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. Author manuscript; available in PMC: 2008 Aug 2.
Published in final edited form as: Neuron. 2007 Aug 2;55(3):435–447. doi: 10.1016/j.neuron.2007.06.038

Drosophila Ebony Activity is Required in Glia for the Circadian Regulation of Locomotor Activity

Joowon Suh 1, F Rob Jackson 1,*
PMCID: PMC2034310  NIHMSID: NIHMS28216  PMID: 17678856

Summary

Previous studies suggest that glia may be required for normal circadian behavior, but glial factors required for rhythmicity have not been identified in any system. We show here that a circadian rhythm in Drosophila Ebony (N-β-alanyl-biogenic amine synthetase) abundance can be visualized in adult glia and that glial expression of Ebony rescues the altered circadian behavior of ebony mutants. We demonstrate that molecular oscillator function and clock neuron output are normal in ebony mutants, verifying a role for Ebony downstream of the clock. Surprisingly, the ebony oscillation persists in flies lacking PDF neuropeptide, indicating it is regulated by an autonomous glial oscillator or another neuronal factor. The proximity of Ebony-containing glia to aminergic neurons and genetic interaction results suggest a function in dopaminergic signaling. We thus suggest a model for ebony function wherein Ebony glia participate in the clock control of dopaminergic function and the orchestration of circadian activity rhythms.

Keywords: Drosophila, ebony, circadian, glia, locomotor activity

Introduction

Because life has evolved in the presence of daily geophysical cycles, most organisms have acquired the ability to adapt the timing of physiological processes to external cycles using an intrinsic time-keeping device called a circadian clock. Both forward genetic and molecular screens in Drosophila and other organisms have identified genes encoding integral components of the circadian oscillator (Dunlap and Loros, 2005;Hardin, 2005;Reppert and Weaver, 2002;Young and Kay, 2001). In the fruit fly, the core oscillator mechanism governing behavioral rhythmicity is comprised of two interconnected molecular loops that result in circadian changes in PER and TIM clock protein abundance and the cyclical feedback repression of clock gene transcription (Cyran et al., 2003). In addition to the core transcriptional loops, posttranscriptional factors have been identified that are required for the modulation of clock protein stability, activity or nuclear entry (Akten et al., 2003;Grima et al., 2002;Ko et al., 2002;Lin et al., 2002a;Martinek et al., 2001;Price et al., 1998;Sathyanarayanan et al., 2004). Although there has been significant progress in delineating clock mechanisms, less is known about the molecular and cellular output pathways that control organismal physiology and behavior (Jackson et al., 2005).

Two behaviors are widely employed to assay circadian rhythmicity in Drosophila: eclosion (the emergence of the adult from the pupal case) and adult locomotor activity (Jackson et al., 2005). Mutation of a clock element affects rhythms in both eclosion and activity, as the same or a molecularly similar clock regulates both behaviors. In contrast, several mutations have been reported to affect only one of these two behaviors (Majercak et al., 1997;Myers E.M.. et al., 2003;Newby and Jackson, 1991;Newby and Jackson, 1993); i.e., to have rhythm-specific effects on circadian behavior. These findings indicate that genetically separable output pathways mediate the circadian control of the two different processes. Mutations in ebony, for example, selectively perturb the locomotor activity rhythm, causing arrhythmicity, but have no effect on the adult eclosion rhythm (Newby and Jackson, 1991). Such a rhythm-specific effect suggests that Ebony acts downstream of the clock mechanism to orchestrate the circadian control of locomotor activity.

Multiple microarray-based studies have identified Drosophila transcripts exhibiting rhythmic daily changes in abundance (Ceriani et al., 2003;Claridge-Chang et al., 2001;Lin et al., 2002b;McDonald and Rosbash, 2001;Ueda et al., 2002). These studies verified cycling for all of the known clock genes and, importantly, identified hundreds of other genes that show robust circadian changes in abundance within head tissues. Of note, ebony RNA was shown to exhibit robust circadian cycling in two independent studies (Claridge-Chang et al., 2001;Ueda et al., 2002). These results are consistent with the behavioral studies discussed above which suggest that Ebony protein functions in a clock output pathway.

The most obvious phenotype of ebony mutants is defective sclerotization and cuticle pigmentation although they also exhibit altered rhythms, vision (Hotta and Benzer, 1969) and courtship behavior (Kyriacou et al., 1978). Consistent with these phenotypes, Ebony protein can be detected in the hypodermis (which produces the cuticle), the visual system and other brain regions (Richardt et al., 2002). In the fly visual system, Ebony is localized exclusively to glia including neuropile and epithelial glia (Richardt et al., 2002) and it is thought that Ebony functions in a novel metabolic pathway (Borycz et al., 2002;Richardt et al., 2003) that may terminate the action of histamine, the photoreceptor cell neurotransmitter. Based on studies of the pigmentation phenotype of ebony mutants, it was shown that Ebony protein has β-alanyl-dopamine (DA) synthase (BAS) enzymatic activity (Hovemann et al., 1998), and consequently mutants are lacking N-β-alanyl-dopamine (NBAD) in peripheral and neural tissues (Perez et al., 2004) and have elevated levels of DA and β-alanine in both types of tissues (Hodgetts and Konopka, 1973;Ramadan et al., 1993). Recently, it was reported that the Ebony enzyme has a broader substrate specificity than anticipated from previous studies: purified Ebony can conjugate β-alanine to several different biogenic amines, including DA, serotonin (5-HT), histamine, tyramine, and octopamine (Richardt et al., 2003); hence, it is now considered a β-alanyl-biogenic amine synthase.

It is known that DA, 5-HT and other biogenic amines have neuromodulatory activity in Drosophila and other insects (Kume et al., 2005). Together with the behavioral defects of ebony mutants, these findings suggest a model for the circadian function of Ebony, in which clock output regulates Ebony (BAS) activity, and consequent changes in biogenic amine-related signaling within a specific group of neural cells of the fly brain. Here we show that Ebony-containing glia are localized close to clock cell projections, that there is a PER/TIM-dependent control of rhythmic ebony expression within a discrete population of glial cells, and that Ebony enzymatic activity is required within glia for the clock control of locomotor activity. Cellular and molecular analyses indicate that Ebony acts downstream of the clock to control locomotor activity and that Ebony-containing glia are positioned near DA and 5-HT neurons of the larval and adult brains, consistent with the idea that these glia are required for the modulation of aminergic functions. A genetic interaction between ebony1 and an allele of the fly dopamine transporter gene (dDAT) suggests that dopaminergic transmission has a role in rhythmicity in vivo. That glia may function in rhythmicity is consistent with a previously published genetic mosaic study (Ewer et al., 1992) that implied a role for PER/TIM-containing glia in the regulation of activity rhythms.

