Circadian Regulation of Gene Expression Systems in the Drosophila Head

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provided by Elsevier - Publisher Connector Neuron, Vol. 32, 657–671, November 20, 2001, Copyright 2001 by Cell Press Circadian Regulation of Gene Expression Systems in the Drosophila Head

Adam Claridge-Chang,1,5 Herman Wijnen,1,5 PER and TIM proteins physically associate to form a Felix Naef,2 Catharine Boothroyd,1 repressor of CLK/CYC. VRI negatively regulates per and Nikolaus Rajewsky,3 and Michael W. Young1,4 tim expression through a separate autoregulatory path, 1 Laboratories of Genetics, but also determines cycling expression of a key neuro- 2 Mathematical Physics, and peptide, PDF (Blau and Young, 1999). Cycling accumula- 3 Theoretical Condensed Matter Physics tion of this peptide hormone is required for normal pat- The Rockefeller University terns of rhythmic locomotor activity (Helfrich-Forster et 1230 York Avenue al., 2000; Renn et al., 1999). New York, New York 10021 VRI’s contribution to PDF expression and behavioral signaling indicated a potential mechanism for creating extensive pathways of circadian regulation. Since CLK/ Summary CYC activity oscillates, patterns of diverse gene expression might be influenced by the clock through transcrip- Mechanisms composing Drosophila’s clock are con- tional controls using this circadian activator. Evidence served within the animal kingdom. To learn how such for such a strategy has also come from independent clocks influence behavioral and physiological rhythms, studies of the mammalian clock, which is highly homolo- we determined the complement of circadian tran- gous to that of the fly. Vasopressin, a well-known modu- scripts in adult Drosophila heads. High-density oligo- lator of neuroendocrine function, oscillates due to direct nucleotide arrays were used to collect data in the form regulation by orthologs of CLK/CYC in mammals (Jin et of three 12-point time course experiments spanning al., 1999). a total of 6 days. Analyses of 24 hr Fourier components Known clock-controlled functions in Drosophila in- of the expression patterns revealed significant oscilla- clude eclosion, vision, olfaction, courtship, locomotion, transcripts. Based on secondary filters and sleep. In the face of such functional variety, a single 400ف tions for and experimental verifications, a subset of 158 genes transcriptional strategy for generating rhythmic gene showed particularly robust cycling and many oscilla- expression might be considered restrictive. In fact, strict tory phases. Circadian expression was associated adherence to the patterns of CLK/CYC control de- with genes involved in diverse biological processes, scribed above could predict gene expression rhythms including learning and memory/synapse function, vi- with a limited number of phases, even a preponderance sion, olfaction, locomotion, detoxification, and areas of correlated expression patterns overlapping that of of metabolism. Data collected from three different per, tim, and vri. To address such hypotheses, and to identify a comprehensive set of clock-controlled ef- clock mutants (per0, tim01, and ClkJrk), are consistent with both known and novel regulatory mechanisms fectors of rhythmic behavior, we conducted RNA assays controlling circadian transcription. of adult fly heads using high-density oligonucleotide arrays. Circadian resolution of gene expression was achieved by sampling adult Drosophila heads over six Introduction 24 hr intervals. Our survey included profiles of gene expression obtained in light/dark cycles and in constant Daily rhythms of behavior and physiology are established darkness. Fourier analysis of the data allowed us to by circadian clocks. Intracellular biochemical oscillators identify genes with oscillatory expression patterns. Veri- have been linked to circadian rhythms in bacteria, fungi, fication of the microarray results by extensive Northern plants, and animals. Genetic screens have allowed com- blot analyses showed a significant enrichment of genes ponents of such clocks to be identified and have pro- displaying periodic expression in a set of about 400 vided significant insights into their mechanisms (Allada candidates. We further distinguished a subset of genes et al., 2001; Cermakian and Sassone-Corsi, 2000; Loros (158) that exhibited particularly robust patterns of circa- and Dunlap, 2001; Reppert and Weaver, 2001; Ripperger dian expression. This latter group contains genes with and Schibler, 2001; Williams and Sehgal, 2001; Young a wide variety of oscillatory phases and molecular func- and Kay, 2001). tions including learning and memory, vision, olfaction, In Drosophila, several genes form an autoregulatory locomotion, detoxification, and areas of metabolism. In network that allows self-sustained molecular rhyth- parallel to the time-resolved experiment, we used addi- micity and perception/adjustment to daily environmen- tional microarrays to assess profiles of gene expression tal cycles (Allada et al., 2001; Edery, 2000; Williams and in arrhythmic clock mutants. This experiment indicated Sehgal, 2001; Young and Kay, 2001). The transcription that Drosophila’s circadian clock regulates gene activity factors CLOCK (CLK) and CYCLE (CYC) bind to homolo- through both known and novel transcriptional mecha- gous sequences (E boxes) in the promoters of period nisms. (per), timeless (tim), and vrille (vri), coordinately inducing their expression. After a delay that appears to be medi- Results and Discussion ated by the casein kinase I⑀ ortholog double-time (dbt), Experimental Overview 4 Correspondence: [email protected] We performed a genome-wide expression analysis 5 These authors contributed equally to this work. aimed at identifying all transcripts from the fruit fly head Neuron 658

