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A UAS-pdf line was generated using a Romalea cDNA kindly provided by J. Klein (University of Bonn). The cDNA was inserted into the pUAST vector using an EcoRI/XhoI fragment. When the UAS-pdf flies are crossed to flies that carry the gal4 sequence under control of certain enhancers or promotors, GAL4 binds to the UAS, and the transcription of the pdf gene is activated. Consequently, the pdf gene is switched on in all of the cells that express gal4. We always crossed UAS-pdf males to females of the different gal4 lines in the present experiments.
The Journal of Neuroscience, May 1, 2000, 20(9):3339-3353
Ectopic Expression of the Neuropeptide Pigment-Dispersing Factor Alters Behavioral Rhythms in Drosophila melanogaster
Charlotte Helfrich-Förster1, Marcus Täuber2, Jae H. Park3, Max Mühlig-Versen2, Stephan Schneuwly2, and Alois Hofbauer2 1 Zoological Institute/Animal Physiology, University of Tübingen, 72076 Tübingen, Germany, 2 Zoological Institute/Developmental Biology, University of Regensburg, 93053 Regensburg, Germany, and 3 Department of Biology, Brandeis University, Waltham, Massachusetts 02454
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
To study the function of the neuropeptide pigment-dispersing factor (PDF) in the circadian system of Drosophila, we misexpressed the pdf gene from the grasshopper Romalea in the CNS of Drosophila and investigated the effect of this on behavioral rhythmicity. pdf was either ectopically expressed in different numbers of neurons in the brain or the thoracical nervous system or overexpressed in the pacemaker neurons alone. We found severe alterations in the activity and eclosion rhythm of several but not all lines with ectopic pdf expression. Only ectopic pdf expression in neurons that projected into the dorsal central brain severely influenced activity rhythms. Therefore, we conclude that PDF acts as a neuromodulator in the dorsal central brain that is involved in the rhythmic control of behavior. Overexpression of pdf in the pacemaker neurons alone or in the other neurons that express the clock genes period (per) and timeless (tim) did not disturb the activity rhythm. Such flies still showed a rhythm in PDF accumulation in the central brain terminals. This rhythm was absent in the terminals of neurons that expressed PDF ectopically. Probably, PDF is rhythmically processed, transported, or secreted in neurons expressing per and tim, and additional PDF expression in these cells does not influence this rhythmic process. In neurons lacking per and tim, PDF appears to be continuously processed, leading to a constant PDF secretion at their nerve terminals. This may lead to conflicting signals in the rhythmic output pathway and result in a severely altered rhythmic behavior.
Key words: circadian rhythms; pigment-dispersing factor; neuropeptides; gene misexpression; Drosophila; insects
INTRODUCTION
Internal clocks organize the temporal structure of physiological and behavioral functions in probably all organisms. They consist of a circadian pacemaker center that generates an endogenous rhythm of ~24 hr, an entrainment pathway for the synchronization with Zeitgebers, and output pathways to effector organs. In recent decades, surprising similarities have been revealed between the pacemaker centers of mammals and insects: both the mammalian suprachiasmatic nucleus (SCN) (Klein et al., 1991
) and the insect accessory medulla (for review, see Helfrich-Förster et al., 1998
In insects, the most abundant neuropeptide in the accessory medulla is the "pigment-dispersing factor" (PDF), an ortholog of the crustacean "pigment-dispersing hormone" (PDH) family (Rao and Riehm, 1993
PDF itself might be involved in the regulation of insect circadian rhythms. Ablation of the PDF neurons in the cockroach caused the rhythm of locomotor activity to disappear (Stengl and Homberg, 1994
To study the function of PDF in the circadian system of D. melanogaster we misexpressed the pdf gene of the grasshopper Romalea microptera in the CNS of the fly and investigated the effect of this misexpression on behavioral rhythmicity. We show that ectopic pdf expression in specific neurons led to severe alterations in activity and eclosion rhythms, suggesting that PDF is an integral part of the output pathway of the pacemaker.
