The crayfish Procambarus clarkii CRY shows daily and circadian variation
Laboratorio de Neurofisiología Comparada, Departamento de Biología, Facultad de Ciencias, Universidad Nacional Autónoma de México, México DF 11000
* Author for correspondence (e-mail: mlfm{at}hp.fciencias.unam.mx)
Accepted 27 January 2004
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Summary |
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Key words: rhythm, Procambarus clarkii, pacemaker, cryptochrome, circadian photoreceptor
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Introduction |
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The crayfish is a nocturnal crustacean that displays a variety of circadian
rhythms controlled by periodic function of the nervous system
(Fanjul-Moles and Prieto-Sagredo,
2003). Although some of these rhythms are well described, there is
scant information about the circadian photoreceptors and the entrainment
pathways that couple the clock to the daily light changes. Some authors have
proposed that the photoreceptors participating in the entrainment are
extra-retinal and located in the supra-esophageal ganglion (Page and Larimer,
1972
,
1976
;
Sandeman et al., 1990
), while
others have proposed the sixth abdominal ganglion as a locus of circadian
photoreception (Bernal-Moreno et al.,
1996
; Prieto-Sagredo and
Fanjul-Moles, 2001
). Interestingly, experiments have demonstrated
that the crayfish circadian photo-entrainment depends on the quality of light
(Fanjul-Moles et al., 1992
;
Bernal-Moreno et al., 1996
;
Miranda-Anaya and Fanjul-Moles,
1997
), suggesting that this phenomenon rests on different photo
proteins and inputs that converge on the circadian pacemakers, the eyestalk
(retina and optic lobe) and the brain
(Aréchiga et al.,
1993
).
The effect of blue monochromatic light (=440 nm) on the
electroretinogram (ERG) and activity rhythms of crayfish
(Fanjul-Moles et al., 1992
;
Miranda-Anaya and Fanjul-Moles,
1997
), as well as the features of the phaseresponse curves
constructed for the ERG rhythm
(Inclán-Rubio, 1991
;
Bernal-Moreno et al., 1996
),
confirm the photo entrainment action of blue light, indicating the presence of
a photo pigment that absorbs light in the blue spectrum (400500 nm).
This pigment could be a cryptochrome (CRY), blue/UV-A absorbing photo protein,
originally discovered in plants, which has homologues in the animal kingdom
(insects, mice and human; Sancar,
2003
) and is associated with the circadian clock. Mounting
evidence from genetic and molecular studies indicates that in insects, light
acts directly on the clock through CRY
(Emery et al., 1998
;
Stanewsky et al., 1998
);
although to date in mammals a photoreceptive function for these pigments has
not been proved, they do participate in the feedback loop of the circadian
genes constituting the machinery of the clock
(Stanewsky, 2003
).
In crayfish, the molecular mechanisms involved in the generation and synchronization of circadian rhythms are practically unknown, although there is abundant but controversial information about the physiological and behavioral mechanisms underlying the generation and entrainment of the clock. The object of the present study is to contribute to this knowledge, investigating whether CRY is expressed in the putative circadian pacemakers of crayfish, the eyestalk and the brain, and whether this protein may be considered as an element of the circadian clock.
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Materials and methods |
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Whole-mount immunocytochemistry
The whole-mount technique was a modification of that previously described
(Galizia et al., 1999).
Dissection of the whole eyestalk and brain was performed in cold (4°C)
physiological saline (Van Harreveld). The tissue was fixed with 10%
formaldehyde in phosphate-buffered saline (PBS) for 12 h at 4°C and rinsed
in PBS, followed by a series of alcohol solutions (50%, 70%, 90, 96% and 100%
ethanol for 15 min each, xylol 5 min and 100%, 96%, 70%, 50% ethanol 15 min
each). Subsequently the tissue was washed with PBS-Tween 20, pH 7.6 for 20
min, and then protein-blocked and incubated in the primary antibody diluted
1:1500 (v/v) with PBS for 48 h at 4°C. We used a commercially available
antiserum generated in rabbit immunized with Drosophila CRY (Alpha
Diagnostic International Inc., San Antonio, TX, USA). Tissues were washed in
PBS for 2 h and incubated in 1:50 (v/v) secondary antiserum (goat anti-rabbit
IgG labeled with Texas Red; Rockland, Gilbertsville, PA, USA) for 24 h at
4°C, followed by several washes in PBS for 2 h. Finally, the tissue was
dehydrated in increasing ethanol solutions (50%, 70%, 90%, 100%, 15 min each),
mounted in methyl salicylate (ICN Biomedicals, Inc., Irvine, California, USA)
and viewed with a confocal Bio-Rad MRC-1024 (Bio-Rad, Hercules, California,
USA) attached to a Nikon Diaphot 300 microscope (Nikon, Tokyo, Japan).
