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Pattern of distribution and cycling of SLOB, Slowpoke channel binding protein, in Drosophila

Pattern of distribution and cycling of SLOB, Slowpoke channel binding protein, in Drosophila Background: SLOB binds to and modulates the activity of the Drosophila Slowpoke (dSlo) calcium activated potassium channel. Recent microarray analyses demonstrated circadian cycling of slob mRNA. Results: We report the mRNA and protein expression pattern of slob in Drosophila heads. slob transcript is present in the photoreceptors, optic lobe, pars intercerebralis (PI) neurons and surrounding brain cortex. SLOB protein exhibits a similar distribution pattern, and we show that it cycles in Drosophila heads, in photoreceptor cells and in neurosecretory cells of the PI. The cycling of SLOB is altered in various clock gene mutants, and SLOB is expressed in ectopic locations in 01 jrk tim flies. We also demonstrate that SLOB no longer cycles in the PI neurons of Clk flies, and that SLOB expression is reduced in the PI neurons of flies that lack pigment dispersing factor (PDF), a neuropeptide secreted by clock cells. Conclusions: These data are consistent with the idea that SLOB may participate in one or more circadian pathways in Drosophila. SLOB/dSlo complex dramatically shifts the channel volt- Background Five independent groups [1-5] recently conducted age range of activation to more depolarized potentials [6]. genome-wide microarray analyses to identify Drosophila transcripts that display circadian oscillations. Each group The circadian system consists of an input pathway, a cen- uncovered slob as a robustly cycling RNA transcript. SLOB, tral clock, and an output pathway [7]. The clock itself is Slowpoke binding protein, is a key component of the Dro- comprised of the transcription factors CLOCK (CLK) and sophila Slowpoke/SLOB/Leonardo dynamic protein com- CYCLE (CYC), which bind to the promoters of period (per) plex [6]. This complex is thought to affect membrane and timeless (tim), inducing their expression. The PER and excitability, as electrophysiological recordings reveal that TIM proteins heterodimerize and feed back to repress SLOB binding to the channel results in an increase in activity of CLK/CYC. Although the expression patterns of channel activity, whereas the addition of Leonardo to the CLK and CYC are not known, the localization of PER and Page 1 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 TIM has been important with respect to identifying neu- for three days in 12:12 hour light/dark cycles (LD). We rons relevant for circadian behavioral rhythms [8]. This found that SLOB protein cycles (Figure 1A) with a peak at molecular loop must transduce signals to surrounding Zeitgeber Time (ZT) 10–14, and a trough around ZT 2 (ZT cells to generate rhythmic behavior [9]. Altering mem- 0 = lights on; ZT 12 = lights off in a 12 hour:12 hour light/ brane excitability is a key mechanism for transducing neu- dark cycle). ronal information, and thus it is reasonable to suspect that the molecular feedback loop might communicate To ensure that rhythms of SLOB expression are circadian with ion channels. and not driven only by external LD cycling we analyzed SLOB protein cycling in conditions of constant darkness. Ion channels may be under direct transcriptional control Flies were entrained for three days in LD, and then placed of the clock genes or modulated by clock-controlled genes in constant darkness (DD) for two days. SLOB protein still [10,11]. Mounting evidence supports the importance of cycles under these conditions, with a slightly dampened electrical activity for the propagation of circadian oscilla- amplitude, but with the same overall phase (Figure 1B). A tions. For example, electrical silencing of clock neurons similar decrease in amplitude has been described for oscil- through targeted expression of potassium channels stops lations of other clock genes in DD [18,19]. the oscillation of PER and TIM proteins and causes arrhythmicity in flies [12]. The cyclic release of neuropep- Expression of SLOB protein is influenced by clock genes tides from clock cells [13] may be a direct consequence of To determine if the clock genes control the cycling of a rhythmic fluctuation in membrane potential [14]. Diur- SLOB, we assayed its expression in different mutant