MATERIALS AND METHODS
The upstream activation sequence/GAL4 system
The upstream activation sequence (UAS)/GAL4 system is a powerful technique for expressing any cloned gene under the control of specific enhancers or promotors (Fischer et al., 1988gal4 lines
The following gal4 lines were used: Mz1366-gal4, Mz1525-gal4, Mz1172-gal4 (gift from K.-F. Fischbach, University of Freiburg, and G. Technau, University of Mainz), elav-gal4 (P{w[+mW.hs] = GawB}elav[C155], Bloomington stock center), gmr-gal4 (P{gmr-gal4} UF815) (Freeman, 1996Recording of locomotor activity rhythms
Locomotor activity of individual flies (males and females) was recorded photoelectrically at 20 ± 1°C as described previously (Helfrich-Förster, 1998Estimation of circadian parameters
At the end of an experiment, the raw data from each recorded fly were transformed to actograms to allow visual judgment of the activity pattern of individual flies. To compare the activity pattern of the flies under LD, an average day was calculated and plotted as a histogram for each fly. Furthermore, the phase relationship of peak activity to lights on was calculated for each individual fly as described elsewhere (Helfrich-Förster, 20002 test with 5% significance level (Sokolove and Bushell, 1978
Monitoring of eclosion rhythms of elav-gal4;UAS-pdf flies
elav-gal4 (female) × UAS-pdf (male) crosses were set up along with elav-gal4 and UAS-pdf parental lines and kept under 12:12 LD conditions at 20 or 25°C. Flies were transferred to a new bottle every 2-3 d. After the first bottle had seeded for 9 d, adult flies were removed from the last bottles. Pupae were collected and glued to disks that subsequently were placed onto the eclosion monitor as described previously (Konopka et al., 1994Statistics
The different rhythm parameters of control flies and flies with ectopic pdf expression were compared with an orthogonal two-way ANOVA or t test. Significantly different data sets were then tested for normal distribution by the Kolmogorov-Smirnov test. Periods and evening peaks were not normally distributed only in the case of gmr-gal4;UAS-pdf flies. Therefore, the values of probability (p > 0.01 in both cases) were adapted according to Glaser (1978)Histology
Experimental animals. After the end of the recording, the flies were transferred from DD to light for 1-2 hr before dissection. The animals were then dissected, and brain whole mounts were immunostained with an antiserum against crab PDH by the peroxidase-anti-peroxidase method exactly as described previously (Helfrich-Förster, 1997
RESULTS
Misexpression of the pdf gene in virtually all neurons
In a first attempt we tried to misexpress the pdf gene in all neurons to determine whether such a strong ectopical expression has any effect on the eclosion and the locomotor activity rhythm.
We did this with the help of the elav-gal4 line. The elav gene is expressed in all neurons shortly after their differentiation starts and continues to be expressed (Robinow and White, 1988
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Figure 1. PDH-immunoreactive neurons in the brain of older elav-gal4; UAS-pdf flies. Frontal reconstruction of the anterior (A), medial (B), and posterior (C) brain. The ventral lateral neurons (LNvs) that express natural PDF are shown together with their arborizations (arrows); the varicose network that they form on the surface of the medulla (Me) is only partly shown. The neurons with ectopic PDF are numbered, and only their somata are drawn. The numbers refer to the cell descriptions given in Table 1 and Figure 2. A, In the anterior central brain, prominent PDF labeling was found in the neuropil of the antennal lobes (AL) and in the -,
-, and
-lobes of the mushroom bodies (MB). The fine network of fibers in the glomeruli of the antennal lobes may arise from neurons of cluster 3 and/or cluster 4. The dense staining in the mushroom bodies belongs to the Kenyon cells that are located near the calyces (Ca). The somata of these cells were stained during a short period in midpupal development but not in adults. One large cell was labeled anteriorly in the tritocerebrum (cluster 5), and many cells were stained in the anterior and medial subesophageal ganglion (cluster 6; see also Fig. 2D). Most of these cells seem to run toward the esophageal foramen. For a detailed description of the other neurons stained in the anterior brain (clusters 1 and 2 and DGI), see Results and Figure 2B. B, Medially in the central brain, prominent staining was found in neurons of the pars intercerebralis (cluster 9) and in a layer in the fan-shaped body (FB) of the central complex (see also Fig. 2C). Ventrolaterally in the central brain just at the border to the optic lobe, a large neuron was always strongly stained (cell 8). This neuron made connections to its counterpart in the contralateral brain hemisphere and shows conspicuous wide arborizations into the anterior as well as the posterior dorsolateral brain (see Figs. 2D, 6A,B, 10). Neurons of cluster 7 in the ventral medial