Results

Diurnal changes in ebony RNA and protein

In order to confirm and extend previously published studies of ebony RNA abundance, we carried out a quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) study using oligonucleotide primers for ebony that flank exons 2 and 3. Consistent with previous results, we found that ebony RNA exhibits diurnal (in LD) and circadian (in DD) oscillations in abundance in adult head tissues, with peak abundance occurring at the beginning of the photoperiod or subjective day (Fig.1A). In both conditions, there is an approximate 4-fold difference between the times of peak and trough RNA abundance. Although the abundance of ebony RNA seems to decrease slightly during DD, there is nonetheless still a clear rhythm in abundance with a peak at the beginning of subjective day. Ebony RNA cycling is eliminated in tim01 and per01 mutants, in both LD and DD, indicating that it is governed by a PER/TIM-based circadian oscillator (Fig. 1A; data not shown for per01).

Figure 1. Ebony gene expression is under clock control.

Figure 1

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(A) Ebony mRNA cycles in abundance in a light:dark (LD) cycle and in constant darkness (DD). Total RNA samples were prepared from heads of control yw and yw;tim01 flies collected every 4 hours in LD and DD. Real-time quantitative RT-PCR was performed on RNA samples using primers specific for ebony (see Methods). Relative abundance indicates the amount of ebony transcript normalized to rp49 mRNA abundance which is known to remain constant throughout the cycle. Ebony RNA cycles in yw but not in tim01(A) flies. (B) Ebony protein abundance changes in a diurnal manner in wild-type (yw) brains. Hand-dissected brains were collected every 4 hours in an LD cycle. Protein blots were immunostained with rabbit anti-MAPK antibodies. Each gel lane contained approximately 5μg protein. The graph shows the ratio of Ebony to MAPK protein as a function of time of day. The horizontal bars in panels A and B indicate the light:dark schedules.

To determine if Ebony abundance is under clock control, we examined abundance at different times of day in wild-type head extracts. Changes in protein amount were evident in some experiments but we did not reproducibly see obvious rhythms in protein abundance, in LD or DD, using head extracts and protein blotting techniques (data not shown). The lack of cycling might reflect a high abundance of non-cycling Ebony protein in the head cuticle. To examine Ebony abundance in neural tissues, we prepared protein extracts from hand-dissected adult brains in LD conditions. In these experiments, there were obvious changes in abundance, with high Ebony levels observed at the beginning of day and low levels seen at night (Fig. 1B), consistent with the phase observed for the ebony RNA rhythm. Given the laborious nature of these experiments, which require many hand-dissected brains for the preparation of protein extracts from each time of day, we decided to examine the circadian control of Ebony protein abundance by immunostaining of individual adult brains (see below).

Ebony Protein is localized exclusively to glial cells of the larval and adult nervous systems

It has previously been reported that Ebony protein can be detected in the larval nervous system and the adult visual system; in the visual system, Ebony was found to be exclusively localized to neuropile and epithelial glial cells (Richardt et al., 2002). We wished to determine the spatial localization of Ebony protein within the adult nervous system, including the visual system and protocerebrum, in order to examine the abundance of the protein at different times of day. To determine the locations of Ebony-containing cells throughout the brain, immunostaining was performed using an anti-Ebony antibody and whole mounts of the adult or third-instar larval nervous systems. These studies indicated that the protein was localized to many regions of the brain and ventral nervous system at both developmental stages (Fig. 2). As previously reported, Ebony can be detected in larvae within defined cell populations of the brain lobes and ventral nervous system (Fig. 2C) (Richardt et al., 2002). In adults, Ebony was detected in the optic lobes, protocerebrum and thoracic ganglia (Fig. 2A, B) of wild-type animals, whereas Ebony-positive cells were not seen in tissues obtained from an e1 mutant (Fig. 2E, F), a known amorph. We note that development of the nervous system appeared to be grossly normal in the e1 mutant, and the spatial distribution of Tyrosine Hydroxylase, PDF and TIM proteins were similar in the e1 mutant and the wild type (Supplemental Fig. 2 and data not shown). Double labeling with anti-Ebony and anti-Repo (Reversed Polarity, a glia-specific antigen) antibodies demonstrated that all Ebony-containing cells of the larval and adult nervous systems are glia (Fig. 2C, D). A similar analysis, using anti-Ebony and anti-Elav (a neuronal marker), did not detect any co-labeled cells (data not shown), consistent with the idea that Ebony is exclusively localized to glia. Previous reports demonstrated Ebony localization in glia of the optic lobes (Richardt et al., 2002), and consistent with these published results, we detected approximately 120 Ebony-containing cells in each adult medulla. Within the adult protocerebrum, we detected approximately 100 Ebony-positive glial cells, and these are located in several different areas of the dorsal and lateral protocerebrum, regions that are known to contain clock neurons (see later sections). The positions of Ebony glia suggest that most correspond to the neuropile class of glia, as reported by Richardt et al (2002), but certain Ebony-positive cells are in close proximity to neuronal cell bodies (see later section) and may represent cell body glia (Hartenstein et al., 1998).

Figure 2. Ebony protein is localized exclusively to glia of the larval and adult brains.

Figure 2

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(A-F) Ebony can be detected with a cytoplasmic distribution in cells of the wild-type (Canton-S) adult brain (A), the adult ventral nervous system (B), and the larval brain and ventral nervous systems (C), but is not specifically detected in the e1 mutant (E, F; note the lack of cell body staining in the mutant). Panel D shows co-localization of Ebony and Repo proteins in glial cells of the optic lobes. Repo can be detected in every Ebony-containing glial cell. Staining with the neuronal marker ELAV did not detect any Ebony-positive cells in brain tissues (data not shown).

Cyclical changes in Ebony protein abundance in different brain regions

To determine where in the adult brain Ebony might show circadian changes in abundance, we examined Ebony immunoreactivity in whole mounts of the adult brain at two times of day that correspond to the peak and trough of abundance as determined by protein blotting experiments (see Fig. 1C). As shown in Figure 3, anti-Ebony immunofluorescence signals were higher at the beginning of the day (or subjective day) (ZT2 or CT3.5) than during the late night or subjective night (ZT21 or CT21). Rhythmic changes in Ebony immunofluorescence were observed in all regions of the adult brain, although they were most apparent in the dorsal protocerebrum and the optic medulla (Fig. 3A, D; arrows indicate sites of prominent rhythmicity). Whereas robust circadian fluctuations in anti-Ebony staining were apparent in wild-type (WT) brains, this was not observed for brains for the tim01 mutant (Fig. 3C, D), consistent with a circadian regulation of Ebony protein abundance.

Figure 3. The Ebony protein oscillation can be detected by immunostaining in wild-type brains but is damped or eliminated in the tim01 mutant.

Figure 3

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(A) Ebony immunofluorescence at two times of the cycle in wild-type brains. Brains were dissected at ZT2 and ZT21 from flies maintained in LD or at CT3.5 and CT21 in DD conditions. Ebony fluorescence was observed to be more intense during the day (or subjective day) than at night (Arrows indicate sites of prominent rhythmicity) (B) Ebony fluorescence intensity is weaker in tim01 than in the wild type during the day, and rhythmicity is damped in the mutant. (C) Average normalized fluorescence (pixel) intensity for different brain regions (optic lobes and protocerebrum) in wild type and tim01. In all cases, Ebony fluorescence was normalized to Repo antibody staining intensity. * p<0.01, ** p<0.05 for differences in fluorescence intensity at two times of day in wild type (two-tailed student’s t-test). Differences were not significant for tim01.

