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The Journal of Neuroscience, January 15, 1998, 18(2):741-750
Drosophila Photoreceptors Contain an Autonomous
Circadian Oscillator That Can Function without period mRNA
Cycling
Yuzhong
Cheng1 and
Paul
E.
Hardin2
1 Department of Biology, Texas A & M University,
College Station, Texas 77843, and 2 Department of Biology,
University of Houston, Houston, Texas 77204
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ABSTRACT |
Circadian oscillations in period
(per) mRNA and per protein (PER)
constitute, in part, a feedback loop that is required for circadian
pacemaker function in Drosophila melanogaster.
Oscillations in PER are required for oscillations in per
mRNA, but the converse has not been rigorously tested because of a lack
of measurable quantities of per mRNA and protein in the
same cells. This circadian feedback loop operates synchronously in many
neuronal and non-neuronal tissues, including a set of lateral brain
neurons (LNs) that mediate rhythms in locomotor activity, but whether a
hierarchy among these tissues maintains this synchrony is not known. To
determine whether per mRNA cycling is necessary for PER
cycling and whether cyclic per gene expression is tissue
autonomous, we have generated per01
flies carrying a transgene that constitutively expresses
per mRNA specifically in photoreceptors, a cell type
that supports feedback loop function. These transformants were tested
for different aspects of feedback loop function including
per mRNA cycling, PER cycling, and PER nuclear
localization. Under both light/dark (LD) cycling and constant dark (DD)
conditions, PER abundance cycles in the absence of circadian cycling of
per mRNA. These results show that per
mRNA cycling is not required for PER cycling and indicate that
Drosophila photoreceptors R1-R6 contain a tissue autonomous circadian oscillator.
Key words:
Drosophila; circadian clock; photoreceptors; period gene; transgene; gene expression
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INTRODUCTION |
Genetic screens for mutations that
affect circadian rhythms in Drosophila melanogaster have
identified two genes that encode components of the circadian
timekeeping apparatus, period (per) and
timeless (tim). Mutations in these genes can
shorten (perS,
timSL), lengthen
(perL), or abolish
(per01,
tim01) circadian rhythms in locomotor
activity and eclosion (Konopka and Benzer, 1971
; Sehgal et al., 1994;
Rutila et al., 1996). In parallel with these behavioral rhythms are
molecular rhythms in the abundance of per and tim
gene products; per and tim RNAs peak early in the
evening, whereas per protein (PER) and tim
protein (TIM) peak several hours later (Siwicki et al., 1988; Hardin et al., 1990; Zerr et al., 1990; Edery et al., 1994b). Induced alterations in PER or TIM levels can shift the phase of behavioral rhythms (Edery
et al., 1994a; Lee et al., 1996; Myers et al., 1996; Zeng et al.,
1996), showing that behavioral rhythms are dependent on molecular
rhythms.
To account for these molecular rhythms, a negative feedback loop model
was proposed in which PER and/or TIM suppress per and tim gene transcription (Hall, 1995; Hardin and Siwicki,
1995; Sehgal, 1995). After the lights are turned off, PER and TIM
accumulate in the cytoplasm as heteromeric complexes (Lee et al., 1996;
Zeng et al., 1996) and enter the nucleus over the course of a few
hours, starting ~6 hr after the lights are turned off (Curtin et al., 1995). In the nucleus, PER and/or TIM feedback to repress
per and (probably) tim gene transcription (Hardin
et al., 1990; Zeng et al., 1994; Sehgal et al., 1995). Destruction of
PER and TIM proteins, in turn, coincides with the accumulation of
per and tim transcripts during midday and the
start of another circadian cycle.
Little is known about how PER-TIM complexes regulate transcription
after they enter the nucleus. However, sequences that mediate PER- and
TIM-dependent transcriptional cycling have been localized to a 69 bp
fragment lying ~500 bp upstream of the per transcription start site (Hao et al., 1997). Surprisingly, per genomic
fragments lacking a promoter (Hamblen et al., 1986; Frisch et al.,
1994) or driven by constitutively active promoters (Ewer et al., 1988, 1990; Vosshall and Young, 1995) drive PER cycling and rescue locomotor activity rhythms in per01 flies. Although
PER cycling was only measured in 12 hr light/dark (LD) cycles in these
studies, such cycling presumably persists because behavioral rescue was
measured in constant darkness. Behavioral rescue by these transgenes,
however, raises the intriguing possibility that per RNA
cycling is not necessary for PER cycling and locomotor activity
rhythms.
The per gene is expressed in a variety of neuronal (i.e.,
photoreceptors, antennae, brain neurons and glia, and thoracic
ganglion) and non-neuronal (i.e., gut, Malpighian tubules, testes, and
ovary) tissues (Liu et al., 1988; Saez and Young, 1988; Siwicki et al., 1988; Ewer et al., 1992). The circadian feedback loop operates in all
of these tissues except the ovary, where per is
constitutively expressed (Hardin, 1994). In adults, lateral neurons
(LNs) are the only cells within the per expression pattern
known to control autonomously a rhythmic output, locomotor activity
(Ewer et al., 1992; Frisch et al., 1994). Oscillator autonomy has also
been observed in the pupal prothoracic gland (Emery et al., 1997), but
whether oscillators operate autonomously in other adult tissues is
unknown.
In this report we drive per from a crippled rhodopsin
1 (Rh1) promoter to determine whether per
mRNA cycling is required for PER cycling and whether photoreceptors
contain an autonomous circadian oscillator. PER produced by this
transgene cycles in abundance under LD and constant dark (DD)
conditions. Because the transgene expresses constitutive levels of
per mRNA, the rhythms in PER show that an oscillator is
running through some post-transcriptional mechanism. In addition, this
oscillator is operating exclusively in photoreceptors R1-R6, which
suggests that these cells contain an autonomous oscillator.