subesophageal ganglion appear to send fibers through the cervical connective to the thoracic nervous system. C, In the posterior brain, many neurons were stained additionally to the per and tim expressing dorsal neurons (DN1 and DN2). Two large conspicuous cells were stained in the pars lateralis. The more dorsolateral-located neuron (cell 10) projected toward the pars intercerebralis and then down the median bundle parallel to the projections of the dorsal neurons of cluster 9 (see Fig. 6C showing Mz1172-gal4; UAS-pdf). The adjacent more ventromedial-located second large neuron (cell 11) also projected down the median bundle but additionally arborized in the dorsolateral brain (see Fig. 10). The projections of this neuron were only weakly labeled. A third neuron with small soma was consistently stained ventral to the just described large cells at about the level of the ellipsoid body (cell 12). This cell projected dorsally into the medial dorsolateral brain as well as into the anterior part of the brain where it seems to terminate close to the most dorsal end of the
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Figure 2. Ectopic PDF in the brain and thoracic nervous system of elav-gal4; UAS-pdf flies revealed by PDH immunocytochemistry. In the anterior central brain (A), strong labeling was found in the
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Table 1. PDH-immunoreactive neurons in young (2-10 d) and old (20-30 d) flies of the different gal4 lines PDH immunohistochemistry on adult elav-gal4;UAS-pdf flies
Ectopic PDF was found in most of the neurons that express the clock genes period and timeless but normally don't contain PDF (Kaneko et al.,. 1997
Prominent PDF labeling was found in the neuropil of the antennal lobes, in the
In the thoracic NS, PDF was present in several superficially located neurons (Fig. 2F-H). Most of these were located in the third thoracic segment and in the abdominal segments close to the natural PDF-abdominal (PDFAb) neurons (Fig. 2G,H) (cf. Helfrich-Förster and Homberg, 1993
Comparison of elav-gal4-driven GFP and PDF
The previous experiment showed that pdf gene expression is not necessarily reflected in PDF peptide expression. We made similar observations when we used GFP as a reporter for gal4 expression and compared elav-gal4-driven GFP with elav-gal4-driven PDF. Not all cells that were GFP positive were also PDF positive. No PDF was found in the photoreceptor cells and in the optic lobe, although much GFP was detected in these cells in elav-gal4;UAS-gfp flies (Fig. 3A). Similarly, PDF was not found in the ellipsoid body, although elav-gal4;UAS-gfp flies showed GFP in ring neurons (Fig. 3C). In situ hybridization with complementary pdf mRNA on the retina of gmr-gal4;UAS-pdf revealed massive pdf mRNA in this tissue, although no mature peptide was detected (data not shown). All of these findings indicate that the pdf gene is expressed in all GAL4-containing neurons but that only specific cells provide the functional apparatus to process PDF into its mature peptide form. As a consequence, PDF could be revealed only in a subset of the gal4-expressing cells in the different gal4 lines.
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Figure 3. Confocal microscopy to compare GAL4-mediated expression visualized with the reporter GFP and with PDF. The spatial distribution of GAL4-driven GFP and PDF was often different. In the elav-gal4; UAS-gfp line, GFP was prominently revealed in the photoreceptor cells (A, the arrow points to their axonal terminals in the first optic ganglion) and other cells in the optic lobe (OL), whereas no PDF was found in these cells (B). The only PDF labeling present in the optic lobes stemmed from the processes of the LNv that formed a varicose network (arrow in B) on the surface of the medulla. Similarly, no PDF was found in ring neurons of the ellipsoid body (ebo), although these cells strongly expressed GFP (arrowheads in C). Even the spatial distribution of GFP and PDF in the same neuron was often different: GFP labeling was found in the Kenyon cells (D, arrowheads) and their corresponding dendrites in the Calyx (Ca) of the mushroom body. However, ectopic PDF was only revealed in the
Furthermore, the spatial distribution of GFP and PDF within the neurons was often different. The elav-gal4 line showed strong labeling in the somata of the Kenyon cells of the mushroom bodies when GFP was used as a reporter (Fig. 3D). When crossed to UAS-pdf, no labeling at all was seen in the somata of these cells in adults, but the neurites of the Kenyon cells that constitute the pedunculus,
In summary, we observed differences between overall expression of pdf mRNA and the mature peptide PDF, as well as between localization of PDF and GFP within the cell. This suggests that the spatial and temporal expression of the peptide is controlled post-transcriptionally or even post-translationally. PDF appears to be modified, transported, accumulated, and secreted from the neurite independently from pdf gene expression.