that exhibit circadian oscillations in their expression. Northern sampling throughout the interval p-F24 0.05– Our experiments and analyses were designed to take 0.1 (see Conclusions). full advantage of two defining properties of circadian Organization of the Subset of 158 Circadian hr periodicity and (2) self-sustained Transcripts by Phase of Peak Expression 24ف (rhythms: (1 propagation in a constant environment. By taking time Figure 1A shows an arrangement of the circadian ex- points every 4 hr, we obtained a data set that has a high pression patterns identified by our selection criteria or- enough sampling rate to reliably extract 24 hr Fourier dered by peak phase. Each column represents a time components. We decided to perform time course experi- point, and each row represents a gene. The colors repre- ments spanning a day of entrainment followed by a sent the magnitude of the log ratios for the measured day of free-running to take advantage of both the self- average difference values normalized per experiment, sustaining property of circadian patterns and the im- with shades of red indicating an increase and shades proved amplitude and synchrony of circadian patterns of green indicating a decrease (Eisen et al., 1998). Oscil- found during entrainment. We analyzed 36 RNA isolates lating patterns of all possible phases are represented. from wild-type adult fruit fly heads, representing three When organized into 4 hr phase groups ranging from 2 day time courses, on high-density oligonucleotide ZT0 (Zeitgeber Time 0; lights on) through ZT23 (just arrays. Each array contained 14,010 probe sets (each before lights on), the 158 genes show the following distri- composed of 14 pairs of oligonucleotide features) in- bution of peak phases: 30 genes in ZT0-4 (e.g., Clk, to, genes annotated from complete se- ninaA, Pdh), 19 in ZT4-8 (e.g., e, Ugt35b), 17 in ZT8-12 13,600ف cluding quence determination of the Drosophila melanogaster (e.g., trpl, ATPCL, Zw), 34 in ZT12-16 (e.g., tim, vri, per, genome (Adams et al., 2000). To identify different regula- Slob), 34 in ZT16-20 (e.g., Cyp4d21, Cyp18a1), and 24 tory patterns underlying circadian transcript oscilla- in ZT20-24 (e.g., Rh5, amphiphysin). tions, we collected four-point time course data from Hierarchical Clustering three strains of mutant flies with defects in clock genes To organize our circadian transcripts in a way that was (per0, tim01, and ClkJrk) during a single day of entrainment. informed by the data from the mutants and grouped, Because all previously known clock-controlled genes we performed hierarchical clustering. We included both cease to oscillate in these mutants but exhibit changes the log ratio wild-type data (normalized per experiment) in their average absolute expression levels, we focused and the log ratios for each of the three clock mutants our analysis of the mutant data on changes in absolute (normalized to the entire data set) to achieve clusters expression levels rather than on evaluations of period- that have both a more or less uniform phase and a icity. uniform pattern of responses to defects in the circadian Identification of Circadian Transcripts clock. Figure 1B shows the organization of our 158 genes In a simultaneous analysis of the three separate time after hierarchical clustering with an equal weighting for courses, we extracted the 24 hr Fourier component (F24) all included log ratio measurements (see also Supple- for all of the observed expression patterns. We used mental Figure S1 at http://www.neuron.org/cgi/content/ the p value associated with F24 (p-F24) to rank the full/32/4/657/DC1). interrogated transcripts according to the perceived ro- One of the most interesting clusters generated by this bustness of their oscillation. A set of filters was applied organization is the per cluster (displayed in Figure 4A). to select against unreliable oscillations. We demanded This cluster contains genes that have an expression that the measured range of oscillation and the measured peak around ZT16 and a tendency to be reduced in expression values exceeded noise thresholds and that expression in the ClkJrk mutant. Strikingly, all genes pre- circadian transcripts showed a positive 24 hr autocorre- viously known to show this pattern of oscillation (per, lation in at least one of the experiments. According to tim, vri; Allada et al., 1998; Blau and Young, 1999) are this procedure, 293 genes emerged as significantly found in this cluster. In fact, the tim gene, which has rhythmic with 24 hr periodicity (p-F24 Ͻ 0.05; see Experi- multiple representations on our oligonucleotide arrays, mental Procedures and supplemental data). has two independent representations in this cluster. To- In order to further define a set of circadian transcripts gether with the novel oscillator CG5798, per, tim, and based on independent experimental data in addition to vri form a subcluster (average phase ZT14) that shows the statistical predictions derived from our microarrays, upregulation in both the per0 and tim01 mutants. The fact we performed Northern blots for a sample of genes that per, tim, and vri all function in the central circadian taken from ordered segments of the p-F24 Ͻ 0.05 set. clock raises the possibility that several other genes from For the interval p-F24 Ͻ 0.02, each of 16 sampled genes, this cluster, including the ubiquitin thiolesterase gene distributed over the full range of the interval, was verified CG5798 and the gene coding for the channel modulator as strongly rhythmic with a phase concordant with that Slowpoke binding protein (Slob; discussed further be- predicted from the microarray data. This cutoff defined low), may function in the circadian clock or directly a set of 158 genes and included six known circadian downstream of it. genes beyond the 16 sampled (cf. Figures 1–3). Given The genes in a second cluster (Clk cluster, Figure the limited resolution of Northern blots and the extension 4B) are primarily grouped together based on their peak of the verification to greater than 10% of this p-F24 phase (average phase ZT2). By virtue of the mutant ex- class, the results indicate that periodic gene expression pression data, we can recognize several subclusters patterns are efficiently identified by our experimental within this phase group. The known circadian genes Clk and statistical method. Northern sampling also indi- and takeout (to) are part of this cluster. Clk is found in cated substantial enrichment for circadian gene expres- a clustered pair with the leucyl aminopeptidase gene sion in the p-F24 0.02–0.05 interval; in fact, enrichment CG9285. In terms of chromosomal organization, to, for cycling gene expression continued to be evident by CG11891, and CG10513 map closely together on chro- Circadian Gene Expression in the Drosophila Head 659

Figure 1. Organization of 158 Circadian Expression Patterns by Phase and by Hierarchical Clustering In both diagrams (A and B), columns represent experimental time points, and rows represent genes. The colors in an ascending order from green to black to red represent the magnitude of the normalized log ratios for the measured average difference values. The data collected from wild-type flies in three 12-point time courses of 2 days each is normalized per experiment and shown in the phase diagram (A) and in the first 36 columns of the clustergram (B). Data collected from three arrhythmic mutants, per0, tim01, and ClkJrk, in four-point time courses spanning 1 day each is normalized to all 48 data points and displayed in the last 12 columns of the clustergram (B). The blocked horizontal bars below the phase diagram and the clustergram correspond to the light-dark entrainment schedule used in the time course experiments. White blocks indicate periods of exposure to light, whereas black blocks correspond to periods of darkness, and the gray blocks indicate periods of subjective light under the free-running condition of constant darkness. The vertical bar on the right of the phase diagram indicates the peak phase for the genes in each of the rows relative to the light-dark entrainment schedule. The time of lights-on is indicated by ZT0 and 24 (Zeitgeber time 0 and 24), whereas lights-off is denoted by ZT12. The clustergram in (B) was generated by hierarchical clustering using the uncentered average linkage method from the “CLUSTER” program giving each time point equal weight. The tree on the left of the clustergram indicates the pairwise similarity relationships between the clustered expression patterns. The italicized gene names on the right of the clustergram (ap, apterous; per, period; e, ebony; Clk, Clock) correspond to the positions of four clusters that each contain one of these genes. mosome 3R. If we consider all genes with p-F24 lower members of the Clk cluster (CG17524, CG8993, CG3174, than 0.05 we find two additional circadian genes in this Cyp6a21). It may also be noteworthy that the genes chromosomal region (CG11852, CG10553). Interestingly, for three oxidoreductases found in this group (Pdh, the Clk cluster contains three pairs of homologous CG15093, CG12116) have almost identical phases (ZT3). genes with very similar expression patterns: the UDP- All genes of the apterous (ap) cluster are defined by glycosyl transferase genes Ugt35a and Ugt35b, the en- both the oscillatory phase of their expression pattern teropeptidase genes CG9645 and CG9649, and the (average phase ZT17) and by a distinct expression pro- long-chain fatty acid transporter genes CG6178 and file in the three clock mutants (see Figure 4C). Although CG11407. In the first two cases, the homologous genes the 6 hr sampling interval in the mutant data makes it are also directly adjacent to each other on the chromo- difficult to reliably detect oscillations, it seems that the some. An overview of the map positions of all circadian majority of the genes in this cluster shows some degree genes in our study is available as supplemental informa- of periodicity in the three mutant light-dark regime (LD) tion online (http://www.neuron.org/cgi/content/full/32/ time courses. Although we cannot rule out that there 4/657/DC1). Apart from Ugt35a and Ugt35b, several are circadian oscillations independent from the known other genes with a predicted function in detoxification are clock genes, we favor the hypothesis that there may be Neuron 660

