|Molecular Vision 2001;
Received 20 July 2001 | Accepted 23 August 2001 | Published 29 August 2001
Three cryptochromes are rhythmically expressed in Xenopus laevis retinal photoreceptors
Carla B. Green
NSF Center for Biological Timing, Department of Biology, University of Virginia, Charlottesville, VA
Correspondence to: Dr. Carla B. Green, Department of Biology, Gilmer Hall 264, University of Virginia, Charlottesville, VA, 22904; Phone: (804) 982-5436; FAX: (804) 982-5626; email: email@example.com
Purpose: To clone Xenopus laevis cryptochromes (crys) and to understand their role in the Xenopus retinal clock.
Methods: We designed degenerate PCR primers based on homology between mouse and human crys. DNA fragments generated from these PCR reactions were used to screen a Xenopus retinal cDNA library. Three independent clones were identified and sequenced. The temporal and spatial expression of these genes in retina were studied by Northern blot analysis and in situ hybridization.
Results: We cloned three cry homologs from Xenopus laevis retina. We named them xcry1, xcry2a, and xcry2b based on their high homology to the mouse crys. Sequence analysis shows that these Xenopus CRYs have more than 85% identity to mouse CRYs at the amino acid level. Northern blot analysis demonstrated that all three xcrys are rhythmically expressed in the retina with peaks at different times of the day. The xcrys are expressed in a variety of tissues. In retina, they are expressed predominantly in photoreceptor cells.
Conclusions: Our finding of cry expression in Xenopus photoreceptor cells further supports the idea of independent circadian oscillators being present in these cells. The sequence similarities to mouse crys suggest similar functions in the circadian clock. However, their distinct temporal expression patterns suggest some unique role for xCRY in the Xenopus retina.
Organisms adapt to the 24-h light/dark cycle by maintaining internal circadian clocks that oscillate in close synchronization with the environment. These clocks, while oscillating independently, can be reset by entraining signals such as light and temperature changes. CRYs are proteins that play important roles in circadian clocks in plants, insects, and vertebrates, although their roles in these organisms appear to be distinct . Sequence analyses show that CRYs are very similar to the photolyase protein family that function in the repair of DNA damage by UV light. Like photolyase, CRYs are bound to two chromophores, pterin and flavin, however, CRYs can not repair DNA damage .
CRYs were first discovered in Arabidopsis thaliana as blue light photoreceptors  and were subsequently implicated in circadian photoreception [4,5]. Likewise, Drosophila CRY (dCRY) is involved in light-mediated resetting of the circadian clock that controls behavior . This resetting is thought to be mediated by light-dependent interaction of dCRY with one of the central clock components, TIMELESS (TIM) , resulting in a relief of repression of the transcription of the period (per) and tim genes. Light activation of dCRY is presumed to be through the chromophores and this is supported by isolation of a mutant (crybaby), which contains a single amino acid substitution in the conserved flavin-binding domain . Flies with this mutation still have a functional clock, but the clock's sensitivity to light is greatly reduced [6,8,9].
In contrast, in mammals, the two CRY proteins are components of the central clock mechanism, and are involved in repression of CLOCK/BMAL1 activation of the per genes [10-12]. Knockout mice missing either cry still maintained locomotor rhythms, but with aberrant periods. Mice missing both CRYs are completely arrhythmic [13,14]. Unlike dCRY, mammalian CRY functions have not been shown to be directly light sensitive , although in mice lacking crys, acute light responses in the suprachiasmatic nuclei (SCN), the site of the mammalian clock controlling locomotor activity rhythms, are diminished [15,16].
The Xenopus laevis retina contains a robust circadian clock [17,18]. Isolated photoreceptor layers continue to oscillate and can be reset by light, implying the presence of a clock and a circadian photoreceptor within this layer of tissue . Xenopus homologs of known central clock components are expressed within the retina with temporal expression patterns similar to the mouse SCN [20-23].