Eclosion and locomotor activity rhythms in elav-gal4;UAS-pdf flies
The analysis of eclosion revealed clear circadian eclosion profiles in the elav-gal4 and UAS-pdf parental lines by visual inspection, periodogram analysis, and MESA (Fig. 4A,B). In contrast to this, visual inspection of the eclosion profiles of the elav-gal4;UAS-pdf flies showed that eclosion occurred only initially in a rhythmic manner in these flies. Approximately 2-3 d after transfer into DD, elav-gal4;UAS-pdf flies appeared to eclose in an arrhythmic fashion (Fig. 4C,D). This was similar at 20 and 25°C. Periodogram analysis and MESA revealed some rhythmicity in the eclosion profiles of these flies at 20°C but none at 25°C. Therefore, we conclude that the rhythm of eclosion is disturbed in elav-gal4;UAS-pdf flies.
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Figure 4. Eclosion rhythm in UAS-pdf flies (A), elav-gal4 flies (B), and elav-gal4; UAS-pdf flies (C, D). The parental lines (UAS-pdf and elav-gal4 flies) were recorded at 25°C. The elav-gal4; UAS-pdf cross was monitored at 25°C (C) and at 20°C (D). At both temperatures only two clear eclosion peaks (arrows) could be seen by visual inspection in these flies; then eclosion slowly became arrhythmic. In the parental lines, eclosion remained clearly rhythmic throughout the 6 d of monitoring. Periodogram analysis and MESA revealed strong rhythmicity in the parental lines and some rhythmicity in elav-gal4; UAS-pdf flies at 20°C (D), but no rhythmicity at 25°C (C). the cervical connective and that of the thoracic NS (arrowheads in F). The cell pair number 8 appeared to be contralaterally connected (arrows) and formed conspicuous wide arborizations in the anterior and dorsolateral brain that are shown in Figure 6, A and B. In the posterior central brain (E), a large number of cells with ectopic PDF were found; only some of these were numbered (clusters 10-14). In the third thoracic neuromere of dorsal thoracic NS (F), ectopic PDF was revealed in many superficially located cells that were arranged in a horseshoe-shaped manner. Whether the varicose network of fibers on the surface of the thoracic NS (arrowheads) stems partly from these cells or entirely from those located in the subesophageal ganglion of the brain (cluster 7) is unclear. In the ventral thoracic NS (G), several large cells were stained in the midline of the second thoracic neuromere (large arrow in G and H) plus several smaller ones were stained at the borders between the three neuromeres (arrowheads). In the abdominal neuromeres, many cells (right arrow in H) were stained in addition to the natural PDFAb cells. A sagittal view of the thoracic NS (H) shows that many of the ventrally located cells projected dorsally (arrowheads). Scale bars: A-E, 20 µm; (shown in F) F-H, 100 µm.
For judgement of the locomotor activity rhythm, 87 elav-gal4;UAS-pdf flies (43 males, 44 females) were compared with 71 control flies (33 males, 38 females). Although all controls (Fig. 5A) were clearly rhythmic, only 44.19% of the male and 29.54% of the female elav-gal4;UAS-pdf flies showed such a clear rhythmicity. Most flies were either complex rhythmic or completely arrhythmic (Fig. 8, first two columns). Most of the complex rhythmic flies showed two simultaneously free-running components: one with a period of ~22 hr and one with a period of ~25 hr (Fig. 5C). Both free-running components seem to arise from the evening peak of activity. The component with the longer period was generally more dominant than that with the short period, and the 32 flies (19 males, 13 females) with clear rhythmicity showed the long period component. The periods of both components were highly significantly different from the periods of the controls (Fig. 9A, first two columns).
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Figure 5. Locomotor activity rhythms in elav-gal4; UAS-pdf and control flies. All flies were first recorded for 7 d under a 12 hr light/dark cycle (LD) and subsequently for 26 d under continuous dark conditions (DD). For every fly actogram, average days during LD and periodograms of the free-running rhythm in DD are shown. The first panels (A) illustrate the rhythmic behavior of a typical male control (wild-type) fly. During LD, the fly's activity pattern was clearly bimodal: a morning and an evening peak of activity could be distinguished in the average day. Activity starts ~0.5 hr before lights on, but most activity was restricted to the light phase. During DD the fly was clearly rhythmic and showed a more or less stable, free-running period. A slight lengthening in period occurred from day 20 onward. Such smaller period changes were frequently observed in wild-type flies and were not considered further in this study. B and C show the activity pattern of two typical male elav-gal4; UAS-pdf flies. During LD both flies had a bimodal activity pattern similar to the wild type, but their activity was less restricted to the light phase. Especially the fly in C showed a considerable amount of activity during the dark phase. During DD, both flies had a complex activity pattern. The fly in B showed a main free-running component with a long period, but shortly after transfer into DD a second free-running component with a short period appears to arise from this main component (arrow). Both components clearly originate from the evening peak of activity. From day 22 onward, the fly became arrhythmic (lower periodogram). The fly in C showed two simultaneously free-running components throughout the recording time in DD.