Figure 2. Log Ratio-Plotted Expression Data for 22 Transcripts in the Drosophila Head Plots with six peaks represent 36 time points of array data collected over 6 days; the dashed lines indicate the boundary of separate 2 day experiments. Plots with two peaks represent 12 time points of Northern blot data collected over 2 days; these are adjacent to the corresponding array data. In each case, the raw data are coplotted with a 24 hr guideline (red). We emphasize that the shown guidelines are not sinusoidal fits but show the 24 hr component in the pattern. The amplitude of that component is equal to the amplitude of the data only when the latter follow perfect sinusoidal behavior, and necessarily smaller otherwise. Estimated phases and log ratio amplitudes are noted for each gene. Genes are grouped by biological process. Circadian Gene Expression in the Drosophila Head 661

Figure 3. Organization of a Subset of the 158 Circadian Genes (p-F24 Ͻ 0.02) by Molecular Function Category Green print corresponds to published oscillators, red print denotes Northern-verified oscillators, blue print denotes a Northern-verfied oscillator Peak phases in black print correspond to times of (subjective) light, whereas those in white print .(0.05ف latheo) outside of the 158 (p-F24) correspond to times of (subjective) darkness. a light-driven response underlying the observed mutant genes for the transcription factor moira, the synaptic expression pattern. The genes in this group may, there- regulator syndapin, two septins (Sep1, CG9699), and fore, be regulated not only by the circadian clock, but two ATP binding cassette (ABC) transporters (CG6162, also by a direct light-dependent mechanism. It should CG9990). In terms of chromosomal organization, CG6166, be mentioned that we specifically searched for evidence the gene adjacent to CG6162 on chromosome 3R is of gene expression patterns that were purely light-driven homologous to CG9990 and coregulated with CG6162 in wild-type flies, but found little indication of such regu- and CG9990. lation. Instead, we encountered genes with both a strong The founding member of the fourth cluster (Figure 4D; light-driven oscillation and a weak circadian component average phase ZT9), ebony (e), encodes ␤-alanyl-dopa- (data not shown). apterous (ap) encodes a LIM-homeo- mine synthase and has roles in both cuticle tanning and box transcription factor, which is known to act both regulating circadian locomotor behavior (Newby and in neural development and in neuropeptide expression Jackson, 1991). Among the other cluster members are (Benveniste et al., 1998). The ap cluster includes the six genes that function in protein cleavage (CG9377,

(A) Array data for known clock genes showing robust circadian expression, establishing the technique as a powerful way to detect circadian expression. (B) Circadian transcripts connected to synaptic function, including transmission and plasticity. (C) Genes connected to amine neurotransmitter transduction and metabolism. (D) Five genes expressed in the eye. Rh5, Rh4, trpl, and ninaA all play a role in visual transduction. (E) Circadian proteases. (F) Oscillating transcripts for antioxidation and detoxification proteins. Neuron 662 Circadian Gene Expression in the Drosophila Head 663

Ser99Da, SP1029, CG7828, CG11531, BcDNA:GH02435), (e.g., Pdh, CG11891). Type I and II match the two known three transcription factor genes (CG15632, CG17257, modes of regulation for circadian genes. The behavioral CG6755), as well as two genes each that act in signal and molecular phenotypes of the per0 and tim01 muta- transduction (pn, loco), the cytoskeleton (TpnC47D, tions are almost identical (Sehgal et al., 1994, 1995). It Chd64), and lipid metabolism (ATPCL, CG1583). may, therefore, be relevant that we find no circadian Regulation of Circadian Transcripts genes that are significantly upregulated in per0 and sig- in Arrhythmic Flies nificantly downregulated in tim01 or vice versa. Apart The per0, tim01, and ClkJrk mutations affect genes that are from genes that were a priori predicted to have expres- essential for maintaining circadian rhythms and result in sion patterns of type I (vri, tim, to)orII(Clk), we find both molecular and behavioral arrhythmicity. In addition novel genes for each of these two expression patterns. to abrogating the oscillations of the per, tim, vri, to, The average phases for the type I and type II subclusters and Clk transcripts, these mutations also affect their depicted in Figure 5 are, respectively, ZT12 and ZT7, absolute levels of expression. per0 and tim01 flies have but there is large variation in phase among the members intermediate or somewhat elevated levels of per, tim, vri, of each of these subclusters. to is in the type I subcluster and to transcript, and decreased levels of Clk transcript and has a phase peak at ZT2, whereas CG15447 is in whereas ClkJrk mutants have the opposite effect (Hardin the type II subcluster and peaks at ZT10. This phenome- et al., 1990; Sehgal et al., 1994, 1995; Allada et al., 1998; non of phase differences among transcripts with a simi- Blau and Young, 1999; Glossop et al., 1999; So et al., lar response to clock defects has been described pre- 2000). Based on these observations, we gathered ge- viously for type I regulation in the case of to (So et al., nome-wide expression data from per0, tim01, and ClkJrk 2000). Here, we detect a similar phenomenon for genes mutant flies in three separate four-point time course with Clk-like type II regulation. Type III and IV predict a experiments. We employed a rank-sum Wilcoxon test novel and unexpected response to the circadian mu- to determine if any of the interrogated transcripts were tants. significantly up- or downregulated in any of the mu- Enrichment for Known Circadian tants when compared to our total wild-type expression Regulatory Elements data set. We tested the promoter sequences of our set of 158 Out of the 14010 probe sets on our arrays, 4865 genes for the presence of known and candidate circa- showed up- or downregulation in one or more of the dian enhancer motifs (see Experimental Procedures; see three mutants with a p value lower than 0.05, 2544 were also supplemental data online at http://www.neuron. significantly changed in the tim01 flies, 1810 were signifi- org/cgi/content/full/32/4/657/DC1). The results suggested cantly different in per0, and 2181 in ClkJrk. It is unclear that in fact this set is enriched in such elements. For what proportion of these changes depends on the actual example, the frequency of E boxes in our set of oscilla- mutations themselves, because (1) our three mutant fly tors (42 hits in total) is significantly higher than the fre- strains have different genetic backgrounds and (2) we quency in random selections of genes. The significance collected data for only one population of each mutant of this result does, however, depend on the inclusion of strain. Although there are known examples of noncirca- well-studied genes (per, tim, vri). Some novel oscillators dian genes whose expression is affected by clock muta- with remarkable frequencies of “circadian transcription tions, we decided that it would be more informative to elements” are loco (1 PDP1 site; 3 W boxes; 8 CRE consider effects of the clock mutations only with respect elements; 3 TER elements), trpl (1 E box; 1 PDP1 site; to the subset of 158 strong oscillators. Among this set, 9 W boxes; 6 CRE elements; 12 TER elements). Rh5 (5 we find 72 genes with one or more significant expression W boxes; 5 CRE elements; 6 TER elements), and Slob changes in the three clock mutant strains (See Figure (1 E box; 1 PERR element; 2 PDP1 sites; 6 W boxes; 5; *p Յ 0.05, **p Յ 0.01). 15 TER elements). It is noteworthy that many robustly Included in our regulated set are tim (twice indepen- cycling genes have no known circadian transcription dently), vri, to, and Clk, and their patterns agree with elements in their promoters or first introns. previously published observations. The hierarchical clustergram of Figure 5 shows four basic patterns of regulation: (type I) increased in per0 and tim01 but de- Molecular Function of Genes Expressed creased in ClkJrk (e.g., vri, CG5798), (type II) decreased in with a Circadian Rhythm per0 and tim01 but increased in ClkJrk (e.g., Clk, CG15447), We have also organized our set of 158 circadian genes (type III) decreased in all three mutants (e.g., Ugt36Bc/ according to annotated or predicted molecular function CG17932, ea), and (type IV) increased in all three mutants (see Figure 3). Several of these functional classes may

