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Recent studies have demonstrated that astrocytes cooperate with neurons of the brain to mediate circadian control of many rhythmic processes including locomotor activity and sleep. Transcriptional profiling studies have described the overall rhythmic landscape of the brain, but few have employed approaches that reveal heterogeneous, cell-type specific rhythms of the brain. Using cell-specific isolation of ribosome-bound RNAs in Drosophila, we constructed the first circadian “translatome” for astrocytes. This analysis identified 293 “cycling genes” in astrocytes, most with mammalian orthologs. A subsequent behavioral genetic screen identified a number of genes whose expression is required in astrocytes for normal sleep behavior. In particular, we show that certain genes known to regulate fly innate immune responses are also required for normal sleep patterns.

Glial astrocytes of vertebrates and invertebrates are important modulators of nervous system development, physiology, and behavior. In all species examined, astrocytes of the adult brain contain conserved circadian clocks, and multiple studies have shown that these glial cells participate in the regulation of circadian behavior and sleep. This short review summarizes recent work, using fruit fly (Drosophila) and mouse models, that document participation of astrocytes and their endogenous circadian clocks in the control of rhythmic behavior. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Nervous System Development > Flies.

The analysis of adult astrocyte glial cells has revealed a remarkable heterogeneity with regard to morphology, molecular signature, and physiology. A key question in glial biology is how such heterogeneity arises during brain development. One approach to this question is to identify genes with differential astrocyte expression during development; certain genes expressed later in neural development may contribute to astrocyte differentiation. We have utilized the Drosophila model and Translating Ribosome Affinity Purification (TRAP)-RNA-seq methods to derive the genome-wide expression profile of Drosophila larval astrocyte-like cells (hereafter referred to as astrocytes) for the first time. These studies identified hundreds of larval astrocyte-enriched genes that encode proteins important for metabolism, energy production, and protein synthesis, consistent with the known role of astrocytes in the metabolic support of neurons. Comparison of the larval profile with that observed for adults has identified genes with astrocyte-enriched expression specific to adulthood. These include genes important for metabolism and energy production, translation, chromatin modification, protein glycosylation, neuropeptide signaling, immune responses, vesicle-mediated trafficking or secretion, and the regulation of behavior. Among these functional classes, the expression of genes important for chromatin modification and vesicle-mediated trafficking or secretion is overrepresented in adult astrocytes based on Gene Ontology analysis. Certain genes with selective adult enrichment may mediate functions specific to this stage or may be important for the differentiation or maintenance of adult astrocytes, with the latter perhaps contributing to population heterogeneity.

Summary Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. In mammalian and invertebrate models, well-characterized neuronal circuits and evolutionarily conserved mechanisms regulate circadian behavior and sleep [ 1 , 2 , 3 , 4 ]. In Drosophila, neuronal populations located in multiple brain regions mediate arousal, sleep drive, and homeostasis (reviewed in [ 3 , 5 , 6 , 7 ]). Similar to mammals [ 8 ], there is also evidence that fly glial cells modulate the neuronal circuits controlling rhythmic behaviors, including sleep [ 1 ]. Here, we describe a novel gene (CG14141; aka Nkt) that is required for normal sleep. NKT is a 162-amino-acid protein with a single IgC2 immunoglobulin (Ig) domain and a high-quality signal peptide [ 9 ], and we show evidence that it is secreted, similar to its C. elegans ortholog (OIG-4) [ 10 ]. We demonstrate that Nkt-null flies or those with selective knockdown in either neurons or glia have decreased and fragmented night sleep, indicative of a non-redundant requirement in both cell types. We show that Nkt is required in fly astrocytes and in a specific set of wake-promoting neurons—the mushroom body (MB) α’β’ cells that link sleep to memory consolidation [ 11 ]. Importantly, Nkt gene expression is required in the adult nervous system for normal sleep, consistent with a physiological rather than developmental function for the Ig-domain protein.

