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Volume 17, Number 2,
Issue of January 15, 1997
pp. 676-696
Copyright ©1997 Society for Neuroscience
Temporal and Spatial Expression Patterns of Transgenes Containing
Increasing Amounts of the Drosophila Clock Gene
period and a lacZ Reporter: Mapping Elements of
the PER Protein Involved in Circadian Cycling
Ralf Stanewsky1,
Brigitte Frisch1,
Christian Brandes1,
Melanie J. Hamblen-Coyle1,
Michael Rosbash1, 2, and
Jeffrey C. Hall1
1 Department of Biology and 2 Howard Hughes
Medical Institute, Brandeis University, Waltham, Massachusetts
02254
This article is dedicated to
Brigitte Frisch. She found delight in her work and life. She believed
that everyone had something important to offer and made us all feel
special. Her humor and strength will always be remembered.
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Rhythmic oscillations of the PER protein, the product of the
Drosophila period (per) gene, in
brain neurons of the adult fly are strongly involved in the control of
circadian rhythms. We analyzed temporal and spatial expression patterns
of three per-reporter fusion genes, which share the
same 4 kb regulatory upstream region but contain increasing amounts of
per's coding region fused in frame to the bacterial
lacZ gene. The fusion proteins contained either the
N-terminal half (SG), the N-terminal two-thirds (BG), or nearly all
(XLG) of the PER protein. All constructs led to reporter signals only
in the known per-expressing cell types within the
anterior CNS and PNS. Whereas the staining intensity of SG flies was
constantly high at different Zeitgeber times, the in situ signals in BG and XLG flies cycled with ~24 hr
periodicity in the PER-expressing brain cells in wild-type and
per01 loss of function flies. Despite
the rhythmic fusion-gene expression within the relevant neurons of
per01 BG flies, their locomotor
activity in light/dark cycling conditions and in constant darkness was
identical to that of per01 controls,
uncoupling protein cycling from rhythmic behavior. The XLG construct
restored weak behavioral rhythmicity to (otherwise) per01 flies, indicating that the
C-terminal third of PER (missing in BG) is necessary to fulfill the
biological function of this clock protein.
Key words:
circadian rhythms;
period gene;
lacZ
reporter;
Lateral Neurons;
fusion proteins;
locomotor behavior;
immunochemistry
INTRODUCTION
The period (per) gene is
thought to be a central component of the circadian clock in
Drosophila. Mutations in this gene abolish (per01), shorten
(perS, perClk, and
perT), or lengthen
(perL) daily locomotor activity rhythms
that persist in constant darkness (DD) in wild-type flies (Konopka and
Benzer, 1971
; Dushay et al., 1992; Konopka et al., 1994). Under normal
light/dark (LD) conditions, perS and
perL mutants show phase advances or delays of
their evening activity peaks, whereas
per01 mutants do not entrain at all and
simply react to the LD changes (Hamblen-Coyle et al., 1992; Wheeler et
al., 1993) (see Fig. 12).
Fig. 12.
Locomotor activity of SG, BG, and XLG transgenics
and their controls in LD cycles. Data were plotted from the entrainment portions (the first 7 or 14 d, 12:12 hr LD cycle) of the locomotor activity runs performed in this study (see Materials and Methods, Table
1). Histograms were generated by first superposing locomotor data from
a given (male) fly (see inset in
H), followed by superposing the daily activities
of all flies from the same genotype (for additional details about
preparation of these plots, see Hamblen-Coyle et al., 1989). The
open bars indicate activity exhibited in the light
portion, and the solid bars activity monitored in the
dark portion of the cycle. A,
per+ SG3 males (n = 40)
and ry506 control males
(n = 46). B,
per+ SG10 males (n = 24)
compared with their w control males
(n = 13). Note the substantially earlier evening
peak in SG transgenic flies. C, BG/TM2
males in the per+ (n = 38) and per01
(n = 34) genetic backgrounds (the latter,
inset). D, BG6 males in the same pair of
backgrounds (n = 29 and 26, respectively). The
phase advance (compared with the ry506
control males shown in A) of the evening peak is not as
strong as for SG (see also Table 1). Note as well that there is no
anticipation of the LD and dark/light (DL) transitions in both of the
per01 BG strains.
E, XLG-A males (n = 47) in a
per+ background showing only a subtle phase
advance of their evening activity, compared with the control strain
Df(1)w (n = 47) shown in the
inset; XLG-A otherwise appeared to behave in a
per+-like manner. F, XLG-B in
a per+ background (n = 46), the behavior of which is very similar to that of
per+ XLG-A. G,
per01 XLG-A
(n = 42). H,
per01 XLG-B
(n = 38). Both G and
H reveal complex patterns of LD behavior; the flies
seemed to anticipate the LD changes, showing increased activity before
the lights are turned off in the evening, with a distinct (not
per+-like) phase advance of nearly 3 hr. In
addition, these per01 XLG
flies clearly reacted to LD and DL transitions as the straight per01 w
sn3 control males do (n = 30;
see inset in G). The complex behavior caused by the simultaneous presence of the XLG transgene and the per01 allele was observed in
the records from individual animals (see inset in
H), and therefore is not a reflection of two
subpopulations of differently behaving flies.
[View Larger Version of this Image (45K GIF file)]
per RNA and PER protein fluctuate in abundance with 24 hr
periods (Hardin et al., 1990; Zerr et al., 1990; Zeng et al., 1994). Expression of the per gene in
per01, perS, and
perL is affected in the same manner as locomotor
behavior, indicating a molecular feedback loop in which the PER protein
regulates its own transcription (for review, see Hall, 1995; Rosbash,
1995). Mutations in a different clock gene, timeless
(tim) also produce arrhythmic (tim01)
and period-altered (timSL) behavior (Sehgal et
al., 1994; Rutila et al., 1996). The products of the tim
gene undergo similar fluctuations as those of per, and both
PER and TIM proteins can dimerize in vivo (Sehgal et al.,
1995; Zeng et al., 1996). tim01 or
per01 mutations result in loss of
rhythmic RNA expression of the other (nonmutated) gene, indicating that
both genes are necessary for establishing the molecular feedback loop
in which the PER-TIM heterodimer is thought to play an important role
(Zeng et al., 1996).
Behavioral analysis of genetic mosaics expressing PER in certain
lateral brain neurons (LNs) of per01
flies is sufficient to restore rhythmic behavior under DD conditions, suggesting that these cells have pacemaker function (Ewer et al., 1992). Analysis of PER coding transgenes showing rhythmic expression in
the LNs of per01 flies was consistent
with these results: the transgenics were able to entrain to LD cycles
and showed rhythmic behavior under DD conditions (Frisch et al., 1994;
Vosshall and Young, 1995).
A genomic 4.2 kb DNA fragment upstream of per's coding
region was shown to drive rhythmic RNA expression of reporter genes fused to this sequence when endogenous PER protein is present in these
flies (Hardin et al., 1992). Fusion of per-regulatory sequences directly to lacZ did not lead to cycling of
-galactosidase (-GAL) levels in a per+
genetic background, whereas this 5
-flanking DNA from the
per locus and half of its coding region were reported to
permit -GAL to fluctuate with a 24 hr period (Zwiebel et al., 1991).
That result did not prove reproducible, which was one of the reasons that prompted our analysis of a series of per-lacZ fusion
constructs, each sharing the same 5 regulatory sequences but with
increasing amounts of per's coding region (Fig.
1). Thus, we mapped regions of the per gene
product that are necessary to allow PER-like protein turnover. To ask
whether the cycling of a certain fusion protein could be independent of
endogenous PER, we also analyzed the temporal expression of these
transgenes in a per01 background.
Analysis of locomotor behavior was performed under LD and DD conditions
to determine which fusion proteins might mediate rhythmic behavior.
Fig. 1.
