gated cation channels located in the plasma membranes and to chronic hyperpolarization of these cells. This hypothesis is supported by the observation that levels of glutamate, the neurotransmitter released by depolarized photoreceptors in the dark, are elevated in GUCY1*B photoreceptors regardless of the light-adapted state of the retina [23]. Chicken pinealocytes and retinal photoreceptors share many structural features [1] and many of the genes that encode the visual phototransduction cascade proteins in photoreceptors are expressed in pinealocytes, including the genes that encode the rod and cone cGMP-gated channel proteins [2]. Studies of the activities of these channels in pinealocytes have shown that they respond to changes in cGMP levels in a manner similar to that observed in photoreceptor cells [3,4]. The precise function of these channels in pinealocytes is not known; however, it is likely that in addition to playing a role in the light response of these cells, these channels may also play a role in controlling [Ca 21 ] i , their activity being modulated by changes in levels of cAMP and cGMP brought about by the actions of cellular G-protein-coupled cascades (for review see Ref. [9]). Based on these observations, it is reasonable to postulate that the absence of GC1 in pinealocytes alters their physiology in a manner similar to that observed in photoreceptors. To date, there is no definitive evidence that the visual phototransduction cascade is active in chicken
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Fig. 2. Comparison of TPH mRNA levels in pineal glands of *B and normal chickens housed under cyclic light and constant dark conditions. (A) Northern blot shown in Fig. 1A hybridized with a TPH probe. (B) Normalized TPH mRNA levels present in the pineal glands of *B (white) and normal (black) chickens housed under 12L:12D conditions. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. (C) Comparison of relative levels of TPH mRNA measured within *B pineal (white) and within normal pineal (black) over the course of a 12L:12D period. The relative amounts of TPH mRNA measured in *B and normal pineal at ZT18 were significantly higher (*P,0.05) than at all other times examined. (D) Normalized TPH mRNA levels present in the pineal glands of *B (white) and normal (black) chickens housed under constant dark conditions. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. Levels of TPH mRNA in *B and normal pineal at CT12 were significantly greater (*P,0.05) than those observed at CT0 and CT6. (E) Comparison of the changes in the relative levels of TPH mRNA within the pineal glands of *B chickens (white) and normal chickens (black) entrained to a 12L:12D cycle and then sacrificed over the course of a 24-h constant dark period at CT0, CT6, CT12 and CT18. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. Levels of TPH mRNA in *B and normal pineal at CT12 were significantly greater (*P,0.05) than those observed at CT0 and CT6. In (B)–(E), the values obtained at ZT0 and CT0 were replotted at ZT24 and CT24, respectively.
pinealocytes. If such a cascade is present in these cells, then we would predict that the absence of GC1 would lead to chronic closure of the cGMP-gated channels in the membranes of these cells, a change that would place these
cells in a chronic physiological state resembling that of light exposed cells. Interestingly, our observation that the levels of pinopsin mRNA in the pineal glands of *B chickens housed in the dark approximate those in the
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pineal glands of normal chickens exposed to 12 h of light is consistent with this prediction. The biochemical mechanism controlling basal levels of pinopsin mRNA in pineal is not known; however, if our hypothesis that elevation of basal levels of pinopsin mRNA in *B pineal is a result of closure of the cGMP-gated channels, then it is possible that intracellular cation levels play an important role in this mechanism. Our analyses of TPH transcripts show that the rhythmic changes observed in TPH mRNA levels in *B and normal pineal in both cyclic light and constant darkness were similar indicating that the circadian oscillators that drive the TPH mRNA rhythm in *B pineal are functional and can be entrained to light in the absence of GC1. This result is consistent with previous analyses of avian pineal that showed that 8-bromo-cGMP had little effect on the acute or phase-shifting effects of light on pineal melatonin rhythms [28]. Currently, there is no evidence linking cGMP to circadian function in the pineal gland; however, the results of a recent study of the circadian clock-regulated iodopsin rhythm in cone photoreceptors of *B chicken suggest that changes in intracellular cGMP levels modulate circadian processes in photoreceptors [12]. In *B photoreceptors, iodopsin rhythms are temporally compressed in cyclic light and the phase of the rhythms is advanced by 6 h in constant darkness. In addition, the rate of entrainment of these rhythms to a reversal of the light / dark cycle is significantly slower in *B photoreceptors than in normal retina. In view of these results, it may be fruitful to examine the entrainment properties of the TPH mRNA rhythm in *B pineal following reversal of the light / dark cycle.
[5] [6]
[7]
[8]
[9] [10] [11]
[12]
[13]
[14]
[15]
[16] [17] [18]
Acknowledgements [19]
We thank Dr. Patrick Larkin for teaching the intricacies of running Northern blots to M.T. This research study was funded by NIH grant EY11388. M.T. received financial support from the University of Florida Scholars program and J.E.C. received partial support from NIH grant EY07132.