Figure 4. Clustergrams for Expression Patterns Grouped by Hierarchical Clustering Columns represent experimental time points (denoted by genetic background, yw, cn bw, tim01, per0,orClkJrk; and time points in Zeitgeber time, ZT, or Circadian time, CT), and rows represent genes (indicated by their symbol and peak phase). The colors in an ascending order from green to black to red represent the magnitude of the normalized log ratios for the measured average difference values. The blocked horizontal bars below the clustergrams correspond to the light-dark entrainment schedule used in the time course experiments. White blocks indicate periods of exposure to light, whereas black blocks correspond to periods of darkness, the gray blocks indicate periods of subjective light under the free-running condition of constant darkness. The trees on the left of the clustergrams indicate the pairwise similarity relationships between the clustered expression patterns. Boxes around sets of genes denote subclusters of genes with closely related expression patterns that are derived from the predicted hierarchy. (A) per (period) cluster; (B) Clk (Clock) cluster; (C) ap (apterous) cluster; (D) e (ebony) cluster. Top row shows average expression pattern for each cluster. Neuron 664

provide insights into pathways influencing rhythmic behavior. Synaptic Transmission and Plasticity An emerging theory of the function of sleep postulates that it is required for neural plasticity, synaptic mainte- nance, and remodelling (Hendricks et al., 2000a). Behav- iorally defined sleep has been identified in the fly, with behavioral recordings in LD indicating increased rest during the dark phase (Hendricks et al., 2000b; Shaw et al., 2000). Our assay for circadian expression uncovered a number of genes known to be involved in synaptic function and synaptic plasticity. Three oscillating trancripts encode synaptic vesicle endocytosis factors: ␤-adaptin (Bap; p-F24 0.007, ZT13), AP-1␥ (p-F24 0.039, ZT18), and syndapin (p-F24 0.007, ZT17). The first two are adaptors between the budding membrane and clathrin lattice, while syndapin is thought to connect vesicle endocytosis to actin (Qualmann and Kelly, 2000; Takei and Haucke, 2001). Clock modulation of the synaptic vesicle pool is consistent with the idea of modulated synaptic function, although it is unclear what effect raising or lowering endocytotic factors would have on synaptic function. The Slob transcript peaks at ZT15 by microarray and Northern blot verification (Figure 2B) and is downregulated in the ClkJrk mutant, suggesting that CLK acts as an activator of Slob. There is an E box 5.4 kb upstream of the Slob transcriptional start site raising the possibility that CLK acts directly on the Slob promoter. If the cycling transcript can be shown to produce oscillating Slob protein, this could indicate a potent mechanism for rhythmic control of synaptic function, including synaptic plasticity: Slob protein binds the calcium-dependent potassium channel Slowpoke (Slo) and has been shown to increase Slo activity and voltage sensitivity (Schopperle et al., 1998). Slob is in turn bound by a second channel regulator, Leonardo. Hypomorphic mutations of leonardo produce defects in learning, and electrophysiological analyses of the larval neuromuscu- lar junction (NMJ) in these mutants show presynaptic function and plasticity is greatly impaired in these animals (Broadie et al., 1997). In contrast to Slob, Leonardo is a strong inhibitor of Slo, but requires Slob for this interaction (Schopperle et al., 1998; Zhou et al., 1999). All three proteins colocalize to the presynaptic bouton at larval NMJs (Zhou et al., 1999). Thus, Slob may contribute to a switching mechanism that ultimately places Figure 5. Clustergram for Expression Patterns Obtained in Three Slo channel activity under circadian control. Clock Mutants Slo channels are widely expressed in the adult fly Columns represent experimental time points (denoted by genetic head, including the eye, lamina, medulla, central brain, background, tim01, per0,orClkJrk; and time points in Zeitgeber time, and mushroom bodies, but it is not known which subset ZT) and rows represent genes with significant regulation in these of these areas contain oscillating Slob expression mutants. The colors in an ascending order from green to black to red represent the magnitude of the normalized log ratios for the (Becker et al., 1995). We performed in situ hybridization measured average difference values. The blocked horizontal bars with larval brains to localize Slob RNA expression. Prom- below the clustergrams correspond to the light-dark entrainment inent staining was observed in a restricted region con- schedule used in the time course experiments. White blocks indicate sistent with placement of the developing mushroom periods of exposure to light, whereas black blocks correspond to body (Figure 6A) (Truman et al., 1993). The staining also periods of darkness. The tree on the left of the clustergram indicates corresponds well with that region of the larval brain the pairwise similarity relationships between the clustered expres- receiving PDF-rich projections of the circadian pace- sion patterns. The symbols preceding the gene names for each row correspond to the significance of the p value of the rank-sum maker cells, lateral neurons (LNs [Helfrich-Forster et al., Wilcoxon test for each of the three mutants (—, not significant; *p Յ 2000]). In future studies, it will be important to determine 0.05; **p Յ 0.01) in the order tim01/per0/ClkJrk. The blocks with Roman whether presence of the innervating LNs is required for numerals indicate four types of expression patterns. cycling Slob expression. leonardo was initially implicated in presynaptic func- Circadian Gene Expression in the Drosophila Head 665