Structure of per-lacZ fusion genes
analyzed for temporal and spatial expression. In the top
panel, the structure of a 13.2 kb genomic DNA fragment
containing the per gene is shown. This construct
restores rhythmic behavior after transformation into per01 mutants (Citri et al., 1987).
The solid line represents upstream untranscribed
regulatory sequences as well as introns and 3 untranscribed DNA. The
bars reflect exon sequences; solid parts
designate untranslated and open bars protein-coding DNA
sequences. Striped portions represent the PAS domain,
which can function as an intermolecular protein dimerization motive
(residues 240-496) (Burbach et al., 1992). Shaded areas
represent the C domain involved in intramolecular protein interactions
(residues 524-685, Huang et al., 1995), and checked
areas represent the GT repeats, which vary in number among different Drosophila melanogaster strains (e.g., 20 Gly-Thr pairs in the wild-type strain Oregon-R) (Yu et
al., 1987). Potential PEST sequences, identified by the program
PESTFIND (Rechsteiner et al., 1987) are indicated by
asterisks and extend from residues 135 to 162 (exon 3),
652 to 664 (exon 5), 1184 to 1215 (exon 6), and 1242 to 1256 (also
showing the highest PEST score, exon 7). The arrow
indicates per's transcription start site
(+1). Translation starts at the NcoI site
and stops 32 bp 3 of the NciI site. The different
per-lacZ fusion genes contain various amounts of this genomic per DNA fragment, fused in frame to the
E. coli lacZ gene. They all share the same 5 regulatory
region (from the BamHI site at 
4200) and transcribed
sequences up to the SacI site in exon 5 (including the
PAS domain), where the SG fragment is fused to lacZ. SG
contains only one complete PEST sequence and is missing parts of the C
domain and all of the GT repeat. The BG construct contains additional
per-coding DNA up to the BamHI site in
exon 5, including two PEST sequences as well as the complete C domain and GT repeat. XLG extends to the NciI site in exon 8, including two additional PESTs that are missing in BG. Note that this
construct does not quite encode the full-length PER protein (see
Materials and Methods).
[View Larger Version of this Image (12K GIF file)]
MATERIALS AND METHODS
Generation of per-lacZ fusion constructs
The structure and generation of the SG construct has been
described previously (Liu et al., 1988). It contains a 4.2 kb
5-flanking region that is part of a 13.2 kb genomic per
fragment; the latter almost completely rescues the effects of the
arrhythmic per01 mutation (Citri et al.,
1987; Yu et al., 1987; Dushay et al., 1992). In addition to this
upstream region, SG contains per DNA coding for ~50% (638 amino acids) of the N-terminal PER protein (up to the SacI
site, therefore called SG) (Fig. 1) fused in frame to the
bacterial lacZ gene.
The generation of the BG construct is described elsewhere (Dembinska et
al., 1997). It contains the same 4.2 kb 5-flanking region as SG and
encodes 868 amino acids of PER, corresponding to the N-terminal
two-thirds of this protein (up to the BamH1 site and
therefore called BG) (Fig. 1) fused in frame to the lacZ gene (as above).
The XLG construct was generated as follows. First, the CaspeR
lacZ transformation vector (Thummel et al., 1988) was
modified by replacing the BamHI and PstI
restrictions sites with XhoI and NotI sites after
Klenow treatment and blunt-end ligation of the respective linkers. The
XhoI site was created to allow in-frame cloning of genomic
per DNA to lacZ. A 6.4 kb genomic per
DNA fragment containing sequences from 4200 bp to +2267 bp (the bp
numbers are with reference to the transcription start of the
per gene) was cut with NotI and XbaI
and ligated into the NotI and XbaI sites of the
transformation vector (the XbaI site is located between the
XhoI and NotI sites of the modified vector). In a
different step, a clone containing genomic per DNA from the
XbaI site at +2267 to a XhoI site at position
+6658 was generated [the XhoI site was generated after
first filling in the original NciI site at position +6658 with Klenow
and then blunt-ligating it to a filled in XhoI site of
pBluescript II KS(+)]. To create the final construct, this 4.4 kb
XbaI/XhoI fragment was ligated in frame into the
XbaI/XhoI sites of the transformation vector.
This construct contains DNA coding for nearly the whole PER protein,
except 10 amino acids at the C-terminal end, normally encoded by DNA
extending from the NciI site at position +6658 to the
translational stop codon TAG at position +6690. Because the
NciI site was replaced by XhoI and a
XhoI linker was used to create this restriction site in the
lacZ gene, this construct was named XLG.
Stocks and P-element transformation
The genetic variants used for this study are described in
Lindsley and Zimm (1992). Flies were raised on a cornmeal, sugar, yeast, and Tegosept medium (the latter being a mold inhibitor) on a
12:12 hr LD cycle (lights on at 8 A.M.)
Two independently isolated SG lines, carrying this per-lacZ
fusion gene inserted into the X (SG10) or the second
chromosome (SG3) (Liu et al., 1988; Zwiebel et al., 1991) were used in
this study. Both lines were obtained after transforming
per01;ry506
embryos. To generate a per+ SG10 strain, the
per01 SG10 chromosome was recombined with
a per+ w sn3 chromosome
to replace the loss-of-function mutation with per's normal
allele (Ewer et al., 1992).
One transgenic BG line was obtained after transforming
per01;ry506
embryos with the BG construct (X. Liu, personal communication). In this
line, the P-element is inserted on the third chromosome and causes
lethality when flies are homozygous for the insertion; this line is
balanced with the multiply inverted third chromosome In(3LR)TM2,
ry Ubx and was therefore named BG/TM2 (indicating that
flies from this stock carry only one copy of the BG construct). The BG
transposon in BG/TM2 was mobilized by crossing that strain to a transposase-producing 
2-3 strain (Robertson et al., 1988); this
resulted in the homozygous-viable BG6 strain, in which the transgene is
located on the second chromosome.
The XLG-A and XLG-B strains were generated after transforming
Df(1)w embryos with the XLG construct (carrying the
mini-white+ gene as selectable marker).
Transformations were performed using standard techniques (Rubin and
Spradling, 1982); transposase was supplied by co-injection of the
helper plasmid pUChs
2-3 (Laski et al., 1986). Two transformed
lines were recovered, derived from different injected embryos. The
locations of the insertions were determined genetically by crossing
transformant flies to second and third chromosomal balancer
chromosomes. The XLG-A and XLG-B lines carry their (homozygous-viable)
insertions on the second chromosome. To analyze these transgenes in a
per01 background, the Df(1)w
chromosome (which carries per+) was replaced by
an X chromosome carrying
per01 w sn3.
Histochemistry
Transgenic males carrying either the SG-, BG-, or
XLG-lacZ fusion genes were exposed to at least three 12:12
hr LD cycles at 25°C before sectioning. For the cycling experiments,
flies were collected at two different Zeitgeber times (ZTs) in the case of SG and XLG (ZT12 and ZT24 for SG, ZT9 and ZT21 for XLG; ZT24 defines
"lights on" and ZT12 "lights off") and at four different times
in the case of BG (ZT3, ZT9, ZT15, and ZT21). For the time points at
which the flies had to be collected in the dark, vials were moved (in
darkness) from the incubator to a small light-tight container where
they were kept until they were either anesthetized or embedded in a
tissue-freezing medium (TBS).