In this study, we cloned three cry homologs from Xenopus laevis retina. We examined their temporal and spatial expression by using Northern blot analysis and in situ hybridization.
Cloning of the Xenopus cryptochromes
Degenerate PCR primers were designed using CODEHOP techniques  based on the homology between mouse and human cryptochrome sequences. Two sets of primers were designed (Forward-5'-TGATTGTTAGAATTTCTCACACACTGTAYGAYYTNGA-3', Reverse-5'-TGGAGAAGCCAGCAGAGAGTTNGCRTTCAT-3', Forward-5'-CAGGACTGTCTCCATACCTGAGATTYGGNTGYYT-3', and Reverse-5'-CTCCTCTTGTCAGAAAGCAAGCNACNGCRTG-3'). A reverse transcription (RT) reaction was performed on 1 mg of total Xenopus retinal RNA using Superscript II (Life Technology, Gaithersburg, MD). Approximately 1/10 of the product was used for PCR, (in 60 mM Tris-HCl, pH 8.5, 15 mM ammonium sulfate, 2.5 mM MgCl2, 1 mM each dNTP, 0.1 nmol of each primer, and 2.5 units of AmpliTaq Gold polymerase; Perkin-Elmer, Wellesley, MA) using the following parameters: 95 °C for 10 min; 25 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s; 10 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30 s; 72 °C for 10 min; hold at 4 °C. The resulting PCR products were subcloned into pCR2.1-TOPO vector using the TOPO-TA cloning kit (Invitrogen, Carlsbad, CA) and verified by sequencing.
The subcloned DNA fragments were random prime-labeled and used to screen a Xenopus retinal cDNA library (in lHybriZAP vector; Stratagene, La Jolla, CA), prepared from pooled RNA isolated from retinas at four timepoints throughout the day. Screening was done as previously described  except that the wash solution was 0.1X SSPE, 0.1% SDS. Positive clones were plaque-purified, excised using the ExAssist helper phage (Stratagene, La Jolla, CA), and sequenced.
Eyecup preparation and culture
Xenopus laevis (5-6.5 cm) were purchased from Nasco (Fort Atkinson, WI) and were maintained in 12 h light: 12 h dark cyclic light. Animals used in these experiments were entrained in these lighting conditions for at least 2 weeks prior to use. Animal care and use was in accordance with the Guide for the Care and Use of Laboratory Animals published by the Institute for Laboratory Animal Research. Dissected eyecups (including retina, pigment epithelium, choroid, and sclera) were cultured in defined culture medium , in a humidified atmosphere of 95% O2/5% CO2.
Incubations were carried out on a rotary shaker (60 rpm), in a constant temperature incubator at 21±0.1 °C under the indicated light conditions. All times are expressed as Zeitgeber Time (ZT) in hours (h), with respect to the original entraining light cycle, in which ZT 0 is defined as the time of normal light onset (dawn) and ZT 12 is defined as time of normal dark onset (dusk).
RNA preparation and Northern blot analysis
At the appropriate times, retinas were isolated and quickly frozen on dry ice for subsequent isolation of RNA using Trizol Reagent (Life Technology, Gaithersburg, MD). Northern blot analyses were carried out in QuickHyb Hybridization solution (Stratagene, La Jolla, CA) using random primed probes made from fragments of xcry cDNA clones. To avoid cross-hybridization, only the 3' portion of each clone (including the 3' UTR) was used. The lengths of the probes were: xcry1, 736 bp; xcry2a, 769 bp; xcry2b, 640 bp. Sequence identity between xcry1 and xcry2a fragments is 33%; between xcry1 and xcry2b is 31%; between xcry2a and xcry2b is 54%. Filters were stripped by boiling twice for 10 min in 0.01X SSPE, 0.1% SDS and rehybridized with random primed probes made from b-actin clones for normalization.