have been mapped, but no studies have been done of either 5-HT receptor localization or receptor mutant phenotypes (Valles and White, 1988). Neither of these receptors are orthologs of the mammalian 5-HT receptor implicated in photic clock entrainment; this would be represented by the Drosophila 5-HT7 gene (Tierney, 2001), which we find with p-F24 0.067. 5-HT1A belongs in a class of receptors that respond to agonists by de- creasing cellular cAMP, while 5-HT2 is homologous to mammalian receptors whose main mode of action in- volves activation of phospholipase C, a function involved in synaptic plasticity (Tierney, 2001; Kandel et al., 2000). The ebony transcript was found to oscillate by microarray and Northern blot, showing a peak of expression around ZT5 (Figure 2C). ebony oscillation connects with a body of earlier evidence linking ebony to circadian acitivity rhythms. ebony hypomorphs show severe defects in circadian rhythm including arrhythmicity/aberrant periodicity in the free-running condition, as well as abnormal activity patterns in LD conditions (Newby and Jackson, 1991). Ebony is a putative ␤-alanyl dopamine synthetase, and hypomorphs show elevated levels of dopamine (Hovemann et al., 1998; Walter et al., 1996). Dopamine has been implicated in control of motor behavior, as it induces reflexive locomotion in decapitated flies, and this response is under circadian control (An- dretic and Hirsh, 2000). Our results suggest that oscillations of ebony contribute to the assembly of rhythmic locomotor behavior. Other evidence points to a role in clock resetting. In addition to impaired entrainment in LD, ebony flies show an abnormal ERG, and ebony is Figure 6. Localization of Novel Cycling Genes by In Situ Hybrid- strongly expressed in the lamina and the medulla optic ization neuropile, a region associated with vision rather than (A) In situ hybridization of Slob antisense probe to larval brains motor control (Newby and Jackson, 1991; Walter et al., is consistent with mushroom body staining, and the brain region 1996). innervated by PDF-expressing pacemaker cells (LNs). Vision (B) In situ hybridization of CG13848 antisense probe to adult fly The Drosophila eye is both a likely target of clock control head slices shows staining specific to basal cells in the eye, which and partly responsible for photic input to the central is high at ZT2 and low or absent at ZT14. pacemaker (Helfrich-Forster et al., 2001; Yang et al., 1998). Several genes found to oscillate by our microarray assay are components of visual processes. tion by the effect of mutations on learning (Broadie et Photoreceptor cells contain peripheral clocks, sug- al., 1997). Mutations of latheo also cause learning de- gesting that visual function may be regulated by the fects, and this protein is also found at larval NMJs. clock. In vertebrates, the synthesis of various visual Lowered latheo function has been associated with ele- components is known to be under circadian control vated synaptic transmission and reduced synaptic plas- (Hartman et al., 2001; Korenbrot and Fernald, 1989). In ticity (Rohrbough et al., 1999). latheo showed cycling Drosophila, electroretinogram (ERG) measurements of expression with peak accumulation at ZT12-15 in our visual sensitivity revealed a 4-fold cycle in sensitivity, microarray analysis (p-F24 0.05) and in Northern assays with a minimum at ZT4 and a broad peak around lights (Figure 2B). We also detect rhythmicity in the expression off (ZT12) (Chen et al., 1992). This suggests that some of dunce (p-F24 0.04) and Calpain-B (p-F24 0.03) (Figure of the fly visual components are clock controlled. How- 2B) genes involved in learning and synaptic long-term ever, a previous study of five major phototransduction potentiation, respectively (Dudai et al., 1976; Lynch, components found no cycling of either mRNA or protein 1998). (Hartman et al., 2001). In our genome-wide assay, the trpl Amine Neurotransmitter-Related Functions transcript was found to oscillate with peak expression at Two serotonin receptor transcripts, 5-HT2 and 5-HT1A, ZT11 (p-F24 0.0026; Figure 2D). TRPL is one of two ion were found to oscillate with phases of ZT15 and ZT18, channels in the visual transduction pathway, along with respectively (see Figure 2C for 5-HT2 data). Serotonin TRP, a paralog. TRPL and TRP open in response to is known to be involved in a variety of neuronal pro- a G protein-coupled phosphoinositide cascade that is cesses in animals, including synaptic plasticity, clock initiated by the isomerization of rhodopsin by light (Har- entrainment, and mating behavior (Ehlen et al., 2001; die and Raghu, 2001). Their opening produces the light- Horikawa et al., 2000; Kandel et al., 2000; Lee et al., sensitive conductance in the photoreceptors. Amorphic 2001). The 104 serotonergic neurons in the adult CNS mutants of each channel show visual defects, while the Neuron 666