Immunohistochemistry. Flies were anesthetized, their wings
and legs removed, and the specimens transferred to an Eppendorf tube
containing 1 ml ice-cold fixing solution (4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.0). After fixing them for 4 hr at 4°C on a rotating mixer, the fixing solution was removed and
the flies were washed four times for 10 min in 0.1 M
phosphate buffer, pH 7.4, at 4°C. After the last wash, flies were
incubated overnight in 25% sucrose in 0.1 M phosphate
buffer, pH 7.4, at 4°C. Flies were frozen in tissue-freezing medium,
and horizontal sections (10 µm) of individual specimens were made on
a cryostat microtome (SLEE). Sections were then transferred to glass
slides, dried for 1 hr, and rinsed in 0.1 M phosphate
buffer, pH 7.4, for 10 min and in PBS two times for 10 min each at room
temperature. Each slide was preincubated for 1 hr at room temperature
with 400 µl PBS containing 3% normal horse serum (NHS), 0.1% Triton X-100, and 0.1% BSA. Sections were then incubated overnight at 4°C
with a monoclonal mouse anti--GAL antibody (Promega, Madison, WI)
diluted 1:2000 in PBS containing 3% NHS, 0.03% Triton X-100, and
0.1% BSA. On the next day, slides were rinsed three times for 10 min
each at room temperature (as for all the following washes and
incubations) in PBS containing 0.03% Triton X-100 and 0.1% BSA and
then incubated for 1 hr with a 1:200 dilution (see above) of a
secondary biotinylated horse anti-mouse antibody (Vector Laboratories,
Burlingame, CA). After rinsing the sections three times for 10 min each
in PBS containing 0.03% Triton X-100 and 0.1% PBS, then two times for
10 min each in the same solution without Triton X-100, the Vectastain
ABC ELITE Kit (Vector) was used to amplify the staining signal. Slides
were washed again two times for 10 min each in PBS containing 0.1% BSA
and three times for 10 min each in PBS before they were developed with
DAB, as described in Siwicki et al. (1988) with minor modifications. All sections included in the cycling analysis were incubated with 0.3 mg/ml DAB solution for exactly 3 min. Slides were then baked with
Crystal Mount (Biomeda), and coverslips were mounted with DPX (Fluka,
Ronkonkoma, NY). Sections were viewed and photographed using Nomarski
optics and a Zeiss Axiophot light microscope.
Anti-PER stainings were performed by incubating fly sections (see
above) with a polyclonal rabbit anti-PER antibody, which was raised
against full-length PER protein expressed in a baculovirus expression
system (Liu et al., 1992). Anti-PER antibodies were used in a final
concentration of 1:6000 after preabsorbing the serum against
per01 embryos. Signals were visualized
after incubation with a secondary biotinylated donkey anti-rabbit
antibody (1:200) and DAB, as described above.
X-gal staining. Stainings with
5-bromo-4-cloro-3-indolyl--D-galactopyranoside (X-gal) were carried
out on 10 µm frozen horizontal sections, as described in Liu et al.
(1988), except that slides were mounted and photographed as described
above (for the antibody stainings).
Scoring of staining intensities. In the case of the BG
construct, transgenic males from the BG/TM2 strain in a
per+ and per01
background were collected at four different time points during a 24 hr
cycle (see above). Five males (per+
genetic background) or six males
(per01 background) per time point
were analyzed. After staining and mounting, the slides were coded and
scored by three different investigators, who inspected the sections at
160× magnification under the microscope. Levels of staining were
subjectively scored using an intensity scale of 1 to 4, in increments
of 0.5 (1 = no detectable staining above background, 4 = most
intense staining). Scoring was carried out separately for the following
three per-expressing tissues in the head: photoreceptor (PR)
cells, glia, and per-expressing LNs (cf. Zerr et al., 1990).
Final (to be plotted) scores were calculated as follows. First, the
mean for each animal was determined as the average of the value given
by each of the three investigators, then the five or six means for all
animals sectioned at the same time were averaged to calculate the final
mean (± SEM) for a given genotype, tissue, and time (see Results; see
Fig. 8). In the case of the SG construct type, males from the two
transgenic lines SG3 and SG10 [the former in a
per+ (n = 10) and a
per01 (n = 8) background,
the latter in a per+ background only
(n = 10)] were sectioned at two opposite ZTs (see
above) and stained with X-gal. Collections were made from incubators
that were kept on opposite phases [LD and dark/light (DL)] so that
pairs of flies with the same genotype but entrained to opposite ZTs
could be collected and processed at the same time (each pair was
processed on a given experimental day). To standardize the scoring of
the staining intensity, photographs (all at the same exposure) of each
fly brain, at the level of the esophagus, were taken. These images of
fly brains, from flies sectioned on the same day, were then ranked by
three different observers (each fly was given one rank, representing an
average staining intensity of PRs, glia, and the ventral group of
per-expressing LNs). These photographs were then decoded,
and ranks given to a pair of flies, with the same genotype and opposite
ZT, were compared. If the fly at ZT12 was given a higher rank (e.g.,
stronger staining intensity) compared with its partner at ZT24, the
pair was classified as "higher staining at ZT12"; and vice versa.
If both flies of a pair turned out to have the same rank, they were
classified as "equal" (see Fig. 6).
Fig. 8.
Quantification of BG-mediated staining
intensities. Sections from per+ or
per01 BG/TM2
males were stained with anti--GAL-antibody. Flies were entrained and collected under 12:12 hr LD conditions. Five
(per+) or six
(per01) flies were
sectioned and stained at each time point. The staining intensities in
the LNs, glia, and PRs of each fly were subjectively scored by three
observers (who were unaware of either genotype or ZT of the preparations), using an intensity scale from
1 to 4. The means for each genotype are plotted as a function of ZT (see Materials and Methods, including the procedure of SEM
calculation). A, Staining intensities in LNs in a
per+ background. B,
per01 background; in both
genetic backgrounds, the staining intensities show circadian
fluctuations, although cycling is less robust and with lower amplitude
in the mutant background. C, Staining of glia in a
per+ background. D,
per01 background. Only in the
per+ background were strong circadian
fluctuations of staining intensities detectable. E, PR
staining in a per+ background.
F, per01
background. Again, circadian changes in staining intensities were
detectable only when per+ was
expressed.
[View Larger Version of this Image (20K GIF file)]
Fig. 6.
Quantification of staining intensities for the SG3
and SG10 per-lacZ transformants. Three observers were
asked to rank (X-gal-mediated) staining intensities of fly brains
sectioned at ZT24 and ZT12 on the same day (see Materials and Methods).
The intensity for a given pair of flies (with the same
per genotype) was classified as "higher at lights
on" (ZT24), "higher at lights off" (ZT12), or "equal." For
the per+ SG3/SG3 and
per+ SG10 transgenics, five pairs of flies
were scored, and for per01
SG3/SG3, four pairs (resulting in a total of 15 classifications for the
per+ genetic background and 12 classifications in the per01
background). The majority of fly pairs from all genotypes showed equal
staining at both time points or higher staining at lights off (in
contrast to wild-type PER or BG- and XLG-per-lacZ
fusion proteins, which stain most intensely around lights on) (Zerr et al., 1990; present study).