[20]
[21]
References [1] M.B. Bischoff, Photoreceptoral and secretory structures in the avian pineal organ, J. Ultrastruct. Res 28 (1969) 16–26. [2] W. Bonigk, F. Muller, R. Middendorff, I. Weyand, U.B. Kaupp, Two alternatively spliced forms of the cGMP-gated channel alpha-subunit from cone photoreceptor are expressed in the chick pineal organ, J. Neurosci. 16 (1996) 7458–7468. [3] S.E. Dryer, D. Henderson, A cyclic GMP-activated channel in dissociated cells of the chick pineal gland, Nature 353 (1991) 756–758. [4] S.E. Dryer, D. Henderson, Cyclic GMP-activated channels of the
[22]
[23]
[24]
[25]
57
chick pineal gland: effects of divalent cations, pH, and cyclic AMP, J. Comp. Physiol. A 172 (1993) 271–279. J. Falcon, Cellular circadian clocks in the pineal, Prog. Neurobiol. 58 (1999) 121–162. J.C. Florez, K.J. Seidenman, R.K. Barrett, A.M. Sangoram, J.S. Takahashi, Molecular cloning of chick pineal tryptophan hydroxylase and circadian oscillation of its mRNA levels, Mol. Brain Res. 42 (1996) 25–30. C.B. Green, J.C. Besharse, M. Zatz, Tryptophan hydroxylase mRNA levels are regulated by the circadian clock, temperature, and cAMP in chick pineal cells, Brain Res. 738 (1996) 1–7. T. Kasahara, T. Okano, T. Yoshikawa, K. Yamazaki, Y. Fukada, Rod-type transducin alpha-subunit mediates a phototransduction pathway in the chicken pineal gland, J. Neurochem. 75 (2000) 217–224. U.B. Kaupp, Family of cyclic nucleotide gated ion channels, Curr. Opin. Neurobiol. 5 (1995) 434–442. S. Kawamura, S. Yokoyama, Molecular characterization of the pigeon P-opsin gene, Gene 182 (1996) 213–214. P. Larkin, W. Baehr, S.L. Semple-Rowland, Circadian regulation of iodopsin and clock is altered in the retinal degeneration chicken retina, Mol. Brain Res. 70 (1999) 253–263. P. Larkin, S.L. Semple-Rowland, A null mutation in guanylate cyclase-1 alters the temporal dynamics and light entrainment properties of the iodopsin rhythm in cone photoreceptor cells, Mol. Brain Res. 92 (2001) 49–57. A. Matsushita, T. Yoshikawa, T. Okano, T. Kasahara, Y. Fukada, Colocalization of pinopsin with two types of G-protein alphasubunits in the chicken pineal gland, Cell Tissue Res. 299 (2000) 245–251. M. Max, P.J. McKinnon, K.J. Seidenman, R.K. Barrett, M.L. Applebury, J.S. Takahashi, R.F. Margolskee, Pineal opsin: a nonvisual opsin expressed in chick pineal, Science 267 (1995) 1502– 1506. M. Max, A. Surya, J.S. Takahashi, R.F. Margolskee, B.E. Knox, Light-dependent activation of rod transducin by pineal opsin, J. Biol. Chem. 273 (1998) 26820–26826. H. Meissl, Photic regulation of pineal function. Analogies between retinal and pineal photoreception, Biol. Cell 89 (1997) 549–554. T. Okano, T. Yoshizawa, Y. Fukada, Pinopsin is a chicken pineal photoreceptive molecule, Nature 372 (1994) 94–97. S.L. Semple-Rowland, K.M. Cheng, rd and rc chickens carry the same GC1 null allele (GUCY1*), Exp. Eye Res. 69 (1999) 579– 581, letter. S.L. Semple-Rowland, P. Larkin, J.D. Bronson, K. Nykamp, W.J. Streit, W. Baehr, Characterization of the chicken GCAP gene array and analyses of GCAP1, GCAP2, and GC1 gene expression in normal and rd chicken pineal, Mol. Vis. 5 (1999) 14. S.L. Semple-Rowland, N.R. Lee, J.P. Van Hooser, K. Palczewski, W. Baehr, A null mutation in the photoreceptor guanylate cyclase gene causes the retinal degeneration chicken phenotype, Proc. Natl. Acad. Sci. USA 95 (1998) 1271–1276. J.S. Takahashi, M. Zatz, Photic regulation of cyclic nucleotide levels and N-acetyltransferase activity in the cultured avian pineal, Soc. Neurosci. Abs. 8 (1982) 546. Y. Takanaka, T. Okano, M. Iigo, Y. Fukada, Light-dependent expression of pinopsin gene in chicken pineal gland, J. Neurochem. 70 (1998) 908–913. R.J. Ulshafer, D.M. Sherry, R. Dawson Jr., D.R. Wallace, Excitatory amino acid involvement in retinal degeneration, Brain Res. 531 (1990) 350–354. T. van Veen, R. Elofsson, H.G. Hartwig, I. Gery, M. Mochizuki, V. Cena, D.C. Klein, Retinal S-antigen: immunocytochemical and immunochemical studies on distribution in animal photoreceptors and pineal organs, Exp. Biol. 45 (1986) 15–25. S.D. Wainwright, Diurnal cycles in serotonin acetyltransferase activity and cyclic GMP content of cultured chick pineal glands, Nature 285 (1980) 478–480.