Quantification of message levels was done directly from the radiolabeled filter using a phosphoimager (Molecular Dynamics, Sunnyvale, CA). Total counts per xcry band (minus background) were divided by the total counts per band of b-actin (minus background) resulting in numbers that were normalized for differences in lane loadings. Final results are expressed relative to the mean sample quantity and the results from three independent experiments were averaged.
In situ hybridization
Xenopus eyes were dissected from adult frogs at ZT 2 and eyecups were prepared and fixed overnight in 4% paraformaldehyde in phosphate buffered saline (PBS) at 4 °C. The tissue was cryoprotected by incubation in 30% sucrose in PBS for 2-3 h at 4 °C and then embedded in Tissue-Tek O.C.T. compound (Ted Pella, Redding, CA) and cryosectioned (14 mm).
Digoxygenin-labeled antisense and sense riboprobes were prepared from the cDNA of xcrys. Again, to avoid cross hybridization, only the 3' region of each clone was used to generate the antisense probe (see above for description). These riboprobes were hydrolyzed to an average length of 100-250 bases. In situ hybridization protocol was adapted from .
Identification of three cry homologs in Xenopus laevis retina
We performed degenerate RT-PCR using CODEHOP primers  and cloned two distinct cry-like fragments from Xenopus laevis retinal RNA. Both fragments contained a single open reading frame. The deduced amino acid sequences showed that one of the fragments was more similar to mouse cry1 (79% identical to mcry1 and 67% to mcry2) while the other fragment showed higher similarity to mcry2 (61% identical to mcry1 and 71% to mcry2). These data suggested that at least two different homologs of crys are expressed in Xenopus retina.
These fragments were then used to screen a Xenopus retinal cDNA library. Three independent clones were identified. Their deduced amino acid sequences are shown in Figure 1 in alignment with mCRY sequences. Based on the similarity to mCRY sequences, we named these three clones: xcry1, xcry2a, and xcry2b. The amino acid sequence of mcry1 and xcry1 is 86% identical, 92% similar. Between mcry2 and xcry2a, the identity is 82% and the similarity is 90%, while the identity and similarity between mcry2 and xcry2b is 81% and 90%, respectively. xcry2a and xcry2b are very similar to each other (93% amino acid identity). However, our xcry2a clone is incomplete and missing a portion of the 5'-end. We cannot exclude the possibility that xcry2a and xcry2b are two different alleles of the same gene or a product of the pseudotetraploid nature of the Xenopus genome. Like all other identified cry genes [1-3,9], the N-terminal two thirds of the proteins are highly similar and contain the conserved flavin and pterin binding domains. The C-termini of the proteins are highly variable between all identified cry genes [1-3,9].
xcrys are expressed in photoreceptor cells
The 3' UTR regions of the xcry cDNA clones, which contain unique sequence in each of the three xcrys, were used as probes to investigate the expression pattern of xcry within the retina by in situ hybridization. Our results showed intense staining within the cell bodies of photoreceptor cells by all three probes (Figure 2). Other cell types also show minimal staining that is slightly higher than the sense control. These results indicate that xcrys are expressed predominantly in the photoreceptor cells but some cells within both the inner nuclear layer and the ganglion cell layer may also express small amounts of xcry mRNA. There is no distinctive difference in expression patterns between the three xcrys.
xcrys are expressed rhythmically at low amplitudes
The temporal expression of xcry mRNA in the retina was studied by northern blot analysis, using probes specific for each xcry (Figure 3). Both xcry2 probes hybridized to a single band of approximately 2.5 kb. The xcry1 probe hybridized to a band of 2.5 kb and also to a larger message of 4.5 kb and both messages were expressed at comparable levels. This is very similar to the cry1 in both mouse and human where two messages are also expressed . xcry1 mRNA showed a robust rhythm in light/dark (LD) conditions with a peak at ZT 16. The rhythm was also observed in constant darkness (DD) but with lower amplitude. Both xcry2a and xcry2b show low amplitude rhythms in LD and DD with peaks at ZT 0 (xcry2a) and ZT 20 (xcry2b), respectively.