[View Larger Version of this Image (24K GIF file)]
Immunoblotting
Flies from the XLG-A and XLG-B transgene strains, in a
per+ and a per01
background, were entrained for at least 3 d in 12:12 hr LD cycles, as described for the histochemistry experiments. Animals from all four
genotypes were collected and immediately frozen on dry ice at six
different ZTs (ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22). Total fly-head
protein extracts were prepared from 100 to 200 heads of each
collection, as described in Edery et al. (1994). Equal amounts of each
extract (~100 µg) were mixed with 5× SDS sample buffer (Laemmli,
1970) and separated on 5.7% polyacrylamide (29.6:0.4,
acrylamide:bisacrylamide ratio)/SDS gels. After electrophoresis, proteins were electroblotted to nitrocellulose membranes (12-14 hr
with 150 mA in a buffer containing 20% methanol, 0.1% SDS, 14.2 mM Tris, 192 mM Glycin). To ensure that the
transfer of the 300 kd XLG fusion proteins had been complete, gels were
stained with Coomassie brilliant blue. The quality of protein transfer and the (intended) equal loading were checked by staining the proteins
on the nitrocellulose membrane, using the reversible Ponceau S stain
(Sigma, St. Louis, MO). After blocking with 1% BSA in a solution
containing 140 mM NaCl, 10 mM Tris/HCl, ph 7.5, 0.05% Tween 20 (TBST) membranes were incubated with a polyclonal rabbit anti-PER (see above) or a mouse monoclonal anti--GAL
(Promega) antibody (both 1:10,000 diluted in 5% nonfat dry milk in
TBST) for 2 hr. Filters were washed one time for 15 min and three times for 5 min in TBST and incubated for 30 min with a secondary HRP-coupled anti-rabbit or anti-mouse antibody (Amersham, Arlington Heights, IL),
respectively. After washing (see above), proteins were visualized using
the Enhanced Chemi-Luminescence Kit (Amersham) followed by
autoradiography. Typically, exposures were for 1-60 sec. Band intensities were quantified after exposing membranes to
chemiluminescence sensitive screens (usually 10 times longer than for
x-ray exposures) and by subsequent imaging of exposed screens in a
phosphoimager (Bio-Rad, Hercules, CA). After staining a membrane with
the anti-PER antibody, the same membrane was stripped (in 100 mM -mercaptoethanol, 2% SDS, 62.5 mM
Tris/HCl, pH 6.7, for 30 min at 50°C), blocked, and incubated with
anti--GAL antibody, as described above. Amplitudes of protein
cycling were calculated after first setting the highest expression
values (as obtained from the phosphoimager data) in each experiment
equal to 1, followed by dividing the mean of the two highest values
(usually ZT18 and ZT22) by that of the two lowest values (usually ZT2
and ZT6).
Circadian behavioral rhythms
Locomotor activity of adult males was monitored automatically,
as described in Hamblen et al. (1986). Data were processed and
analyzed, as described in Hamblen-Coyle et al. (1992).
Flies were kept under 12:12 hr LD conditions at 25°C for 7 or 11 d, depending on the experiment. Recording of the locomotor activity
data began after 1 d in LD conditions. On day 8 or 12, respectively, the lights stayed off, such that the flies were subsequently monitored in DD conditions for the next 12-14 d. To
determine the period of free-running rhythms, data collected under DD
conditions were searched for periodocities using the 
2
periodogram (Sokolove and Bushell, 1978). Significant periods were
determined as described by Hamblen et al. (1986).
To determine the exact position of the morning and evening activity
peaks in LD conditions, a program called Phase was applied (for
details, see Hamblen-Coyle et al., 1992). It allows an objective determination of an activity peak (with respect to times of
environmental transitions in LD cycles) for each fly on a given day;
then a mean (per day) value is computed for an individual for which
successive cycles involve the same environmentally cycling conditions.
These (per fly) mean phase values were used in a (nonparametric)
Mann-Whitney U test to perform statistical comparisons
among the different genotypes. The software (Hamblen-Coyle et al.,
1992) was also used to compute group phase values for all the animals
of a given genotype tested (see Table 1).
Table 1.
Entrained and free-running behavior of SG, BG, and XLG
transgenics
| Genotype |
No.
of experiments (n) |
Morning phase (hr ± SEM) |
Evening phase (hr ± SEM) |
Rhythmic/ tested (%) |
Tau ± SEM |
|
| per01
SG10/Y;ry506 |
2 (13) |
| w
SG10/Y |
2 (24) |
+0.90
± 0.1 |
1.80 ± 0.05 |
19
/23 (82.6) |
22.3
± 0.1 |
| per01/Y;SG3;ry506 |
3 (43) |
| +/Y;SG3;ry506 |
3 (40) |
+0.35
± 0.1 |
2.00 ± 0.1 |
23 /31 (74.2) |
23.8
± 0.1 |
| per01/Y;BG/TM2ry506 |
2 (34) |
| +/Y;BG/TM2ry506 |
2 (38) |
+0.75
± 0.05 |
1.05 ± 0.05 |
29 /37 (76.3) |
23.7
± 0.1 |
| per01/Y;BG6;ry506 |
2 (26) |
| +/Y;BG6;ry506 |
2 (29) |
+0.25
± 0.1 |
0.80 ± 0.1 |
29 /29 (100) |
23.3
± 0.1 |
| per01 w
sn/Y;XLG-A |
3 (42) |
0.40 ± 0.1 |
2.25
± 0.15 |
10 /41 (24.4) |
24.6
± 1.5* |
| Df(1)w/Y;XLG-A |
3 (47) |
+0.35
± 0.1 |
0.80 ± 0.1 |
42 /46 (91.3) |
23.3
± 0.1 |
| per01w
sn/Y;XLG-B |
3 (38) |
0.50 ± 0.1 |
2.75
± 0.15 |
7 /35 (20.0) |
21.9
± 1.1* |
| Df(1)w/Y;XLG-B |
3 (46) |
+0.20
± 0.1 |
0.95 ± 0.1 |
43 /45 (95.6) |
22.9
± 0.1 |
| w/Y |
2 (13) |
+0.05 ± 0.3 |
0.70
± 0.1 |
13 /13 (100) |
23.6
± 0.1 |
| Df(1)w/Y |
5 (47) |
+0.10
± 0.15 |
0.20 ± 0.10 |
45 /47 (95.7) |
23.6
± 0.1 |
| +/Y;ry506 |
6 (46) |
+0.60
± 0.1 |
0.00 ± 0.1 |
45 /45 (100) |
24.1
± 0.1 |
| Wild-type male (Canton S) |
1 (9) |
1.05
± 0.2 |
0.50 ± 0.1 |
9 /9 (100) |
23.8
± 0.1 |
|
LD and DD behavior for the different transgenic types and
controls. The latter carried a per+ allele of
the period locus, as well as marker mutations contained in
the genetic backgrounds of the various transgenic types (see Materials
and Methods). The two per01 controls
contained ry506 or w sn3
as marker mutations. All males tested from both strains (n = 53 for per01ry506
and n = 30 for per01 w
sn3 were arrhythmic when tested under DD conditions.
The second column shows the number of independent experiments that were
done with the different transgenics during a time span of 18 months.
n indicates the number of animals tested that entrained to
the 12:12 hr LD cycle; these individual records were therefore used in
the phase analysis. Note that not all animals entrained to this LD
cycle; only between 65% (per01
SG10/Y) and 97%
(per01 w sn/Y)
of all the per01 genotypes were rhythmic
in LD. Overall, 86% of the flies among the 8 per01 genotypes were rhythmic (i.e., had
entrained). In contrast, 99% of all
per+-expressing males tested were rhythmic in
these LD conditions. The morning and evening phase values were
calculated from locomotor behavior recorded in the LD portion of the
experiment using the Phase program (see Materials and Methods). Values
(in hours) are given relative to lights-on (ZT0) for the morning phase
and to lights off (XT12) for the evening phase. The last two columns tabulate the data collected in DD. The numbers of rhythmic individuals compared with the total tested (and the percentages), as determined by
periodogram analysis (see Materials and Methods). From those analytical
plots, "power" values are obtainable, as are the numbers of 0.5 hr
time bins crossing the "significance line" (cf. Ewer et al., 1992)
(see also Fig. 13). Only flies showing periods in combination with a
power of  20 and a time-bin width of 2 were considered rhythmic. To
determine whether the phase advances and period shortenings, observed
in all transgenic strains in a per+ background,
were significant, nonparametric Mann-Whitney U tests were
performed, comparing each P-element strain and its relevant control. In
all cases, the observed differences were significant (p < 0.05).
|
|
*
Although the average periods of the
per01 XLG-A or XLG-B flies were within
the circadian range, the individual periods of the rhythmic flies were
distributed over a broad range (between 18.5 and 27.5 hr for XLG-A and
between 18 and 28 hours for XLG-B).
|
|
RESULTS
Spatial expression pattern of SG, BG, and XLG transgenes at a high
time point
In situ expression of the per gene has been
revealed by using per-lacZ fusion genes and by anti-PER
antibody stainings (Liu et al., 1988, 1991, 1992; Siwicki et al., 1988;
Ewer et al., 1992). In Drosophila heads, per is
expressed in certain neurons, in PRs R1-R8, in the ocelli, and in glia
cells of the optic lobes and the central brain. The
per-expressing neurons consist of two classes, known as
Lateral Neurons (LNs), which are located in the cortex between the
dorsal anterior brain and the medulla, and dorsal neurons (DNs),
located in the posterior dorsal-most cortex. The LNs were reported to
consist of two groups: a more dorsally located cluster of approximately
three to seven cells on each side of the brain called the
dorsal LNs (LNd) and another, more ventral cluster of at least eight cells called the ventral LNs
(LNv). For the DNs, between 2 and 10 cells can be found on
each side of the brain (Ewer et al., 1992; Helfrich-Förster and
Homberg, 1993; Frisch et al., 1994; Helfrich-Förster, 1995).
