Current Biology, Vol. 12, 1331–1335, August 6, 2002, 2002 Elsevier Science Ltd. All rights reserved.

PII S0960-9822(02)01036-9

A Role for the Drosophila Fragile X-Related
Gene in Circadian Output
Shunsuke B. Inoue,1,9 Masami Shimoda,2,3,9
Izumi Nishinokubi,3 Mikiko C. Siomi,1Miwako Okamura,1 Akira Nakamura,4Satoru Kobayashi,5 Norio Ishida,3,6,7,8
and Haruhiko Siomi1,81
Institute for Genome Research
University of Tokushima
Kuramoto 3-18-15
Tokushima 770-8503
Japan
2
National Institute of Agrobiological Sciences
Ohwashi 1-2
Tsukuba, Ibaraki 305-8634
Japan
3
Clock Cell Biology
National Institute of Advanced Industrial Scienceand Technology (AIST)IMCB 6-5
Higashi 1-1-1
Tsukuba 305-8566
Japan
4
Institute of Biological Sciences
University of Tsukuba
Ten-noudai 1-1-1
Tsukuba, Ibaraki 305-8572
Japan
5
Center for Integrated Bioscience
Okazaki National Research Institutes
OkazakiAichi 444-8585
Japan
6
Department of Biomolecular Engineering
Tokyo Institute of Technology
Nagatsuta 4259
Yokohama 226-8501
Japan
7
Center for Interdisciplinary Research
Tohoku University
AramakiAobaku, Sendai 980-8578
Japan

Summary
Mutations that abolish expression of an X-linked gene,
FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental
retardation [1, 2]. To understand the normal functionof the FMR1 protein, we have produced fly strains
bearing deletions in a Drosophila homolog of FMR1
(dfmr1). Since fragile X patients show a number ofabnormal behaviors including sleep problems [3, 4],
we investigated whether a loss-of-function mutationof dfmr1 affect circadian behavior [5–8]. Here we showthat under constant darkness (DD), a lack of dfmr1
8

Correspondence: n.ishida@aist.go.jp (N.I.), siomi@genome.tokushimau.ac.jp (H.S.)
9
These authors contributed equally to this work.

expression causes arrhythmic locomotor activity, but
in light:dark cycles, their behavioral rhythms appear
normal. In addition, the clock-controlled eclosion
rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in thecircadian output pathway regulating locomotor activity in Drosophila.
Results and Discussion
The fruit fly Drosophila has proven to be a powerful tool
for the genetic dissection of biochemical pathways forhuman neurological diseases [9, 10]. Recently, a Drosophila homolog of FMR1 (the abbreviation “dfmr1” will
be used hereafter) has been identified [11]. Drosophila
and vertebrate FMR1 proteins share a number of topographical landmarks, including two types of RNA binding motifs [11]. To obtain fly strains bearing mutations
in dfmr1, we mobilized a P{EP} element [12] inserted
near the dfmr1 transcriptional start site on the EP(3)3422
chromosome to produce several partial deletions ofdfmr1 (Figure 1A). One such mutation was selected forfurther characterization. We refer to this mutation as
dfmr1B55. In this mutant, imprecise excision of the EP
element generated a 2.5 kb deletion of genomic DNA,
which included exons 2, 3, and 4 of the dfmr1 gene. The
deletion removed the translational start codon and thefirst 59 codons (Figure 1A). The homozygous dfmr1B55
mutant proceeds into adulthood without expressing a
discernible morphological defect. Western blot analysis
with anti-DFMR1 antibodies revealed that the DFMR1
protein was expressed throughout development in wildtype controls (Figure 1B). In contrast, there is no DFMR1
protein of the expected size, 85 kDa, in homozygous
dfmr1B55 mutant flies (Figure 1B). These results showthat dfmr1B55 is a null mutation of the dfmr1 gene.
To test for behavioral effects of the mutation, dfmr1B55
flies were assayed for circadian locomotor activity [13].
Over the course of 24 hr in light:dark (LD) cycles, wildtype flies are entrained to (or synchronized with) LD
cycling and exhibit a substantial locomotor activity riseduring the second half of the day (e.g., [14]). Homozygous dfmr1B55 flies appeared to behave in this manner(i.e., before lights-off) and exhibited 24 hr periodicity
under such LD (data not shown). Clear anticipations oflights-off suggest that the LD behavior of dfmr1B55 flies
is truly clock dependent and not simply masking behavior. In contrast, locomotor behavior of dfmr1B55 in constant darkness was arrhythmic. Actograms of dfmr1B55
individuals showed that most mutants lost rhythmicity
within a couple of days after transfer to DD (Figure 2B).
By periodogram analysis, 86% of dfmr1B55 flies exhibitedarrhythmicity in DD for 15 days (Table 1). Of the 50 dfmr1
mutant flies tested, the 7 “escapers” that were rhythmic
appear to have a wild-type 24 hr rhythm (Table 1). Although period length of escapers in clock mutants areusually affected (e.g., see [15, 16]), the dfmr1B55 mutation
does not appear to affect period length of the escapers.
To confirm that disruption of the dfmr1 gene is directlyCurrent Biology
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Figure 1. Characterization of the dfmr1 Deletion Mutant dfmr1B55(A) Structure of the dfmr1 locus is shown. Exons are indicated with
open boxes, and the closed portion is the protein coding region.
The translation initiation start site is located in exon 3. The positionof EP(3)3422 is represented as triangle with an arrow pointing in
the direction of GAL4-induced transcription. The deleted genomic
region of the dfmr1B55 chromosome is shown. Sequence informationfrom the Berkeley Drosophila Genome Project (BDGP; [37]) reveals
that the dfmr1 locus is positioned in a small region on the cytologicallocation 85F11-12, on the right arm of the third chromosome. Thereis no other gene that overlaps in the dfmr1 locus.
(B) Developmental Western blot analysis with anti-DFMR1 antibodies reveals that there is no DFMR1 protein of the expected size of
85 kDa in homozygous dfmr1B55 mutant flies.