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[26] R.B. Yang, S.W. Robinson, W.H. Xiong, K.W. Yau, D.G. Birch, D.L. Garbers, Disruption of a retinal guanylyl cyclase gene leads to cone-specific dystrophy and paradoxical rod behavior, J. Neurosci. 19 (1999) 5889–5897. [27] M. Zatz, Photoendocrine transduction in cultured chick pineal cells: IV. What do vitamin A depletion and retinaldehyde addition do to the effects of light on the melatonin rhythm?, J. Neurochem. 62 (1994) 2001–2011. [28] M. Zatz, D.A. Mullen, Photoendocrine transduction in cultured chick pineal cells. II. Effects of forskolin, 8-bromocyclic AMP, and
8-bromocyclic GMP on the melatonin rhythm, Brain Res. 453 (1988) 51–62. [29] M. Zatz, D.A. Mullen, Two mechanisms of photoendocrine transduction in cultured chick pineal cells: pertussis toxin blocks the acute but not the phase-shifting effects of light on the melatonin rhythm, Brain Res. 453 (1988) 63–71. [30] X. Zhao, K. Yokoyama, M.E. Whitten, J. Huang, M.H. Gelb, K. Palczewski, A novel form of rhodopsin kinase from chicken retina and pineal gland, FEBS Lett. 454 (1999) 115–121.
Research report
Pinopsin mRNA levels are significantly elevated in the pineal glands of chickens carrying a null mutation in guanylate cyclase-1 Susan L. Semple-Rowland*, Miguel Tepedino, Jason E. Coleman University of Florida McKnight Brain Institute, Department of Neuroscience, 100 S. Newell Drive, Bldg. 59, Rm L1 -100, Gainesville, FL 32610 -0255, USA Accepted 16 October 2001
Abstract The purpose of this study was to determine if the absence of guanylate cyclase-1 (RetGC1, GC1), a key visual phototransduction cascade enzyme that is expressed in both retinal photoreceptors and pinealocytes, disrupts light regulation of pinopsin mRNA levels in the chicken pineal gland. In this series of experiments, we compared levels of pinopsin and tryptophan 5-hydroxylase mRNA in the pineal glands of GUCY1*B (*B) and normal chickens housed under either cyclic light or constant dark conditions. The *B chicken carries a null mutation in the gene encoding guanylate cyclase-1 that results in blindness in these animals at hatching. The results of our experiments show (1) that the amount of pinopsin mRNA in *B pineal is significantly higher than the amount in normal pineal in both light and dark conditions, (2) that light induces an increase in pinopsin mRNA levels in *B pineal, (3) that the relative magnitude of the light-induced increase in pinopsin mRNA in *B pineal is not significantly different from that observed in normal pineal, and (4) that the changes in the regulation of pinopsin mRNA levels in *B pineal gland are not accompanied by changes in the circadian expression of tryptophan 5-hydroxylase mRNA. These results show that the absence of guanylate cyclase-1 expression in the *B pineal gland leads to a significant increase in basal levels of pinopsin mRNA in this gland but does not alter the magnitude of the increase in pinopsin mRNA levels that is observed as a result of light stimulation. 2001 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Retina and photoreceptors Keywords: Circadian; Transcription; Light-induced; Guanylate cyclase-1; GUCY1*B chicken
1. Introduction Light plays an important role in regulating gene transcription, enzyme activity and the function of circadian oscillators in chicken pinealocytes. Studies of light regulation of melatonin production in avian pineal gland suggest that at least two light transduction cascades are present in this organ, the first suppresses melatonin release and involves a pertussis toxin-sensitive G-protein and the second messenger, cAMP, and the second entrains the pineal oscillators that drive the melatonin rhythm and does not appear to involve either of these cascade components
*Corresponding author. Tel.: 11-352-392-3598; fax: 11-352-3928347. E-mail address: [email protected] (S.L. Semple-Rowland).
[27–29]. Prompted by the structural similarities exhibited by pinealocytes and retinal photoreceptors, several studies have been conducted to determine if pinealocytes possess a light transduction cascade similar to that present in retinal photoreceptors, the results of which suggest that such a cascade is present in avian pinealocytes [5] (for reviews see Ref. [16]). The primary pigment molecule expressed in avian pineal is pinopsin (P-opsin), a member of the opsin protein family [10,14,17]. Pinopsin forms a light-sensitive pigment with an absorption maximum of |470 nm when bound to 11-cis retinal that is capable of activating rod transducin in vitro [15]. Recent studies show that both the rod transducin alpha subunit and Gq / 11 are expressed in chicken pinealocytes [8,13], either of which could participate in light transduction cascades initiated by pinopsin. Several additional retinal phototransduction cascade proteins are
0169-328X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 01 )00297-2
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expressed in avian pineal including arrestin [24], guanylate cyclase-1 (RetGC1, GC1), guanylate cyclase activating protein-1 (GCAP1) and GCAP2 [19], cGMP-gated channel proteins [2–4] and rhodopsin kinase [30]. The observations that levels of cGMP exhibit a diurnal rhythm with levels peaking during the dark phase of the light / dark cycle [25] and that cGMP levels in chicken pineal are reduced by light [21] provide additional support for the hypothesis that a functional cGMP-mediated light transduction cascade similar to that found in retinal photoreceptors is present in avian pinealocytes. A role for cGMP in pineal physiology has not yet been established. Studies using the cGMP analogue, 8-bromo cGMP, suggest that the effects of light on melatonin production and circadian oscillator function in avian pineal are not dependent upon cGMP [28]. The recent observation that levels of pinopsin mRNA in the chicken pineal gland are regulated by light, independent of the pineal circadian system [22], is of interest in this regard, and raises the question of whether a cGMP-mediated light transduction cascade might not be involved in this process. The GUCY1*B (*B) chicken carries a null mutation in the gene encoding GC1, an enzyme that is normally expressed in retinal photoreceptors and pineal gland [18–20]. GC1 plays a critical role in the retinal phototransduction cascade and its absence severely compromises the ability of photoreceptors to transduce light for vision [20,26]. Interestingly, although GC1 is not expressed in the retinas and the pineal gland of the GUCY1*B chicken, only the retinas appears to degenerate [19]. In this series of experiments, we examined levels of pinopsin mRNA in *B and normal pineal under cyclic light and constant dark conditions to determine if the absence of GC1 alters light regulation of pinopsin mRNA levels. Levels of tryptophan 5-hydroxylase mRNA (TPH), a circadian clock-regulated transcript [6,7], were monitored as a control.