To compare the spatial distribution of the three different PER--GAL
fusion proteins used in this study with that of wild-type PER protein,
sections of SG, BG, and XLG transgenic flies were stained by
application of anti--GAL antibodies (Fig. 1). To allow comparisons
of expression levels among the different transgenes, all flies were
fixed for sectioning late at night (ZT21), when PER is expressed at its
maximum levels (Zerr et al., 1990). In agreement with earlier studies
(Liu et al., 1988; Ewer et al., 1992), the SG fusion protein is
expressed in all known per-expressing cells, although
several differences (quantitative and qualitative) were observed.
Figure 2 demonstrates head expression of the SG3 transgenic
line, which shows the same staining pattern and intensity differences
in the various cell types that were reported previously for the SG10
strain (per--gal) (see Materials and
Methods; see also Ewer et al., 1992). Compared with PER expression, SG
signals appear to be more intense in glia (in Fig. 2C,
arrowheads) and PRs, whereas staining in the LNs
(arrows) is much fainter (compare Fig. 2 with Ewer et al.,
1992). Also, the number of stained glial cells in the optic lobes and
especially in the central brain seems to be larger compared with that
of PER positive cells (Fig. 2) (cf. Ewer et al., 1992). In addition the
fusion protein is detectable in both nuclei and cytoplasm of LNs and
PRs (glial cells are too small to make this distinction), whereas PER
is predominantly nuclear late at night (Fig. 2) (cf. Ewer et al., 1992;
Liu et al., 1992, Curtin et al., 1995).
Fig. 2.
Distribution of anti--GAL immunoreactivity in a
per+ SG3 transgenic adult male.
A, Staining pattern at the level of the esophagus, in a
horizontal section (the others depicted here are in the same plane).
Scale bar, 40 µm. B, Magnification of
A; arrow points to a group of the
LNv (relatively ventral LNs; see Ewer at al., 1992);
arrowheads point to glia cells in the outer rim of the medulla, in the cortex, and to glia located at the border between the
cortex and the neuropil of the central brain. Scale bar, 8 µm.
C, A different section located just ventral to that in
A and B; the arrow points
to LNv, arrowheads to glia cells located in the lamina and the outer rim of the medulla. Scale bar, 12.5 µm. D, A section showing not only LNv
(arrow), but also glia cells in the second optic chiasm
(arrowhead). Scale bar, 12.5 µm.
[View Larger Version of this Image (102K GIF file)]
The larger BG fusion protein is also expressed in all known
per-expressing cells; however, in contrast to SG, its
expression seems more closely to reflect the wild-type expression
pattern (Fig. 3). All types of neurons (DN, LNd,
LNv) were stained intensely. This fusion protein fills up
the somata, which made it possible to distinguish the different sizes
(and shapes) of the neurons. The DNs are a loose cluster of cells. The
two neurons shown in Figure 3, E and F, have a
diameter of ~8 µm. The LNd usually form a tight cluster
of ~six cells; their diameter is ~6 µm (Fig.
3A,B). This group is usually
difficult to identify in the SG strains, probably because of the weak
SG expression in the LNs in general (see above). The LNv
consist of two different-sized cell types: four large oval neurons
(diameter, ~13 µm) and four to six smaller cells (diameter, 6 µm)
(Fig. 3C,D) (cf. Helfrich-Förster and Homberg, 1993; Helfrich-Förster, 1995). In most samples, the locations of the LNv span 30-40 µm in the dorsal to
ventral direction. Usually the large neurons are slightly dorsal to the
smaller neurons, but there is variability in the arrangement of these
cells.
Fig. 3.
Pattern of per-expressing cells in
a per+ BG transgenic adult at ZT21. This
male carried the BG construct heterozygous with a balancer chromosome
(TM2; see Materials and Methods) and was stained with
anti--GAL antibody. A, The open arrow
points to LNd (relatively dorsal LNs; see Ewer et al.,
1992). Scale bar, 25 µm B, High-magnification view of
A; four to five LNd (diameter, ~6 µm)
lie close together; arrowheads point to glia in the
cortex, adjacent to the neurons, and to glia bordering the medulla.
Scale bar, 12.5 µm. C, A large oval cell of the
LNv group (large arrow); the smaller
arrow points to four neurons of smaller diameter (~6 µm).
D, Higher magnification of C;
arrowheads point to glial cells at the outer rim of the
medulla and to glia in the second optic chiasm. E, DNs.
F, Higher magnification of E, showing two
overlapping DNs present in this section (diameter, ~8 µm); the
arrowhead indicates cortical glia. G, PR
staining (left part of the image); the
arrowheads point to glia in the lamina and in the layer
between the PRs and lamina. Scale bar, 8 µm. H,
Staining in the cardia. Magnifications in C,
E, and H as in A; in
D and F as in B.
[View Larger Version of this Image (110K GIF file)]
By focusing through the soma of the LNs, one can usually
distinguish the nucleus from cytoplasm by slightly
lighter staining in the former compartment (see Fig. 3B for
the LNd and D for the LNv). Thus,
the BG fusion protein can enter the nucleus, but it is clearly not
predominantly nuclear as is PER at this time of the LD cycle (Curtin et
al., 1995). Glial staining in the BG transgenic type was generally less
intense and restricted to fewer cells than glial staining in SG flies
(compare Figs. 2A and 3C). Strong glial
staining was only observed along the outer rim of the medulla (Fig.
3D). In contrast to SG, only weak glial staining was
observed in the lamina (Figs. 2C and 3D), the
cortex, including neuropil borders (Figs. 2A and
3C), and the inner optic chiasm (Figs. 2D and 3D). Glial staining of BG transgenics therefore closely
reflects PER expression in this cell type (cf. Ewer et al., 1992).
The nuclei of the PRs in BG flies showed robust reporter expression.
The cytoplasm of the PR cells is also labeled but less strongly than in
SG flies (Figs. 2A,C,D,
3C,D,G); this might
contribute to the weaker signal in the lamina in BG flies (see above).
Expression of the BG fusion protein in the thorax and abdomen was also
inspected and found to be similar to that of PER (for an overview of
per-expressing tissues in the whole fly, see Hall, 1995). As
an example, nuclear BG staining in the outer layer of epithelial cells
in an alimentary structure (the cardia) is shown (Fig.
3H).
The XLG fusion protein was expected to exhibit an expression
pattern very similar to that of PER, because this transgene encodes nearly the full-length protein (Fig. 1). Yet the observed stainings in
PR cells and glia were different from the wild-type and BG patterns, at
least in terms of intensity of the signals (Fig. 4); XLG
expression in the PRs is not restricted to the nucleus but is equally
distributed through the whole cells (Fig. 4E). Whereas prominent glial expression was observed at the outer rim of the
medulla in SG and BG, XLG flies showed very low expression in these
cells (Fig. 4A-C). Moreover, these glial
cells and few additional ones in the cortex of the optic lobe (Fig.