Under LD conditions, the elav-gal4;UAS-pdf flies extended their activity into the dark period, whereas the activity of controls was more restricted to the light period (Fig. 5). The phase determination of morning and evening peak revealed that the elav-gal4;UAS-pdf flies had a highly significant earlier morning peak and later evening peak than the controls (Fig. 9C, first two columns). This was true for both sexes.
Under DD conditions, the elav-gal4;UAS-pdf flies showed a significantly higher activity level than the controls (Figs. 5, 9B, first two columns).
As described previously (Helfrich-Förster, 2000
In summary, misexpression of the pdf gene in the elav-gal4;UAS-pdf flies resulted in severe alterations in eclosion and activity rhythms. The latter were visible as an increase in activity level, an extension of activity into the dark-period, a period increase, and a destabilization of the free-running rhythm becoming evident in the frequent occurrence of complex rhythmic or arrhythmic activity patterns. Therefore, we conclude that PDF strongly affects behavioral rhythms.
Misexpression of PDF in specific groups of neurons
To reveal what neurons with ectopic PDF are responsible for the alterations in the activity rhythm, we misexpressed PDF only in subgroups of the PDF-positive cells of the elav-gal4;UAS-pdf flies (Table 1, Fig. 6). Four different gal4 lines were used for this purpose: Mz1366-gal4;UAS-pdf, Mz1525-gal4;UAS-pdf, Mz1172-gal4;UAS-pdf, and gmr-gal4;UAS-pdf. In gmr-gal4;UAS-pdf, the pdf gene was expressed in all photoreceptor cells. Nevertheless, we did not find any mature PDF in these cells by PDH immunohistochemistry, and the flies showed a normal locomotor activity rhythm (Figs. 8, 9). In the other gal4 lines, PDF was restricted to specific subsets of neurons in the brain and thoracic NS (Table 1). Generally, more PDF cells were detected in pupae and younger flies than in old ones. Furthermore, the intensity of PDH immunoreactivity decreased with increasing age of the flies. This was most evident in the thoracic NS of the lines Mz1366-gal4 and Mz1525-gal4 (Table 1). Especially in older Mz1525-gal4;UAS-pdf flies, PDF was virtually absent from the thoracic NS. Similarly, PDF was restricted to very few brain neurons in older flies of this line. Strong PDF labeling was found only in neurons 8, 11, and 12 (Fig. 6A,B) and in the LNv (Fig. 3F-H). Nevertheless, Mz1366-gal4;UAS-pdf and Mz1525-gal4;UAS-pdf flies exhibited a severely altered locomotor activity rhythm that was very similar to elav-gal4;UAS-pdf flies (Figs. 7-9). The majority of flies were either arrhythmic or showed complex rhythmicity that was composed of two activity components, the activity level of the flies under DD was increased, and the phase of the morning peak was advanced.