responsible for the circadian phenotype, we transformed mutant flies with a P element containing wildtype dfmr1 genomic sequences. The arrhythmic locomotor activity phenotype in DD was ameliorated by an
introduction of a dfmr1 minigene (Figure 2C and Table1). This demonstrates that the arrhythmic phenotype iscaused by the dfmr1B55 mutation rather than a secondsite mutation elsewhere on the chromosome. The “rescued” flies have altered periods, with 1 hr longer thanwild-type (Figure 2C and Table 1). This might be due todosage effects on period of the dfmr1 locus as is often
the case for many clock mutants (e.g., [17, 18]). In fact,
expression levels of dfmr1 mRNA in heads of the rescued flies appear to be significantly higher than thosein wild-type flies (data not shown).
To investigate whether other rhythms are also affectedin flies bearing DFMR1 deficiencies, homozygous
dfmr1B55 flies were tested for an independent manifestation of circadian output, eclosion (emergence of theadult fly from the pupal case) [5]. Although eclosion
occurs only once in the lifetime of an individual fly, it
occurs repeatedly and rhythmically in a population offlies of diverse ages [19, 20]. In stark contrast to what
was found for locomotor activity (Figure 2), homozygous
dfmr1B55 flies emerged from their pupal cases in a circadian-gated manner with a phase and amplitude very
similar to those observed in normal flies (Figure 3). Under
the experimental conditions used in this study we clearlyobserved normal eclosion rhythm even at day 5 of DD.
Therefore, rhythmic eclosion in the dfmr1B55 mutant persists with a high amplitude after prolonged incubations

Figure 2. dfmr1B55 Flies Are Arrhythmic
Representative locomotor activity records of three individual flies,(A) one yellow-white wild-type (wt), (B) one dfmr1B55, and (C) one
dfmr1B55 with a dfmr1 minigene (P[w⫹: dfmr1]; dfmr1B55), in constantdarkness (DD). Adult flies were entrained to cycles of 12 hr light:12
hr dark cycle (12L:12D) for at least 2 days, and their locomotor
activity was measured subsequently in DD. The data are double
plotted: activity from days 1 and 2 is on the first line, days 2 and 3
on the second, and so on. The dark bars indicate the subjective
night and the gray bars indicate the subjective day. The results of
chi-square periodogram analysis are shown in each right panel. Theline indicates 99% confidence level. During DD, most dfmr1B55 flies
lost locomotor activity rhythm and the rhythmicity was rescued byan introduction of a dfmr1 minigene. The genomic rescue constructcontained a 12 kb BamHI-KpnI fragment of the dfmr1 locus.

in DD, showing that the period of eclosion rhythm is notaffected by the mutation. Thus, these results indicatethat DFMR1 is not universally required for the manifestation of overt circadian rhythms.
To investigate whether the dfmr1B55 mutation changes
the molecular oscillation of known clock components