Histological procedures
Both the eyestalk and the brain of six organisms were separately fixed in
10% formaldehyde in PBS for 12 h. The fixed material was progressively
dehydrated in 50%, 70%, 98% and 100% ethanol, incubated in Paraplast for 12 h
andembedded in a block for sectioning. Both organs were cut into longitudinal
sections (4 µm thick) using a calibrated microtome. Serial sections were
collected, deparaffinized in xylene, mounted on gelatine-coated glass slides
and progressively rehydrated (100%, 96%, 70%, 50% ethanol, water). To localize
CRY, the brain and eyestalk sections were processed by immunofluorescence. The
sections were incubated for 12 h at room temperature in the same polyclonal
rabbit anti-Drosophila CRY serum (dilution 1:1500 v/v). To visualize
the primary immunoreaction the sections were incubated for 1 h in goat
anti-rabbit IgG-Texas Red (Rockland, 1:50 v/v) at room temperature. The slices
were preserved with fluorescent mounting medium (Biogenex, San Ramon, CA,
USA). Control sections were (i) treated in the same way but with the antiserum
omitted and (ii) treated by preadsorption of the CRY antiserum with
Drosophila CRY peptide (Alpha Diagnostic International, Inc.). To
localize CRY, the sections were examined using a Nikon Labophot 2
epifluorescence microscope.
Image analysis and confocal microscopy
The immunofluorescent sections were studied by stereological analysis as
described elsewhere (Escamilla-Chimal et
al., 2001). The sections were studied using a Nikon Labophot 2
epifluorescence microscope. For each histological section (at least three
sections were examined), three video images of the structures were captured
using the 40x objective, and digitalized by means of an image processor
system (Argos 20, Hamamatsu, Hamamatsu City, Japan), captured with MGI Video
Wave software (Roxio, Santa Clara, California, USA.) and analyzed
stereologically using Sigma Scan Pro (vs. 4.01, SPSS Inc., Chicago,
IL, USA).
Confocal images of whole-mount preparations were obtained using an MCR 1024 Bio-Rad laser-scanning system equipped with an Ar Kr/Ar air-cooled laser attached to an inverted Nikon TMD 300 microscope. Images were collected with a Nikon 40x objective (numerical aperture 1.0). Neurons stained with Texas Red were excited with the 568 nm line of the laser, and emitted light was band-passed with a 605 nm filter. Serial optical sections were taken at intervals of 15 µm. The stacks of images were processed into stereo pairs of movies, saved as three-dimensional projections and converted to TIF format with Todd Clark's program Confocal Assistant 4.2. Further analysis to adjust brightness and contrast was performed using Adobe Photoshop 5.0 (Adobe Systems Inc., San José, CA, USA).
Biochemical determination
Protein sample preparation
Brain and eyestalk, including the retina, were carefully homogenized in 100
µl of ice-cold PBS, pH 7.4. Then, the homogenates were centrifuged at 11
000 g for 25 s at room temperature. Supernatants were stored
at 71°C until analyzed. Samples were thawed at room temperature and
the protein concentration was determined using the method of Bradford
(1976) and standards of 3.75,
11.25, 18.75, 26.25, 37.5 µg µl1 bovine serum albumin
(Sigma-Aldrich Co.; St Louis, MO, USA).
Western blotting
Proteins were separated by denaturing polyacrylamide gel electrophoresis
(SDS-PAGE; Laemmli, 1970) with
a 10% polyacrylamide separating gel. Each lane was loaded with 40 µg of
protein except for the positive control (control peptide; Alpha Diagnostic
International, Inc.) and the molecular mass standards.