RNA-binding proteins mediate posttranscriptional functions in the circadian systems of multiple species. A conserved RNA recognition motif (RRM) protein encoded by the lark gene is postulated to serve circadian output and molecular oscillator functions in Drosophila and mammals, respectively. In no species, however, has LARK been eliminated, in vivo, to determine the consequences for circadian timing. The present study utilized RNA interference (RNAi) techniques in Drosophila to decrease LARK levels in clock neurons and other cell types in order to evaluate the circadian functions of the protein. Knockdown of LARK in timeless (TIM)– or pigment dispersing factor (PDF)–containing clock cells caused a significant number of flies to exhibit arrhythmic locomotor activity, demonstrating a requirement for the protein in pacemaker cells. There was no obvious effect on PER protein cycling in lark interference (RNAi) flies, but a knockdown within the PDF neurons was associated with increased PDF immunoreactivity at the dorsal termini of the small ventral lateral neuronal (s-LNv) projections, suggesting an effect on neuropeptide release. The expression of lark RNAi in multiple neurosecretory cell populations demonstrated that LARK is required within pacemaker and nonpacemaker cells for the manifestation of normal locomotor activity rhythms. Interestingly, decreased LARK function in the prothoracic gland (PG), a peripheral organ containing a clock required for the circadian control of eclosion, was associated with weak population eclosion rhythms or arrhythmicity.

A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein.

Brain glial cells, in particular astrocytes and microglia, secrete signaling molecules that regulate glia–glia or glia–neuron communication and synaptic activity. While much is known about roles of glial cells in nervous system development, we are only beginning to understand the physiological functions of such cells in the adult brain. Studies in vertebrate and invertebrate models, in particular mice and Drosophila, have revealed roles of glia–neuron communication in the modulation of complex behavior. This chapter emphasizes recent evidence from studies of rodents and Drosophila that highlight the importance of glial cells and similarities or differences in the neural circuits regulating circadian rhythms and sleep in the two models. The chapter discusses cellular, molecular, and genetic approaches that have been useful in these models for understanding how glia–neuron communication contributes to the regulation of rhythmic behavior.

Sleep and arousal are known to be regulated by both homeostatic and circadian processes, but the underlying molecular mechanisms are not well understood. It has been reported that theDrosophilarest/activity cycle has features in common with the mammalian sleep/wake cycle, and it is expected that use of the fly genetic model will facilitate a molecular understanding of sleep and arousal. Here, we report the phenotypic characterization of aDrosophilarest/activity mutant known asfumin(fmn). We show thatfmnmutants have abnormally high levels of activity and reduced rest (sleep); genetic mapping, molecular analyses, and phenotypic rescue experiments demonstrate that these phenotypes result from mutation of theDrosophila dopamine transportergene. Consistent with the rest phenotype,fmnmutants show enhanced sensitivity to mechanical stimuli and a prolonged arousal once active, indicating a decreased arousal threshold. Strikingly,fmnmutants do not show significant rebound in response to rest deprivation as is typical for wild-type flies, nor do they show decreased life span. These results provide direct evidence that dopaminergic signaling has a critical function in the regulation of insect arousal.