4A) were the only positively stained non-neuronal
cells that we were able to identify (for example, no staining could be
detected in the lamina and the inner optic chiasm) (Fig.
4C,E). In contrast, XLG-mediated signals in
per-expressing neurons were prominent and similar to those
observed for BG. The XLG fusion protein also fills up the neuronal
perikarya, making it possible to distinguish cell sizes and shapes.
Figure 4, A and B, shows six cells of the
LNd group. In addition, two large cells from the dorsal
subset of the LNv are visible. Figure 4D
illustrates staining of smaller DNs (diameter, ~3-5 µm) compared
with the DNs shown in Figure 3F (diameter, ~8 µm). In
both cases, these neurons (usually two) are located at similar lateral
positions in the posterior dorsal brain and were the only DNs found in
that region. This indicates that they belong to the same group of cells
that shows variable cell size rather than representing cells of two
different groups, as was observed for the LNv (Fig.
3C,D). A different group of five to
eight DNs expressing the BG fusion protein was found to be located in
more central regions of the dorsal brain (data not shown).
Fig. 4.
per-Expressing cells in the head of
a per+ XLG-B transgenic adult at ZT21. The
sections of the male were stained with anti--GAL antibody (staining
of this line is more intense than in XLG-A, but the spatial
pattern is identical; compare Fig. 10) (also data not shown).
A, Six LNd cells (open arrow)
are visible in this section (diameter, ~5-6 µm); the solid
arrow points to two large cells (diameter, ~11 µm) from the
relatively dorsal region of the LNv group;
arrowheads indicate glia cells bordering the medulla. Scale bar, 12.5 µm. B, Higher magnification of
A. Scale bar, 8 µm. C, The solid
arrow points to LNv; four cells of smaller size (diameter, ~4-5 µm) are visible in this plane. D,
DNs (arrow); note that the nucleus seems stained lighter
than the surrounding cytoplasm (diameter, ~3-5 µm). Magnifications
in C and D are as in A. E,
PR staining, which appears mainly cytoplasmic (the nuclei cannot be
distinguished from the soma); the open arrow points to a
group of LNd. Scale bar, 25 µm.
[View Larger Version of this Image (107K GIF file)]
The XLG fusion protein is present in the nucleus and the cytoplasm, and
the majority of the signal seems to be in the latter compartment (Fig.
4A-D). This indicates that the C-terminal
part of PER, not present in BG, is not responsible for proper nuclear localization of PER--GAL fusion proteins (see Discussion). We have
no explanation for the relatively low expression of XLG in glia cells.
Two independent transgenic lines (XLG-A and XLG-B) gave similar
results, arguing against chromosomal position effects. Furthermore,
Western blot experiments show that both transgenic lines express the
fusion protein in comparable amounts to endogenous PER (see Fig.
11).
Fig. 11.
Western blots of XLG transgenics. Males carrying
the A and B insert location of this fusion gene and either
per+ or
per01 had protein extracted
from their heads, which was electrophoresed and subjected to
immunoblotting as a function of ZT (i.e., different times within a
12:12 hr LD cycle when the adults were killed for protein extraction).
These times are indicated above each lane in A and
B. B, XLG-B based Western blots after
incubation with polyclonal anti-PER antibody; in both
per+ and
per01 genetic backgrounds, the
PER--GAL fusion protein undergoes similar changes in abundance and
mobility as the endogenous PER protein does. For quantification of this
experiment, see C and D. As controls, equal protein amounts of head extracts from Df(1)w
(per+) and
per01 w
sn3
(per01) flies were
blotted on the same gel. Note the absence of endogenous PER in all
genotypes for which the genetic background is
per01. B,
Signals obtained after application of a monoclonal anti--GAL antibody [the same membrane (see A)] was stripped and
subsequently incubated with this reporter-detecting reagent) and showed
the same mobility shifts and fluctuations in abundance as in
A, in both per+ and
per01 genetic backgrounds. See
E for quantification. In addition, a 116 kd protein band
is detected by the anti--GAL antibody in all head extracts from the
XLG transgenics but not in those from the two control strains. Note
that this band runs at a similar position in the gel as does the
bacterial -GAL protein, which was included in the MW marker.
C-F, Quantification of a set of Western
blot experiments using a phosphoimager (see Materials and Methods). To
allow amplitude and phase comparisons between different protein curves,
the highest expression values for each protein in each experiment were
set equal to 1. C, XLG-A and XLG-B proteins in the
per+ background. D, XLG-A and
XLG-B in the per01 background
(for C and D, the proteins were detected
with anti-PER). To compare the cycling of both fusion proteins with
that of PER, endogenous PER abundance (from
per+ XLG-A flies in C and
per+ XLG-B flies in D) is
also plotted. Amplitudes of protein cycling in these experiments were
as follows: PER in XLG-A [strain B], 4 (6.5)-fold; XLG-A [B] in
per+, 4.5 (5)-fold; XLG-A [B] in
per01, 1.5 (2)-fold (for how
the amplitudes were calculated, see Materials and Methods). For
E, the same blots used to generate the data for
C and D were subsequently incubated with
anti--GAL (see above) and quantified; amplitudes were 11 (9.5)-fold
for XLG-A [B] in the per+ and 3.5 (3)-fold
for XLG-A [B] in the per01
background. In F, quantification of a second,
independent Western blot experiment involving the XLG-A and XLG-B lines
(in the per+ and
per01 backgrounds) probed with
anti--GAL was performed. Again, the XLG fusion proteins show robust
abundance and mobility fluctuations in both genetic backgrounds
(amplitudes for XLG-A [B] were 8 (9)-fold in the
per+ and 2 (3)-fold in the
per01 background. The
open bars represent the light, and the solid bars the dark portion of the LD cycle, respectively.
[View Larger Version of this Image (35K GIF file)]
Temporal pattern of transgene expression in
per+ and per01
genetic backgrounds
The detailed analysis of the spatial expression patterns allowed
us to study temporal changes in expression in the different cell types
expressing the various fusion proteins.
To determine whether the different PER--GAL fusion proteins
undergo PER-like circadian fluctuations in abundance, we performed X-gal and anti--GAL antibody stainings at different times of the day
(see Materials and Methods). Flies were entrained for at least 3 d
in 12:12 hr LD cycles before they were sectioned at a variety of ZTs.
The transgene-mediated signals were also assessed in flies for which
only (endogenous) per gene was a loss-of-function mutation
(per01) to ask whether
cycling could be controlled by the PER sequences in a
given fusion protein.
It was reported that -GAL enzyme activity shows circadian
oscillations in head extracts of per+ SG flies,
whereas the activity was constant in a
per01 background (Zwiebel et al.,
1991). Because this aspect of the study proved irreproducible (see
Discussion), we performed X-gal stainings to reexamine this finding.
When pairs of SG3 and SG10 transgenic flies, collected at opposite ZTs
(ZT12 and ZT24), were stained in a per+
background, no differences in staining intensities could be observed. At both time points, strong staining in PR and glial cells was observed, the only difference being a generally weaker signal in the
per+ SG10 flies (Fig. 5,
middle and bottom rows). In contrast, PER protein
shows its maximum abundance at ~ZT24 and at most ~20% of that
expression level at ZT12 (Zerr et al., 1990; Zeng et al., 1994). Thus,
the SG fusion protein (or at least its -GAL activity) does not
undergo circadian fluctuations after all. Note that in this regard it
is possible to detect a mere twofold difference in per
expression levels using X-gal staining on head sections of
per-lacZ transgenics (Cooper et al., 1994) and that the PER protein cycles with a ~10-fold amplitude in biochemical experiments (Zeng et al., 1994). In agreement with the earlier study and as expected from the results just described, no cycling of the SG fusion
could be detected in a per01
background (Fig. 5, top row) (Zwiebel et al.,
1991).