back- jrk nal modulation of pacemaker potentials and calcium cur- grounds. Clk flies, flies mutant for CLK, were entrained rent, intracellular calcium levels and NMDA-evoked in LD cycles for three days and then collected at six time calcium currents have all been observed within a mamma- points. Some flies were transferred from LD to DD for two lian central clock, the suprachiasmatic nucleus (SCN) additional days and likewise collected at six time points. [11,15,16]. In addition, microarray screens have detected Western blot analysis of Drosophila heads shows that jrk the cycling transcripts of ion channels such as Shaker, trpl SLOB protein continues to cycle in Clk adults in LD, with and slowpoke [1,3], and flies mutant in slowpoke have weak a phase and amplitude similar to wild type (Figure 2A). In locomotor rhythms [1]. These observations suggest that DD, the western blot analysis shows SLOB expression to ion channels and their modulators may participate in cir- have no discernable peak indicating that it does not cycle cadian regulation. (Figure 2B). Claridge-Chang et al. [2] noted that slob RNA jrk is down regulated in the Clk mutant, suggesting that CLK We explored a role for SLOB in the circadian system and acts as a transcriptional activator of slob expression. We found that SLOB protein cycles in Drosophila heads during did not observe a dramatic decrease in protein levels in jrk both light/dark and constant darkness conditions. SLOB Clk flies; the level was between the trough and peak lev- oscillates in at least two discrete areas of the fly head, the els seen in wild type flies. photoreceptor cells and the PI neurons. The photorecep- 01 01 tors have their own peripheral circadian oscillator [17], The cycling of SLOB was also analyzed in per and tim whereas the PI neurons, large neurosecretory neurons, are mutants during LD (Figure 2A) and DD (Figure 2C,2D). suspected to play a role in the output pathway that drives In both mutant lines, SLOB continues to cycle in LD with rest:activity rhythms (Kaneko and Hall, 2000). Our results a phase similar to that of wild type (Figure 2A). SLOB may reveal differential effects of clock mutations on SLOB be regulated by a direct light-dependent mechanism, expression and cycling in these two regions. There is a sig- obviating the need for these clock genes in LD. As in the 01 jrk nificant decrease in SLOB levels in the PI neurons of Pdf case of Clk , there appears to be either dampened or no flies, thus implicating PDF as an upstream regulator of protein cycling in per flies under DD conditions (Figure SLOB. SLOB also no longer cycles in the PI neurons of 2C). However, in tim flies SLOB still cycles, but there is jrk Clk flies, supporting the idea that SLOB is a clock con- a shift in the phase of the oscillation in DD (Figure 2D). trolled protein. Together with the observation that flies Instead of peaking at Circadian Time (CT) 10–14, SLOB overexpressing SLOB exhibit a breakdown of rest:activity now peaks at CT 2. This suggests that the regulation of patterns, these data are consistent with the idea that SLOB SLOB by TIM may be different from that by PER, which is participates in circadian rhythms. not unprecedented [20]. Expression pattern of slob transcript in Drosophila heads Results SLOB protein cycles in Drosophila heads To determine the expression pattern of slob transcript, we To determine whether SLOB protein cycles, we performed performed in situ hybridizations on cryosections of Dro- western blot analysis on Drosophila head lysates from wild sophila yellow-white (y w) fly heads. Digoxygenin-labeled type Canton S (CS) or yellow-white (y w) flies entrained antisense and sense RNA probes of slob were used on Page 2 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 A B 2 6 10 14 18 22 2 6 10 14 18 22 SLOB SLOB MK MK Zeitgeber Time (hrs) Circadian Time (hrs) 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 2 6 10 14 18 22 2 6 10 14 18 22 Zeitgeber Time (hrs) Circadian Time (hrs) SLOB protein Figure 1 cycles in both LD and DD SLOB protein cycles in both LD and DD. (A) Canton S flies were entrained in LD for three days and collected at six time points over a 24 hour period. Fly head lysates were prepared and an anti-SLOB antibody was used to identify the 58 kDa band representing SLOB on the top western blot. The bottom blot was probed