Proteins resolved by SDS-PAGE were electrophoretically transferred from the gels to nitrocellulose membrane Hybond ECL (Amersham Pharmacia Biotech, Little Chalfont, Bucks, England) by routine methods, using a Bio-Rad Mini Trans-Blot system at 100 V for 45 min. Protein loading and localization for molecular mass were revealed by staining with Coomassie Blue. Prior to immunodetection the membranes were incubated with a blocking solution containing 3% gelatin diluted in TBS (Immuno-Blot Assay Kit, Bio-Rad) for 1 h followed by two rinses with TTBS (350 µl Tween-20 diluted in 700 ml TBS). Later the blots were incubated for 12 h at room temperature with the previously described rabbit anti-CRY antiserum diluted 1:800 (v/v) in a 1% gelatin solution. Immunoblots were revealed using peroxidase-labelled anti-rabbit antibodies (Immuno-Blot Assay Kit, Bio Rad) diluted in 1% gelatin (1:3000 v/v) for 2 h. To test the specificity of the antibody it was incubated with the peptide control at 4°C for 24 h and afterwards the antibody was used for western blotting.
Gel and blots were scanned and digitalized using a HP 3400 C Scanjet
scanner (Hewlett Packard, Palo Alto, CA, USA). Quantifications of the bands
were performed in a computerized analyzer system using the software Sigma Scan
Pro (SPSS Inc., vs. 4.01) and GeneTools (vs. 3.00.22; Syngene Division,
Synoptics Group, Cambridge, UK). Briefly, the scanned images of the bands of
the blots were framed to fill the stained areas on the image, the dark areas
measured and the average intensity of each band determined. The criterion for
selecting the immunoreactivity targets was a minimum ratio of background 0
pixels (white) and 255 pixels (black). For each experiment the data (average
intensity of the immunoreactive area of the band) obtained for each time point
were averaged, expressed as mean ±
S.E.M. of CRY relative abundance, normalized
to the maximal value obtained for the experiment, and plotted as chronograms.
The raw data were statistically analyzed using a single cosinor analysis
(Nelson et al., 1979) by mean
of the software program COSANA (Menna
Barreto et al., 1993
).
Cosinor analysis
The software COSANA utilizes the cosinor statistical method described
elsewhere (Castañon-Cervantes et
al., 1999). On the basis of a test period (
), cosinor
analysis adjusts data to a sinusal curve and provides an objective test of
whether the amplitude of the rhythm differs from zero, i.e. whether the rhythm
is validated for an assumed
. This method provides descriptive estimators
for a number of different parameters of a rhythm, i.e. acrophase, mesor,
amplitude and percentage of rhythmicity (PR). The acrophase is the crest time
of the best-fitting mathematical function approximating data, the mesor
(M) is the value about which oscillation occurs, and when the
interval of time between data is constant, it equals the arithmetic mean of
the rhythmic oscillation. Hence, in the present work M corresponds to
the arithmetic mean of the rhythmic oscillation of CRY abundance throughout 24
h. The period is the duration of one complete cycle of the oscillation and it
is expressed in units of time. In the cosinor method, the amplitude (A) is
equal to half the difference between the highest and lowest values of the
oscillation, i.e. it is the crest-to-trough difference, and the percentage of
rhythmicity (PR) is the percentage of the data included within the 95%
confidence limits of the best-fitting cosine function. The cosinor test allows
objective testing of the hypothesis that the rhythm amplitude differs from
zero using different trial period lengths. Several periods were tested to
analyze whether the temporal profiles observed in both structures were indeed
circadian.