Expression of Ebony exclusively in glia rescues the behavioral phenotype of ebony mutants

We propose that Ebony functions within glia to regulate behavioral rhythmicity. To test this hypothesis, we selectively expressed the product in glial cells of ebony mutants and wild-type animals using the Gal4/UAS binary expression system (Brand and Perrimon, 1993). To determine if increased ebony+ expression perturbed the activity rhythm of wild-type individuals, the glial-specific driver Repo-Gal4 was employed to over express the gene in all glia. As shown in Figure 4 and Table 1, there were no obvious differences between repo-Gal4; UAS-ebony flies and UAS-ebony control individuals with regard to the robustness of rhythmicity or the percentage of rhythmic flies. We conclude that Ebony product is not limiting for determination of rhythmicity.

Figure 4. Expression of ebony+ in glia is sufficient to rescue the rhythm phenotype of the e1 mutant. Overexpression of ebony+ in a wild-type background has no obvious effects.

Figure 4

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(A) Actograms for representative control flies, e1 mutants, rescued mutants, and individuals overexpressing Ebony in an e+ background. Data are from the DD portions of the records starting with the first day after lights-off. The horizontal bars beneath records indicate subjective day and night. The overexpression of Ebony in an e1 mutant background, using a glial cell driver (repo-Gal4), rescued circadian behavior. Overexpression of Ebony in a wild-type background had no effect on rhythmicity. All flies were in a w1118 genetic background. Repo > ebony+ = w1118; repo-Gal4, e1/UAS-ebony+, e1. (B) Body color phenotypes of various genotypes. i) w1118. ii) w1118; e1/e1. iii) w1118; actin-Gal4/+; UAS-ebony+, e1/e1. iv) w1118; repo-Gal4, e1/UAS-ebony+, e1.

Table 1.

Genotype Period* Std. err. % Rhythmicity RI RI std. err. n
w1118 24.1 0.1 96 0.57 0.02 80
yw 23.8 0.2 88 0.53 0.03 41
CS 23.8 0.06 92 0.42 0.08 22
CS e1/+ 23.3 0.11 90 0.49 0.04 27
CS e1/e1 23.9 0.07 35 0.27 0.03 26
e1/e1 23.4 0.3 28 0.17 0.02 28
yw e1/e1 23.5 0.24 20 0.12 0.01 24
ebony overexpression
w;repo-Gal4/UAS-e+ 24.5 0.1 92 0.50 0.04 59
w;+;repo-Gal4/+ 24 0.1 78 0.40 0.04 32
w;+;UAS-e+/+ 24.1 0.1 83 0.32 0.03 31
w;+;actin-Gal4/UAS-e+ 24.4 0.1 97 0.48 0.03 32
w;+;actin-Gal4/TM3,e1 24.1 0.08 94 0.50 0.04 17
w;+;UAS-e+/TM6b,e1 24.4 0.23 93 0.46 0.04 15
w;actin-Gal4/+;UAS-e+/+ 23.8 0.08 100 0.40 0.02 30
w;Cy/+;UAS-e+/+ 23.6 0.06 100 0.55 0.04 16
w;actin-Gal4/+;TM3,e1/+ 24.1 0.06 100 0.47 0.04 17
elav-Gal4/y;;UAS-e+/+ 24.2 0.09 100 0.41 0.04 25
elav-Gal4/y;+;+ 24.1 0.12 100 0.50 0.04 24
Rescue
w;+;repo-Gal4,e1/UAS-e+,e1 23.9 0.1 81 0.40 0.02 70
w;+;repo-Gal4,e1/TM6,e1 24 0.2 23 0.22 0.03 22
w;+;UAS-e+,e1/TM6,e1 24.2 0.2 27 0.23 0.02 59
w;+;UAS-e+,e1/e1 24.1 0.2 24 0.21 0.04 17
w1118;+;repo-Gal4.eAFA/UAS-e+.eAFA 24.1 0.04 95 0.46 0.03 23
w1118;+;UAS-e+.eAFA/TM6,e1 24.1 0.08 39 0.26 0.03 28
w1118;+;repo-Gal4.eAFA/TM6,e1 24.3 0.03 24 0.22 0.02 29
w1118;;UAS-eS5,eAFA/repo-Gal4,eAFA 25.4 0.48 48 0.39 0.03 28
w1118;;UAS-eS5,eAFA/TM6,e1 24.6 0.59 32 0.33 0.03 23
w1118;actin-Gal4/+;eAFA/UAS-e+.eAFA 24.2 0.03 92 0.51 0.02 31
w1118;actin-Gal4/+;eAFA/eAFA 24.3 0.05 33 0.25 0.03 27
w;tim-Gal4/+;UAS-e+,e1/e1 24.3 0.1 86 0.55 0.03 29
w;tim-Gal4/+;TM6,e1/e1 24.2 0.2 27 0.27 0.03 26
w;tim-Gal4/+;e1/e1 24.5 0.2 15 0.24 0.03 20
elav-Gal4/y;+;UAS-e+,e1/e1 23.7 0.01 59 0.42 0.03 32
elav-Gal4/y;+;TM6,e1/e1 23.8 0.1 41 0.26 0.03 60
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Importantly, the glial-specific expression of ebony+ in an ebony null background, using repo-Gal4, completely rescued rhythmicity, demonstrating that expression within glia is sufficient for normal behavior (Repo > ebony+, e1/e1 in Fig. 4A; Table 1). Furthermore, Ebony BAS activity is required for normal behavior as the glial expression of an enzymatically dead form of Ebony [a Ser to Ala change in residue 611 of the active site (Richardt et al., 2003)] did not rescue behavioral rhythms (w;actin-Gal4;UAS-eS5,In(3R)eAFA in Table 1). Interestingly, the behaviorally rescued flies still had a dark body color similar to e1 control flies (Fig. 4B), directly demonstrating anatomically separable requirements for ebony in pigmentation and behavioral rhythmicity. As expected, actin-Gal4; UAS-ebony+; eAFA/ eAFA flies, which express ebony+ ubiquitously, had normal pigmentation (Fig. 4B) and locomotor activity rhythms (Table 1).

Although Ebony protein normally cycles in abundance in wild-type individuals, we surmised that pan-glial over expression, using repo-Gal4, might eliminate the protein cycle. To determine if this was the case, we examined Ebony cycling in repo-Gal4; UAS-ebony+ and control flies using immunostaining procedures. Whereas Ebony protein abundance showed circadian oscillations in control flies, the protein seemed to be constitutively high in repo-Gal4; UAS-ebony individuals (Supplemental Figure 1). Thus, it would appear that a constitutively high level of Ebony in glia can rescue behavioral rhythmicity, even though the protein normally shows circadian changes in abundance in wild-type animals. We cannot exclude the possibility, however, that Ebony cycling persists in a limited glial cell population of repo-Gal4; UAS-ebony+ flies (see Discussion).