Fig. 5.
Temporal expression in heads of SG transgenic
flies. Pairs of males carrying the same per allele were
sectioned and stained with X-gal at opposite ZTs. In contrast to PER,
which is highly abundant late at night (ZT24) and nearly undetectable
by the end of the day (ZT12), the SG fusion protein shows no such
fluctuations (compare ZT12 with ZT24). The reporter activity was at a
constantly high level in PR cells, glia, and LNs in
per+ and
per01 genetic backgrounds, for
both the SG3 and SG10 transgenic flies (data not shown for
per01 SG10). The same results
were obtained after staining SG transgenics with anti--GAL antibody
(data not shown).
[View Larger Version of this Image (109K GIF file)]
To quantify the staining data, sections of fly pairs with the same
genotype, but opposite ZTs, were ranked blindly by three different
observers (see Materials and Methods). After decoding these results,
each pair was classified either as "equal staining at both ZTs,"
"higher staining at ZT24," or "higher staining at ZT12" (Fig.
6). Cycling seemed to occur only in one case (open bar), but this was in a per01
background, and the phase was opposite to that of PER cycling in wild
type. In all other cases, the majority of the pairs showed equal
staining intensities at both time points (Fig. 6). We also performed
anti--GAL antibody stainings at different ZTs on SG3 flies (in a
per+ genetic background); again, the results
indicated noncycling (data not shown).
In the case of the BG fusion protein, oscillations in the reporter
signals were clearly detectable in a per+
background (Figs. 7, 8, 9). Strong staining in PRs, glia, and LNs was observed in BG/TM2 flies at ZT3 and ZT21, whereas
only weak signals in these tissues were observed at ZT9 and ZT15 (Fig. 7, left column). Interestingly, it seemed that the signal
intensity in the LNs of per01
BG/TM2 flies was also cycling; whereas prominent staining in the eyes
and in glia was observed at all times, neuronal staining was clearly
reduced at ZT9 (Fig. 7, right column). To confirm these
observations, coded sections were scored blindly by three observers
according to the strength of the staining signal in the three different
per-expressing cell types. The result of this quantification
revealed robust cycling in all three cell types in the
per+ genetic background (Fig.
8A,C,E) and
appreciable (but relatively low-amplitude) fluctuations of LN
expression in the per01 background
(Fig. 8B). The peaks of expression in all three cell types (in the per+ background) were at ZT21 and
ZT3, whereas the trough values were observed at ZT9 and ZT15. This is
in good agreement with PER's immunohistochemically determined cycling
in wild type; Zerr et al. (1990) reported peak expression at ~ZT24
and minimal expression at ~ZT12. In
per01, the weakest BG-mediated
staining in the LNs was observed at ZT9; but in contrast to the results
from per+ BG flies, staining was again prominent
at ZT15 (Figs. 7 and 8B). In PR and glia cells, the
staining intensities in per01 BG
were constantly high (~three-fourths of the peak values from the
per+ flies) (Fig.
8D,F).
Fig. 7.
Cycling immunoreactivity of the BG
fusion protein. Males carrying this transgene heterozygous with the
TM2 balancer and, expressing either
per+ or
per01, were sectioned and
stained with anti--GAL antibodies. All sections shown are at the
level of the esophagus, where strong staining of the ventral group of
LNs (LNv) can be observed at a high time point (compare
Fig. 3). LNv are marked by arrows, glia
cells by arrowheads. The left column
shows staining in a per+ background at four
different ZTs; at ZT3, strong staining of the LNv, glia,
and PR cell nuclei can be observed. Scale bar, 20 µm. At ZT9, only
weak staining in all three cell types is detectable. Scale bar, 40 µm. At ZT15 staining is still weak in the LNv and glia cells, although the signal in the
cytoplasm of the PRs seems stronger compared with ZT9 (the
magnification for both of these time points is the same); staining in
all three cell types is again very prominent at ZT21. Scale bar, 12.5 µm. The temporal pattern in the
per01 background (right
column) differs from that just described. At ZT3, prominent
staining in the LNv, glia, and nuclei of the PRs is
visible, in contrast to the per+-expressing
flies, and the staining intensity in the PRs and glia remains at a high
level at ZT9. Only the LNv show a decrease in signal
strength at that time point. At ZT15, the staining became stronger
again in the LNv and remained high in PRs and glia. At ZT21, staining intensities in all three cell types was as high as in
the per+ background. Magnifications for all
per01 time points are as in
per+ at ZT3.
[View Larger Version of this Image (108K GIF file)]
Fig. 9.
Temporal staining pattern of another BG transgenic
strain. Males from the BG6 line (see Materials and Methods) for which
the genetic backgrounds were either per+ or
per01 were sectioned and
stained with anti--GAL. In per+, only
weak staining of the LNv (arrow) and PRs was
observed at ZT9. Staining in glia was nearly undetectable at this time
point. At ZT21, strong staining in the LNv
(arrow) and PRs, as well as weak staining in glia (in
the lamina and bordering the medulla), can be observed. In
per01, only light staining of
the LNv is observed at ZT9 (arrow). In contrast to the expression of BG6 in the
per+ background, there is also prominent
staining in the nuclei of the PRs and glia cells
(arrowhead). At ZT21, LNv are strongly labeled, whereas the signal intensities in the PRs and glia seem to be
only slightly stronger compared with ZT9. Scale bar, 25 µm.
[View Larger Version of this Image (102K GIF file)]
To ask whether fusion protein cycling apparently can be controlled by
BG itself or is attributable to position effects (i.e., operating in
the BG/TM2 strain), additional experiments were performed using an independent transgenic line (BG6) in both
per+ and
per01 backgrounds. The fusion
protein expression pattern was very similar to that described for
BG/TM2 except for the reduced number of glial cells that
were stained in the BG6 line (compare ZT21 in Figs. 7 and
9). Temporal expression analysis revealed clear differences in staining intensity between flies sectioned at ZT9 and ZT21 (Fig. 9).
In the per+ background, staining was stronger at
ZT21 in all three cell types (Fig. 9, left column). In
per01, cycling of the BG fusion
protein was detectable in the LNs; in addition, and
unlike what was observed in the experiments using the BG/TM2
strain, the signal appeared to fluctuate in the PRs as well (Fig. 9,
right column).
We conclude that the additional 230 amino acids present in BG are
sufficient to drive rhythmic oscillations of that fusion protein in a
PER-like manner. In a per+ background, cycling
seems to occur in the same cells and with similar phase as for PER
fluctuations in wild-type flies. Cycling in a
per01 background is less robust
and may be restricted to neurons; yet, the BG fusion protein fluctuates
in a circadian fashion, independently of PER function.
To determine whether the additional per-coding sequences
that are present in XLG flies influence protein cycling, the
polypeptide encoded by this fusion gene was monitored temporally. First
we stained sections from both XLG transgenic lines (XLG-A and XLG-B) at
ZT9 and ZT21. As expected, a strong increase in staining intensity could be observed in the LNs late at night (Fig.
10A). Staining in the PRs was weak at ZT9
and increased only slightly at ZT21 (note also that the staining is not
restricted to the PR nuclei; compare Fig. 4). Glial staining was not
detectable at the low time point and was faintly visible at ZT21; this
was expected, given the expression pattern of this transgene late at
night (Fig. 4). In a per01
background, cycling occurred in the LNs in both transgenic lines. In
the weaker-expressing XLG-A line, no staining was detectable in the LNs
at ZT9 (Fig. 10B). In contrast to the
per+ background, there appeared to be more
robust fluctuations of staining intensity in the PRs; in addition,
staining of glia in the outer rim of the medulla became clearly
detectable at ZT21 (Fig. 10B, bottom row).