were approved by the Institutional Animal Care and Use Committee and followed all federal guidelines. For these experiments, normal and *B chicken eggs were incubated on a 12 h light:12 h dark (12L:12D) cycle in separate incubators that were located in light-proof boxes illuminated by 20 W cool white fluorescent bulbs (90 lux ambient). Lights were controlled by electronic timers and were turned on at 07:00 and off at 19:00 h, times designated as Zeitgeber time (ZT) 0 and ZT12, respectively. On day 1 post-hatch, the chickens were transferred from the incubators to separate noise- and light-resistant holding pens that were on the same light / dark cycle as the incubators. The ambient light level in the pens was 90 lux. Animal care was administered on a random schedule and food and water were available to the animals ad libitum. For the cyclic light experiments, normal and *B chickens were maintained on a 12L:12D cycle for the first 4 days after hatching. On day 5 post-hatch, normal and *B chickens were sacrificed at ZT0, ZT6, ZT12 and ZT18 and the pineal glands were quickly removed and frozen in liquid nitrogen. The samples at ZT0 were taken just after the lights came on and the samples at ZT12 were taken just after the lights went off. For the constant dark experiments, normal and *B chickens were entrained to a 12L:12D cycle for the first 4 days after hatching and on day 5 post-hatch, the lights were not turned on at the normal time. The animals in these experiments were sacrificed at circadian time (CT) 0, CT6, CT12 and CT18 during the first 24 h in constant darkness. The samples at CT0 were taken just after the lights would have come on and samples at CT12 were taken just after the lights would have gone off. A low intensity red safe light (15 W bulb with Kodak [2 filter) was used to facilitate all dissections that were carried out in darkness. Twelve chickens were sacrificed at each time point for the cyclic light and for the constant dark experiment.
2.2. Northern blot analyses 2. Materials and methods
2.1. Animals and tissue collection Breeding colonies of normal and *B Rhode Island Red chickens are maintained at the University of Florida. The *B and normal Rhode Island Red chickens used in this study were genetically related as a result of a series of conventional matings. Firstly, the *B chickens were outcrossed to normal Rhode Island Red chickens. The F1 heterozygotes were then backcrossed to the *B chickens. Finally, the resulting *B mutants from the backcross were bred and the progeny were used in these experiments. The normal chickens used in the study were the progeny of matings between members of the normal Rhode Island Red colony. All experimental protocols involving these animals
Total RNA was extracted from the pineal glands using an RNeasy kit (Qiagen, CA). Four pineal glands were pooled to obtain each RNA sample and three samples (n53) were analyzed at each time point. Northern blots were prepared (8 mg RNA / lane) and hybridized consecutively with radiolabeled cDNA probes specific for pinopsin, TPH and 18S rRNA as described previously [11]. The template for the pinopsin probe was a 1.36 kb EcoRI / HindIII fragment of the pinopsin cDNA clone, V2L1 (clone obtained from Dr. T. Okano, University of Tokyo). The cDNA templates for the TPH and 18S rRNA probes were obtained using PCR and the following primers: TPH, 59-AGG GAA CAA CTG AAT GAG AT and 59-GAC TGG GCG AAT GGT GAA AC; 18S rRNA, 59-GGT TGA TCC TGC CAG TAG CA and 59-CCC CCI GCC GTC CCT CTT A. Hybridization signals were quantita-
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tively evaluated using a BioRad Molecular Imager FX system. Analyses of pinopsin and TPH mRNA levels were carried out in two ways. Each blot was prepared so that it contained samples from both normal and *B pineal. Blots for the 12L:12D experiments and constant dark experiments contained samples that were taken at ZT0, 6, 12 and 18 and CT0, 6, 12 and 18, respectively. Running the samples in this manner made it possible to examine the relative changes in the levels of pinopsin and TPH transcripts that occurred within normal and *B pineal gland as a function of time, and to obtain normalized values for the amounts of each transcript in normal and *B pineal glands at each time point. Relative changes in pinopsin and TPH mRNA levels were calculated by dividing the values obtained for the pinopsin and TPH hybridization signals in each sample by the value obtained for the 18S rRNA in that same sample and expressing these values relative to the average pinopsin or TPH value obtained across time. To determine the normalized values for the amount of pinopsin and TPH mRNA present in each sample, the pinopsin and TPH hybridization signal values obtained for each sample were corrected for loading differences by multiplying the values by an18S rRNA correction factor that was determined by dividing each sample 18S rRNA hybridization signal value by the average of the 18S rRNA hybridization signal values obtained from all of the samples run on the blot. The resulting pinopsin and TPH values, corrected for differences in sample load, were expressed relative to the average of the pinopsin and TPH values obtained across time, respectively. Data were analyzed using one-way ANOVA of the ranked values (Kruskal–Wallis) and twoway ANOVA, balanced design (SigmaStat, Jandel, CA).