for MAPK, the protein level of which does not cycle [37] and was used as a loading control. SLOB oscillation has a trough at ZT 2 and a peak between ZT 10–14. The graph below represents the mean ± SEM for each time point from a minimum of three independent experiments. (B) Canton S flies were entrained in LD for three days and transferred to constant darkness. They were collected on the second day of DD at six time points, and SLOB cycling was determined as in (A). frontal sections and the antisense revealed a widespread SLOB is expressed in the photoreceptors and the brain distribution of slob. Expression of slob was detected in the Previously, we demonstrated that SLOB protein is present brain cortex (Figure 3A), photoreceptors, lamina and at the neuromuscular junction of Drosophila larvae [6]. In medulla (Figure 3B) and the PI region of the brain (Figure order to locate SLOB protein in the adult fly, frontal head 3C). A similar pattern of transcript distribution, particu- sections and wholemounts of the brain were prepared larly in the photoreceptors, optic lobes and brain cortex, from Canton S and y w flies. Immunostaining of sections is seen for timeless, clock and cycle [21]. Figure 3D,3E,3F reveals intense staining in the nuclei of the photoreceptor shows the respective areas of Figures 3A,3B,3C using the cells and the basal cells of the eye (Figure 4A). The brain sense RNA probe for slob. wholemounts reveal very bright and discrete cytoplasmic staining of 6–8 PI neurons, with widespread but less intense staining elsewhere in the optic lobe and brain cor- tex (Figure 4B,4C). Although the overall patterns of Page 3 of 14 (page number not for citation purposes) Normalized SLOB Normalized SLOB BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 per Canton S Canton S 0.2 jrk 01 0.2 jrk Clk tim Clk 0.0 0.0 2 6 10 14 18 22 2 6 10 14 18 22 Zeitgeber Time (hrs) Circadian Time (hrs) C D 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 Canton S Canton S 0.2 01 0.2 per tim 0.0 0.0 2 6 10 14 18 22 2 6 10 14 18 22 Circadian Time (hrs) Circadian Time (hrs) SL Figure 2 OB cycling is altered under DD conditions in clock mutants SLOB cycling is altered under DD conditions in clock mutants. (A) Western blot analysis of SLOB cycling in Canton S, per , 01 jrk tim and Clk flies under LD conditions. Oscillations in all flies are in phase with one another. The graphs represent the mean ± SEM for each time point from a minimum of three experiments for each genotype. (B, C) Western blot analysis of Canton jrk 01 jrk 01 S, Clk and per flies in DD conditions. There is no obvious cycling of SLOB in Clk and per flies. (D) Western blot analysis 01 01 of Canton S and tim flies in DD conditions. There is a shift in the phase of SLOB cycling in tim flies. The peak is no longer at CT 14 but at CT 2. The same Canton S data are illustrated in panels (B-D). distribution of mRNA and protein are comparable, there identified three distinct subsets of PI neurons (PI 1–3) are apparent differences in the abundance of protein rela- that innervate the corpora cardiaca (a glandular tissue) tive to that of mRNA in some regions. This may simply be and the aorta. To determine which subset of PI neurons is due to the histological differences between head sections SLOB positive we used four of their enhancer trap lines. (Figure 3) and brain wholemounts (Figure 4B), or to the Mai 301, Kurs 58 and Kurs 45 GAL4 lines express GAL4 different detection methods used. It is also possible that exclusively in subsets PI-1, PI-2 and PI-3, respectively. Mai SLOB protein does not accumulate everywhere the mRNA 281 GAL4 expresses GAL4 in two subsets, PI-2 and PI-3. is found. SLOB protein is also expressed in other head tis- We crossed these GAL4 flies to a UAS-GFP transgenic line, sues such as the antenna and proboscis (data not shown). and found by immunostaining that SLOB is expressed exclusively in subset PI-3 (Figure 4F,4G). SLOB does not Siegmund and Korge [22] performed a large scale analysis colocalize with GFP from the enhancer fly lines that of peptidergic neurons of Drosophila larvae. Their study express GFP only in subsets PI-1 or -2 (Figure 4D,4E). Page 4 of 14 (page number not for citation purposes) Normalized SLOB Normalized SLOB Normalized SLOB Normalized SLOB BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 A B DF E Distribution