Thus, it seems as if the general expression level might be higher in
the mutant genetic background. The fact that this fusion protein
consistently cycled in PR and glia cells in a
per01 background (in contrast to
BG) suggests that the additional C-terminal sequences are necessary to
achieve a rhythmic expression pattern that better reflects the
wild-type pattern.
Fig. 10.
Temporal expression pattern of XLG transgenic
flies. Males carrying the XLG construct (lines XLG-A and XLG-B) each
with per+ (A) or
per01 (B) were
sectioned and stained with anti--GAL. A, In XLG-A, only weak cytoplasmic staining in the PRs is visible at ZT9. Scale bar,
40 µm. At ZT21, prominent staining in the LNv
(arrows) is detectable, whereas staining in PRs seems
only slightly increased compared with ZT9 and is mainly cytoplasmic.
Glia staining at this high time point is just above the limit of
detection. Magnification is as at ZT9. For the XLG-B line, similar
expression patterns were observed. In LNv at ZT9, the
signal was barely detectable (solid arrow), whereas
prominent staining of the dorsal group of LNs (LNd) appears
at ZT21 (open arrow). Scale bars: ZT9, 25 µm; ZT21,
12.5 µm. B, In the
per01 background, no
XLG-A-mediated expression was detectable in LNs and glia at ZT9. As in
per+, there was only weak cytoplasmic PR
staining at this time point. At ZT21, strong staining in the
LNv (arrows) as well as in glia and PRs is
visible. Magnifications are the same as for XLG-A
(per+). For the XLG-B line, similar
signal fluctuations were observed, although weak staining of the
LNd (open arrow) was detectable at ZT9. The
solid arrow at ZT21 points to a group of LNv
showing strong expression of the XLG-B fusion protein. Magnifications are as in XLG-B (per+) at ZT9.
[View Larger Version of this Image (99K GIF file)]
To quantify XLG-encoded protein levels, we performed immunoblottings
with head extracts from both transgenic strains. XLG-A and XLG-B flies,
carrying per+ or
per01 in their genetic
backgrounds, were collected at six different ZTs. Head protein extracts
were separated on polyacrylamide gels. The ensuing Western blots, to
which an anti-PER antibody was applied, were developed and the signals
quantified (see Materials and Methods). This anti-PER reagent revealed
circadian fluctuations of endogenous PER protein in abundance in both
XLG transgenic lines. As has been reported for wild-type flies, maximum
amounts of PER were detected between ZT18 and ZT22, and minimal levels
were present between ZT6 and ZT10 (Fig.
11A,C,D)
(cf. Zeng et al., 1994). Temporally dependent protein mobility shifts
were also observed, again as reported previously (Edery et al., 1994).
These shifts are caused by phosphorylation; the faster-migrating form
(~155 kDa) becomes phosphorylated, resulting eventually in the most
slowly migrating one (~190 kDa) (Edery et al., 1994). In agreement
with the earlier study, only the faster migrating form of PER is
present at the beginning of the accumulating phase (ZT10-ZT14) (Fig.
11A). At ZT18, the first slower-migrating
(phosphorylated) forms become visible, becoming predominant at ZT22. At
ZT2, only the phosphorylated forms are present and at ZT6, both forms
are present at very low levels (Fig. 11A).
The PER antibody also detected the XLG fusion protein, which is
expressed at similar levels and shows circadian fluctuations in
abundance as PER in both per+ and
per01 genetic backgrounds (Fig.
11A,C,D). In addition to
cycling with identical phase, the turnover of XLG occurs with a
PER-like amplitude in a per+ background (see
legend to Fig. 11C,D). In contrast, the amplitude is reduced by >50% in a per01
background (XLG-A, 1.5-fold; XLG-B, twofold) (Fig.
11D), indicating that endogenous PER is necessary to
achieve full-amplitude cycling of the XLG fusion protein. The lower
amplitude is attributable to higher protein concentrations at trough
times of expression rather than lower levels at peak times (compare ZT2
and ZT6 of per+ with the same
per01-based time points) (Fig.
11A). Mobility shifts of the XLG proteins were also detectable in both
genetic backgrounds, similar to those described for PER (Fig.
11A). The shifts were not as drastic as observed for
PER, perhaps because the resolution of small mobility differences for
the ~300 kDa XLG fusion protein is not as good as for the smaller PER
polypeptide. (However, the shifts were still obvious; compare ZT2 and
ZT14 in Fig. 11A; the faster-migrating form is
clearly absent at ZT2 in both genetic backgrounds.)
When the same blots were incubated with anti--GAL antibodies,
similar fluctuations in abundance and mobility shifts of the fusion
protein were observed (Fig. 11B). For unknown
reasons, the amplitude of the molecular rhythm was higher in both
genetic backgrounds, compared with those resulting from application of
anti-PER (see legend to Fig. 11E). Nevertheless, the
reduction of amplitude in the
per01 background was in the same
range as observed with anti-PER antibody (>50%) (Fig.
11E), indicating that the differences obtained with both antibodies were only relative. To confirm these results, a second,
independent experiment was performed using a different collection of
flies and anti--GAL antibody. In this experiment, cycling in XLG-A
(XLG-B) flies occurred with eightfold (ninefold) amplitude in
per+ and twofold (threefold) amplitude in a
per01 genetic background; thus,
the amplitude reduction observed in a
per01 background is real (Fig.
11F).
In addition to the ~300 kDa XLG fusion protein band, a smaller ~116
kDa band was present in blots incubated with anti--GAL antibody.
These smaller band comigrates with the actual Escherichia coli -GAL protein, included as a molecular weight marker (Fig. 11B). Because this band is present in all lanes
containing extracts from XLG transgenic flies, but absent in
per+ and
per01 control flies, it is very
likely derived from the fusion protein. It is possible that the 116 kDa
band is a degradation product resulting from the cyclical turnover of
the PER--GAL fusion protein. As expected from the long half-life of
-GAL activity in Drosophila (Monsma et al., 1988), no
daily fluctuations of its abundance could be observed (Fig.
11B; also data not shown). Note also that the
relatively small polypeptide is more abundant than suggested by the
blot shown in Figure 11B; to transfer the large XLG
proteins quantitatively, unusually long protein transfers were
necessary, resulting in a substantial loss of the smaller -GAL
protein (data not shown) (see also Materials and Methods). High amounts
of this protein are usually present in protein extracts of flies
carrying fusion genes encoding PER--GAL fusion proteins that
undergo circadian fluctuations (e.g., in BG flies). In contrast, in
transgenics for which the fusion proteins do not cycle the relative
abundance of -GAL is much lower (e.g., SG), indicating that rhythmic
fluctuations of PER--GAL fusion proteins result in increased
amounts of free -GAL, probably attributable to circadianly regulated
protein degradation (Dembinska et al., 1997).
Given these results, the anti--GAL antibody stainings might
not reveal the intracellular distribution of the fusion proteins, because the observed staining patterns probably result from detection of both the intact fusion proteins and -GAL, which would not necessarily be co-localized. To address this matter, we performed stainings of BG and XLG flies (in which the -GAL is abundant on
Western blots) with anti-PER antibodies in a
per01 mutant background, detecting
only the intact fusion proteins. per01 XLG-B males were stained at
ZT15 (n = 4) and ZT21 (n = 4) and per01 BG/TM2 males at ZT16
(n = 3) and ZT20 (n = 2), respectively. The results of these preliminary experiments indicate that the fusion
proteins were present in both the cytoplasm and the nucleus at times
when staining should be restricted to either one of these compartments
(cytoplasmic at ZT15/ZT16, nuclear at ZT20/ZT21) (Curtin et al., 1995).
Therefore, we conclude that the anti-