3. Results
3.1. Analyses of pinopsin mRNA levels in pineal glands of * B and normal chickens Light induces increases in the amount of pinopsin mRNA in the pineal gland through a mechanism independent of the circadian oscillators present in this gland [22]. In this series of experiments we sought to determine if the absence of GC1 alters light regulation of pinopsin mRNA levels in the pineal glands of *B chickens. To address this question, we compared the levels of pinopsin mRNA in the pineal glands of normal and *B chickens that were entrained to a 12L:12D cycle and were then either maintained on this cycle or were placed in constant dark conditions. In our first experiment, we analyzed pinopsin mRNA levels over the course of a single 12L:12D cycle taking samples at ZT0, 6, 12 and 18. Examination of the Northern
53
blots showed that light produces an increase in the levels of the pinopsin transcript (|1.6 kb) in both normal and *B pineal glands and suggested that the light-induced increase in pinopsin mRNA in the *B pineal glands is much greater than that observed in normal pineal glands (Fig. 1A). Formal analyses of the normalized pinopsin mRNA levels in *B and normal pineal confirmed these observations. Two-way ANOVA revealed that the increase in mean pinopsin levels over the course of the 12-h light period was significant (F56.08, df53, P,0.01) and that the mean pinopsin mRNA levels in *B pineal were significantly greater than those in normal pineal (F526.14, df51, P, 0.001) (Fig. 1B). Post-hoc tests (Student–Newman–Keuls) revealed that the pinopsin levels in *B pineal at ZT6 and ZT12, which were not significantly different from each other, were significantly greater (P,0.05) than those measured at all times in normal pineal and at ZT0 and ZT18 in *B pineal. Examination of the levels of pinopsin mRNA in *B and normal pineal during the 12-h dark period suggested that the basal or dark levels of pinopsin mRNA in *B pineal glands were higher than those in normal pineal glands (Fig. 1A and B, compare ZT0 and ZT18). A comparison of the combined pinopsin mRNA values obtained under dark conditions (ZT0 and at ZT18) for *B pineal (0.8660.12; mean6S.E.M.) and normal pineal (0.4460.10) showed that the mean dark level of pinopsin mRNA in *B pineal was significantly higher than that observed in normal pineal (t522.76, df510, P5 0.02). In addition to the comparison of normalized pinopsin mRNA in *B and normal pineal, we also examined the relative changes in the levels of pinopsin mRNA that occurred over the course of the 12L:12D cycle within normal and within *B pineal gland (Fig. 1C). Analyses of these data using two-way ANOVA revealed that the relative increases in pinopsin mRNA in *B and in normal pineal glands were not significantly different from each other (F50.0, df51, P51.0). In both *B and normal pineal there was a significant increase in the relative levels of pinopsin mRNA during the 12-h light period (F510.81, df53, P,0.001). Post-hoc tests (Student–Newman– Keuls) revealed that the relative amount of pinopsin mRNA measured in *B pineal at ZT6 was significantly higher (P,0.05) than the levels measured at ZT0 and ZT18. In normal pineal, the relative amount of pinopsin mRNA measured at ZT12 was significantly higher (P, 0.05) than that measured at ZT0. In our second experiment, we examined pinopsin mRNA levels in pineal glands of *B and normal chickens entrained to a 12L:12D cycle and then placed in continuous darkness. No significant changes were observed in the levels of pinopsin mRNA in *B and in normal pineal glands over the course of the constant dark period; however, a comparison of the mean dark levels of pinopsin mRNA measured in *B pineal (1.1060.11; mean6S.E.M.) and normal pineal (0.5660.04) (Fig. 1D) showed that the
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Fig. 1. Comparison of pinopsin mRNA levels in pineal glands of *B and normal chickens housed under cyclic light and constant dark conditions. (A) Representative Northern blot hybridized consecutively with pinopsin and 18S rRNA probes. Each sample (8 mg total RNA) contained RNA extracted from four pineal glands. The experiment was repeated three times with similar results. Normal and *B chickens entrained to and housed under a 12L:12D cycle were sacrificed at ZT0, ZT6, ZT12 and ZT18. (B) Normalized pinopsin mRNA levels in the pineal glands of *B (white) and in normal (black) chickens housed under 12L:12D conditions. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. The values obtained at ZT0 were replotted at ZT24. Mean levels of pinopsin mRNA in *B pineal at ZT6 and ZT12 were not significantly different from each other but were significantly (*P,0.05) higher than those measured at all times in normal pineal and at ZT0 and ZT18 in *B pineal. (C) Comparison of the changes in the relative levels of pinopsin mRNA within the pineal glands of *B chickens (white) and normal (black) chickens over the course of a 12L:12D period. The values obtained at ZT0 were replotted at ZT24. The relative amount of pinopsin mRNA measured in *B pineal was significantly higher (*P,0.05) at ZT6 than at ZT0 and ZT18. The relative amount of pinopsin mRNA measured in normal pineal was significantly higher (*P,0.05) at ZT12 than at ZT0. (D) Comparison of mean normalized pinopsin mRNA levels in pineal glands of normal and *B chickens maintained in constant darkness. Each bar represents the mean6S.E.M. of the pinopsin mRNA levels in 11 normal and 12 *B pineal samples (four pineal glands in each sample). Mean pinopsin mRNA in *B pineal were significantly higher (*P,0.05) than those measured in normal pineal. Pineal glands were procured at CT0, CT6, CT12 and CT18 from chickens that had been entrained to a 12L:12D cycle and then placed in constant darkness.
level of pinopsin mRNA in *B pineal was significantly higher than that observed in normal pineal (t524.57, df521, P,0.001). This result is consistent with our
comparison of the mean levels of pinopsin mRNA measured in *B and normal pineal during the 12-h dark period of the 12L:12D cycle.