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MATERIALS AND METHODS |
Plasmid construction. The
Rh1(
250)-LacZ,
Rh1(180)-LacZ, and
Rh1(120)-LacZ transformation constructs were
made as follows. Rh1 promoter fragments starting 250,
180, and 120 bp upstream of the Rh1 transcription start
site, respectively, and extending to the Rh1 translation
initiation site were generated by PCR using Rh1-per (Zeng et al., 1994) as a template.
The Rh1 upstream region primers included the
Rh1(250)+XhoI sense primer
(5
-GCCTCGAGACTCAAGAATAATAC-3), the
Rh1(180)+XhoI sense primer
(5-GCCTCGAGCCCATTGCGATGTG-3), and the
Rh1(120)+EcoRI sense primer
(5-CCGAATTCGCGGCCGCGGTACCTGTCGACACTTT-3). The antisense
primer at the Rh1 translation initiation site was Rh1(ATG)+BamHI (5-GCGGATCCATTGTGTTTTGGTTAC-3).
The
Rh1(250)+XhoI/Rh1(ATG)+BamHI and
Rh1(180)+XhoI/Rh1(ATG)+BamHI
amplification products were digested with XhoI and
BamHI, and the
Rh1(120)+EcoRI/Rh1(ATG)+BamHI amplification product was digested with EcoRI and
BamHI and cloned into the pCaSpeR-
-gal transformation
vector (Thummel et al., 1988).
The Rh1(180)-per transformation construct was
generated as follows. A 6.2 kb SalI-XbaI DNA
fragment from 13.2 (HA/C) (Rutila et al., 1992), consisting of genomic
sequences from the SalI site in exon 3 to an XbaI
site ~2 kb downstream of per transcribed sequences and
including a C-terminal hemagglutinin tag, was cloned into Bluescript
KS. A 630 bp DNA fragment from the SalI site at 120 bp
of the Rh1 promoter to the SalI site in exon 3 of
the per genomic sequence was removed from
Rh1-per (Zeng et al., 1994) and inserted at the
SalI site upstream of the SalI to XbaI
per fragment in Bluescript KS. The orientation of the
SalI fragment was checked via PCR and restriction enzyme
digests, and the plasmid having the per coding region driven
by the Rh1120 promoter fragment was named
Rh1(120)-perKS. To make the
Rh1(180)-perKS, we first removed an
XhoI site close to the XbaI site at the 3-end of
the per genomic sequence by digestion with XhoI,
then filling in with the Klenow fragment of DNA polymerase I. An
XhoI-SpeI fragment starting 180 bp upstream of
the Rh1 transcription initiation site to the SalI
site of per exon 3 was generated by PCR using the
Rh1(180)+XhoI sense primer (see above) and the
ex3SalI antisense primer (5-GCAACGCGTTGTCGACCTTCTGGC-3)
and then was digested with XhoI and SpeI. This
fragment was inserted into the Rh1(120)-perKS plasmid after Rh1 sequences between 120 bp of the
transcription start site and the SpeI site in the
Rh1 leader sequence were removed, thereby forming
Rh1(180)-perKS. The inserts from
Rh1(180)-perKS were removed by digestion with
XhoI and XbaI and were inserted into the CaSpeR4
transformation vector (Thummel and Pirotta, 1991) to form the
Rh1(180)-per transformation plasmid.
Fly stocks and germ line transformation. The wild-type
D. melanogaster strain Canton-S and transgenic fly strains
were raised on a cornmeal, sugar, agar yeast, and Tegosept (a mold
inhibitor) medium at 25°C. P-element-mediated transformation was
performed as described previously (Hao et al., 1997). Transformant
lines with inserts on the second or third chromosomes were balanced with In(2LR)Cyo or In(3LR)TM2, respectively, and
were crossed into a y,per01,w
genetic background.
Behavioral analysis. Locomotor activity of adult male
Canton-S;
y,per01,w;Rh1(180)-per-1;
y,per01,w;Rh1(180)-per-2;
and y,per01,w flies were
monitored and analyzed as described by Hamblen et al. (1986). Flies
were entrained in 12 hr light/dark cycles at 25°C for 72 hr; then the
lights stayed off for 7 d. Locomotor activity was monitored from
the first day of entrainment, and data collected during constant
darkness were analyzed to determine the period and strength of the
rhythm (Hamblen et al., 1986). Flies were designated rhythmic or
arrhythmic based on the criteria of Ewer et al. (1992).
RNase protection assays. Flies used for time course analyses
were entrained at 25°C in 12 hr light/dark cycles for at least 96 hr
before collection. For each time point, RNA was extracted as described
from either whole heads (Hardin et al., 1990) or eyes (Hardin et al.,
1992b; Zeng et al., 1994), and 10 µg of whole-head RNA or 5 µg of
eye RNA was used for RNase protection assays as described (Hardin et
al., 1990). To make an RNase protection probe, we cloned a
per cDNA fragment from the SpeI site in exon 2 to the SalI site in exon 3 into Bluescript KS vector. The
RNase protection probe was linearized by SpeI digestion, and
an RNA probe was transcribed with T3 RNA polymerase. It protects an
endogenous per fragment of 324 nucleotides (nt) and a
Rh1(180)-per-derived transcript of 285 nt.
Protected per bands were quantitated using a Fuji BAS50
phosphorimager. As a control for the amount of RNA in each lane, an
antisense ribosomal protein 49 (RP49) probe was used in each RNase
protection assay (Hardin et al., 1990).