The lark gene was initially identified in a behavioral screen for flies that displayed circadian rhythm defects (Newby and Jackson, 1993, 1996). It encodes an RNAbinding protein of the RNA Recognition Motif (RRM) class. Whereas lark heterozygotes exhibit a rhythm defect, homozygotes die during embryonic development because of a zygotic requirement for the gene product. Subsequently, it was shown that there is also a maternal requirement for lark function during oogenesis and early embryonic development (McNeil et al., 1999). Currently, several fly labs are pursuing studies of LARK function as related to oogenesis and embryonic development. LARK protein contains three potential RNA binding domains, two RNA recognition motifs, and a single retroviral type zinc finger, that have been shown to be important for LARK function during development but appear to play functionally redundant roles in the circadian regulation of eclosion (Newby and Jackson, 1996; McNeil et al., 2000). The LARK protein is widely expressed in most if not all tissues and throughout development (Zhang et al., 2000). In the majority of cells that express LARK, the protein has a nuclear localization. A notable exception occurs in a subset of neurosecretory cells that express the neuropeptide crustacean cardioactive peptide (CCAP), in which LARK is cytoplasmic. Of interest for the rhythm phenotype, CCAP cells exhibit circadian changes in LARK abundance (Zhang et al., 2000). In order to further characterize LARK protein function we generated pP{UAS-lark T:Ivir }(UAS-lark-3HA, Fig. 1, panel a). To distinguish GAL4 driven expression of the UAS-lark transgene from endogenous LARK expression, sequences encoding a triple hemagglutinin (HA) epitope were incorporated into the construct immediately prior to the LARK stop codon. Seven independent UAS-lark3HA transgenic lines were generated and here we report the preliminary characterization of three of these lines. We employed one line carrying a second chromosome insertion (w; P{w mC UAS-lark }94A) and two lines with independent third chromosome insertions (w; P{w mC UAS-lark }9A and (w; P{w mC UAS-lark }23A). All three lines are homozygous viable and fertile. To examine the effect of LARK overexpression, we first used the strong ubiquitous Act5C-GAL4 driver to drive expression of UAS-lark-3HA transgenes. Flies carrying an Act5C-GAL4 third chromosome insert (balanced over TM6b, Tb) were crossed to flies homozygous for a UAS-lark-3HA transgene. Adults carrying both the Act5C-GAL4 driver and any of the three UAS-lark-3HA transgenes were never observed; i.e., the only adult progeny that resulted from these crosses carried the TM6b, Tb chromosome (Table 1). In the case of two of the UAS-lark-3HA lines, 94A and 9A, some of the LARK overexpressing flies were able to develop to the early pupal stage (as indicated by a few Tb pupae in the vials; data not shown). However, these individuals ceased to develop soon after pupal formation and no brown or black Tb pupae were observed in these crosses. In the case of crosses with the 23A UAS-lark-3HA line, Tb pupae were never observed, suggesting that ubiquitous overexpression of LARK from this insertion is lethal prior to pupation. To determine if the observed lethality was caused by increased LARK protein expression in the nervous system, we used an elav-GAL4 driver (c155) to express UAS-lark-3HA transgenes in all differentiated neurons. Such a pan-neural overexpression of LARK caused lethality, the severity of which was dependent on the particular UAS-lark-3HA line employed in crosses. For example, crosses of males bearing the X-linked elav-GAL4 driver to females homozygous for the 23A UAS-lark-3HA transgene failed to produce any female offspring (Table 1). In similar crosses with elav-GAL4 and either the 94A or 9A UAS-lark-3HA transgene, GAL4/UAS flies were observed to eclose at a reduced frequency compared to UAS sibling controls (Table 1). Of the GAL4/UAS flies that do eclose, all display a cuticle-tanning defect characterized by a significant delay in cuticle hardening and pigmentation (not seen in control flies). In addition, the GAL4/UAS flies do not show normal wing expansion (Fig. 1, panel b). With the 94A UAS-lark-3HA transgene, none of the GAL4/UAS flies ever expanded their wings. Approximately 2% of the GAL4/UAS flies displayed nor