of Figure 3 slob mRNA Distribution of slob mRNA. Frontal sections of Drosophila heads were probed with digoxigenin labeled RNA probes for slob. (A) Low magnification to survey entire head section. Antisense slob probes hybridized to the photoreceptors, optic lobe, PI neurons and the surrounding brain cortex. (B) High levels of expression detected in the photoreceptors, lamina and medulla. (C) slob message appears in the PI region. The arrows point to the dorsal medial PI neurons. (D-F) Digoxigenin labeled sense probes of slob reveal no significant labeling. The three panels correspond to the head sections of (A-C). Figure 4H highlights components of the circadian system DNs are immunostained red indicating the presence of in order to illustrate the location of the PI neurons in rela- PER. Projections from the LN s are green due to PDF tion to other clock gene expressing cells. The lateral neu- immunostaining. The PI neurons are the SLOB positive rons (LNs) as mentioned before consist of three clusters, a green cells. cluster of cells located dorsally (LN s), and two other ven- The phase of SLOB protein cycling is different in the trally located clusters differing in the size of their somata, large LN s and small LN s. Another group of clock gene photoreceptor and PI neurons v v expressing cells are the dorsal neurons (DNs), which con- Immunohistochemistry was used to determine whether sists of three subsets as well, DN . This wholemount was SLOB cycles in the photoreceptor and PI neurons. Flies 1–3 immunostained for SLOB, PER and PDF. The LNs and were entrained for three days in LD and collected at four Page 5 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 time points; others were then transferred to DD for two A B additional days and likewise collected at four time points. Antibody against SLOB was used on whole head sections and wholemounts of the brain. We find that SLOB protein cycles in the nuclei of the photoreceptor neurons with a trough at ZT 2 and a peak at ZT 14–21 (Figure 5A). During constant darkness there is a peak at around CT 14 (Figure 5A). This correlates with western blot data collected from the entire Drosophila head (see Figure 2). Intriguingly, in the PI neurons, SLOB cycles with a peak at ZT 3 and a trough at ZT 15–21 (Figure 5B), out of phase to the oscil- lation in the photoreceptor neurons. SLOB continues to oscillate during constant darkness conditions in both the photoreceptor and PI neurons (Figure 5A,5B). DN /DN DN 01 Ectopic expression of SLOB in tim flies PI We noticed an increase in SLOB protein levels, at all time DE LN points examined by western blot, in tim flies. To locate LN v 01 where the increase in SLOB occurs, we stained tim heads with anti-SLOB antibody, and found considerable SLOB F G H expression in the optic lobe, specifically in the outer edges of the lamina (Figure 6A). Samples shown here are at ZT SLOB is n Figure 4 eurons expressed prominently in photoreceptor cells and PI 14. Expression in these regions is much more limited in SLOB is expressed prominently in photoreceptor cells and PI the wild type controls. No increases in SLOB expression neurons. (A) Head frontal section from y w (wild type) flies were detectable in the PI neurons of tim flies (Figure 6B). immunostained for SLOB protein. SLOB is expressed in the nuclei of the photoreceptor cells. (B) Brain wholemounts Since SLOB protein normally appears to be expressed at from y w flies immunostained for SLOB protein. SLOB is relatively low levels in the optic lobe, we infer that the loss expressed prominently in the PI neurons of the protocere- of TIM leads to elevated or ectopic expression of SLOB in brum (box), as well as elsewhere in the brain. (C) Enlarge- specific cells. We cannot exclude the possibility that the ment of the boxed area in (B) to illustrate the cytoplasmic SLOB signal from tim flies seen on western blots repre- localization of SLOB in the PI neurons. Typically 6–8 PI neu- sents other head tissues as well. This elevated expression rons are SLOB positive. (D-G) GAL4 flies, specific for the of SLOB in tim flies further demonstrates the regulation three subsets of PI neurons, were crossed to UAS-GFP flies of SLOB expression by the clock genes. and immunostained for SLOB. (D) Mai 301 GAL4 is expressed in PI-1 neurons [22], shown by GFP expression. SLOB is regulated by a