Immunohistochemistry. For LD experiments, homozygous
per01;Rh1(180)-per-1
flies were entrained in 12 hr LD cycles for at least 4 d and
collected at 4 hr intervals. For each time point, flies were
immediately embedded into OCT compound (Tissue-Tek) on dry ice, and
10-12 µm horizontal cryostat sections were prepared. A rabbit
polyclonal anti-PER antiserum (kindly provided by Ralf Stanewsky)
preabsorbed against per01 embryos was
used for immunostaining. Immunostaining detection was performed using a
goat anti-rabbit secondary antibody conjugated to horseradish
peroxidase as described (Siwicki et al., 1988). For DD experiments,
heterozygous
per01;Rh1(180)-per-1/+
flies were entrained for 4 d in 12 hr LD cycles; the lights were
then left off, and eight samples were collected every 4 hr starting at
circadian time 4 (CT4) during the first and second days of constant
darkness. Sample preparation and immunostaining for flies collected in
DD conditions were performed as described for flies collected in LD
conditions.
Western blotting and quantitation. Fly head extracts were
prepared from either homozygous transgenic flies for LD experiments or
from heterozygous transgenic flies for DD experiments. Flies were
entrained and collected in LD and DD conditions as described above for
the immunohistochemistry and then were subjected to Western blotting
analyses (Edery et al., 1994b) with the following modifications: the
first antibody is the same used for immunohistochemical staining and
was diluted to 1:20,000, and the secondary antibody is anti-rabbit IgG
horseradish peroxidase-conjugated antibody (Amersham, Arlington
Heights, IL) diluted 1:5000 in blocking solution. X-ray film exposures
of Western blots were scanned using OFOTO software and quantitated
using National Institutes of Health Image 1.6 software. The level of
PER at each time point was taken as the PER signal minus the background
in each lane. In each independent time course, the highest PER signal
was set to 1.0, and all other time points were normalized to this
value. The normalized PER values from three independent time courses of
Canton-S and
per01;Rh1(180)-per-1/+
and from two independent time courses of
per01;Rh1(180)-per-2/+
were added together to yield pooled data for each genotype. The peak
PER value from the pooled data was then set to 1.0. The normalized
pooled data from each genotype were used to generate a curve based on
its fit to a polynomial function using NIH Image 1.6 software.
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RESULTS |
A minimal rhodopsin 1 gene promoter drives constitutive
expression in photoreceptors R1-R6
To test whether circadian oscillator function requires
per mRNA cycling and operates autonomously in peripheral
tissue (i.e., cells that are not capable of driving locomotor activity
rhythms), we had to find an appropriate promoter to drive
per expression. Such a promoter should meet the following
criteria: (1) it directs gene expression in a tissue that normally
expresses per, (2) it expresses a constant level of mRNA,
and (3) it drives expression at a level comparable with that of
per. One promoter that meets the first two criteria is the
ninaE (rhodopsin 1, Rh1) promoter that
constitutively expresses mRNA in the six outer photoreceptor cells of
each ommatidium (i.e., R1-R6) but not in the inner photoreceptors R7
and R8. However, this promoter expresses high levels of mRNA and was
used to show that PER overexpression represses the endogenous per RNA cycling (Zeng et al., 1994).
To make this promoter useful for our purposes, we sought to reduce its
level of expression while maintaining its tissue specificity. An
earlier study had identified an Rh1 promoter fragment
[Rh1(120/+67)] with these properties (Mismer and Rubin,
1987); however, this promoter fragment was not specific to
photoreceptors R1-R6 in our hands (H. Hao, Y. Cheng, and P. E. Hardin, unpublished observations). Therefore, we tested the expression
level and tissue specificity of two progressively larger portions of
the Rh1 promoter in transgenic flies using a lacZ
reporter gene (Fig. 1). Flies transformed
with the Rh1(250)-LacZ and
Rh1(180)-LacZ constructs, which contain 250 or
180 bp of Rh1 upstream sequences, respectively, and leader sequences up to the initiating ATG, were sectioned and stained for
-galactosidase (-gal) activity. Both constructs showed specific staining in the eye, but Rh1(250)-LacZ showed
considerably higher levels of expression than did
Rh1(180)-LacZ (Fig. 1). Although each
Rh1(180)-LacZ line was expressed specifically
in the eye, expression in two of the lines was patched (data not
shown). Such a pattern may be caused by position effects because the
Rh1 promoter is adjacent to the 5-end of the P-element
vector. Because we wanted to minimize expression levels, we used the
Rh1(180) promoter for subsequent experiments.
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Figure 1.
Top. A crippled Rh1
promoter produces low levels of -gal in eyes. A,
Schematic representation of the
Rh1(250)-LacZ and
Rh1(180)-LacZ DNA constructs. The
Rh1(250) and Rh1(180) promoter
fragments, which contain 250 and 180 bp of upstream sequences, respectively, and leader sequences up to the translation initiation site, are represented by black bars. These promoter
fragments were fused to lacZ sequences (white
bars) at the lacZ translation initiation site.
B, Spatial expression and -gal staining intensity in
Rh1(250)-LacZ transgenic flies.
-Gal activity was detected via X-gal staining for 12 hr in a
horizontal section through a whole head oriented with the anterior end
up. -Gal activity (blue stain) is present throughout
the eyes of this and four other lines. C, Spatial
expression and -gal staining intensity in
Rh1(180)-LacZ transgenic flies.
-Gal activity was detected in heads as described in
B. -Gal activity is present throughout the eyes of
this and other lines. Two other lines showed eye-specific staining
present in patches within the eye.
Figure 2.
Bottom. Rh1(180)-per
expresses specifically in photoreceptors R1-R6. A,
Schematic representation of the
Rh1(180)-per DNA construct. The
Rh1(180) promoter fragment, which contains sequences
from 180 bp upstream of the transcription start site to the
translation initiation site, is represented by a black bar. The Rh1(180) promoter was fused to
per genomic sequences at the per
translation initiation site. White bars symbolize
per protein coding exons 2-8, the hatched
bar denotes the per 3-untranslated sequence,
thin lines represent per intron
sequences, and the dashed line denotes genomic sequences
downstream from per. HA refers to the
hemagglutinin tag inserted at the C terminus of PER (Rutila et al.,
1992). B, Spatial expression of PER in
per01;Rh1(180)-per
transgenic flies. PER immunoreactivity was detected in flies collected
at Zeitgeber time 23 (ZT23), sectioned, and incubated with anti-PER
antibodies. A horizontal section of a whole head is oriented with the
anterior end up. PER immunoreactivity (dark brown stain)
is only present in the nuclei of photoreceptors R1-R6, which are
indicated by small arrows. No immunoreactivity is seen
in the area of the LNs, indicated by the large arrows. Photoreceptor specificity was obtained in a total of five transgenic strains.