Table 1. dfmr1B55 Causes Arrythmicity
Genotype␶ ⫾ SD% AR

nWild-typew; ⫹; dfmr1B55
w; P[w⫹: dfmr1]; dfmr1B55

23.5 ⫾ 0.3
23.8 ⫾ 0.5
24.8 ⫾ 1.6

7.9
86.0
28.8

63
50
52

The genotypes of normal, mutant, and transgenic flies are listed atleft. The y w flies were used as a wild-type (wt). Flies were entrainedin a 12 hr light:12 hr dark cycle (12L:12D) for at least 2 days andthen transferred to constant darkness (DD) for 15 days. Period length
was calculated from all data collected using chi-square periodogram
analysis (ClockLab, Actimetrics, Inc.) The genomic rescue constructcontained a 12 kb fragment of the dfmr1 locus (see Figure 1A). ␶
indicates free-running period, means ⫾ SD in DD. Percent AR indicates percentage of flies that are arrhythmic.

Brief Communication
1333

Figure 3. Normal
dfmr1B55 Mutants

Eclosion

Rhythmin

Eclosion rhythm is not affected by thedfmr1B55 mutation. Eclosion profiles of yellowwhite wild-type (WT) and dfmr1B55 flies in DD
are shown. The number of flies eclosing fromtheir pupal cases is plotted as a function of
circadian time. Black and hatched horizontal
bars at the bottom of the panels represent
subjective night and day, respectively. For
eclosion assays, larvae were reared in
12L:12D, then pupae were transferred to DD
at 25⬚C. Pupae were glued to disks that subsequently were placed onto the eclosion
monitor as described previously [19]. Emerging flies were counted using an eclosion monitor (TriKinetics, Inc.) in DD. The activity period
was determined by chi-square periodogram
analysis (ClockLab, Actimetrics, Inc.).

[5–8], we looked at PER and TIM protein time courses on
Western blots using protein extracts that were preparedfrom heads of flies [21, 22]. Flies were entrained for three
LD cycles and then transferred to DD conditions. We
assayed the last day of LD and subsequent 3 days of
DD for wild-type and dfmr1B55 genotypes (Figure 4). In
wild-type flies, PER and TIM undergo daily fluctuationsin abundance and electrophoretic mobility (Figure 4A).
These results are similar to those previously observed
for wild-type flies [7, 8]. Differences in the apparent molecular weight of PER are due to the circadian regulationof its phosphorylation [7, 8]. In LD, both proteins oscillated in dfmr1B55 flies as they did in wild-type controls(Figure 4A). These results, together with the observationthat the LD behavior of the dfmr1B55 flies appears normal,
suggest that DFMR1-deficient flies have an active circadian pacemaker that can be entrained by light-dark cycles. However, in DD, phase differences of PER and TIM
protein accumulation between the genotypes could be
detected (Figure 4B). For dfmr1B55 in DD, transitions fromhigh-molecular type to low-molecular type PER are notas sharp as they are for wild-type controls. For TIM, thelevels of expression of the protein are not grossly altered
for the two genotypes, and the oscillation in dfmr1B55
flies remains quite robust even in the third day of DD
conditions when most of the flies display arrhythmic
locomotor activity (Figure 2). However, TIM oscillateswith a slight phase delay in dfmr1B55 in DD, indicatingthat dfmr1B55 alters expression of TIM as for PER. Alteredcycling of PER and TIM in dfmr1B55 flies in DD could be
due to a measure of the developing desynchrony among
many fly heads because most flies are behaviorally arrhythmic (Figure 2). However, we could not exclude thepossibility that there might be feedback effects of theDFMR1-mediated output pathway on the pacemaker.
To determine the pattern of expression of DFMR1, thelevels of DFMR1 protein accumulating during a circadiancycle were examined by Western blot analysis (Figure4C). No significant difference of DFMR1 protein levels