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Results |
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Biochemical analysis
Analysis of the extracts of crayfish brain and eyestalks revealed the
presence of a protein immunoreactive to anti-CRY antibody. This protein
matches the molecular mass of the cryptochrome reported for Drosophila
melanogaster, approximately 60 kDa
(Emery et al., 1998)
(Fig. 3).
|
Chronograms showing the temporal changes in CRY relative abundance in the eyestalk are depicted in Fig. 4AC. Western blot showed that levels of CRY oscillate daily attaining maximal values at late subjective night (03:00 h) with a deep trough coincident with the onset of light (07:00 h). Throughout the subjective day and night, the CRY protein increased steadily with only a slight decrement after the offset of light (Fig. 4A). Interestingly, when the lights were turned off, and the crayfish were submitted to 24 h darkness, levels of CRY relative abundance increased almost twofold, oscillating with a bimodal rhythmic oscillation due to the two troughs corresponding to the previous offset and onset of light (Fig. 4B). After 72 h of darkness, a very damped unimodal rhythm appears showing a 4 h phase advance (maximal peak at 2300). Cosinor analysis shown in Table 1 revealed that the level of activity (mesor) and amplitude rhythmic parameters are modified by the different experimental conditions. The mesor of the rhythm obtained under LD (50±6) increases after 24 and 72 h darkness (89±2 and 71±4.3), when the period value (see Materials and methods) changes from 24 h to 12 hand 24 h, respectively.
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Temporal changes of CRY abundance in the brain under the same experimental conditions are shown in the chronograms of Fig. 4DF. When crayfish are subjected to LD, the CRY protein in brain tends to increase during the subjective day and decrease in the subjective night, showing maximal abundance at 19:00 h. This rhythm oscillates with a 24 h statistically significant rhythm that shows a higher activity level than the eyestalk rhythm (M=66±5) (Table 1). After 24 and 72 h of darkness the zenith of the CRY oscillation delays for 8 h, shifting to the late subjective night and adjusting to statistically significant unimodal and bimodal rhythmic oscillations with a period value equal to 25 h and 11.5 h, respectively. After darkness, as shown in Table 1, the mesor of the rhythm increases to 70±2 and 81±2, respectively. Interestingly, when the crayfish changes from LD to DD the brain rhythm's mesor value increment is about half the value of the eyestalk rhythm (22% and 48%, respectively). Cosinor analysis detected that in the three experimental conditions the brain showed statistically significant circadian and bimodal rhythms but the eyestalk did not show any statistically significant rhythm (Table 1).
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Discussion |
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This latter cluster of cells could be associated with the extraretinal
brain photoreceptor reported elsewhere in the crayfish C. destructor
(Sandeman et al., 1990). These
photoreceptor cells are reported to specifically bind to a rhodopsin-antibody,
showing maximal response at 540 nm. The findings of the present study suggest
that the cryptochromes located in different cells of this cluster, together
with this photoreceptor, may contribute to the wide spectral response of the
circadian system of P. clarkii (400700 nm) shown elsewhere
(Fanjul-Moles et al., 1992
).
The activity rhythm of this species is able to entrain to blue and red
monochromatic light in the absence of retina and lamina
(Miranda-Anaya and Fanjul-Moles,
1997
). This extraretinal synchronization is probably mediated by
brain photoreceptors; the cryptochromes should be responsible for the blue
spectrum of the circadian response to light. Unexpectedly, some of the lateral
protocerebral neurons, those basal to the hemiellipsoid body, showed a CRY
immunoreaction. These neurons apparently communicate with the medulla
terminalis through a neurite (Fig.
2A) and seem to correspond to the interneurons described elsewhere
(McKinzie et al., 2003
;
Mellon, 2003
). In P.
clarkii these cells are multimodal sensory neurons that receive sensory
input of distinct sensory systems, among them a photic pathway from the
ipsilateral eye (Mellon,
2000
). Our results suggest that cryptochromes could be elements of
the light input to the clock of crayfish, as has been proposed for other
species (Stanewsky et al.,
1998
; Emery et al.,
1998
).