Ebony glia are in close proximity to clock cells and aminergic neurons

We wondered how the molecular clock regulated the rhythmic expression of ebony in glia. To determine the locations of Ebony glia, relative to clock cells, we performed co-immunostaining experiments using anti-Ebony and anti-PER (or TIM) antibodies. This analysis documented co-localization of Ebony and clock proteins in certain glia and a close association of other Ebony glia with PER/TIM-containing neurons or glia. Interestingly, there are Ebony glia adjacent to the small ventral lateral neurons (sLNvs), the dorsal lateral neurons (LNds), and the dorsal neuron 1 (DN1) and dorsal neuron 3 (DN3) groups (Fig. 5A-C). In the optic medulla, Ebony and PER (or TIM) co-localize within certain glia - in this region, approximately 60% of the Ebony glia contain PER and TIM; Fig. 5D-E). This suggests that ebony expression might be directly regulated by a PER/TIM-based oscillator within certain glial populations.

Figure 5. Many Ebony-containing cells stain positive for PER or are close to PER-containing neurons.

Figure 5

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(A-C) Double labeling for Ebony and PER shows that Ebony glia are adjacent to the sLNv, LNd, DN1, and DN3 neurons. (D, E) In the optic medulla, Ebony protein is localized to PER-containing glia (Red = PER, Green = Ebony); these two panels show different regions within the optic medulla. TIM protein was also co-localized with Ebony in many optic lobe glial cells (data not shown). (F-I) Localization of Ebony relative to PDF. (F) pdf-Gal4-driven expression of UAS-mCD8GFP in the adult brain. Ebony antibody was used to visualize the localization of Ebony protein relative to PDF-expressing cells and their projections. (G-H) Ebony-containing cells are in close proximity to PDF cell projections in the dorsal brain (G, arrow shows one Ebony cell) or in the medulla (H). (I) a high magnification picture of the region in panel H indicated by the dotted rectangle (Red = Ebony, Green= PDF).

Given that many Ebony glia contain PER and TIM, we wondered whether expression of Ebony in all clock cells (both glia and neurons) would restore normal behavioral rhythmicity in ebony mutants. As shown in Table 1, this is indeed the case; tim-Gal4-driven expression of ebony+ restored normal behavior in e1 homozygotes. We presume that the expression of ebony in a TIM-containing subset of the Ebony glia restores rhythmicity, although expression in neurons (using the elav-Gal4 driver) also weakly rescues rhythmicity (Table 1). Neuronal rescue might be due to production and release of β-alanyl-amine conjugates from BAS-containing neurons.

To determine whether Ebony glia might be positioned to regulate pacemaker neuronal function or to be regulated by the pacemaker cells, we looked more closely at the anatomical relationship between Ebony- and PDF (Pigment Dispersing Factor)-containing cells. PDF neuropeptide is released from the dorsal projections of the small ventral lateral neurons (sLNvs) according to a circadian rhythm, and it is essential for the circadian regulation of locomotor activity (Helfrich-Förster et al., 2000;Renn et al., 1999). As illustrated in Figure 5, co-immunostaining with anti-Ebony and anti-PDF antisera demonstrates that Ebony-containing glia are in close proximity to PDF cell bodies and their projections in the adult brain (Fig. 5F-I). It would appear that certain processes of the PDF neurons are closely apposed to Ebony-containing glia (Fig. 5G, H, I), suggesting the possibility of neuronal-glial communication.

Because Ebony’s BAS activity can conjugate β-alanine to biogenic amines, including DA and 5-HT, we also examined the distribution of Ebony glia relative to dopaminergic and serotonergic neurons. Studies in mammals and insects demonstrate roles for biogenic amines in the regulation of locomotor activity; a dramatic example is the hyperlocomotion phenotype of dopamine transporter (DAT) mutants (Giros et al., 1996;Kume et al., 2005). Thus, an interesting hypothesis is that the Ebony BAS may terminate biogenic amine transmitter action by sequestering amines in N-β-alanyl-biogenic amine (NBAA) conjugates (Richardt et al., 2003), and thus play a role in regulating locomotor activity. As shown in Figure 6, co-immunostaining with anti-Ebony and anti-tyrosine hydroxylase (TH) or anti 5-HT antibodies indicated that most or all DA (Fig. 6A) and 5-HT (Fig. 6B) neurons are close to Ebony glia within the larval and adult brains. Indeed, it would appear that these Ebony glia lie in close proximity to projections from DA or 5-HT neurons (insets in Fig. 6). This pattern of localization supports the hypothesis that Ebony enzymatic activity, which is restricted to glial cells, may modulate synaptic biogenic amine levels by generating NBAA conjugates. It is an intriguing hypothesis that NBAA release from glia functions as a feedback mechanism to regulate biogenic amine release from aminergic neurons.

Figure 6. Ebony glia are located near aminergic neurons.

Figure 6

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Ebony-containing cells are in close proximity to TH (Tyrosine Hydroxylase)-positive (A) or serotonergic (B) neurons of the larval and adult brains. In panel A, green = Ebony, Red = TH; in panel B, green = Ebony, Red = serotonin). Panel A shows a whole mount of the entire larval brain. Panel B shows the larval ventral nervous system (VNS) minus the brain lobes. The proximity of Ebony glia and 5-HT neurons is more easily visualized in the VNS. Insets show high magnification views of Ebony glia in proximity to TH-or 5-HT-positive neuronal processes of the adult brain.

Ebony acts downstream of the clock to regulate behavioral rhythmicity

If activity feeds back to regulate clock function, in some way, then the molecular oscillator might be perturbed by a lack of Ebony product. To ask whether ebony flies have normal oscillator function, we examined per or tim RNA abundance in wild-type and e1 mutant flies. Those studies showed that the temporal pattern of per and tim expression is similar in the two strains (Fig. 7A-B); however, tim transcript abundance seemed to be slightly reduced relative to the wild type in DD conditions. We note, however, that the TIM protein abundance rhythm was similar in the wild type and e1 as assayed by Western analysis (Fig. 7C-D). Furthermore, TIM levels cycled normally in both genotypes in several different TIM-containing cell types (Supplemental Fig. 5), indicating normal clock function.

Figure 7. Per/tim RNA and protein cycling are normal in the e1 mutant.

Figure 7

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(A-B) Quantitative RT-PCR shows that per and tim RNAs cycle in abundance in head tissues of wild-type (C-S) and e1 mutant flies. Values for per or tim RNA abundance were normalized to rp49 values (see Methods). Each curve represents 5 independent measures of RNA abundance from two independent fly head preparations (two measures from one, three measures from the other). Error bars indicate standard error. (C-D) TIM protein oscillates in abundance in C-S and e1 heads. TIM protein amounts are normalized to MAPK values for all timepoints. Each curve in panel D represents 3 independent measures of protein abundance from two independent fly head extracts. Error bars indicate standard error.