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Figure 6. Ectopic PDF in the central brains of three different gal4; UAS-pdf lines that express PDF only in subsets of the neurons that were found in the elav-gal4; UAS-pdf line. A typical Mz1525-gal4; UAS-pdf brain is depicted from an anterior (A) and posterior (B) plane of focus. This brain stemmed from the experimental animal with arrhythmic activity pattern shown in Figure 7C. Ectopic PDF was found in only a few neuron pairs in this brain. The most prominent one was neuron 8, which showed arborizations in the anterior brain (arrowheads in A) as well as in the dorsolateral posterior brain (arrowheads in B). The somata of neuron pair 11 were also strongly stained, but their arborizations into the dorsolateral brain and down the median bundle (see Fig. 10A) were not revealed. Weak staining was found in the soma of neuron 12 and in neurons of cluster 4 plus their putative arborizations in the antennal lobes (AL). In the line Mz1172-gal4; UAS-pdf (C), neuron pair 10 was prominently revealed together with neurons in the pars intercerebralis (cluster 9). All of these cells projected down the median bundle (MBu). The other arborizations (arrow) in this brain stemmed from the dorsal giant interneuron (DGI). The arrowheads point to the characteristic bend of the neurite close to its soma (cf. Ito et al., 1997
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Figure 7. Locomotor activity rhythms in three different gal4; UAS-pdf lines. The recording schedule is as in Figure 5. The Mz1366-gal4; UAS-pdf line (A) and the Mz1525-gal4; UAS-pdf line (B, C) showed a behavior that was very similar to that of the elav-gal4; UAS-pdf line (Fig. 5B,C), although fewer neurons showed ectopic PDF expression. The flies were either complex rhythmic (A, B) or arrhythmic (C). The brain of the arrhythmic Mz1525-gal4; UAS-pdf fly (C) is depicted in Figure 6, A and B. When PDF was overexpressed in the LNvs only (pdf-gal4; UAS-pdf fly), locomotor activity rhythm was wild-type-like (D).
Some minor differences were observed in the overall activity pattern between these two lines and those of the line elav-gal4;UAS-pdf: in Mz1366-gal4;UAS-pdf flies with complex rhythmicity, the components with short and long periods were of equal strength, and in Mz1525-gal4;UAS-pdf flies, the short period component was the more prominent one. (In the first line, three of the five clearly rhythmic flies had a long period; the remaining two had short periods. In the latter line, six of such flies had short periods, and only two had long ones.) Furthermore, the effect of PDF on the phase of the evening peak was different in males and females of Mz1366-gal4;UAS-pdf and Mz1525-gal4;UAS-pdf flies: ectopic PDF induced a phase delay in the evening peak only in female flies (Fig. 9C). This interaction between sex and evening peak was significant in both lines, whereas we did not observe such an effect in the line elav-gal4;UAS-pdf (Fig. 9C).
Despite these minor differences among Mz1366-gal4;UAS-pdf, Mz1525-gal4;UAS-pdf, and elav-gal4;UAS-pdf flies, the most important observation is that all three lines exhibited a severely altered activity rhythm, suggesting that the latter is attributable to pdf misexpression in only a few neurons common to all three lines (see below).
Adult flies of line Mz1172-gal4;UAS-pdf showed an almost complementary PDF expression pattern to the two lines that were just described. Older flies of this line revealed strong PDF labeling in the thoracic NS, in neurons 9 and 10, and in the DGI (Table 1, Fig. 6C). These flies showed a normal rhythmic behavior; we did not observe an increase in the number of complex and arrhythmic flies (Fig. 8). Nevertheless, the period was slightly shortened and the phase of the morning peak a bit advanced, as compared with the controls (Fig. 9A,C). These effects were much less dramatic, however, than those observed in the previous lines. Therefore, we conclude that misexpression of PDF in the thoracic NS and the neurosecretory cells of the pars intercerebralis (plus neuron 10 and the DGI) has little influence on the rhythm of locomotor activity and does not disturb general rhythmicity at all.
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Figure 8. Distribution of locomotor activity patterns in the different lines with ectopic PDF. The percentages of normal rhythmic flies (black bar), complex rhythmic flies (gray bar), and arrhythmic flies (white bar) are shown for males (m) and females (f) of the different groups. Numbers of tested animals are given on top of the bars. Most control flies (95-100%) were normally rhythmic; therefore, these were not included in the present graph. Data of controls and of the corresponding flies with ectopic PDF were arranged in contingency tables for