circadian output molecule, PDF UAS-GFP tends to have a nuclear localization. SLOB positive The neuropeptide, PDF, accumulates at dorsal axon termi- PI neurons are immunostained with Texas Red, and SLOB has a cytoplasmic expression pattern. SLOB does not localize nals of the small LN in a cyclic fashion, indicative of vs within PI-1 neurons. (E) Kurs 58 GAL4 is expressed in PI-2 regulated release from these terminals [13]. After two to neurons. SLOB is not found within the PI-2 neurons. (F) Mai three days in constant darkness, the majority of Pdf null 281 GAL4 is expressed in two of the three subsets of PI neu- flies are behaviorally arrhythmic supporting a role for PDF rons (PI-2 and PI-3). SLOB is localized within some of these in clock output [23]. The location of SLOB in PI neurons PI neurons and because SLOB was not found within PI-2 this makes it a candidate for an output molecule. To deter- suggests that the SLOB positive subset is PI-3. (G) Kurs 45 mine whether SLOB is a component of the output path- GAL4 is expressed only in PI-3 neurons. SLOB positive cells way, we analyzed SLOB expression in the PI neurons of a colocalize with the GFP expressing PI-3 cells. (H) Brain jrk 01 clock mutant, Clk , and the output mutant, Pdf . Flies wholemount from y w flies highlighting components of the were entrained and collected at four time points. Western circadian system – the small ventral lateral neurons (LN ), the dorsal lateral neurons (LN ), the dorsal neurons (DN ) analysis of head lysates during DD indicated that SLOB d 1–3 jrk and the pars intercerebralis neurons (PI). The wholemount cycling is dampened or absent in Clk flies (Figure 3B). was immunostained for SLOB, PER and PDF. The red com- Similarly, SLOB cycling in DD is diminished in the PI neu- ponent is PER staining; the green staining in the PI neurons jrk rons in Clk flies (Figure 7A). This suggests that CLK is at corresponds to SLOB, and the green staining elsewhere is least indirectly required for SLOB oscillation in the PI neu- PDF. Previous staining in Pdf flies reveals no PDF in the PI rons. Wholemounts of brains from Pdf flies reveal a neurons (data not shown). PDF filled projections end close decrease in SLOB expression and dampened cycling in the to, but appear to stop short of, the PI neurons. The projec- PI neurons (Figure 7B), consistent with a role for SLOB tions of the DN (not visible) may synapse on the PI neurons. downstream of the PDF-secreting neurons. Page 6 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 ZT2 ZT9 ZT14 ZT21 4 4 ** 3 3 2 2 1 1 0 0 ZT14 ZT2 ZT9 ZT21 CT2 CT9 CT14 CT21 CT3 CT9 CT15 CT21 4 4 3 3 ** 2 2 1 1 0 0 ZT3 ZT9 ZT15 ZT21 CT3 CT9 CT15 CT21 SLOB cycles in the photorec Figure 5 eptor cell and the PI neurons SLOB cycles in the photoreceptor cell and the PI neurons. y w flies were entrained in LD for three days and collected at four time points over a 24 hour period. Some flies, after three days entrainment, were transferred to DD for two additional days. At least 5 heads were assayed per time point. (A) Head frontal sections were immunostained for SLOB. The bars depict mean ± SEM staining intensity scores (*p < 0.05, ** p < 0.001). The left bar graph is for LD and the right is for DD. SLOB cycling in the photoreceptors closely follows that of the western blots for both LD and DD. (B) Brain wholemounts from y w flies immu- nostained for SLOB. SLOB cycles in the PI neurons with an altered phase compared to the photoreceptors. The left bar graph is for LD and the right is for DD. Statistical analysis was done as in (A). Page 7 of 14 (page number not for citation purposes) Relative Intensity Score Relative Intensity Score Relative Intensity Score Relative Intensity Score BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 Optic lobe ** ry jrk Clk CT9 CT14 CT14 CT9 CT9 CT14 jrk Clk y w ry tim Brain wholemount ** y w Pdf CT9 CT15 CT9 CT15 CT9 CT15 y w tim y w Pdf 01 jrk 01 Figure 6 Elevated or ectopic expression of SLOB in tim flies Figure 7 SLOB expression is altered in Clk and Pdf flies jrk 01 Elevated or ectopic expression of SLOB in tim flies. (A) SLOB expression is altered in Clk and Pdf flies. (A) Brain jrk Head sections from y w and tim flies immunostained for wholemount from wild type rosy (ry) and Clk flies. Two time points, taken on the second day of DD, show that SLOB SLOB. There is greater