As an assay of clock output, we compared rhythms of PDF immunostaining in e1 and the wild type. In both types of flies, we observed robust rhythms of PDF immunostaining in the dorsal projections of the sLNv neurons, indicative of rhythms of PDF release (Supplementary Fig. 2). We conclude that molecular clock function and PDF synaptic output are normal in ebony mutants and that Ebony acts downstream of the clock to regulate circadian behavioral rhythms.

Ebony cycling is not regulated by PDF neuropeptide release

To test the hypothesis that PDF release controls the ebony transcriptional rhythm, we examined Ebony immunofluorescence at different times of day in wild-type flies and pdf01 mutants, which lack PDF peptide (Renn et al., 1999). Ebony protein cycling, as assessed by immunostaining for the protein at the peak and trough of the cycle, was found to be normal in the pdf01 mutant (Supplemental Fig.4A, B). Furthermore, quantitative RT-PCR measurements using RNA samples from wild-type and pdf01 mutant heads revealed significant cycling of Ebony in both genotypes (during LD and several days of DD) but no differences between the two genotypes (Supplemental Fig. 4C). These results indicate that ebony cycling is regulated by a mechanism not involving the neuronal release of PDF.

Genetic interactions indicate that Ebony modulates dopaminergic signaling

We examined rhythmicity of the e1 mutant in two other genetic backgrounds to determine if alterations of DA signaling might contribute to the arrythmicity of ebony mutants. It was first postulated years ago that the Yellow gene product functions in the melanization pathway (Wright, 1987). More recently, it has been suggested that y mutants might have elevated DA levels (Drapeau et al., 2003), as the disruption of DOPA-melanin synthesis in yellow mutants is predicted to lead to elevated DA levels, similar to that observed in ebony flies (Hodgetts and Konopka, 1973). As shown in Table 1, double mutants carrying e1 and a yellow mutation (y1) exhibited increased arrhythmicity relative to e1 single mutants, suggesting an additive effect of the two mutations (Table 1). Although this might be due to a further increase in DA levels in the double mutant, the precise biochemical role of Yellow is still quite controversial (Drapeau, 2003).

In contrast, it is quite likely that the Drosophila dDAT mutant (fumin, fmn), with decreased dopamine transporter function (Kume et al., 2005) has elevated synaptic DA levels. Thus, we also examined rhythmicity in double mutants homozygous for both DATfmn and ebony (DATfmn; e1), and those studies revealed an interesting genetic interaction between the two genes. Whereas e1 mutants seem to have normal levels of activity, DATfmn flies are known to exhibit significantly elevated activity levels (Fig. 8) (Kume et al., 2005). Not surprisingly, flies carrying both the e1and DATfmn mutations are arrhythmic, similar to e1, but unexpectedly the double mutant exhibited activity levels more similar to those of e1 flies than to DATfmn individuals (Fig. 8); i.e., e1 suppresses the hyperactivity phenotype of DATfmn. Given that fly DAT is localized exclusively to DA neurons (Porzgen et al., 2001), this result strongly suggests that Ebony enzymatic activity is relevant for dopaminergic neuronal function (see Discussion).

Figure 8. e1 suppresses the hyperactivity of the DATfmn mutant.

Figure 8

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Mean daily activity for e1, DAT fmn and the double mutant. Plots show population averages (w n=32, w; e1 n=32, w; DATfmn n=63, w; DATfmn; e1 n=70). Flies were entrained to LD 12:12 at 23°C for 5-6 days and then transferred to constant darkness (DD) at the same temperature for an additional 10-14 days. Average activity level per 30 minute bin was calculated for each genotype. Error bars indicate standard error.

In the course of quantitating activity in single and double mutants, we carefully measured activity levels in wild-type flies and ebony mutants at different times of day. Measurements of daily activity levels indicated that e1 flies are slightly less active than wild-type individuals in both LD and DD conditions (Supplemental Figure 3A). Interestingly, however, the reduction in activity selectively occurred during the day portion of the cycle (Supplemental Figure 3B), indicating the Ebony function is required for high levels of daytime activity. Such a result is consistent with the notion that Ebony (and perhaps NBAD) promotes locomotor activity during the daytime (see Discussion).

Discussion

Studies of Ebony indicate that glia have an essential role in the orchestration of circadian locomotor activity. It is of interest that previous studies in both mammals and insects have suggested that glia might be important for the control of rhythmic physiological events. Cultured cortical astroglia that express per-luciferase transgenes, for example, show circadian rhythms of bioluminescence that may depend on diffusible signals from neurons of the suprachiasmatic nuclei (Prolo et al., 2005); these studies suggest that such glia contain autonomous oscillators that can be reset by environmental stimuli or by interactions with clock neurons. In Drosophila, previous investigations have shown that the clock proteins PER and TIM can be detected in neurons and glia of the optic lobes and protocerebrum (Siwicki et al., 1988;Zerr et al., 1990), and PER protein abundance fluctuates according to a circadian rhythm in both cell types (Zerr et al., 1990). Consistent with roles for PER in neurons and glia, genetic mosaic analysis has suggested that per expression in either cell type might be sufficient for rhythmicity (albeit weak rhythmicity with glial expression)(Ewer et al., 1992). Our results indicate that Ebony is localized to glia of at least two types: those containing PER and TIM and a second class in which clock protein expression is not detectable. In the first class of cells, it seems likely that rhythmic ebony expression is controlled by an intracellular PER/TIM-based oscillator. In the latter class, ebony expression is most likely regulated by direct or indirect interactions with clock cells.

Neuron-glia interactions in circadian behavior?

It is now an accepted axiom that neuron-glia interactions are critical for neuronal development and function (Haydon, 2001). In addition to serving support roles in the mature nervous system, glial cells influence the developmental specification of neurons, migration, myelination, synapse number and synaptic transmission (Fields and Burnstock, 2006;Freeman, 2006;Fricker-Gates, 2006;Ullian et al., 2001). In Drosophila, studies have provided a detailed understanding of glial cell development and revealed the transcriptional mechanisms underlying the differentiation of this class of neural cells (Halter et al., 1995;Hartenstein et al., 1998;Jones, 2005). Previous studies have shown that glial cells function in the phagocytosis of neuronal debris during development (Cantera and Technau, 1996) and documented roles for glia in injury-induced neuronal degeneration(Custer et al., 2006). Of note, a new study in Drosophila has described a glial-specific receptor known as Draper that is part of a neuron-glia signaling mechanism mediating such injury-induced responses (MacDonald et al., 2006). Insect glia have also been implicated in neurotransmitter uptake/recycling, based on studies of GABA, acetylcholine or glutamate uptake (Campos-Ortega, 1974;Carlson and Saintmarie, 1990). Finally, studies of Drosophila repo mutants have demonstrated that glial support is important for neuronal survival in insects, similar to results obtained in mammals (Xiong and Montell, 1995).