The results of the biochemical and immunocytochemical analyses performed
under 12:12 LD conditions in the current work are in agreement. The
immunoreaction found at the four time points tested, 11:00 h, 19:00 h, 23:00 h
and 03:00 h, coincides with the relative abundance of CRY at these times,
determined biochemically (Figs
3,
4). There is no statistical
difference in levels in the eyestalk or the brain between 11:00 h and 23:00 h,
but there was a significant difference between 19:00 h and 03:00 h, and the
maximal CRY imunoreaction and abundance in both structures is
semi-phase-locked, showing a mirror-image relationship
(Fig. 2). The biochemical
results of this study demonstrate daily and circadian changes in the CRY
relative abundance in the eyestalk and the brain, respectively
(Table 1). In the brain, these
rhythmic changes appear to be endogenously driven, since they continue to run
freely after 72 h of darkness, changing phase and period and running with
statistically significant circadian periods in LD and DD conditions. However,
the abundance of CRY in the eyestalk showed a non-statistically significant
daily rhythm under LD, which was dampened 24 and 72 h after darkness, and
revealed no statistically significant circadian rhythms. This indicates that
the daily oscillation could be due to a masking effect of the LD cycle. The
significance of the rhythms, the mirror image of their phases, as well as the
effect of light on CRY abundance in eyestalk and brain, raise the possibility
that this protein has a dual function: one in the MT-HB, acting as a
photopigment able to absorb light and translating that information to the
master oscillator, and the other in the median protocerebrum, proposed by some
authors as a master oscillator of crayfish
(Barrera-Mera and Block, 1990),
where it participates in the rhythm-generating mechanisms. Both possibilities
exist, as demonstrated in flies and mice, two species in which both genetic
and molecular circadian mechanisms are well documented. In
Drosophila, the latest evidence suggests that CRY is a photopigment
that acts in the entrainment pathway of the clock in the brain, and also a
protein that participates in the circadian rhythm-generating process of the
compound eye and peripheral body tissues
(Stanewsky, 2003
). In mice
most evidence indicates that CRY is only involved in the clock rhythmic
generation, but it has recently been proposed that, as with insect
cryptochrome, mammalian CRYs function pleiotropically in circadian rhythm
generation, photic entrainment and behavioral responses such as masking
(Van Gelder et al., 2002
). In
crayfish, and generally in crustaceans, our knowledge on the genetic and
molecular mechanisms underlying the circadian clock is scant, although the
conserved nature of the clock genes could lead us to presume that all groups
share similar genetic proprieties. Hence, knowledge of the molecular and
physiological features of circadian mechanisms in different species will help
us to understand the biological perception of time.
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Acknowledgments |
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References |
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---|
Aréchiga, H., Fernández-Quiroz, F., Fernández-de-Miguel, F. and Rodríguez-Sosa, L. (1993). The circadian system of crustaceans. Chronobiol. Int. 10,1 -9.[Medline]
Barrera-Mera, B. and Block, G. D. (1990). Protocerebral circadian pacemakers in crayfish, evidence for mutually coupled pacemakers. Brain Res. 522,241 -245.[CrossRef][Medline]
Bernal-Moreno, J. A., Miranda-Anaya, M. and Fanjul-Moles, M. L. (1996). Phase shifting the ERG amplitude circadian rhythm of juvenile crayfish by caudal monochromatic illumination. Biol. Rhythm Res. 27,299 -301.[CrossRef]
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye-binding. Ann. Biochem. 7, 248-254.
Castañón-Cervantes, O., Battelle, B. A. and
Fanjul-Moles, M. L. (1999). Rhythmic changes in the serotonin
content of the brain and eyestalk of crayfish during development.
J. Exp. Biol. 202,2823
-2830.
Escamilla-Chimal, E. G., Van Herp, F. and Fanjul-Moles, M.
L. (2001). Daily variations in crustacean hyperglycemic
hormone and serotonin immunoreactivity during the development of crayfish.
J. Exp. Biol. 204,1073
-1081.
Emery, P., So, W. V., Kaneko, M., Hall, J. C. and Rosbash, M. (1998). CRY, a Drosophila clock and light-regulated cryptochrome, is a major contributor to circadian rhythm resetting and photosensitivity. Cell 95,669 -679.[Medline]
Fanjul-Moles, M. L., Miranda-Anaya, M. and Fuentes Pardo, B. (1992). Effect of monochromatic light upon the ERG circadian rhythm during ontogeny in crayfish Procambarus clarkii. Comp. Biochem. Physiol. 102A,99 -106.