double null genotype results in a blind fly (Reuss et al., ZT16, and each produces a ubiquitin specific protease 1997). A cycling trpl transcript could contribute to the (Ubp; cleaves ubiquitin from ubiquitin-protein conju- visual sensitivity cycle. First, the two phenomena are in gates) that may act to prevent the degradation of spe- the same phase with both sensitivity and trpl expression cific protein targets (Chen and Fischer, 2000; Huang et peaking around lights-off. Second, it is estimated that al., 1995). Two metalloprotease genes, two aminopepti- TRPL contributes about half of the wild-type conduc- dase genes, and five serine peptidases show circadian tance, allowing for a substantial range of modulation by oscillation. Four of the five serine peptidase genes cycle reducing TRPL function (Reuss et al., 1997). Another with a peak phase between ZT4-7. This profusion of possible role for oscillating TRPL function is connected oscillating proteases suggests that circadian proteoly- with circadian entrainment. In addition to being blind, sis may represent a broad mechanism of clock control, trp/trpl double null mutants show reduced circadian be- both of clock components themselves, as well as output havioral resetting and less TIM degradation in response factors. to light pulses (Yang et al., 1998). We note that the While circadian transcriptional mechanisms are rela- established entrainment factor CRY is known to cycle tively well understood, less is known about posttransla- and that interactions of CRY and TIM are essential for tional mechanisms of circadian regulation. Proteases light-dependent TIM degradation (Ceriani et al., 1999; are known to be involved in circadian control of the Emery et al., 1998). The oscillating, clock-related protein degradation of some central clock components, and the VIVID has also been shown to regulate photo-entrain- clock proteins PER, TIM, CLK, CRY, and VRI are all ment in Neurospora (Heintzen et al., 2001). known to undergo daily cycles of protein accumulation. Our microarray experiments show that two opsin Degradation of TIM is responsible for photic resetting genes are under circadian control: Rh5 and Rh4. The of the Drosophila clock. This is thought to be mediated Rh5 mRNA rhythm (p-F24 0.0098) was confirmed by by interaction with CRY, followed by ubiquitination and .(ZT 21, while the Rh4 array proteasome-dependent loss of TIM (Naidoo et al., 1999ف Northern blot to peak at data show a circadian pattern (p-F24 0.037) with a peak Nothing is known about the factors mediating TIM deg- 4 hr later, at ZT1 (Figure 2D). Rh5 is a blue-absorbing radation in the dark, yet patterns of CG5798 expression rhodopsin expressed in a subset of R8 cells at the base may be of interest in this regard as it encodes a cycling of the retina, while Rh4 is a UV-absorbing pigment ex- Ubp whose peak expression (ZT14) immediately pre- pressed in apical R7 cells (Chou et al., 1996; Salcedo cedes an interval of rapid TIM accumulation in pace- et al., 1999). Rh5 is never expressed in an R8 cell underly- maker cells (Figure 2E). ing an Rh4-expressing R7 cell, so in this way all omma- A likely clock-related target of one or more proteases tidia would contain one cycling rhodopsin (Chou et al., is the neuropeptide PDF, whose regulation may be cru- 1996). In terms of regulating sensory receptiveness to cial to linking the clock to behavior. While pdf RNA light, it is unclear why these two opsins should be targets is expressed constitutively, the peptide accumulates for clock control. The major blue rhodopsin Rh1 does rhythmically under indirect control of the clock gene not cycle so it seems unlikely that an oscillation in these vri (Blau and Young, 1999; Park and Hall, 1998). This two minor pigments would produce overall tuning of the mechanism has not been further explored, but a clear sensitivity of the fly visual system (Hartman et al., 2001). possibility is that a PDF propeptide is cleaved rhythmi- NinaA is a rhodopsin chaperone and is required to cally, allowing cyclical release of active PDF (Helfrich- move Rh1 from the endoplasmic reticulum (ER) to the Forster et al., 2000). Of the 15 cycling proteases sug- rhabdomeric membrane (Colley et al., 1991). ninaA mu- gested by our study (Figure 3), CG4723 may be of special tants display aberrant accumulation of Rh1 protein in interest due to its inclusion in a class of proteases known the ER. ninaA mRNA shows cycling expression in fly to cleave neuropeptides (Isaac et al., 2000). The phase heads by both array and Northern blot, with peak ex- of CG4723 expression, ZT4, also coincides with that of pression around ZT2 (Figure 2D). It is tempting to hy- PDF immunoreactivity in the Drosophila head (Park et pothesize that early morning ninaA upregulation would al., 2000). have the effect of releasing a reservoir of Rh1 from the Detoxification and Oxidative Stress ER, making it available for use in visual transduction. One hypothesis of sleep characterizes it as a cellular However, a previous assay of NinaA levels in an LD detoxification and repair process (Inoue et al., 1995; Yin, regime revealed no oscillation in protein levels (Hartman 2000). We find twelve genes that have a predicted role et al., 2001). If true, this would represent a surprising in detoxification. Three additional redox enzymes may example of a robustly oscillating mRNA producing a also participate in this process. Toxins are initially modi- constitutive cognate protein. Finally, although its func- fied into reactive species by reductases/dehydrogenases tion is still unknown, Photoreceptor dehydrogenase and cytochrome P450 molecules. Then glutathione-S- (Pdh) is a robustly oscillating transcript (see Figure 2D transferases (GSTs) and UDP-glycosyl transferases add and Figure 3) with an extremely high level of expression polar groups to the substrates to render them hydro- in the screening pigment cells of the eye (Brunel et al., philic for elimination by secretion (Salinas and Wong, 1998). 1999). Firstly, following this pathway, we find the genes Rhythmic Proteases and Accessory Factors for three circadian dehydrogenases: Pdh, CG10593, and Fifteen of the identified oscillatory genes are implicated CG12116. Secondly, we find both morning and night in protein cleavage (see Figure 2E and Figure 3). CG7828 cytochrome P450 genes: Cyp6a21 and Cyp305a1 peak and BcDNA:GH02435 (peak phase ZT6 and ZT10, respec- early in the day (ZT0 and ZT5), whereas Cyp18a1 and tively) mediate ubiquitination of protein substrates, thus Cyp4d21 (see Figure 2F and Figure 3) peak at approxi- targeting them for degradation. Conversely, CG5798 and mately the same time late at night (ZT18 and ZT19). CG7288 cycle with a peak at respectively, ZT14 and Thirdly, while CG17524 is the only GST gene found in Circadian Gene Expression in the Drosophila Head 667