Perhaps more relevant for behavior, recent studies have shown that certain types of mammalian glia (astrocytes) can regulate the excitability of neurons through the regulated release of “gliotransmitters” (glutamate, ATP, adenosine, cytokines, and growth factors), and it has become apparent that there are reciprocal neuron-glia signaling systems that regulate neuronal excitability (Fields and Burnstock, 2006;Haydon, 2001;Haydon and Carmignoto, 2006). Although certain aspects of this dynamic communication system are beginning to be understood, clearly much remains to be learned about the specific factors that regulate neuron-glia communication. Our studies have identified a glial-specific factor (Ebony) and a sub-population of glia within the fly nervous system that function with clock neurons to regulate circadian activity rhythms. It seems likely that intercellular communication between the neuronal and glial elements of the fly circadian system is important for the temporal coordination of activity.

PDF and ebony expression

Does PDF release contribute to the control of rhythmic ebony expression? Our co-localization studies, using antibodies for Ebony and PDF, show that greater than 80% of the Ebony-containing glia reside in close proximity to PDF neuronal somata or projections - this is the case for glia that reside in the lateral and dorsal protocerebrum and the optic medulla of the adult brain. Previous immuno-electron microscopy results show that certain PDF-containing varicosities are adjacent to glial cells of the optic medulla(Miskiewicz et al., 2004). We observe Ebony-containing glia near varicosities of the PDF neuronal projections, which probably contain dense core vesicles (DCVs), and adjacent to other regions of the projections. Surprisingly, however, it appears that PDF is not essential for the regulation of the ebony rhythm (Fig.8). We postulate, therefore, that communication between Ebony glia and clock-neurons, if it occurs, is mediated by factors other than the PDF neuropeptide.

BAS activity and biogenic amine action

A transgene expressing an enzymatically dead form of Ebony does not provide behavioral rescue for ebony mutants (Fig. 4); thus, BAS activity is essential for Ebony’s circadian function. As indicated previously, BAS can conjugate β-alanine to many different aminergic neurotransmitters, including DA, 5-HT, histamine, octopamine, and tyramine (Richardt et al., 2003, Perez et al., 2004). Interestingly, it has been demonstrated that Ebony glia are situated near histamine release sites of photoreceptor cells in the lamina, and it has been suggested that BAS activity conjugates histamine to β-alanine to terminate action of the transmitter (Richardt et al., 2002). We have shown that Ebony-containing cells are in close proximity to dopaminergic and serotonergic neurons of the larval and adult brains, suggesting a role for BAS in terminating DA and 5-HT action. A genetic interaction between e1 and DATfmn, a dopamine transporter mutant, strongly suggests that Ebony has a role in dopaminergic signaling. The rhythmic production of Ebony (BAS) may result in a circadian modulation of DA action and in turn rhythmic regulation of locomotor activity. Alternatively, circadian change in BAS activity may result in the rhythmic production and release of N-β-alanyl-dopamine (NBAD) with high levels of NBAD driving locomotor activity. Two lines of evidence support this idea: 1) NBAD is presumably highest during the day, the time of maximal activity. 2) the e1 mutation, a protein null, eliminates NBAD and this mutation suppresses the hyperactivity of DATfmn flies even though the double mutant is predicted to have high synaptic levels of DA.

An explicit model for Ebony function in rhythmicity

We show that Ebony glial expression is regulated in a circadian manner and that the protein is required within glia for normal behavioral rhythmicity. The localization of Ebony-containing glia near clock cells and aminergic neurons suggests an explicit model for Ebony regulation and function in the circadian system (Fig. 9). According to this model, ebony transcription is regulated either directly by a PER/TIM-dependent oscillator within glia (for those glia containing PER and TIM) or by the release of an unidentified output factor from clock neurons. Consequently, diurnal changes in ebony-encoded and glial-localized BAS activity lead to rhythms in the conjugation of biogenic amines to β-alanine and generation of NBAA product (NBAD in glia near DA neurons). Such a diurnal modulation of amine action may help shape the temporal organization of the daily bouts of locomotor activity. This model, of course, implies the existence of a glial amine transporter that mediates the uptake of synaptic amines into glia, although such a system has not yet been identified in Drosophila.

Figure 9. Hypothetical model for the control of the Ebony molecular rhythm and the regulation of adult locomotor activity.

Figure 9

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The model postulates that ebony expression may be regulated by autonomous glial oscillators and by output from clock neurons. In turn, rhythms in Ebony activity are postulated to control the excitability of dopaminergic or other neurons that regulate locomotor activity, perhaps by rhythmic production of NBAD. Presumably, dopamine (DA) and NBAD are actively synthesized during the daytime, as a consequence of rhythmic TH and Ebony activities, both of which are high during the day. According to the model, DA is released from dopaminergic terminals and taken up by ebony-containing glial cells. Ebony (BAS) activity then conjugates dopamine to β-alanine to produce N-β-alanine-dopamine (NBAD) which may be released from glia to regulate neuronal excitability and the activation of circuits controlling locomotor activity.

Furthermore, we postulate that the production of NBAD, which is high during the subjective day, serves as a bioactive compound to drive locomotor activity during the daytime. The observation that e1 mutants exhibit selective daytime deficits in locomotor activity is consistent with this idea (Supp. Figure 3). According to this model, NBAD is released from glia and acts on dopaminergic or other neurons to regulate excitability and/or transmitter release. To our knowledge, there is no evidence in the literature that β-alanyl-amine conjugates have bioactivity, but this is certainly a possibility given that many other glial compounds have such activity. Obviously, NBAD may not regulate locomotor activity, by itself, as it is presumably high throughout the day, given the profile of Ebony production, whereas locomotor activity is bimodal, with bouts occurring at dawn and dusk. An alternative model for the role of Ebony in the regulation of activity is that the unconjugated amine (i.e., DA) provides excitatory drive for behavior and that its modification by BAS activity decreases such excitation. However, such a model is not consistent with the presumption that NBAD levels are highest during the daytime, the time of maximal activity, nor with the observation that the DATfmn; e1 mutant, which probably has high DA levels, is not hyperactive.

Finally, it is known that Drosophila tyrosine hydroxylase (TH) RNA is transcribed according to a circadian rhythm (Ceriani et al., 2003), with high abundance occurring during the subjective day, and it is thus a good assumption that TH enzymatic activity and DA production is maximal during the day. Such a profile of DA production may explain why a high constitutive expression of Ebony (BAS) in glia can restore rhythmic behavior (Fig. 4, Supp. Fig. 1). Because TH production is presumably still rhythmic in ebony mutants, high NBAD levels would be expected to occur in such flies only during the daytime, thus permitting behavioral rhythmicity.