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The Rh1(180) promoter and its leader sequence were fused
to per genomic sequences at the translation initiation site
to drive low level per expression specifically in
photoreceptors R1-R6 (Fig. 2). Eight
independent transgenic lines were obtained and crossed into a
per01 background to eliminate endogenous
PER expression. The spatial expression pattern of PER in these
transgenic strains was examined by anti-PER immunohistochemical
staining. Six of the eight strains showed undesirable per
expression patterns; one expresses PER at high levels, two show PER
expression in photoreceptors R1-R6 and the central brain, and three
express per in only a subset of R1-R6 cells (data not
shown). The other two transgenic strains have the desired PER
expression pattern: photoreceptor R1-R6-specific expression at levels
similar to that of PER. To confirm that PER is not expressed at levels
too low to detect by immunohistochemical methods in the central brains
of these two transgenic lines, we monitored the lines for locomotor
activity rhythms. Both lines are arrhythmic, indicating that they do
not have a functional circadian pacemaker in their brain (Table
1). These two lines, designated
per01;Rh1(180)-per-1
and
per01;Rh1(180)-per-2,
were used for subsequent molecular analyses.
Rh1(180) promoter activity is constitutive in DD
conditions and fluctuates in LD cycles
Previous studies showed that Rh1 mRNA in wild-type
flies is expressed at high levels throughout the circadian cycle (Zeng et al., 1994). To ensure that per RNA from this crippled
Rh1(180) promoter does not cycle in abundance, we
entrained
per01;Rh1(180)-per-1
flies in 12 hr LD cycles for 4 d and collected flies every 4 hr
during the fifth LD cycle. Total head RNA was prepared from each time
point, and levels of Rh1(180)-per mRNA were
determined by RNase protection assays. Whole heads were used in these
assays because the Rh1(180)-per transgene is
specifically expressed in eyes (Fig. 2; Table 1), and the
transgene-derived transcript can be monitored independently of the
endogenous per01 transcript. In contrast
to wild-type Rh1 mRNA levels,
Rh1(180)-per mRNA levels are ~2.5-fold
higher during the day than during the night (Fig.
3). To determine whether these mRNA
fluctuations are attributable to the Rh1(180) promoter or
clock regulatory sequences within, or downstream of, the per
coding region, we examined mRNA levels from
Rh1(180)-LacZ transgenic flies under LD
conditions. As seen with Rh1(180)-per mRNA,
Rh1(180)-LacZ mRNA was found to be ~2.5-fold
higher during the day than during the night (data not shown),
indicating that the Rh1(180) promoter, and not PER coding
or downstream sequences, mediates these mRNA fluctuations.
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Figure 3.
Rh1(180) promoter activity is constitutive
in DD conditions and fluctuates in LD cycles. RNase protection assays
were performed on total head RNA samples prepared from
per01;Rh1(180)-per
flies. A, Endogenous and transgene-driven
per RNA levels measured in samples collected during LD
cycles at the time points indicated.
Rh1(180)-per represents the
transgene-driven per-protected fragment,
per01 represents the endogenous
per-protected fragment, and RP49
represents the ribosomal protein 49 mRNA-protected fragment. The
white and black boxes represent times
when lights were on or off, respectively. B,
Quantitation of the data in A. Relative RNA abundance
refers to the values of endogenous
per/RP49 (filled
squares) or transgene-driven per/RP49 (open squares),
when the peak value of endogenous per mRNA was adjusted
to 1.0. The white and black boxes
represent times when lights were on or off, respectively. Similar
results were obtained from six independent time courses.
C, Endogenous and transgene-driven per
RNA levels measured in samples collected during the first day of DD at
the time points indicated. The protected fragments are as designated in
A. The hatched and black
boxes represent times when lights would have been on or off,
respectively, if the light cycle were continued. D,
Quantitation of the data in C. Relative RNA abundance is
described in B. The hatched and black boxes represent times when lights would have been
on or off, respectively, if the light cycle were continued. Similar results were obtained from three independent time courses.
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To determine whether the Rh1(180) promoter is sensitive to
light or is under clock control, we measured
Rh1(180)-per mRNA under constant dark
conditions. Flies were entrained in 12 hr LD cycles for 4 d,
transferred to constant darkness, and collected every 4 hr for one
circadian cycle. Head RNA extracted from each time point was analyzed
by RNase protection assays, and unlike the previous results in LD
conditions, Rh1(180)-per mRNA levels do not
fluctuate under constant dark conditions (Fig. 3). Likewise, the levels
of per01 transcripts are constant (Fig.
3), because of the lack of oscillation in noneye head tissue where more
per01 mRNA is expressed. Identical
results were obtained when Rh1(180)-LacZ mRNA
levels were measured under constant dark conditions (data not
shown).
Rh1(180)-per mRNA levels are approximately
fivefold higher than endogenous per01
transcript levels in these transgenic lines (Fig. 3). This difference is mainly caused by the Rh1(180) promoter because
Rh1(180)-LacZ mRNA levels are two- to
threefold higher than wild-type per mRNA levels (data not
shown). In addition, per01 mRNA levels
are two- to threefold lower than that of wild-type per mRNA
(Hardin et al., 1990), which would further magnify per mRNA
levels for the Rh1(180)-per transgenics. These
measurements were made on homozygous transgenic lines; however, the
levels of Rh1(180)-per mRNA may be lower in
heterozygotes because one of the dosage compensation elements (i.e.,
intron 1) is missing in these constructs (Cooper et al., 1994).