our core set, we noticed that two other members of the participating in a NAD(P)H-mediated autoregulatory GST type III gene cluster on chromosome 2R show 24 loop of the clockworks (Schibler et al., 2001). hr periodicity (CG17523, p-F24 0.025; CG17527, p-F24 Nucleic Acid Metabolism 0.031). Fourthly, UDP-glycosyl transferase genes are We find a subset of 15 genes involved in nucleic acid represented in our circadian set by Ugt35a, Ugt35b, metabolism (Figure 3). This includes five genes encoding and Ugt36Bc. Of these, Ugt35b (Figure 2F) encodes specific RNA polymerase II transcription factors. Four an antennal specific transcript with a potential role in of these encode parts of the circadian clock itself (per, olfaction, whereas Ugt35a is expressed more uniformly tim, vri, and Clk), whereas the fifth one, apterous (ap), (Wang et al., 1999). generates a homeobox transcription factor with a role CG13848 (see Figures 2F and 6B) is an ␣-tocopherol in neurogenesis and the expression of neuropeptides transfer protein (␣-TTP) that is strongly expressed in (Benveniste et al., 1998; Hobert and Westphal, 2000). certain basal cells of the eye, showing peak expression Taf30␣2 is a subunit of the general transcription factor around dawn. Humans with mutations in the homolo- TFIID, while moira (mor) is part of the SWI-SNF chroma- gous ␣-TTP show neurodegenerative ataxia that is asso- tin-remodeling complex (Aoyagi and Wassarman, 2000; ciated with a deficiency in ␣-tocopherol (vitamin E) incor- Crosby et al., 1999). One splicing factor gene (DebB) poration into lipoprotein particles (Ouahchi et al., 1995). and one DNA repair gene (CG4049) are also found Vitamin E acts as an antioxidant, and it is thought that among this set of oscillating transcripts. this activity allows it to protect neurons from damage Cytoskeleton by free radicals (Ouahchi et al., 1995). Also from human Circadian genes with a role in the cytoskeleton include studies, it is known that photoreceptor cells are particu- those encoding two actin binding proteins (Chd64, larly susceptible to oxidative damage due to high levels CG11605), a troponin C (TpnC47D), and two septins of polyunsaturated fatty acids in the photoreceptor (Sep-1, CG9699). Chd64 (phase peak ZT4) and TpnC47D membrane, and their exposure to visible light (Beatty et (phase peak ZT7) function specifically in muscle con- al., 2000). Thus, we propose that the dawn phase of traction. Another Drosophila Troponin C, TpnC73F, is CG13848 represents an upregulation of ␣-TTP for in- found to peak at ZT6 and has a p-F24 of 0.08. creased daytime transfer of photoprotective vitamin E into the photoreceptor membrane. Also in our circadian Conclusions set, Catalase (encoded by Cat) and a thioredoxin (en- We have recognized a set of 158 genes expressed with coded by CG8993) are both involved in neutralizing reac- a robust circadian rhythm in the adult Drosophila head tive oxygen species (Gutteridge and Halliwell, 2000). by microarray screening. These encompass a wide vari- Metabolism ety of molecular functions, and expression patterns rep- Different aspects of metabolism are represented among resented essentially all circadian phases. A larger set the selected set of oscillating transcripts: lipid metabo- of genes was identified (393 entries, p-F24 Ͻ 0.05; 293 lism (five genes), amino acid metabolism (three), carbo- entries after secondary filters), and our statistical ap- hydrate metabolism (three), and glycoprotein biosynthe- proach again indicated significant circadian rhythmicity sis (two). Intriguingly, Zw encodes glucose-6-phosphate for these, but they were characterized by somewhat less 1-dehydrogenase of the pentose-phosphate pathway robust oscillations than those of p-F24 Ͻ 0.02. Indepen- (PPP), while CG10611 encodes fructose-bisphospha- dent verifications indicated substantial enrichment for tase in gluconeogenesis. The two pathways have antag- cycling gene expression in this larger set and beyond, p-F24 Ͻ 0.1. At a p-F24 cutoff of 0.1, 532 genes passف onizing roles in glucose metabolism; both genes are to key control points in their respective pathway and are our secondary filters for 24 hr autocorrelation, noise, maximally expressed in opposite phases (Voet and Voet, and the range-to-noise measure. From the frequency of 1990). Zw transcripts are at their zenith just before dusk Northern-verified oscillations detected in this larger pool (ZT11), whereas CG10611 transcripts peak at dawn of candidate genes, we believe we can usefully predict (ZT0). Thus, the antiphase oscillation of these two genes that the total complement of circadian genes would in- .in the adult head 500–400ف may produce daily alternation between glucose anabo- clude lism and catabolism. Zw and CG10611 transcript levels There are important factors that might lead us to un- also respond in opposite fashion to clock defects: Zw derestimate the total complement of circadian genes. levels are significantly decreased in per0 and tim01 mu- Our approach will favor genes that are homogeneously tants, whereas, CG10611 is significantly upregulated in expressed in the head. If the same gene is expressed tim01 (Figure 5). with varied phases in different head tissues, this will In more direct relation to the clock itself, Zw is the lessen the robustness of the apparent oscillation and first committed step in the PPP and generally thought phase. Similarly, if only a restricted portion of the head to control flux through this pathway (Stryer, 1988; Voet generates the cycling gene pattern, but constitutive ex- and Voet, 1990). The PPP is the major pathway of NAD pression is found elsewhere in the head, amplitude of (or NADP) conversion to NAD(P)H (Stryer, 1988; Voet the signal will be diminished. Differences of this sort and Voet, 1990). It was recently shown that NAD(P)H might be expected in cases where a cycling gene prod- can bind homologs of CLK and CYC, promoting their uct produces a limited physiological effect. Regulation dimerization and DNA binding (Rutter et al., 2001). Maxi- of this type might be expected in the antennae, where, mal Zw expression at ZT11—and therefore presumably for example, electrophysiological responses to odorants NAD(P)H production via the PPP—is coincident with vary with a circadian rhythm (Krishnan et al., 1999). We maximal per and tim transcription by CLK/CYC (So and should also stress that we have sampled only the fully Rosbash, 1997). This information is consistent with Zw differentiated adult head. If transient patterns of circa- Neuron 668

