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Molecular Vision 2001; 7:210-5 <http://www.molvis.org/molvis/v7/a29>
Received 20 July 2001 | Accepted 23 August 2001 | Published 29 August 2001
© 2001 Molecular Vision
Three cryptochromes are rhythmically expressed in Xenopus laevis
retinal photoreceptors
Haisun Zhu, Carla B. Green
NSF Center for Biological Timing, Department of Biology, University of Virginia, Charlottesville, VA
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 laevisretina. 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 Xenopusphotoreceptor 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 [1]. 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 [2].
CRYs were first discovered in Arabidopsis thaliana as
blue light photoreceptors [3] 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 [6]. This resetting is
thought to be mediated by light-dependent interaction of dCRY
with one of the central clock components, TIMELESS (TIM)
[7], 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 [6]. 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 [1012]. 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 [10], 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 [19]. 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.
METHODS
Cloning of the Xenopus cryptochromes: Degenerate PCR
primers were designed using CODEHOP techniques [24] based
on the homology between mouse and human cryptochrome
sequences. Two sets of primers were designed (Forward-5'TGA TTG TTA GAA TTT CTC ACA CAC TGT AYG AYY
TNG A-3', Reverse-5'-TGG AGA AGC CAG CAG AGA GTT
NGC RTT CAT-3', Forward-5'-CAG GAC TGT CTC CAT
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 protected]
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ACC TGA GAT TYG GNT GYY T-3', and Reverse-5'-CTC
CTC TTG TCA GAA AGC AAG CNA CNG CRT G-3'). A
reverse transcription (RT) reaction was performed on 1 µg 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.
© 2001 Molecular Vision
The subcloned DNA fragments were random prime-labeled and used to screen a Xenopus retinal cDNA library (in
λHybriZAP vector; Stratagene, La Jolla, CA), prepared from
pooled RNA isolated from retinas at four timepoints throughout the day. Screening was done as previously described [25]
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
Figure 1. Comparison of amino acid sequences between Xenopus and mouse crys. The deduced amino acid sequences of the xcrys (Genbank
accession numbers AY049033, AY049034, and AY049035) were aligned with the mouse sequences using the ClustalW algorithm (MacVector;
Oxford Molecular, Campbell, CA). Identical amino acids are highlighted in dark gray and similar ones are highlighted in light gray. Introduced
gaps are shown as dashes.
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© 2001 Molecular Vision
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 [18], 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
β-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 βactin (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 µm).
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 [26].
ment 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 mcry2and 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 Xenopusgenome. 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.
Figure 2. Spatial analysis of xcry mRNA expression in the Xenopus
retina. Xenopus eyes were dissected and fixed at ZT 2 and 14 µm
cryosections were prepared. In situ hybridization analysis was done
with digoxygenin-labeled antisense (left three) and sense (right) xcry
probes. (Only the sense of xcry1 was shown here. Similar results
were obtained for both xcry2a and xcry2b sense probes.) The white
arrowhead emphasizes the heavy in situ labeling in the inner segments of the photoreceptor cells. Retinal cell layers are labeled on
the left: RPE, retinal pigment epithelium; OS, photoreceptor outer
segments; IS, photoreceptor inner segment; ONL, outer nuclear layer
(photoreceptor cell nuclei); INL, inner nuclear layer; and GCL, ganglion layer.
RESULTS
Identification of three cry homologs in Xenopus laevis retina:
We performed degenerate RT-PCR using CODEHOP primers
[24] 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 frag212
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© 2001 Molecular Vision
mRNA in muscle. In addition, the xcry1expression level seems
much higher in brain than either of the xcry2messages. 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.
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 [27]. 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
xcry2bexpression 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 µg of total retinal RNA were loaded, while 6
µg of RNA from all other tissues were used) and were also
high in heart and liver. We were not able to detect any cry
DISCUSSION
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 [28]. 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 [29]. Our photoreceptor localization conforms to the previous finding that the
Xenopus photoreceptor cells contain a circadian clock that
drives melatonin rhythms [18]. Also, we have observed similar photoreceptor expression patterns for other central clock
genes such as xClock [20] and xBmal1 [23]. 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
Figure 3. Temporal analysis of xcry expression in retina. Total RNA
was isolated from Xenopus retina collected at 4-h intervals throughout the day from eyecups cultured in either 12L/12D (light/dark)
cycles or constant darkness. Each lane contains 2 µg RNA. The ZT
time of each sample is shown above. (White bars-day; dark bar-night;
hatched bars-subjective day.) Matching actin results are shown below each blot. On the right, quantifications of the Northern results
are shown. Each sample represents the average of three independent
experiments. Error bars indicate the SEM.
Figure 4. xcry expression in body tissues. RNA was isolated from
several Xenopus tissues at ZT 4. Each lane was loaded with 6 µg
RNA with the exception of retinal RNA where only 2 µg was loaded.
The samples were loaded from left to right: R, retina; B, brain; H,
heart; L, liver; M, skeletal muscle; S, spleen; and T, testis. The three
filters represent xcry1, xcry2a, and xcry2b, as labeled.
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expression pattern is consistent with that seen in mice [30]
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 Xenopusretina 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 [31]. 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.
[15] 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.
ACKNOWLEDGEMENTS
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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The print version of this article was created on 31 August 2001. This reflects all typographical corrections and errata to the article through that
date. Details of any changes may be found in the online version of the article.
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