Consequently, heterozygotes were used in experiments performed under
constant conditions (see below).
PER abundance and nuclear localization cycle during
LD conditions
Because TIM is light sensitive and is required for PER
accumulation and nuclear localization (Vosshall et al., 1994; Price et
al., 1995; Hunter-Ensor et al., 1996; Myers et al., 1996; Zeng et al.,
1996), we expected that both PER abundance and nuclear localization
would cycle under LD conditions in these transgenic lines. To determine
whether PER levels cycle, we entrained
per01;Rh1(180)-per-1
flies to 12 hr LD cycles and collected flies at 4 hr intervals during
the final cycle. Protein extracts were prepared from fly heads and
analyzed on Western blots with anti-PER antiserum (see Materials and
Methods). The results show that PER cycles under these conditions with
a lower level during the day than during the night (Fig.
4A). The cycling
profile under these conditions is different from that of wild-type
flies in two respects; it peaks at ZT15 rather than ZT21, and PER is
never completely absent during the day. These differences may be caused
by either the relatively high levels of per RNA from the
transgene or a premature (light-driven) rise in mRNA levels from the
transgene.
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Figure 4.
PER abundance and nuclear localization cycles in
Rh1(180)-per flies under LD conditions.
A, Head protein extracts were prepared from
per01;Rh1(180)-per-1
transgenic flies collected at the time points indicated, from
per01 flies collected at ZT23, and
from wild-type Canton-S (CS) flies collected at ZT23.
These protein extracts were used to prepare Western blots, which were
probed with anti-PER antibodies. The gel strip shows the
level of PER immunoreactivity for each sample. The white
and black boxes represent times when lights were on or
off, respectively. Similar results were obtained from five independent
time courses. B,
per01;Rh1(180)-per
flies were entrained for 4 d in 12 hr LD cycles and collected at
the time points indicated during the fifth LD cycle. Horizontal
sections were prepared and probed with anti-PER antibodies. One
hemisphere of a head is shown for each time point and is oriented with
the anterior end up. PER immunoreactivity is seen as a dark
brown stain. Two independent time courses showed similar
results.
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Another parameter that can be used to measure oscillator function is
the PER nuclear localization cycle (Curtin et al., 1995). To determine
whether PER derived from the Rh1(180)-per
transgene undergoes nuclear cycling under LD conditions, we measured
PER immunohistochemically on sections of flies collected every 4 hr (Fig. 4B). Little if any nuclear PER is detected
until ZT15, a time point ~2-3 hr earlier than that when PER begins
to enter the nucleus in wild-type flies. Nuclear PER peaks between ZT19 and ZT23 and then begins to decline during the light phase until it
becomes undetectable at ZT11. These results show that PER nuclear localization cycles under LD conditions. However, in
Rh1(180)-per flies, PER accumulation peaks at
ZT15, but the peak level of nuclear PER occurs at ZT23. This is
different than the situation in wild-type flies in which nuclear
localization occurs between ZT18 and ZT20 (Curtin et al., 1995),
~4-5 hr before the peak in protein accumulation (Edery et al.,
1994a). This uncoupling of PER accumulation and nuclear localization
suggests that these two aspects of the circadian cycle are controlled
independently because nuclear localization that is solely dependent on
PER concentration would result in a premature movement of PER into the
nucleus.
Endogenous per01 mRNA cycling
is rescued in the eyes of
per01;Rh1(180)-per
flies
In the circadian feedback loop, nuclear PER suppresses the
transcription of the PER gene, thereby producing fluctuations in mRNA
abundance. From this, we would predict that nuclear PER cycling seen in
per01;Rh1(180)-per
flies should rescue endogenous per01 RNA
cycling. To test this prediction, we performed RNase protection assays
on total RNA isolated from the eyes of homozygous
per01;Rh1(180)-per-1
flies collected every 4 hr during LD cycles (see Materials and
Methods). The results show that endogenous
per01 mRNA cycling is rescued in the eyes
of these transgenic flies and has an amplitude of approximately
fourfold (Fig. 5). The peak in
per01 mRNA abundance occurs at ZT11,
which is 4-6 hr earlier than the per mRNA peak in wild-type
flies (Hardin et al., 1990; Brandes et al., 1996). This early
per01 mRNA peak could be accounted for by
the premature accumulation of nuclear PER (Fig. 4B),
which acts to decrease the levels of per mRNA. Endogenous
tim mRNA also exhibited low amplitude cycling, consistent
with the notion that per and tim transcription
are coregulated. As expected, the levels of
Rh1(180)-per mRNA were higher during the light
phase because of the sensitivity of the Rh1(180) promoter
to light (Figs. 3, 5).
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Figure 5.
Endogenous
per01 and tim mRNA
cycling is rescued in the eyes of Rh1(180)-per
flies. RNase protection assays were performed on total eye RNA samples
(5 µg) prepared from
per01;Rh1(180)-per
flies. A, Endogenous
per01, endogenous tim,
and transgene-driven per RNA levels measured in samples
collected during LD cycles at the time points indicated. Rh1(180)-per represents the
transgene-driven per-protected fragment, per01 represents the endogenous
per-protected fragment, tim represents the endogenous tim-protected fragment, and
RP49 represents the RP49-protected fragment. The
white and black boxes represent times when lights were on or off, respectively. B,
Quantitation of the data in A. Relative RNA abundance
refers to the values of endogenous per/RP49 (open squares),
endogenous tim/RP49 (filled
squares), or transgene-driven
per/RP49 (filled
diamonds), with each peak adjusted to 1.0. The
white and black boxes represent times
when lights were on or off, respectively. Similar results were obtained from three independent time courses.