Experimental Procedures

Fly strains and culture conditions

Fly strains were reared on a modified cornmeal/agar medium (medium 1 of Newby et al. 1991) containing wheat germ. The e1 mutant was crossed to Canton-S (C-S) or w1118 flies for four generations to generate mutants and controls with similar genetic backgrounds. The W15 strain was from Paul H. Taghert (Washington University, St. Louis). UAS-ebony and repo-Gal4 strains were provided by Sean Carroll (Univ. of Wisconsin) and Stefan Thor (Linköping University, Sweden), respectively. UAS-ebonyS5 strain is a gift from Bernhard Hovemann (Ruhr-Universitat Bochum, Germany). Tim-Gal4 strain was from Jeff Hall (Brandies University, Waltham). PDF-Gal4 is given by Jae Park (University of Tennessee, Knoxville). Actin-Gal4, elav-Gal4, and UAS-mCD8GFP strains were obtained from the Bloomington Drosophila Stock Center. For rescue experiments, the Gal4 and UAS transgenes were crossed onto an e1 - containing chromosome using standard genetic recombination procedures. To measure behavioral rescue, homozygous e1 flies carrying both the Gal4 and UAS elements were compared to sibling control flies that carried a balancer chromosome and either the Gal4 or UAS transgene.

Behavioral Analysis

Locomotor activity was assayed using 5-7 day-old males and the Drosophila Activity Monitoring (DAM) system (Trikinetics, Waltham, MA). After loading flies into monitors, they were entrained to LD 12:12 at 23°C for 5-6 days and then transferred to constant darkness (DD) at the same temperature for an additional 10-14 days. Visualization of actograms and the analysis of rhythmicity were performed using a signal processing toolbox (Levine et al., 2002) within the MATLAB software package (MathWorks). Quantitation of activity levels was performed using the software package of K. Kume (RestCalc3.3)(Hendricks et al., 2003).

Quantitative RT-PCR

Strains were reared in LD 12:12 at 23°C prior to collections. Flies were collected every 3 or 4 hours during LD or after transfer to DD, and head samples were prepared using geological sieves. Total head RNA samples were prepared using the Trizol Reagent method (Invitrogen). For each set of extractions, about 50 ∼ 100 μl of heads were suspended in 1ml of TRI Reagent solution and then homogenized using a motorized pestle (Kontes). One μg samples of total RNA were treated with DNAse I (Amplification grade, Invitrogen) to remove genomic DNA and then subjected to reverse transcription using the Superscript II kit (Invitrogen). Real-time quantitative PCR was performed using the 2xSYBR® Green PCR Master Mix (Applied Biosystems) and gene specific primers with a Stratagene MX3000P thermocycler. Each PCR experiment was carried out with at least two independent sample collections. Experiments were repeated at least three times. Primers used in the real-time PCR were as follows: ebony forward, 5′-GACATTATTGTGGCTAGCTTCTATAACAAG-3′; ebony reverse, 5′-CGCTGTAGTCGGTTCTCAAAACT-3′; period forward, 5′-CAGCTGCAGCAACAGCCAGTCG-3′; period reverse, 5′GGCCTGCGTCGAGGGCTTGC-3′; timeless forward, 5′-GGTCAAGCGCAGCAAAAGCAG-3′; timeless reverse, 5′-TGAATTCCTTCAGCAGATTGGAGATG-3′; RP49 forward, 5′-GCCCAAGATCGTGAAGAAGC-3′, RP49 reverse, 5′-CGACGCACTCTGTTGTCG-3′. Amplification and dissociation curves for each reaction were analyzed using the Stratagene MX3000 software. The relative abundances of mRNAs were calculated by reference to the cycle threshold (Ct) values. The values for per, tim, and ebony RNAs were normalized against rp49 RNA, which does not cycle in abundance. Average results were obtained from more than three independent experiments.

Protein Blot and Immunohistochemical analyses

Standard immunoblotting procedures were employed to examine Timeless and Ebony. For the TIM Western analysis, fly heads were homogenized in Head Extraction Buffer (100mM KCL, 20mM HEPES, 5% glycerol, 10mM EDTA, 0.1% Triton X-100) containing 1mM DTT and a 1:100 dilution of protease inhibitor cocktail (Sigma), each added fresh. Thirty μg of protein sample was loaded per time point. For Ebony, protein was isolated using SDS Extraction Buffer (125mM Tris, pH6.8 and 4% SDS). Two μg of protein was loaded per lane to examine diurnal changes in Ebony abundance. Differences in Ebony or TIM abundance between time points were quantified using KODAK 1D Image Analysis Software (Eastman Kodak Company, Rochester, NY). The intensity of each target band was normalized to fly MAP Kinase (anti-MAPK from Sigma). The TIM, Ebony, and MAPK antibodies were used at dilutions of 1:2000, 1:4000 and 1:20000, respectively.

Drosophila adults or larva were entrained to LD and then dissected in LD or DD to obtain neural tissues. The nervous systems were fixed in 4% paraformaldehyde solution and then washed in PBS and PBS-T (0.05% triton X-100). For immunostaining procedures, we employed the primary antibodies at the following dilutions: guinea pig anti-PER (1:1000, I. Edery), guinea pig anti-TIM (1:1000, I. Edery), rabbit anti-PDF (1:2000, K.R. Rao), rabbit anti-Ebony (1:500, S. Carroll), mouse anti-REPO (1:1000, University of Iowa Hybridoma Center), rat anti-Serotonin (1:200, Accurate Chemical & Scientific Corporation) and mouse anti-TH antibodies (1:200,Pel Freeze). Donkey anti-mouse IgG (Cy3-conjugated, Molecular Probes), goat anti-rabbit (Alexa-488 conjugated, Molecular Probes), goat-anti-guinea pig (Cy3-conjugated, Molecular Probes), donkey anti-mouse (Cy3-conjugated, Molecular Probes), and donkey anti-rat (Alexa-488 conjugated, Molecular Probes) second antibodies were all used at dilutions of 1:1000. Confocal Images were acquired from brain whole mounts using a Leica TCS SP2 AOBS microscope within the Tufts/NEMC Imaging Core. Pixel intensities were quantified using Leica Confocal Simulator Software. Net values for fluorescence intensities were determined by subtracting a background value from each acquired image. Normalized pixel intensities were expressed as net fluorescence values per unit area. In experiments designed to detect circadian changes in Ebony abundance, the Ebony intensity values were normalized against REPO staining intensity. Average values were computed from at least four individual brains.

Supplementary Material

01. Supplemental Figure Captions.

Supplemental Figure 1

Ebony is constitutively high in rescued fly brains

The brains of wild-type (w1118) or rescued (Repo>ebony+) flies were hand-dissected and immunostained for Ebony and Repo at ZT3 or ZT15. Wild-type brains show normal rhythmicity whereas Ebony expression is constitutively high in rescued brains. Green=Ebony, Red=Repo.

Supplemental Figure 2

PDF immunostaining in the dorsal projections of the sLNv neurons in wild-type flies and e1 mutants

(A) Anti-PDF staining documents a normal rhythm of immunofluorescence for wild-type and e1 mutants, with highs in the early day (ZT1) and lows in the early night (ZT13).(B) Quantitation of PDF immunofluorescence in the wild-type and e1 at two different times of day (Error bars indicate standard deviation). PDF immunostaining was quantitated in 12 hemispheres for each genotype.