dian expression occur during development, these would real Fourier score with respect to scores obtained from 10,000 ran- go unnoticed in our experiment. Given the plethora of dom permutations of the time points (time points from different time tissues housing autonomous circadian clocks, an ex- courses were not mixed), for each gene separately. All observed expression patterns were ranked according to ascending p-F24, so panded list of rhythmic genes would probably be derived that 188 genes passed a filter of p-F24 Ͻ 0.02. Progressively low- from any related sampling of the body (e.g., wings, legs, ering the cutoff to 0.05, 0.1, and 0.15, respectively, yields 393, 817, excretory, and digestive tissues), especially as this tis- and 1232 entries. sue autonomy may reflect a requirement for tissue-spe- To identify possible false positives that may have passed the cific pathways of circadian control that lie downstream above criteria by accident, we applied several fairly mild secondary from a largely uniform clock mechanism (Plautz et al., filters in series. First, we considered the 24 hr autocorrelation (AC24), which measures the difference in pattern between the LD and DD 1997). parts of the experiment, and has a range in Ϫ1:1. We required that How will the many patterns of cycling gene expression the highest of the three AC24 values was larger than 0. Second, to be further explored? Molecular tools that reveal the im- eliminate genes with too low over all signal intensity, we required portance of oscillating gene activity have already been that more than half of all measured AD values for a given gene be applied to a study of several clock genes in Drosophila. larger than 4Q. Finally, we considered a signal-to-noise like indicator In these studies, oscillating patterns of a target gene’s RN (range-over-noise), defined for each time course as the ratio between the amplitude of oscillation (maximum minus minimum AD expression have been replaced with constitutive activ- over the time course) to the sum of the noise levels Q at the extrema. ity. Central questions related to vri, per, and tim function Oscillations with low RN are likely to be the consequence of noise; have each been explored in this manner (Blau and we, therefore, selected for genes with average RN Ͼ 3.5. Young, 1999; Yang and Sehgal, 2001). The present study allows an expansion of such work to address the molec- Analysis of the Data from Circadian Mutants ular connections between individual behaviors and cir- The significance of differences between AD values estimated for 0 01 Jrk cadian clocks. wild-type flies and AD values for per , tim ,orClk mutant flies was assessed using a rank-sum Wilcoxon test comparing the data for all 36 wild-type time points separately to each set of four mutant Experimental Procedures time points. Fly Strains Wild-type strains used were yw and cn bw. Clock mutant strains Verification of Microarray Results used were per0 (Konopka and Benzer, 1971), tim01 (Myers et al., A sampling of genes that were indicated by the oligonucleotide array 1995), and ClkJrk (Allada et al., 1998). data to have a circadian oscillation were independently assayed by Northern blot. Blots were made with 12 samples of RNA collected Microarray Experiments over 2 days, one LD and one DD, in a similar configuration to the Fly strains were entrained to a 12 hr:12 hr light-dark (LD) regime at array experiments. Probe template was sourced from the Drosophila 25ЊC for 3 days and harvested onto dry ice either at 6 hr intervals Gene Collection (Berkeley Drosophila Genome Project), and each during the following day of light-dark entrainment (mutant strains) clone was sequence verified. Blots were visualized and quantified or at 4 hr intervals during the next day of light-dark entrainment and with a Storm Phosphorimager (Molecular Dynamics). Only band sig- an additional day of free-running in constant darkness (wild-type nals that were measured to be at least 3-fold above the blot back- strains). Frozen flies were broken up and passed through wire mesh ground were considered reliably quantifiable. A gene was consid- to separate the heads from the rest of the animal. This procedure ered to have a confirmed oscillation if it showed two peaks spaced faithfully isolated heads from other body parts and should exclude at 24 hr, with phase of peak expression that was within 4 hr (the contaminating signals from sources outside the head. The heads sampling window) of the array data. So as to have a quantification were used to isolate crude RNA with either Rnazol (Tel-Test) or of the oscillation that is directly comparable with the array result, Trizol (Life Technologies). The yield was approximately 1 ␮g total Fourier analysis was also done on the Northern data. Based on the RNA/fly head. This RNA was then further purified using Rneasy distribution of p-F24 values for the transcripts that were uniformly columns (Qiagen) following the RNA cleanup protocol. 25 ␮g of this verified to cycle by sampling with Northern blots, we decided that material was used to make biotin-labeled cRNA probe following the a p-F24 cutoff of 0.02 would define a subset of 158 genes with methods described in the Affymetrix GeneChip Expression Techni- particularly robust oscillations. cal Manual. cDNA synthesis was carried out with T7-dT24 primer (MWG Biotech), Superscript Choice (Life Technologies), and addi- Clustering tional enzymes from New England Biolabs. In vitro transcription was Hierarchical clustering on the subset of 158 genes was performed done using the ENZO Bioarray HighYield RNA transcript labeling kit. using the “CLUSTER” program (Eisen et al., 1998) on the log ratio Hybridization, washing, staining, and scanning of the target cRNA to values normalized per time course experiment for the wild-type data Affymetrix oligonucleotide arrays were done as per the Affymetrix and normalized to the entire data set for the mutant data, giving GeneChip manual. Uniformity of the hybridization response from each of the arrays equal weight. Before calculating the log ratios one array to another was verified by hybridizing RNA samples from for clustering, average difference values below 4Q were set to 2.8Q. the same strain to duplicate oligonucleotide arrays for several time Clustergrams were visualized with the aid of the “TREEVIEW” pro- points (see supplemental data online). gram. From the set of 158, a subset of 72 genes that showed significant up- or downregulation in at least one of the three mutant data Data Analysis sets was identified. For this set of 72, hierarchical clustering was The circadian tendency of a time course experiment was quantified performed on the mutant data. using the 24 hr Fourier component and its associated p value (F24 and p-F24, respectively). These quantities were determined after Promoter Site Analysis transforming the average difference (AD) scores into log ratios. For The upstream sequences associated with predicted circadian genes each time point, the expression level relative to the mean (taken in were analyzed to test if they were enriched for known circadian log coordinates) over that experiment was computed. AD scores transcription factor binding sites. For each transcript, an upstream below one standard deviation of residual background (Q) were set sequence with a maximum of 10 kb was defined by the translation to Q. Fourier scores were calculated after appending the three nor- start of the transcript and the beginning of the next upstream coding malized time courses, so that phase coherence between the pat- sequence. The following definitions of binding sites were used: E terns in the three experiments was automatically enforced. The re- box, CACGTGAG(C)C with a perfect match for the first six bases sulting F24 scores are independent of the order in which the time and possibly one mismatch for the last three bases; W box, TTG courses were appended. p-F24 was determined from ranking the GCCAGCAA with two mismatches; CRE, TGACGTCA with one mis- Circadian Gene Expression in the Drosophila Head 669

match; PERR, GTTCGCACAA with one mismatch; TER, GCACGTTG of the Drosophila fat facets deubiquitinating enzyme gene. Genetics with one mismatch; PDP1, GAATTTTGTAAC with two mismatches. A 156, 1829–1836. comprehensive description of the promoter site analysis is available Chen, D.M., Christianson, J.S., Sapp, R.J., and Stark, W.S. (1992). online. Visual receptor cycle in normal and period mutant Drosophila: micro- spectrophotometry, electrophysiology, and ultrastructural mor- In Situ Hybridizations phometry. Vis. Neurosci. 9, 125–135. Larval brain in situ hybridization was performed as per Price et al. Chou, W.H., Hall, K.J., Wilson, D.B., Wideman, C.L., Townson, S.M., (1998). Adult fly head in situ hybridization was done as per Schaeren- Chadwell, L.V., and Britt, S.G. (1996). Identification of a novel Dro- Wiemers and Gerfin-Moser (1993). Probe templates were from the sophila opsin reveals specific patterning of the R7 and R8 photore- Drosophila Gene Collection (Berkeley Drosophila Genome Project). ceptor cells. Neuron 17, 1101–1115. Colley, N.J., Baker, E.K., Stamnes, M.A., and Zuker, C.S. (1991). The Acknowledgments cyclophilin homolog ninaA is required in the secretory pathway. Cell 67, 255–263. We thank the staff of the Gene Array facility at The Rockefeller University, in particular Greg Khitrov and Richard Pearson for help Crosby, M.A., Miller, C., Alon, T., Watson, K.L., Verrijzer, C.P., Gold- with the hybridization and processing of oligonucleotide microar- man-Levi, R., and Zak, N.B. (1999). The trithorax group gene moira rays; Stefan Bekiranov for initial help with data analysis; Andrew encodes a brahma-associated putative chromatin-remodeling fac- Eisberg for preparation of Northern blot probes; Mark Schroeder tor in Drosophila melanogaster. Mol. Cell. Biol. 19, 1159–1170. for gene annotation; members of the Young laboratory for helpful Dudai, Y., Jan, Y.N., Byers, D., Quinn, W.G., and Benzer, S. (1976). discussion; and Fernanda Ceriani, Martin Straume, and Steve Kay dunce, a mutant of Drosophila deficient in learning. Proc. Natl. Acad. for sharing results prior to publication. This work was supported by Sci. USA 73, 1684–1688. grants from NSF, Center for Biological Timing, and NIH (GM54339) Edery, I. (2000). Circadian rhythms in a nutshell. Physiol. Genomics to M.W.Y. and NIMH postdoctoral fellowship PHS MH63579 (H.W.). 3, 59–74. Ehlen, J.C., Grossman, G.H., and Glass, J.D. (2001). In vivo resetting Received October 16, 2001; revised November 2, 2001. of the hamster circadian clock by 5-HT7 receptors in the suprachiasmatic nucleus. J. Neurosci. 21, 5351–5357. References Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998). Cluster analysis and display of genome-wide expression patterns. 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