The Drosophila homolog of Casein Kinase I δ/ε, DOUBLETIME (DBT), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of DBT in circadian period determination. However, how DBT expression is regulated remains largely unexplored. In this study, we show that translation of dbt transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative dbt transcripts in clock cells, in particular the dbt-RC transcript. Translation of dbt-RC exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, dbt-RE, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different dbt alleles. Increased LARK delays nuclear degradation of the PERIOD (PER) clock protein at the beginning of subjective day, consistent with the known role of DBT in PER dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of DBT. Our study is the first to investigate translational control of the DBT kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation., Author Summary The CKI family of serine/threonine kinase regulates diverse cellular processes, through binding to and phosphorylation of a variety of protein substrates. In mammals, mutations in two members of the family, CKIε and CKIδ were found to affect circadian period length, causing phenotypes such as altered circadian period in rodents and the Familial Advanced Sleep Phase Syndrome (FASPS) in human. The Drosophila CKI δ/ε homolog DOUBLETIME (DBT) is known to have important roles in development and circadian clock function. Despite extensive studies of DBT function, little is known about how its expression is regulated. In a previous genome-wide study, we identified dbt mRNAs as potential targets of the LARK RBP. Here we describe a detailed study of the regulation of DBT expression by LARK. We found that LARK binds to and regulates translation of dbt mRNA, promoting expression of a smaller isoform; we suggest this regulatory mechanism contributes to circadian period determination. In addition, we have identified a dbt mRNA that exhibits light-induced changes in translational status, in a LARK-dependent manner. Our study is the first to analyze the translational regulation of DBT, setting the stage for similar studies in other contexts and model systems.

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 14–29, 2000

Photic entrainment of insect circadian rhythms can occur through either extraretinal (brain) or retinal photoreceptors, which mediate sensitivity to blue light or longer wavelengths, respectively. Although visual transduction processes are well understood in the insect retina, almost nothing is known about the extraretinal blue light photoreceptor of insects. We now have identified and characterized a candidate blue light photoreceptor gene inDrosophila(DCry) that is homologous to the cryptochrome (Cry) genes of mammals and plants. TheDCrygene is located in region 91F of the third chromosome, an interval that does not contain other genes required for circadian rhythmicity. The protein encoded byDCryis ∼50% identical to the CRY1 and CRY2 proteins recently discovered in mammalian species. As expected for an extraretinal photoreceptor mediating circadian entrainment,DCrymRNA is expressed within the adult brain and can be detected within body tissues. Indeed, tissuein situhybridization demonstrates prominent expression in cells of the lateral brain, which are close to or coincident with theDrosophilaclock neurons. Interestingly,DCrymRNA abundance oscillates in a circadian manner inDrosophilahead RNA extracts, and the temporal phasing of the rhythm is similar to that documented for the mouseCry1mRNA, which is expressed in clock tissues. Finally, we show that changes inDCrygene dosage are associated predictably with alterations of the blue light resetting response for the circadian rhythm of adult locomotor activity.

Previous studies have shown that zygotic expression of the Drosophila lark gene, which encodes an RNA-binding protein, is essential for embryogenesis. We now show that lark mRNA is abundant in preblastoderm (0–2 h) embryos, prior to zygotic transcription, indicative of maternal inheritance. Lark can also be detected within the nurse cells of developing egg chambers, suggesting a function for the protein during oogenesis. To test the hypothesis that the maternal inheritance of lark is required for oogenesis or early development, we employed the “FLP-DFS” technique to eliminate the lark maternal component within the germ line. Unfertilized and fertilized eggs lacking the lark maternal component exhibit a “fragile” phenotype, indicating that the protein functions during oogenesis. Furthermore, differentiation of the fertilized eggs is abnormal—most embryos arrest prior to blastoderm formation and exhibit morphological phenotypes that might reflect underlying defects in syncytial nuclear cycling or cellularization. Mutational analysis of a retroviral-type zinc finger within the lark protein indicates that it is required for the maternal function of the protein: females are completely sterile when their only source of lark protein contains a zinc-finger mutation. The aggregate of our studies shows that the germ-line expression of lark is essential for development, and suggests that the retroviral-type zinc finger mediates important RNA-binding functions during oogenesis and/or early development. Dev. Genet. 25:387–396, 1999. © 1999 Wiley-Liss, Inc.