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PER abundance cycles under constant dark conditions
To determine whether PER cycling in photoreceptors R1-R6 is a
circadian rhythm, we examined both PER levels and nuclear localization in constant darkness.
per01;Rh1(180)-per-1/+
flies were entrained in an LD cycle for 4 d and collected during
the first 2 d of DD at 4 hr intervals. Head protein extracts were
prepared and subjected to Western blotting in parallel with those of
wild-type controls. The results show that PER levels in these
transgenic flies fluctuate over two circadian cycles. Compared with PER
cycling in wild-type flies, in which low levels are present at CT11 and
high levels are present at CT23 (Fig.
6A), the overall PER
levels in
per01;Rh1(180)-per-1/+
flies cycle with a different phase and/or period; PER decreases during
the first subjective day, reaches its trough level at CT15-19,
increases from CT23 to the next CT15, and then decreases (Fig.
6A). This phase and/or period difference in PER cycling is also seen in
per01;Rh1(180)-per-2/+
flies, although the delay in PER accumulation (vs wild-type PER) is
greatly reduced in comparison with that of
per01;Rh1(180)-per-1/+
flies (Fig. 6A).
View larger version (37K):
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Figure 6.
Overall PER abundance cycles under DD
conditions. A,
per01;Rh1(180)-per-1/+
and
per01;Rh1(180)-per-2/+
flies were entrained in LD cycles for 4 d before being transferred
to constant darkness and collected at the circadian time points
indicated. Head protein extracts were made and subjected to Western
blotting as described in Figure 4. The gel strips show the level of PER immunoreactivity for each sample. Head extracts from
CS and per01 flies
collected at ZT23 were used as positive and negative controls for PER
immunoreactivity, respectively (right two lanes). The hatched and black boxes represent times
when lights would have been on or off, respectively, if the light cycle
were continued. Similar results were obtained from three independent
time courses for CS, three time courses for
per01;Rh1(180)-per-1,
and two time courses for
per01;Rh1(180)-per-2.
B, Quantitation of PER cycling under DD conditions is
shown. Data from three independent time courses of CS
(open squares; solid line), three
independent time courses of
per01;Rh1(180)-per-1/+
(open circles; dashed line), and two
independent time courses of
per01;Rh1(180)-per-2/+
(filled triangles; line with
short and long dashes) are plotted. The
peak time point for each time course was set to 1.0, and all other time
points are relative to this value. The curves were generated by fitting
the normalized pooled data for each genotype to polynomial functions
(see Materials and Methods). The circadian time points and the
hatched and black boxes are described in
A.
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|
Quantification of these data shows that PER from both transgenes cycles
over the course of 2 d with a 1.5-2-fold amplitude for
per01;Rh1(180)-per-1/+
flies and an approximately twofold amplitude for
per01;Rh1(180)-per-2/+
flies (Fig. 6B). The cycling amplitudes of these
transgenic lines approach that of the 2-2.5-fold cycling seen in
wild-type flies collected and tested in parallel with the transgenic
lines. This dampened cycling amplitude of wild-type flies in DD
conditions is similar to that seen in previous studies (Edery et al.,
1994b; Zeng et al., 1996). Because PER abundance could only be measured
for a limited number of cycles under DD conditions, it is not possible
to determine whether the differences in peaks and troughs in PER levels
are caused by period differences, phase differences, or a combination
of both. However, it would not be surprising if the higher
per mRNA levels and the lack of per mRNA cycling
in the transgenic lines has a negative effect on PER cycling
amplitude.
To determine whether nuclear PER cycles under constant dark conditions,
we entrained and collected
per01;Rh1(180)-per-1/+
flies as described above. Sections were prepared for each time point
and stained with anti-PER antibodies. The nuclear PER staining signal
is intense as flies enter DD conditions, declines to a minimum value
between CT16 and CT20, and then increases during the next circadian day
(Fig. 7). Unlike the case in LD cycles,
PER staining in
per01;Rh1(180)-per-1/+
flies is never completely absent in the nucleus. The fluctuations in
PER intensity on sections fit well with overall PER abundance
measurements on Western blots (Fig. 6).
View larger version (125K):
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Figure 7.
Top. Levels of nuclear PER cycle under
DD conditions.
per01;Rh1(180)-per-1/+
flies were entrained in LD cycles for 4 d before being transferred
to constant darkness and collected at the indicated time points.
Horizontal sections were prepared and probed with anti-PER antibodies
as described in Figure 4B. One hemisphere of a
head is shown for each time point and is oriented with the anterior end
up. PER immunoreactivity is seen as a dark brown stain.
Two independent time courses showed similar results. Each time point
represents similar results from at least four
flies.
Figure 8.
Bottom. Nuclear
PER level cycles in lateral neurons of 7.2:2 flies under DD conditions.
Homozygous per017.2:2 flies were
entrained in LD cycles for 4 d before being transferred to
constant darkness and collected at CT3, CT9, CT15, and CT21. Immunohistochemical staining of PER was done as described in Figure 4B. One hemisphere of a head is shown for each
time point and is oriented with the anterior end up. The
arrowheads denote anti-PER antibody staining in LNs.