was detected in head extracts from each of the time
points examined in both LD and DD conditions, showingthat levels of DFMR1 protein are not under circadian
control.
The signaling mechanism that mediates output from
central clock proteins to behavior is poorly understood[5]. Several output genes have been identified so far in
Drosophila [23–25]. The circadian phenotype displayedby dfmr1B55 flies is reminiscent of what happen in flies
deficient in either protein kinase A (PKA) or NF1, theprotein product of the neurofibromatosis-1 gene. However, whether mutations in dfmr1 and PKA or NF1 leadto arrhythmic activity by similar or different pathways iscurrently not clear. An intriguing finding is that DFMR1deficient flies manifest normal eclosion rhythms (Figure3), suggesting that the daily timing of developmental
rhythm might not require DFMR1. These results suggestthat eclosion and locomotor rhythms are mediated bydifferent neurons that use the same pacemaker molecules [26].
How, then, might DFMR1 participate in an output
pathway associated with the manifestation of overt locomotor activity rhythms? DFMR1 is a cytoplasmic RNA
binding protein associated with ribosomes, as is thecase for mammalian FMR1 [2, 27]. Therefore, DFMR1
could regulate posttranscriptionally the expression of
specific target mRNAs that control output functions.
Given the recent findings showing that a secreted neuropeptide, pigment-dispersing factor (PDF) is a criticalcircadian mediator that couples a molecular clock tocircadian rhythms in locomotor activity and can in turninfluence function of the clock [28–31], it will be important to follow the fate of this peptide in dfmr1B55 flies.
Alternatively, since in both the human and Drosophila,
the fragile X protein (FMR1 and DFMR1) has been foundto have a role in synaptic growth [32, 33], DFMR1 might
regulate expression of mRNAs required for synaptic
function and structure such as mRNA for microtubuleassociated protein MAP1B [33]. Proteins that affect neu-

Current Biology
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Figure 4. The Timing of PER and TIM Protein
Oscillations Is Altered in dfmr1B55 Adult Heads
in DD(A and B) Western blot analysis of TIM andPER proteins from wild-type (yellow-white:wt)
and dfmr1B55 head extracts, collected at various times (indicated at top) of day during an
LD cycle (A), or collected at various times
over 3 days during an DD cycle (B). The lightcycle is indicated by the open (light) and filled(dark) bars in (A), and bars below in (B) represent prior LD cycles. Note the successive
phase changes in DD for wild-type (wt) anddfmr1B55, respectively. A crossreading nonspecific band (not shown) indicated equalloading. Experiments for wild-type (wt) anddfmr1B55 were performed three times.
(C) DFMR1 protein levels are not under circadian control. DFMR1 protein levels are constant during the course of a daily cycle. Wildtype (wt) fly heads were collected during one
day in LD and after the subsequent 3 days in
DD, as reflected by the bar underneath, withtime shown at top in 24 hr cycles. Blotted
protein extracts were probed with an antiDFMR1 antibody, and with anti-ribosomal
protein P0 [38] to ensure equal loading (datanot shown). DFMR1 levels were constant during days assayed. Identical results were obtained when we used two different antiDFMR1 antibodies (data not shown).

ronal development and/or function are expected to affectcircadian rhythms that are driven by neuronal pacemakers. For example, similar results to those found in
dfmr1B55 flies were obtained when synaptic transmission
was blocked using the tetanus-toxin light chain in per/tim-expressing cells, i.e., less effect on locomotor activity during LD but largely arrhythmic during constant dark
conditions [34].
It is tempting to speculate that sleep problems observed in fragile X patients [4] are attributable to alterations of circadian rhythmicity because sleep propensity
is modulated by a circadian clock [35]. Since the molecular mechanisms involved in the generation of circadianrhythms are remarkably similar between Drosophila andmammals [5, 36], our Drosophila model of fragile X syndrome provides insight into the sleep-wake cycles ofanimals. The discovery of modifiers involved in DFMR1mediated regulation of circadian rhythms reveals additional molecular mechanisms in the fragile X syndrome.
Acknowledgments
We thank G. Dreyfuss and L. Wan for generously providing a dfmr1
cDNA and anti-DFMR1 antibodies; M. Young for anti-TIM antibodies;
J. Hall for anti-PER antibodies; T. Uchiumi for anti-ribosomal protein
P0 antibodies; and A. Beaton and the Berkeley Drosophila Genome
Project for EP lines. We thank members of the Siomi lab and of the
Ishida lab for helpful suggestions and comments on the manuscript.
We are grateful to Kazuko Suzuki and Hiromi Hasegawa for technicalassistance. This work was supported by grants from the FRAXA
Research Foundation, the Cure Autism Now (CAN) Foundation, theJapan Society for the Promotion of Science (JSPS), the Ministry of
Health, Labor, and Welfare of Japan, the Ministry of Education,
Science, Sports, and Culture of Japan, the Competition Research

Brief Communication
1335

Program, AIST, MITI Japan, and the Industrial Technology Research
Grant from New Energy and Industrial Technology Development
Organization (NEDO).
Received: April 29, 2002
Revised: June 6, 2002
Accepted: June 14, 2002
Published: August 6, 2002
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