To investigate the regulation of Drosophila melanogaster behavior by biogenic amines, we have exploited the broad requirement of the vesicular monoamine transporter (VMAT) for the vesicular storage and exocytotic release of all monoamine neurotransmitters. We used the Drosophila VMAT (dVMAT) null mutant to globally ablate exocytotic amine release and then restored DVMAT activity in either individual or multiple aminergic systems, using transgenic rescue techniques. We find that larval survival, larval locomotion, and female fertility rely predominantly on octopaminergic circuits with little apparent input from the vesicular release of serotonin or dopamine. In contrast, male courtship and fertility can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but a complementary role was observed for serotonin. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Further experiments using this model will help determine how multiple aminergic systems may contribute to the regulation of other behaviors. Our data also highlight potential differences between behaviors regulated by standard exocytotic release and those regulated by other mechanisms.

Mutants of the Drosophila miniature-dusky (m-dy) gene complex display morphogenetic phenotypes (miniature or dusky) caused by a change in the size and/or shape of the epidermal cells comprising the adult wing. In addition to a dusky phenotype, certain Andante-type mutants also exhibit lengthened circadian periods for two different behavioral rhythms. If the latter phenotype results from a direct effect on the circadian pacemaker, the Andante function should be required within the brain. In order to define the tissues that require the morphogenetic and behavioral functions, we have carried out a genetic mosaic analysis. This study demonstrates that normal wing morphogenesis is entirely dependent on the genotype of wing cells. Furthermore, temperature-shift experiments with a temperature-sensitive dy mutant indicate that the morphogenetic function is required during adult development, and after the cessation of wing epidermal cell proliferation. At this time in development, a columnar epithelium in the developing wing becomes flattened into the mature wing blade, and we postulate that the cell-size defect of m-dy mutants results from an alteration of this mor-phogenetic process. In contrast to the wing mor-phogenesis phenotype, the characterization of locomotor activity in mosaic adults revealed a strong correlation between the head genotype and the Andante circadian-period phenotype. This result indicates that neural tissues mediate the rhythm function. Thus, the behavioral and morphogenetic functions require gene expression in distinct tissues. Furthermore, the behavioral results are consistent with a requirement for Andante function within circadian pacemaker neurons. © 1995 Wiley-Liss, Inc.

Background Drosophila neurons have dendrites that contain minus-end-out microtubules. This microtubule arrangement is different from that of cultured mammalian neurons, which have mixed polarity microtubules in dendrites. Results To determine whether Drosophila and mammalian dendrites have a common microtubule organization during development, we analyzed microtubule polarity in Drosophila dendritic arborization neuron dendrites at different stages of outgrowth from the cell body in vivo. As dendrites initially extended, they contained mixed polarity microtubules, like mammalian neurons developing in culture. Over a period of several days this mixed microtubule array gradually matured to a minus-end-out array. To determine whether features characteristic of dendrites were localized before uniform polarity was attained, we analyzed dendritic markers as dendrites developed. In all cases the markers took on their characteristic distribution while dendrites had mixed polarity. An axonal marker was also quite well excluded from dendrites throughout development, although this was perhaps more efficient in mature neurons. To confirm that dendrite character could be acquired in Drosophila while microtubules were mixed, we genetically disrupted uniform dendritic microtubule organization. Dendritic markers also localized correctly in this case. Conclusions We conclude that developing Drosophila dendrites initially have mixed microtubule polarity. Over time they mature to uniform microtubule polarity. Dendrite identity is established before the mature microtubule arrangement is attained, during the period of mixed microtubule polarity.

The LARK RNA-binding protein (RBP) has well documented roles in the circadian systems of Drosophila and mammals. Recent studies have demonstrated that the Drosophila LARK RBP is associated with many mRNA targets, in vivo, including those that regulate either neurophysiology or development of the nervous system. In the present study, we have employed conditional expression techniques to distinguish developmental and physiological functions of LARK for a defined class of neurons: the Pigment-Dispersing Factor (PDF)-containing LNv clock neurons. We found that increased LARK expression during development dramatically alters the small LNv class of neurons with no obvious effects on the large LNv cells. Conversely, conditional expression of LARK at the adult stage results in altered clock protein rhythms and circadian locomotor activity, even though neural morphology is normal in such animals. Electrophysiological analyses at the larval neuromuscular junction indicate a role for LARK in regulating neuronal excitability. Altogether, our results demonstrate that LARK activity is critical for neuronal development and physiology.