xcrys are expressed throughout the body
We also examined the mRNA expression of xcrys in other tissues of the body. Our results show that all three xcrys are expressed in retina, brain, heart, liver, spleen, and testis, although xcry2b expression was very weak in many of the tissues (Figure 4). The levels of all xcrys were highest in retina and testis (note that only 2 mg of total retinal RNA were loaded, while 6 mg of RNA from all other tissues were used) and were also high in heart and liver. We were not able to detect any cry mRNA in muscle. In addition, the xcry1 expression level seems much higher in brain than either of the xcry2 messages. This suggests that there might be some tissue-specific functions for xcry1 in brain. Interestingly, the shorter (2.5 kb) xcry1 message was observed only in retina and perhaps testis.
We have cloned and characterized Xenopus cry homologs with high sequence similarity to the mammalian crys. However, in contrast to the two cry genes found in both mouse and human, we identified three distinct clones from the Xenopus retinal cDNA library. One of the clones, xcry1, is most similar to mcry1, while the other two are most similar to mcry2. xcry2a and xcry2b are very similar to each other with the most sequence divergence occurring at the extreme C-terminus. Since Xenopus laevis is a pseudotetraploid animal, it is possible that xcry2a and xcry2b are duplicate versions of the same gene. This kind of genome duplication has been documented in several non-mammalian vertebrates. For example, it has been shown that there are at least seven cry homologs in the zebrafish genome . Since Drosophila and mammalian CRYs have distinct functions, we hypothesized that by studying CRY functions in Xenopus laevis, we would gain information on how cryptochromes and the circadian clock have evolved in animals.
Our studies on xCRYs were focused on the retina. In Xenopus laevis, the retina carries an independent clock that can be directly reset by light. Our results show that within the retina, all three xcrys are expressed predominantly in the photoreceptor cells. This result is distinct from cry expression in the mouse retina, where mcrys are expressed exclusively in the ganglion cell layer and inner nuclear layer . Our photoreceptor localization conforms to the previous finding that the Xenopus photoreceptor cells contain a circadian clock that drives melatonin rhythms . Also, we have observed similar photoreceptor expression patterns for other central clock genes such as xClock  and xBmal1 . The presence of xcrys in these cells further supports the presence of a fully functional molecular clock. In addition to retinal expression, the three xcrys are also widely expressed in many other tissues including brain, heart, liver, spleen, and testis. This wide expression pattern is consistent with that seen in mice  and with the expectation that animal CRYs are involved in regulating both central and peripheral oscillators.
Though the similarity in amino acid sequence suggests that xCRYs have similar functions as mCRYs, our temporal analysis revealed some unexpected results. All three xcry mRNAs exhibited low amplitude rhythms in constant dark in retina, with the peak of xcry1 at ZT 16, xcry2a at ZT 0, and xcry2b at ZT 20. Although the Xenopus retina expresses the per1 and bmal1 genes with phases reminiscent of the mouse SCN, the xcry expression patterns are quite different. In the mouse SCN, mcry1 is rhythmically expressed with the peak of the message at ZT 12. The expression of mcry2 is reported to be arrhythmic by some groups [11,29] or to have low amplitude rhythms that also peak at ZT 12 . These differences in xcry expression patterns suggest subtle differences of the xCRY functions with in the Xenopus photoreceptor clock. The most prominent difference between the Xenopus retinal clock and the mouse SCN clock is that the retinal clock can be reset by light directly and SCN can only be reset indirectly by a signal from the eye. Recent work by Selby et al.  proposes that CRYs may contribute to circadian photoreception within the mammalian retina. It is possible that the different expression patterns of xcrys within the Xenopus retina may reflect additional roles for these proteins in a photoreceptive tissue.
We thank Julie Baggs, Cara Constance, Naoto Hayasaka, and Carl Strayer for their helpful comments on this manuscript. This work was supported by NIH grants EY11489 and MH61461 (CBG).
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