Supplemental Figure 3

Average activity levels for wild-type and e1 flies

Locomotor activity data were collected in 30-min bins for 5 days during LD or 3 days of DD. Day activity represents locomotor activity occurring from ZT(CT)0 to ZT(CT)12, whereas night activity represents the opposite phase. See Methods for details of data analysis. * p<0.01, ** p<0.05 (student’s two tailed t-test).

Supplemental Figure 4

Ebony cycling is not regulated by PDF release

(A-B) Ebony rhythmicity in wild-type and pdf01null mutants in LD and DD1. Brains were hand dissected in the early day (ZT4 or CT4) or night (ZT 16 or CT16). Error bars in B indicate standard deviation. (C) SYBR green real-time RT-PCR was performed to quantitate ebony RNA levels in wild-type (yw) and pdf01 (yw; w15) flies. RNA was extracted from heads collected during LD and DD (DD2 to DD5). Measurements were performed three times for each time point from two different head collections. Error bars indicate standard error. A one-way ANOVA indicates that there is significant Ebony cycling in both genotypes in LD and on DD day 2. Furthermore, there is statistically significant cycling on DD days 3 and 5 in the mutant and on DD day 4 in the wild type. Thus, cycling persists in both genotypes in constant conditions. A two-way ANOVA revealed no significant differences in cycling between the genotypes in either LD or DD conditions.

Supplemental Figure 5

TIM protein cycling is normal in different clock cell groups of the e1 mutant.

(A) Triple immunostaining of adult brains was performed for TIM, PDF and REPO and examined by confocal microscopy. TIM levels in neurons and glia were examined in wild-type (w1118) and e1 brains on the first day of DD at CT1 and CT4 after 5 days of LD entrainment. Fluorescence pixel intensity was measured for each signal, and the value for TIM (red) was normalized to that for REPO (green). PDF (blue) was used as a marker for certain pacemaker neurons. (B) Quantitation of TIM signals relative to REPO. As shown here, TIM levels are not significantly different between the wild type and e1 in either clock neurons or glia. Data were pooled from two independent experiments. Each histogram represents six to ten hemispheres. Error bars indicate standard error. For all cell groups, there were no significant differences between genotypes. For the LNv and DN1 groups, TIM values were significantly different between CT1 and CT4 for both genotypes. For the DN3 group and optic lobe glia, e1 showed significant time-of-day changes whereas the wild type did not. TIM has been shown to cycle in wild-type DN3 cells and optic lobe glia in other studies; we attribute the lack of significant cycling in our study to the small sample sizes or to the fact that we examined cycling during DD1 (TIM cycling in the brain dampens during the first 2 days of DD and then becomes robust again in later days of DD (Peng et al., 2003). * p < 0.01, ** p< 0.003 (Student’s two tailed t-test).

Acknowledgements

We thank Mary Roberts and other Jackson lab members for help with fly collections and statistical analyses. We also thank the Bloomington Stock Center for fly stocks, The University of Iowa Hybridoma Center for monoclonal antibodies, Lai Ding and the Center for Neuroscience Research (CNR) Imaging Core for help with confocal microscopy, Sean Carroll for anti-ebony antibody and the UAS-ebony stock, Stefan Thor for a repo-Gal4 stock, and Bernhard Hovemann for eAFA, UAS-ebonyS5 stocks. This work was supported by NSF IBN 0234724 and NIH R01 HL59873, both to F.R. Jackson, and by a center grant (NIH P30 NS047243; P.I., F. R. Jackson) that funds the Tufts CNR.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01. Supplemental Figure Captions.

Supplemental Figure 1

Ebony is constitutively high in rescued fly brains

The brains of wild-type (w1118) or rescued (Repo>ebony+) flies were hand-dissected and immunostained for Ebony and Repo at ZT3 or ZT15. Wild-type brains show normal rhythmicity whereas Ebony expression is constitutively high in rescued brains. Green=Ebony, Red=Repo.

Supplemental Figure 2

PDF immunostaining in the dorsal projections of the sLNv neurons in wild-type flies and e1 mutants

(A) Anti-PDF staining documents a normal rhythm of immunofluorescence for wild-type and e1 mutants, with highs in the early day (ZT1) and lows in the early night (ZT13).(B) Quantitation of PDF immunofluorescence in the wild-type and e1 at two different times of day (Error bars indicate standard deviation). PDF immunostaining was quantitated in 12 hemispheres for each genotype.

Supplemental Figure 3

Average activity levels for wild-type and e1 flies

Locomotor activity data were collected in 30-min bins for 5 days during LD or 3 days of DD. Day activity represents locomotor activity occurring from ZT(CT)0 to ZT(CT)12, whereas night activity represents the opposite phase. See Methods for details of data analysis. * p<0.01, ** p<0.05 (student’s two tailed t-test).

Supplemental Figure 4

Ebony cycling is not regulated by PDF release

(A-B) Ebony rhythmicity in wild-type and pdf01null mutants in LD and DD1. Brains were hand dissected in the early day (ZT4 or CT4) or night (ZT 16 or CT16). Error bars in B indicate standard deviation. (C) SYBR green real-time RT-PCR was performed to quantitate ebony RNA levels in wild-type (yw) and pdf01 (yw; w15) flies. RNA was extracted from heads collected during LD and DD (DD2 to DD5). Measurements were performed three times for each time point from two different head collections. Error bars indicate standard error. A one-way ANOVA indicates that there is significant Ebony cycling in both genotypes in LD and on DD day 2. Furthermore, there is statistically significant cycling on DD days 3 and 5 in the mutant and on DD day 4 in the wild type. Thus, cycling persists in both genotypes in constant conditions. A two-way ANOVA revealed no significant differences in cycling between the genotypes in either LD or DD conditions.

Supplemental Figure 5

TIM protein cycling is normal in different clock cell groups of the e1 mutant.

(A) Triple immunostaining of adult brains was performed for TIM, PDF and REPO and examined by confocal microscopy. TIM levels in neurons and glia were examined in wild-type (w1118) and e1 brains on the first day of DD at CT1 and CT4 after 5 days of LD entrainment. Fluorescence pixel intensity was measured for each signal, and the value for TIM (red) was normalized to that for REPO (green). PDF (blue) was used as a marker for certain pacemaker neurons. (B) Quantitation of TIM signals relative to REPO. As shown here, TIM levels are not significantly different between the wild type and e1 in either clock neurons or glia. Data were pooled from two independent experiments. Each histogram represents six to ten hemispheres. Error bars indicate standard error. For all cell groups, there were no significant differences between genotypes. For the LNv and DN1 groups, TIM values were significantly different between CT1 and CT4 for both genotypes. For the DN3 group and optic lobe glia, e1 showed significant time-of-day changes whereas the wild type did not. TIM has been shown to cycle in wild-type DN3 cells and optic lobe glia in other studies; we attribute the lack of significant cycling in our study to the small sample sizes or to the fact that we examined cycling during DD1 (TIM cycling in the brain dampens during the first 2 days of DD and then becomes robust again in later days of DD (Peng et al., 2003). * p < 0.01, ** p< 0.003 (Student’s two tailed t-test).

NIHMS28216-supplement-01.pdf (1.3MB, pdf)

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