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Figure 9. The effects of ectopic PDF on locomotor activity. Depending on its expression pattern in the different gal4 lines, PDF affected the free-running period (A), the daily activity level under DD conditions (B), and the phases of morning and evening peaks under LD conditions (C). Phases of morning and evening peaks are given in Zeitgeber time (ZT), whereby lights on is ZT0 and lights off is ZT12 (C). The shaded areas in C indicate the beginning and end of the dark period of the LD cycle, respectively. In many cases the morning peak occurred during darkness before lights on. Mean values (±SE) of the different parameters are shown for the flies with ectopic PDF and their corresponding internal controls of each gal4 line (see description at bottom of Figure). Closed circles and black bars represent mean values for males, and open circles and white bars represent such values for females. Numbers of tested animals are given on top of the bars for males (m) and females (f) separately. (For period determination the numbers of analyzed flies were in some cases lower than the numbers given at the top, because the periods of arrhythmic flies could not be determined. In the case of complex rhythmicity with two simultaneously free-running components, the mean periods of both components are shown in the graph.) The data of controls and experimental animals of each line were compared with an orthogonal two-way ANOVA to reveal the influences of PDF misexpression ( ), of sex (
), and of interactions of both (
) on the different parameters. Three symbols represent a probability for a significant effect of p < 0.001 (F(1,df) > 12.30, df = 77-173); two symbols represent a probability of p < 0.01 (F(1,df) > 7.30, df = 77-173); one symbol represents a probability of p < 0.05 (F(1,df) > 3.90, df = 77-148); and a minus marks the values that were not significantly different (p > 0.05; F(1,df) < 3.84, df = 87-173). In case of complex rhythmicity with two simultaneously free-running components, the periods of both components were compared with the single periods of the controls (A). PDF misexpression had significant effects on period (A), activity level (B), and phases of morning and evening peak (C) mainly in the first three gal4 lines. In the other four lines, PDF had only mild effects on some of the rhythmic parameters. A sexual dimorphism was found in all lines. Female flies had significantly longer periods, a higher activity level (except for Mz1366-gal4; UAS-pdf flies and controls), and a later morning peak than males. In the lines Mz1172-gal4; UAS-pdf and pdf-gal4; UAS-pdf plus controls, we also found an effect of sex on the evening peaks: males had a later evening peak than females (C). Different effects of PDF in both sexes (interaction of PDF and sex) were mainly found in period lengthening or shortening (A) and on the phase of the evening peak (C) in the first three lines. Furthermore, such effects were present in period shortening and activity level changes in the line Mz1172-gal4; UAS-pdf, and on the phase of the morning peak in the line pdf-gal4; UAS-pdf. For other details see Results.
Interestingly, larvae and pupae of Mz1172-gal4;UAS-pdf showed strong PDH immunoreactivity in many more neurons than did adults. Such ectopic PDF during development obviously had no severe effect on adult rhythmicity.
Overexpression of PDF in the LN and DN
Two of the three lines with altered activity rhythm (elav-gal4;UAS-pdf and Mz1525-gal4;UAS-pdf) misexpressed the pdf gene in the LNv in addition to the ectopic expression in other neurons that was described above (Fig. 3G,H). The LNvs are the putative circadian pacemaker neurons of Drosophila, and overexpression of the pdf gene in these cells alone may influence locomotor activity and be responsible for the altered rhythmicity in these two lines. To test this, we overexpressed the pdf gene in the LNv alone (pdf-gal4;UAS-pdf line) or in the LNv, LNd, and DN (per-gal4;UAS-pdf line) (Fig. 6D).
In both lines we did not see any effect on general rhythmicity (Figs. 7D, 8), but as in the line Mz1172-gal4;UAS-pdf, we observed a slight period shortening and a minor advance in the morning peak (Fig. 9A,C). The period shortening was only significant, however, in the pdf-gal4;UAS-pdf line (Fig. 9A), whereas the advance of the morning peak was only significant in the per-gal4;UAS-pdf line (Fig. 9D). Furthermore, both lines showed a lower activity level than the controls (Fig. 9B). In per-gal4;UAS-pdf flies, this was true in LD and DD, whereas in pdf-gal4;UAS-pdf flies, activity was reduced only under LD conditions. These small effects on rhythmic parameters can be attributed to pdf overexpression in the LNv as well as to pdf misexpression in the LNd and DN. Probably both contribute because the observed effects appeared stronger in per-gal4;UAS-pdf flies than in pdf-gal4;UAS-pdf flies. These results also suggest that the pdf gene is indeed overexpressed in the LNv in both lines (although this is hard to confirm by PDH immunohistochemistry) but that the severe alterations in locomotor activity rhythms observed in elav-gal4;UAS-pdf and Mz1525-gal4;UAS-pdf flies are not caused by pdf overexpression in the pacemaker neurons.
To further increase the transcription rate of pdf, we took advantage of the temperature sensitivity of GAL4-mediated ectopic expression, which is stronger at 29°C than at 25 or 18°C (Brand et al., 1994
Where is the site of action of ectopic PDF?