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3.2. Analyses of TPH mRNA levels in pineal glands of * B and normal chickens In view of the changes observed in the regulation of pinopsin mRNA levels in *B pineal, we felt it was important to include in our analyses a gene whose expression in pineal is regulated by the pineal circadian clock. Transcription of these genes would not be expected to change in *B pineal since light entrainment of the pineal circadian oscillator has been shown to occur independent of changes in cGMP levels [28]. Previous studies have shown that the amount of TPH mRNA in chicken pineal is controlled by the pineal circadian clock, the levels of TPH mRNA increasing over the course of the day, reaching peak levels in early night and returning to low levels by the beginning of the next day [6,7]. Based on these results, we chose to measure transcript levels of the TPH gene in the pineal glands of *B and normal chickens that were entrained to a 12L:12D cycle and were then either kept on this cycle or were placed in continuous dark conditions. In our first experiment, we examined TPH mRNA levels in *B and normal pineal glands over the course of a single 12L:12D cycle taking samples at ZT0, 6, 12 and 18 (Fig. 2A). Comparisons of the normalized (Fig. 2B) and relative (Fig. 2C) TPH transcript profiles in *B and in normal pineal using two-way ANOVA revealed that the profiles in *B and normal pineal glands were not significantly different from each other. In both *B and normal pineal gland, the levels of TPH mRNA increased over the course of the 12-h light period, reaching maximum levels |6 h into the 12-h dark period. Post-hoc tests (Student–Newman–Keuls) revealed that the relative levels of TPH mRNA in both *B and normal pineal at ZT18 were significantly higher (P, 0.05) than those measured at ZT0 (Fig. 2C). In our second experiment, we analyzed TPH mRNA levels in the pineal glands of *B and normal chickens entrained to a 12L:12D cycle and then placed in continuous darkness. The results of these analyses showed that the TPH mRNA rhythms observed under cyclic light conditions were maintained in *B and in normal pineal glands in constant darkness (Fig. 2D and E). Comparisons of the normalized (Fig. 2D) and relative (Fig. 2E) TPH transcript profiles in *B and normal pineal using two-way ANOVA revealed that the TPH profiles in *B and normal pineal in constant dark conditions were not significantly different from each other. TPH levels in *B and in normal pineal did vary significantly as a function of time (F513.08, df53, P,0.001), the TPH mRNA levels reaching maximum levels at CT12.
4. Discussion The purpose of this study was to determine if light regulation of pinopsin mRNA levels in chicken pineal gland is dependent on GC1, a key enzyme in the visual
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phototransduction cascade. The results of our study show (1) that light is able to induce an increase in pinopsin mRNA in the pineal gland in the absence of GC1, (2) that the levels of pinopsin mRNA in *B pineal in dark conditions (basal levels) are significantly higher than those observed in normal pineal, (3) that the relative magnitudes of the light-induced increases in pinopsin mRNA are not statistically different in *B and normal pineal, and (4) that the changes observed in the regulation of basal levels of pinopsin mRNA in *B pineal gland are not accompanied by changes in the circadian expression of TPH mRNA. The results of our study show that the absence of GC1 alters basal levels of pinopsin mRNA in pineal without affecting the ability of light to produce increases in pinopsin mRNA. These data suggest that the mechanisms in pineal that regulate basal levels of pinopsin mRNA and mediate the light-dependent increases in pinopsin mRNA levels function independently of each other, and that the signal transduction cascade that mediates the light-driven increases in pinopsin mRNA in pineal is not dependent on GC1. One possible mechanism by which the absence of GC1 could alter regulation of basal levels of pinopsin mRNA in pineal is suggested by our studies of *B retinal photoreceptors. In *B retina, the absence of GC1 produces a significant and permanent reduction in the amount of cGMP in the photoreceptor cells, a condition which prevents these cells from transducing light for vision [20]. We have proposed that reduced cGMP levels in these cells leads to chronic closure of the cGMP-gated cation channels located in the plasma membranes and to chronic hyperpolarization of these cells. This hypothesis is supported by the observation that levels of glutamate, the neurotransmitter released by depolarized photoreceptors in the dark, are elevated in GUCY1*B photoreceptors regardless of the light-adapted state of the retina [23]. Chicken pinealocytes and retinal photoreceptors share many structural features [1] and many of the genes that encode the visual phototransduction cascade proteins in photoreceptors are expressed in pinealocytes, including the genes that encode the rod and cone cGMP-gated channel proteins [2]. Studies of the activities of these channels in pinealocytes have shown that they respond to changes in cGMP levels in a manner similar to that observed in photoreceptor cells [3,4]. The precise function of these channels in pinealocytes is not known; however, it is likely that in addition to playing a role in the light response of these cells, these channels may also play a role in controlling [Ca 21 ] i , their activity being modulated by changes in levels of cAMP and cGMP brought about by the actions of cellular G-protein-coupled cascades (for review see Ref. [9]). Based on these observations, it is reasonable to postulate that the absence of GC1 in pinealocytes alters their physiology in a manner similar to that observed in photoreceptors. To date, there is no definitive evidence that the visual phototransduction cascade is active in chicken
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Fig. 2. Comparison of TPH mRNA levels in pineal glands of *B and normal chickens housed under cyclic light and constant dark conditions. (A) Northern blot shown in Fig. 1A hybridized with a TPH probe. (B) Normalized TPH mRNA levels present in the pineal glands of *B (white) and normal (black) chickens housed under 12L:12D conditions. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. (C) Comparison of relative levels of TPH mRNA measured within *B pineal (white) and within normal pineal (black) over the course of a 12L:12D period. The relative amounts of TPH mRNA measured in *B and normal pineal at ZT18 were significantly higher (*P,0.05) than at all other times examined. (D) Normalized TPH mRNA levels present in the pineal glands of *B (white) and normal (black) chickens housed under constant dark conditions. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. Levels of TPH mRNA in *B and normal pineal at CT12 were significantly greater (*P,0.05) than those observed at CT0 and CT6. (E) Comparison of the changes in the relative levels of TPH mRNA within the pineal glands of *B chickens (white) and normal chickens (black) entrained to a 12L:12D cycle and then sacrificed over the course of a 24-h constant dark period at CT0, CT6, CT12 and CT18. Each point represents the mean6S.E.M. of three samples, each sample containing RNA extracted from four pineal glands. Levels of TPH mRNA in *B and normal pineal at CT12 were significantly greater (*P,0.05) than those observed at CT0 and CT6. In (B)–(E), the values obtained at ZT0 and CT0 were replotted at ZT24 and CT24, respectively.
pinealocytes. If such a cascade is present in these cells, then we would predict that the absence of GC1 would lead to chronic closure of the cGMP-gated channels in the membranes of these cells, a change that would place these
cells in a chronic physiological state resembling that of light exposed cells. Interestingly, our observation that the levels of pinopsin mRNA in the pineal glands of *B chickens housed in the dark approximate those in the
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pineal glands of normal chickens exposed to 12 h of light is consistent with this prediction. The biochemical mechanism controlling basal levels of pinopsin mRNA in pineal is not known; however, if our hypothesis that elevation of basal levels of pinopsin mRNA in *B pineal is a result of closure of the cGMP-gated channels, then it is possible that intracellular cation levels play an important role in this mechanism. Our analyses of TPH transcripts show that the rhythmic changes observed in TPH mRNA levels in *B and normal pineal in both cyclic light and constant darkness were similar indicating that the circadian oscillators that drive the TPH mRNA rhythm in *B pineal are functional and can be entrained to light in the absence of GC1. This result is consistent with previous analyses of avian pineal that showed that 8-bromo-cGMP had little effect on the acute or phase-shifting effects of light on pineal melatonin rhythms [28]. Currently, there is no evidence linking cGMP to circadian function in the pineal gland; however, the results of a recent study of the circadian clock-regulated iodopsin rhythm in cone photoreceptors of *B chicken suggest that changes in intracellular cGMP levels modulate circadian processes in photoreceptors [12]. In *B photoreceptors, iodopsin rhythms are temporally compressed in cyclic light and the phase of the rhythms is advanced by 6 h in constant darkness. In addition, the rate of entrainment of these rhythms to a reversal of the light / dark cycle is significantly slower in *B photoreceptors than in normal retina. In view of these results, it may be fruitful to examine the entrainment properties of the TPH mRNA rhythm in *B pineal following reversal of the light / dark cycle.
[5] [6]
[7]
[8]
[9] [10] [11]
[12]
[13]
[14]
[15]
[16] [17] [18]
Acknowledgements [19]
We thank Dr. Patrick Larkin for teaching the intricacies of running Northern blots to M.T. This research study was funded by NIH grant EY11388. M.T. received financial support from the University of Florida Scholars program and J.E.C. received partial support from NIH grant EY07132.