Three independent time courses showed high intensity staining at CT3
(n = 5 flies) and CT9 (n = 6 flies), low intensity staining at CT15 (n = 6 flies), and medium intensity staining at CT21 (n = 9 flies).
|
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DISCUSSION |
In this study, the Rh1 promoter was modified so that it
would produce low levels of mRNA in photoreceptors R1-R6. This
modified promoter, Rh1(180), is >20-fold less active than
the native Rh1 promoter and produces transcripts
specifically in photoreceptors R1-R6 at levels approximately fourfold
higher than the wild-type per mRNA peak (Figs. 1, 2, 3, 5). The
Rh1(180) promoter is constitutively active in constant
darkness and is light inducible, producing 2.5-fold more mRNA in the
light phase than in the dark phase (Fig. 3). This light inducibility
was not seen in earlier studies with the complete Rh1
promoter (i.e., having 3 kb of upstream sequences), where high levels
of expression may mask differences in mRNA abundance (Zeng et al.,
1994). Such diurnal fluctuations in opsin mRNA abundance have been seen
in certain species of toads and fish; opsin mRNA levels are 4-10-fold
higher during the light phase than in the dark phase and can be induced
by light in the dark phase (Korenbrot and Fernald, 1989). However,
unlike the case in Drosophila, opsin mRNA levels in these
species continue to fluctuate under constant darkness, apparently under
the control of a circadian oscillator (Korenbrot and Fernald,
1989).
In wild-type flies, PER cycling is mediated, in part, via interactions
with the TIM protein. TIM forms heteromeric complexes with PER and is
required for both PER accumulation and nuclear localization (Vosshall
et al., 1994; Gekakis et al., 1995; Price et al., 1995; Lee et al.,
1996; Zeng et al., 1996). Light-induced degradation of TIM, for
example, in natural environmental cycling conditions, seems very likely
to mediate daily resetting of the Drosophila circadian clock
such that the oscillator is in synchrony with the environment (Lee et
al., 1996; Myers et al., 1996; Zeng et al., 1996). In
Rh1(180)-per flies, per mRNA levels
are constantly higher than is the wild-type per mRNA peak.
These high levels of per mRNA nevertheless give rise to
fluctuations in PER abundance and nuclear localization under LD
conditions (Fig. 4). This high amplitude PER cycling is apparently
mediated by TIM; when
per01;Rh1(180)-per
transgenic flies are in LD cycles, light drives TIM cycling in R1-R6
that, in turn, drives both overall PER abundance and nuclear PER to
cycle. This light-mediated cycling of TIM and its effect on PER cycling
have been seen in previous studies (Myers et al., 1996; Zeng et al.,
1996; Dembinska et al., 1997; Stanewsky et al., 1997). However, when
extremely high levels of PER are present, as is the case with the
Rh1-per transgenes, TIM cannot maintain PER
cycling (Zeng et al., 1996).
The phase of PER cycling in
per01;Rh1(180)-per
flies is earlier than that of PER in wild-type flies under LD
conditions. This early peak in PER abundance (~6-8 hr early) is not
followed by an equally early movement into the nucleus, although
nuclear localization is ~3 hr early (Fig. 4). This result shows that
the PER abundance and nuclear localization cycles can be disconnected,
indicating that high levels of PER accumulation alone are not
sufficient for nuclear localization. Because PER moves into the nucleus
as a complex with TIM (Lee et al., 1996; Zeng et al., 1996), the high
levels of PER seen early in the dark phase [in
per01;Rh1(180)-per
flies] may not be complexed with TIM and are therefore unable to move
into the nucleus.
In LD cycles, endogenous per01 mRNA
cycling is rescued in photoreceptors R1-R6 of
per01;Rh1(180)-per
flies. However, this rescued per01 mRNA
cycling is different than the cycling seen for wild-type per
RNA (Hardin et al., 1992b; Zeng et al., 1994). First, the rescued
per01 mRNA cycling amplitude is low. This
may be because PER never drops to zero as it does in wild-type flies
(Fig. 4A), and constant levels of
per01 mRNA are seen in 25% of the
photoreceptor cells that lack transgene expression. Second,
per01 mRNA peaks at ZT11, several hours
earlier than the wild-type per mRNA peak (Fig. 5). This
early mRNA peak probably results from higher than normal PER levels,
which would lead to premature PER nuclear translocation and a decrease
in per gene transcription. Nuclear PER in our transgenic
flies was detected 3 hr earlier than it was in wild-type flies (ZT15 vs
ZT18) (Fig. 4B), indicating that the period of time
between the per mRNA peak and the appearance of nuclear PER
in these transgenic flies is similar to that seen in wild-type flies
(Hardin et al., 1990; Zerr et al., 1990; Hardin and Siwicki, 1995).
Cycling of PER abundance in
per01;Rh1(180)-per
flies persists in constant darkness, showing that per RNA
cycling is not required for PER cycling (Fig. 6). This result is
compelling because expression is restricted to photoreceptors, a cell
type in which expression levels are high enough to be quantitatively
measured. Previous studies in which mRNA expression levels in certain
tissues are inferred, but not directly measured, are consistent with
this finding. The glass promoter, which is constitutively
active in wild-type flies, has been used to drive per
expression in the LNs and photoreceptors of
per01 flies. This
glass-per transgene rescues long-period
(~28-34 hr) locomotor activity rhythms to varying degrees (25-69%
rescue) in each independent line and gives rise to cycling in PER
abundance and nuclear localization under LD conditions in
photoreceptors (Vosshall and Young, 1995). Even though per
RNA cycling was not tested in these transgenic lines, constitutive
expression from the endogenous glass promoter suggested that
PER cycling can occur without per mRNA cycling (Vosshall and
Young, 1995).
Constructs containing a 7.2 kb fragment of per genomic DNA
lacking the promoter, first exon, and most of the first intron were
also used to rescue rhythms in per01
flies. One of the two lines that rescue locomotor activity rhythms (7.2:2) expresses PER exclusively in LNs (Hamblen et al., 1986; Frisch
et al., 1994). In this line, PER abundance and nuclear localization
cycle under LD conditions (Frisch et al., 1994). This cycling is not
simply driven by LD cycles, however, because it persists under DD
conditions (Fig. 8). As seen in wild-type flies, the PER cycling amplitude in 7.2:2 flies dampens under DD
conditions because of PER remaining in the nucleus at times when it
would be undetectable in LD cycles (Fig. 8; Zerr et al., 1990). The
behavioral rescue seen in the two 7.2 lines was attributed to internal
(i.e., within or downstream of the transcribed region) cis-acting
transcriptional cycling elements that are active in a permissive
genomic environment (Frisch et al., 1994). Our results argue against
the existence of internal cycling elements because we see no mRNA
cycling in any of the
per01;Rh1(180)-per
lines, which only differ from 7.2 lines in that they lack the last 343 bp of the first intron and 46 bp of untranslated second exon sequences.