When comparing the pattern of ectopic PDF in the different gal4 lines (Table 1), neurons 8 and 11 were the only cells that showed ectopic PDF expression in all lines with abnormal rhythmicity but not in the ones with normal rhythms. Therefore, ectopic PDF in these two neurons is most likely the cause for the abnormal activity rhythms. Both neurons had extensive arborizations in the dorsolateral brain close to the terminals of the LNv (Fig. 10A), a region that is crucial for the transfer of circadian signals (Helfrich-Förster, 1998
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Figure 10. Arborization pattern of the LNv (black), neuron 8 (red), and neuron 11 (green) in the central brain of D. melanogaster (A), and PDF-staining intensity in the central brain terminals of the LNv in the wild-type and the lines pdf-gal4; UAS-pdf and Mz1525-gal4; UAS-pdf (B) and of neuron 8 (in Mz1525-gal4; UAS-pdf). All three neurons show a partial overlap in their arborization fields in the dorsolateral protocerebrum (A, arrows). Neuron 8 had additional arborizations in the medial and ventral anterior brain (arrowheads). Ped, Pedunculus of the mushroom body; LNv, ventral lateral neuron. The rectangle indicates the terminal area that was used for judgement of staining intensity. Scale bar, 50 µm. Staining intensity (B) was judged at ZT2 and ZT14 and given in gray values (mean gray value of the stained structure minus mean gray value of the background; see Material and Methods). In the LNv terminals, staining intensity was significantly higher at ZT2 than at ZT14 independently of the strain (two-way ANOVA, F(1,54) = 58.912, p < 0.0001 for time; F(2,54) = 0.588, p = 0.559 for strains). In the terminals of neuron 8 (line Mz1525-gal4; UAS-pdf), no significant difference in staining difference was found at either time points (ANOVA, F(1,15) = 0.660, p = 0.429).
DISCUSSION
The aim of the present study was to investigate the function of the neuropeptide PDF in the circadian system of D. melanogaster. Former studies have revealed PDF in a well defined subset of neurons that express the clock genes per, tim, and dbt and are most likely circadian pacemaker neurons (for review, see Kaneko, 1998
Although additional PDF affected behavioral rhythmicity severely, it did not lead to a complete arrhythmicity comparable to that of the known clock-gene rhythm mutants or the anatomical brain mutant disconnected, which lacks the LN (for review, see Hall, 1998
Furthermore, we did not eliminate other possible circadian mediator molecules that might be used as output signals in these neurons. In the accessory medulla of other insects as well as in the SCN of mammals, several neuropeptides were colocalized in the same cells (Reghunandanan et al., 1993
Action site of PDF
Our results showed that the action site of PDF is restricted to the central brain. We could not disturb the activity rhythm by ectopical expression of PDF in the thoracic NS, although some of these fibers even form an extensive plexus at the border between the thoracic NS and the blood and suggest a neurohormonal release of PDF. Neither could we affect the rhythm by expressing PDF in neurons associated with the corpora cardiaca, like the neurosecretory cells of the pars intercerebralis and pars lateralis. This indicates that PDF does not act as a neurohormone in the classical sense. Intriguingly, the existence of such a diffusible substance was predicted by brain transplantation experiments performed a long time ago (Handler and Konopka, 1979
High PDH immunoreactivity may indicate accumulation of PDF combined with little release from the terminals, whereas low PDH immunoreactivity may stand for a strong peptide release. If this is true, PDF should be constantly released from the neurites of neuron 8 and even at a higher constant level than those of neuron 11, because the neurites of neuron 11 were always very weakly labeled. This would lead to a permanent presence of PDF in the dorsolateral protocerebrum, a brain region that might be specially sensitive to PDF. Former observations have already shown that this brain region is crucial for the transfer of rhythmic signals from the LNv terminals via still unknown interneurons toward the locomotor centers (Helfrich-Förster, 1998
Only specific cells are capable of PDF processing
Like most neuropeptides, PDF of D. melanogaster is synthesized from a larger precursor composed of a signal peptide (16 aa), the PDF-associated peptide (63 aa), and PDF itself (18 aa) (Park and Hall, 1998
The effects on rhythmic behavior were also very similar between ectopic Drosophila PDF and Romalea PDF, pointing to an evolutionary well conserved function of PDF (M. Täuber, C. Helfrich-Förster, unpublished results). Drosophila and Romalea PDF differ in 4 of the 18 amino acids (Park and Hall, 1998
Rhythmic control of PDF occurs post-translationally
The pdf gene appears not to be a clock-controlled gene as opposed to other genes with still unknown function t