[20]
[21]
References [1] M.B. Bischoff, Photoreceptoral and secretory structures in the avian pineal organ, J. Ultrastruct. Res 28 (1969) 16–26. [2] W. Bonigk, F. Muller, R. Middendorff, I. Weyand, U.B. Kaupp, Two alternatively spliced forms of the cGMP-gated channel alpha-subunit from cone photoreceptor are expressed in the chick pineal organ, J. Neurosci. 16 (1996) 7458–7468. [3] S.E. Dryer, D. Henderson, A cyclic GMP-activated channel in dissociated cells of the chick pineal gland, Nature 353 (1991) 756–758. [4] S.E. Dryer, D. Henderson, Cyclic GMP-activated channels of the
[22]
[23]
[24]
[25]
57
chick pineal gland: effects of divalent cations, pH, and cyclic AMP, J. Comp. Physiol. A 172 (1993) 271–279. J. Falcon, Cellular circadian clocks in the pineal, Prog. Neurobiol. 58 (1999) 121–162. J.C. Florez, K.J. Seidenman, R.K. Barrett, A.M. Sangoram, J.S. Takahashi, Molecular cloning of chick pineal tryptophan hydroxylase and circadian oscillation of its mRNA levels, Mol. Brain Res. 42 (1996) 25–30. C.B. Green, J.C. Besharse, M. Zatz, Tryptophan hydroxylase mRNA levels are regulated by the circadian clock, temperature, and cAMP in chick pineal cells, Brain Res. 738 (1996) 1–7. T. Kasahara, T. Okano, T. Yoshikawa, K. Yamazaki, Y. Fukada, Rod-type transducin alpha-subunit mediates a phototransduction pathway in the chicken pineal gland, J. Neurochem. 75 (2000) 217–224. U.B. Kaupp, Family of cyclic nucleotide gated ion channels, Curr. Opin. Neurobiol. 5 (1995) 434–442. S. Kawamura, S. Yokoyama, Molecular characterization of the pigeon P-opsin gene, Gene 182 (1996) 213–214. P. Larkin, W. Baehr, S.L. Semple-Rowland, Circadian regulation of iodopsin and clock is altered in the retinal degeneration chicken retina, Mol. Brain Res. 70 (1999) 253–263. P. Larkin, S.L. Semple-Rowland, A null mutation in guanylate cyclase-1 alters the temporal dynamics and light entrainment properties of the iodopsin rhythm in cone photoreceptor cells, Mol. Brain Res. 92 (2001) 49–57. A. Matsushita, T. Yoshikawa, T. Okano, T. Kasahara, Y. Fukada, Colocalization of pinopsin with two types of G-protein alphasubunits in the chicken pineal gland, Cell Tissue Res. 299 (2000) 245–251. M. Max, P.J. McKinnon, K.J. Seidenman, R.K. Barrett, M.L. Applebury, J.S. Takahashi, R.F. Margolskee, Pineal opsin: a nonvisual opsin expressed in chick pineal, Science 267 (1995) 1502– 1506. M. Max, A. Surya, J.S. Takahashi, R.F. Margolskee, B.E. Knox, Light-dependent activation of rod transducin by pineal opsin, J. Biol. Chem. 273 (1998) 26820–26826. H. Meissl, Photic regulation of pineal function. Analogies between retinal and pineal photoreception, Biol. Cell 89 (1997) 549–554. T. Okano, T. Yoshizawa, Y. Fukada, Pinopsin is a chicken pineal photoreceptive molecule, Nature 372 (1994) 94–97. S.L. Semple-Rowland, K.M. Cheng, rd and rc chickens carry the same GC1 null allele (GUCY1*), Exp. Eye Res. 69 (1999) 579– 581, letter. S.L. Semple-Rowland, P. Larkin, J.D. Bronson, K. Nykamp, W.J. Streit, W. Baehr, Characterization of the chicken GCAP gene array and analyses of GCAP1, GCAP2, and GC1 gene expression in normal and rd chicken pineal, Mol. Vis. 5 (1999) 14. S.L. Semple-Rowland, N.R. Lee, J.P. Van Hooser, K. Palczewski, W. Baehr, A null mutation in the photoreceptor guanylate cyclase gene causes the retinal degeneration chicken phenotype, Proc. Natl. Acad. Sci. USA 95 (1998) 1271–1276. J.S. Takahashi, M. Zatz, Photic regulation of cyclic nucleotide levels and N-acetyltransferase activity in the cultured avian pineal, Soc. Neurosci. Abs. 8 (1982) 546. Y. Takanaka, T. Okano, M. Iigo, Y. Fukada, Light-dependent expression of pinopsin gene in chicken pineal gland, J. Neurochem. 70 (1998) 908–913. R.J. Ulshafer, D.M. Sherry, R. Dawson Jr., D.R. Wallace, Excitatory amino acid involvement in retinal degeneration, Brain Res. 531 (1990) 350–354. T. van Veen, R. Elofsson, H.G. Hartwig, I. Gery, M. Mochizuki, V. Cena, D.C. Klein, Retinal S-antigen: immunocytochemical and immunochemical studies on distribution in animal photoreceptors and pineal organs, Exp. Biol. 45 (1986) 15–25. S.D. Wainwright, Diurnal cycles in serotonin acetyltransferase activity and cyclic GMP content of cultured chick pineal glands, Nature 285 (1980) 478–480.
58
S.L. Semple-Rowland et al. / Molecular Brain Research 97 (2001) 51 – 58
[26] R.B. Yang, S.W. Robinson, W.H. Xiong, K.W. Yau, D.G. Birch, D.L. Garbers, Disruption of a retinal guanylyl cyclase gene leads to cone-specific dystrophy and paradoxical rod behavior, J. Neurosci. 19 (1999) 5889–5897. [27] M. Zatz, Photoendocrine transduction in cultured chick pineal cells: IV. What do vitamin A depletion and retinaldehyde addition do to the effects of light on the melatonin rhythm?, J. Neurochem. 62 (1994) 2001–2011. [28] M. Zatz, D.A. Mullen, Photoendocrine transduction in cultured chick pineal cells. II. Effects of forskolin, 8-bromocyclic AMP, and
8-bromocyclic GMP on the melatonin rhythm, Brain Res. 453 (1988) 51–62. [29] M. Zatz, D.A. Mullen, Two mechanisms of photoendocrine transduction in cultured chick pineal cells: pertussis toxin blocks the acute but not the phase-shifting effects of light on the melatonin rhythm, Brain Res. 453 (1988) 63–71. [30] X. Zhao, K. Yokoyama, M.E. Whitten, J. Huang, M.H. Gelb, K. Palczewski, A novel form of rhodopsin kinase from chicken retina and pineal gland, FEBS Lett. 454 (1999) 115–121.