Mounting evidence demonstrates that glial cells might have important roles in regulating the physiology and behavior of adult animals. We summarize some of this evidence here, with an emphasis on the roles of glia of the differentiated nervous system in controlling neuronal excitability, behavior and plasticity. In the review we highlight studies in Drosophila and discuss results from the analysis of mammalian astrocytes that demonstrate roles for glia in the adult nervous system.

Fragile X syndrome (FXS) is the most common form of hereditary mental retardation. FXS patients have a deficit for the fragile X mental retardation protein (FMRP) that results in abnormal neuronal dendritic spine morphology and behavioral phenotypes, including sleep abnormalities. In aDrosophilamodel of FXS, flies lacking the dfmr1 protein (dFMRP) have abnormal circadian rhythms apparently as a result of altered clock output. In this study, we present biochemical and genetic evidence that dFMRP interacts with a known clock output component, the LARK RNA-binding protein. Our studies demonstrate physical interactions between dFMRP and LARK, that the two proteins are present in a complexin vivo, and that LARK promotes the stability of dFMRP. Furthermore, we show genetic interactions between the corresponding genes indicating that dFMRP and LARK function together to regulate eye development and circadian behavior.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP)plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e.,gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

The use of RNA interference (RNAi) techniques, combined with a spatially controlled expression of doublestranded RNA (dsRNA), provides a convenient method for producing cell-type-specific perturbations of gene function (Fortier and Belote, 2000; Martinek et al., 2000). While studying the function of the Drosophila miniature-dusky (m-dy) gene complex (DiBartolomeis et al., 2002), we produced transgenic flies expressing a dsRNA that would permit silencing of the dy gene (P{UAS-dy-dsRNA}; Fig. 1a). The m and dy genes share sequence similarity and both encode fly Cuticulin proteins (DiBartolomeis et al., 2002) that are required at the pupal stage of development (Newby and Jackson, 1995) for normal wing differentiation. In the absence of m-dy products, the wing is reduced in size, due to an autonomous effect on the size and/or shape of wing epidermal cells (Newby et al., 1991). Although a reduction in wing size is the only obvious morphological phenotype in m-dy flies, certain mutants exhibit reduced viability and/or fertility (Newby et al., 1991). To examine the phenotypic effects of the UAS-dy-dsRNA transgene, we ectopically expressed it within wing tissues, taking advantage of four different existing Drosophila GAL4 driver strains: P{GawB}30A, P{GawB}34B, MS1096, and A9. The first two strains are described in Flybase, the last two were obtained from M. O’Connor (Univ. of Minnesota). All four strains provide expression in wing and other imaginal discs. Previously characterized dy mutants have small wings of normal proportion ( 2/3 of the wild-type size), whereas m mutants show a more extreme reduction in wing size and the wings are malproportioned compared to the wild type. The relatively less severe phenotype of dy mutants approximates the null phenotype for this gene, as dy/dy and dy/Df flies exhibit similar wing defects (Newby et al., 1991). Expression of UAS-dy-dsRNA with three out of four of the GAL4 drivers (P{GawB}34B, MS1096, and A9) was associated with a wing phenotype, the severity of which was dependent on the particular driver (Table 1). The fourth driver, P{GawB}30A, caused complete lethality at the late pupal stage (Table 1) and pharate adults were not dissected for examination. We were surprised that ectopic expression of UAS-dydsRNA, using either MS1096 or A9 as a driver, produced a wing phenotype more severe than that observed in existing single dy or m mutants (Table 1; Fig. 1; data not shown). Both of these GAL4 transgenes are known to drive expression in the developing wings, although MS1096 has a slightly broader pattern of expression (M. O’Connor, pers. commun.) that might explain the more severe wing phenotype compared to A9. A wing phenotype was observed with these two drivers using three independent UAS-dy-dsRNA insertions (19-4, 28-1, and 38-1), so the phenotype is not a consequence of transgene insertion. In addition, wing abnormalities were not observed in control siblings carrying any one of the GAL4 drivers or the UAS-dy-dsRNA transgene alone (not shown). Interestingly, the size of the wing in MS1096 or A9; UAS-dy-dsRNA flies is similar to that of a m-dy double mutant called Df(1)m (Fig. 1f), which is known to carry a chromosomal deletion removing only m and dy (Roberts and Jackson, 1997). Given the known sequence similarity between m and dy, such a result suggests that the UAS-dy-dsRNA transgene might target both m and dy RNAs for destruction (see below). Other abnormalities, in addition to wing phenotypes, were observed in our GAL4/UAS-dy-dsRNA crosses. Lethality was observed in populations carrying UAS-dydsRNA with any of the four GAL4 transgenes (Table 1) and the degree of lethality was dependent on the GAL4 driver (see Table 1 notes). In many crosses, much of this lethality appeared to occur at the late pupal stage, although we cannot exclude death at earlier developmental stages. As indicated, the combination of P{GawB}30A and UAS-dy-dsRNA was completely lethal, whereas the other drivers were semilethal in combination with the UAS transgene. Some of this observed lethality is attributable to elimination of m-dy function(s), as Df(1)m also exhibits poor viability (Roberts and Jackson, 1997). Although the general morphology of surviving GAL4; UAS-dy-dsRNA flies is relatively normal, and females are fertile (not shown), these flies nonetheless exhibit additional phenotypes that might reflect targeting of genes other than m and dy. A comparison of m and GAL4; UAS-dy-dsRNA flies, for example, indicates that the mor