Of these differences, the first intron sequence was incapable of
driving cyclic transcription (H. Hao, Y. Cheng, and P. E. Hardin,
unpublished observations). Our results, therefore, suggest that rescue
of PER cycling and behavioral rhythms in 7.2:2 flies occurs without
underlying cycles in per mRNA.
If per mRNA cycling is not required for PER cycling in
general, then what function does transcriptional feedback serve in wild-type flies? We believe that feedback regulation of mRNA cycling is
important for several aspects of circadian clock function. For example,
transgenic strains assumed to express constitutive levels of
per mRNA (i.e., glass-per; 7.2:2;
hsp70-7.2) exhibit period-altered and/or weak locomotor
activity rhythms. Likewise, per01;Rh1(180)-per
flies, which are known to express constant levels of per
mRNA, exhibit an altered phase and/or period of PER cycling compared
with wild type. These examples suggest that per mRNA cycling
affects PER cycling and behavior; however, inappropriate per
mRNA or protein expression (i.e., levels that are too high or too low;
altered or incomplete spatial expression) could also account for these
effects on PER cycling and behavior. Another aspect of clock function
that may require per and/or tim mRNA cycling is
phase resetting. Because light acts to decrease TIM abundance, high
levels of tim mRNA early in the dark phase were proposed to
replenish TIM levels, leading to a phase delay, whereas low levels of
tim mRNA late in the dark phase could not replenish TIM
levels, leading to a phase advance (Hunter-Ensor et al., 1996; Myers et
al., 1996; Zeng et al., 1996). Finally, circadian feedback is believed
to control the expression levels of clock output genes. A group of 20 Drosophila rhythmically expressed genes (Dregs) have been
identified (Van Gelder et al., 1995). Rhythmic expression of several of
these transcripts (i.e., Dregs 5, 6, 9, 10, and 15), as measured by
mRNA cycling, is dependent on per gene function (Van Gelder
et al., 1995; Van Gelder and Krasnow, 1996), indicating that circadian
feedback loop function is important for rhythmic Dreg expression.
Likewise, another clock-regulated gene, Crg-1, has been
identified that exhibits PER-dependent mRNA cycling (Rouyer et al.,
1997).
When per is specifically expressed in the photoreceptors
R1-R6, PER levels show circadian cycling under both LD and DD
conditions. These results demonstrate that Drosophila eyes
contain an autonomous circadian oscillator (i.e., one that operates in
the absence of per expression and circadian feedback loop
function in other tissues). This finding is consistent with earlier
studies on disconnected (disco) flies, which show
that per mRNA and protein cycle in heads under both LD and
DD conditions even though disco flies are behaviorally arrhythmic and lack per-expressing lateral neurons (Zerr et
al., 1990; Dushay et al., 1992; Hardin et al., 1992a). These results suggest that other circadian oscillators operate in the absence of the
pacemaker for locomotor activity.
Like Drosophila photoreceptors, hamster suprachiasmatic
nucleus (SCN) and retina (Ralph et al., 1990; Tosini and Menaker, 1996), Xenopus eyes (Besharse and Iuvone, 1983),
Aplysia eyes (Jacklet, 1969), gypsy moth testes
(Giebultowicz et al., 1989), and chicken pineal glands (Takahashi et
al., 1980) contain tissue autonomous circadian oscillators. This
autonomy extends down to individual isolated basal retinal neurons in
Bulla (Michel et al., 1993), and individual (but not
isolated) rat SCN neurons (Welsh et al., 1995) show circadian
electrical activity. In contrast, the circadian rhythm of lateral eye
sensitivity in horseshoe crab (Limulus polyphemus) is a
slave oscillator controlled by the brain circadian clock (Barlow et
al., 1980; Kaplan and Barlow, 1980; Barlow, 1983; Kass and Barlow,
1992). Thus, depending on the tissue and the organism, circadian
oscillators can operate tissue/cell autonomously or can be dependent on
oscillators in other tissues. In Drosophila, we have shown
that the adult eye contains an autonomous circadian oscillator. This,
along with clock autonomy in the prothoracic gland (Emery et al., 1997)
and lateral neurons (Frisch et al., 1994) and lateral neuron
independent oscillators in Malpighian tubules (Giebultowicz and Hege,
1997; Hege et al., 1997), suggests that the Drosophila
circadian system consists of a collection of autonomous oscillators.
Synchrony among these oscillators could be mediated by the light
sensitivity of TIM because a hierarchical organization is not
apparent.
| |
FOOTNOTES |
Received July 17, 1997; revised Oct. 23, 1997; accepted Oct. 31, 1997.
This study was supported by National Institutes of Health Grant
NS31214. We thank Ralf Stanewsky for providing anti-PER antibody and
Isaac Edery for providing the 13.2 (HA/C) DNA construct. We also thank
Isaac Edery and Ralf Stanewsky for help with the Western blotting
protocol, and Juan Qiu, David Allen, and Bronwyn Morrish for their
assistance with experiments. We are grateful to Haiping Hao for
assistance with fly embryo microinjection, Cai Wu for behavioral
analyses, and Jerry Houl for dissecting fly eyes. We thank Haiping Hao,
Lisa Lyons, and Greg Cahill for comments on this manuscript.
Correspondence should be addressed to Dr. Paul Hardin, Department of
Biology, University of Houston, Houston, TX 77204.
| |
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Top
Abstract
Introduction
per transgenic flies