Fanjul-Moles, M. L. and Prieto-Sagredo, J. (2003). The circadian system of crayfish, A developmental approach. Microsc. Res. Tech. 60,291 -301.[CrossRef][Medline]
Foster, R. G. and Helfrich-Forster, C. (2001). The regulation of circadian clocks by light in fruitflies and mice. Phil. Trans. R. Soc. Lond. 356B,1779 -1789.
Galizia, C. G., McIlwrath, S. L. and Menzel, R. (1999). A digital three-dimensional atlas of the honeybee antennal lobe based on optical sections acquiered by confocal microscopy. Cell Tissue Res. 295,383 -394.[CrossRef][Medline]
Inclán-Rubio, V. (1991). Shifting phase on electroretinogram circadian rhythm induced by monochromatic light stimulus in crayfish Procambarus bouvieri. Comp. Biochem. Physiol. 99A,301 -306.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
McKinzie, M. E., Benton, J. L., Beltz, B. S. and Mellon, D. (2003). Parasol cells of the hemiellipsoid body in the crayfish Procambarus clarkii, dendritic branching patterns and functional implications. J. Comp. Neurol. 462,168 -179.[CrossRef][Medline]
Mellon, D., Jr (2000). Convergence of
multimodal sensory input onto higher-level neurons of the crayfish olfactory
pathway. J. Neurophysiol.
84,3043
-3055.
Mellon, D., Jr (2003). Active dendritic properties constrain input-output relationships in neurons of the central olfactory pathway in the crayfish forebrain. Microsc. Res. Tech. 60,278 -290.[CrossRef][Medline]
Menna-Barreto, L. A., Benedito-Silva, A., Marques, M., Andrade, M. and Louzada, F. (1993). Ultradian components of the sleepwake cycle in babies. Chronobiol. Int. 10,103 -108.[Medline]
Miranda-Anaya, M. and Fanjul-Moles, M. L. (1997). Non parametric effects of monochromatic light on the activity rhythm of juvenile crayfish. Chronobiol. Int. 14, 25-34.[Medline]
Nelson, W., Tong, Y. L., Lee, J. K. and Halberg, F. (1979). Methods for cosinor rhythmometry. Chronobiol. 6,305 -323.
Page, T. L. and Larimer, J. L. (1972). Entrainment of the circadian locomotor activity rhythm in crayfish. The role of the eyes and caudal photoreceptor. J. Comp. Physiol. 78,107 -120.
Page, T. L. and Larimer, J. L. (1976). Extraretinal photoreception in entrainment of crustacean circadian rhythms. Photochem. Photobiol. 23,245 -251.
Prieto-Sagredo, J. and Fanjul-Moles, M. L. (2001). Spontaneous and light evoked discharge of the isolated abdominal nerve cord of crayfish in vitro depicts circadian oscillation.Chronobiol. Int. 18,759 -765.[CrossRef][Medline]
Roenneberg, T. and Foster, R. G. (1997). Twilight times, light and the circadian system. Photochem. Photobiol. 66,549 -556.[Medline]
Sandeman, D. C., Sandeman, R. E. and Aitken, A. R. (1988). Atlas of serotonin-containing neurons in the optic lobes and brain of the crayfish Cherax destructor. J. Comp. Neurol. 269,465 -478.[Medline]
Sandeman, R. C., Sandeman, R. E. and De Couet, H. G. (1990). Extraretinal photoreceptors in the brain of the crayfish Cherax destructor. J. Neurobiol. 21,619 -629.[Medline]
Sancar, A. (2003). Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chem. Rev. 103,2203 -37.[CrossRef][Medline]
Stanewsky, R., Kaneko, M., Emery, P., Beretta, B., Wager-Smith, K., Kay, S. A., Rosbash, M. and Hall, J. C. (1998). The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell 95,681 -692.[Medline]
Stanewsky, R. (2003). Genetic analysis of the circadian system in Drosophila melanogaster and mammals. J. Neurobiol. 54,111 -147.[CrossRef][Medline]
Van Gelder, R. N., Gibler, T. M., Tu, D., Embry, K., Selby, C. P., Thompson, C. L. and Sancar, A. (2002). Pleiotropic effects of cryptochromes 1 and 2 on free-running and light-entrained murine circadian rhythms. J. Neurogenet. 16,181 -203.[CrossRef][Medline]