Neural circadian pacemakers can be reset by light, and the resetting mechanism may involve cyclic nucleotide second messengers. We have examined pacemaker resetting and free-running activity rhythms in Drosophila dunce (dnc) and DC0 mutants, which identify a cAMP-specific phosphodiesterase and the catalytic subunit of cAMP-dependent protein kinase, respectively. dnc mutants exhibit augmented light-induced phase delays and shortened circadian periods, which indicate altered pacemaker function. Interestingly, however, light-induced phase advances are normal in dnc , suggesting a selective effect on one component of the pacemaker resetting response. Furthermore, we demonstrate the presence of circadian rhythms in cAMP content in head tissues and show that dnc mutations increase the amplitude of daily cAMP peaks. These results show that cAMP levels are not chronically elevated in the dnc mutant. A role for cAMP signaling in circadian processes is also suggested by an analysis of DC0 mutants, which have severe kinase deficits and display arrhythmic locomotor activity.

Analysis of the Drosophila lark gene indicates that it encodes an RNA-binding protein that functions as a regulatory element of the circadian clock output pathway controlling adult eclosion. We now demonstrate that the lark RNA-binding protein oscillates in abundance during the circadian cycle; importantly, the phasing of the lark rhythm is consistent with gene-dosage studies, which indicate that the protein behaves as a repressor molecule. The lark protein rhythm persists in constant conditions (continuous darkness and constant temperature) and is eliminated by period gene null mutations, confirming that it is under clock control and suggesting that it acts as an output mechanism that mediates the temporal regulation of adult eclosion. We also show that lark protein oscillates in abundance within a defined group of neuropeptide (CCAP) -containing neurons of the ventral nervous system (VNS), which in other insects are thought to comprise cellular elements of the clock output pathway regulating eclosion.

Molecular genetic analysis has yielded a detailed mechanistic understanding of invertebrate and vertebrate circadian oscillators, but we still know little about how such molecular oscillators are connected to rhythmic physiological processes. Two recent papers in Cell and Neuron now address this scientific issue through the use of gene chip technology to identify clock-regulated genes in an animal species.