expression of SLOB in the optic lobe 01 jrk in tim flies. The arrowheads point to the outer edges of the does not cycle in the PI neurons of the Clk flies. The accom- lamina. (B) Brain wholemounts from y w and tim flies panying bar graph illustrates relative intensity scores. Data immunostained for SLOB. There is no change in SLOB were analyzed as described in Figure 5. (B) Brain wholem- 01 01 ount from wild type y w and Pdf flies. Two time points, expression within the PI neurons of the tim flies. taken on the second day of DD, demonstrate the decrease in SLOB expression and dampened cycling in the PI neurons of Pdf flies. PDF appears to positively regulate SLOB protein. The bar graph represents relative intensity scores. Overexpression of SLOB alters locomotor activity To determine whether manipulation of SLOB levels can alter behavioral rhythms during constant darkness conditions, we created two transgenic UAS (upstream acti- protein results in the breakdown of rhythms suggesting vational sequence) lines. One line, UAS-slob, expresses a that Leonardo binding is not required for this wild type version of the SLOB protein whereas the second, phenomenon. There is some breakdown in rhythmicity in UAS-mtslob, expresses a mutant form of SLOB that renders UAS-slob1/+ flies (Figure 8B), but this is most likely due to it unable to bind Leonardo [6]. The UAS lines were leaky expression of the UAS-slob transgene (inset in Figure C155 crossed to elav (panneuronal), GMR (eye) and Kurs 45 8B). However, breakdown in rhythmicity does not occur (PI-3) GAL4 drivers. Flies were entrained for three days in in the UAS-mtslob/+ flies (Figure 8B), and western blot LD cycles and then monitored for rest:activity in DD for analysis indicates no such leaky expression of the UAS- 14 days. Progeny of the crosses between UAS-slob or UAS- mtslob transgene (inset in Figure 8B). Fly lines carrying c155 mtslob and elav GAL4 are robustly rhythmic for the first either the GMR or K45 driver along with UAS-slob show no seven days, followed by a breakdown in their rhythms decrease in rhythmicity (Figure 8C), suggesting that SLOB during the last seven days (Figure 8A). This breakdown is overexpression in the eye or PI-3 may not be the cause of not seen in control lines. Thus, the lines that overexpress the behavioral breakdown. These data indicate that SLOB exhibit a decrease in the strength of rhythmicity ectopic SLOB expression is capable of altering a behavio- determined by their Fast Fourier Transform (FFT) value ral rhythm produced by the circadian system. during the last seven days, as compared to the control lines (Figure 8B). Overexpression of either form of SLOB Page 8 of 14 (page number not for citation purposes) Relative Intensity Score Relative Intensity Score BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 c155 c155 c155 elav , df(1)w elav ; UAS-slob/+ elav ; UAS-mtslob/+ Days 1-7 Days 8-14 mtslob1/+ slob1/+ 0.16 0.14 0.12 ** 0.10 ** ** 0.08 0.06 0.04 0.02 0.00 slob2/+ mtslob1/+ elav, elav; elav; elav; slob1/+ Df(1)w slob1/+ slob2/+ mtslob1/+ Days 1-7 Days 1-7 Days 8-14 Days 8-14 0.16 0.25 0.14 0.12 0.20 0.10 0.15 0.08 0.06 0.10 0.04 0.05 0.02 0.00 0.00 GMR/ GMR, GMR/ Kurs 45, Kurs 45; Kurs 45; slob1/+ mtslob1/+ Df(1)w Df(1)w mtslob1/+ slob2/+ Overexpressi Figure 8 on of SLOB alters behavioral rhythms c155 Overexpression of SLOB alters behavioral rhythms. Elav , GMR and Kurs 45 GAL4 flies were crossed to UAS-slob, UAS-mtslob and Df(1)w flies. Progeny were entrained for three days in LD cycles and then transferred to a locomotor monitoring device c155 for 14 days in DD. (A) Actograms representing control, UAS-slob and UAS-mtslob lines crossed to elav . There is an apparent breakdown of rhythmicity after 7 days in flies overexpressing wild type and mutant SLOBs (middle and right panels). (B) Sum- c155 mary of behavioral data for elav flies crossed to control and UAS lines. The FFT value is a measure of rhythmic strength and is plotted in the bar graph for the first and second week periods per fly line. Using Student's t test, all overexpressing SLOB lines exhibit significant breakdown of rhythms during the second seven days (*p < 0.05 and **p < 0.001, respectively). Controls show no significant breakdown except for one line, UAS-slob1/+. However, as demonstrated by the inserted western blot, this line expresses transgenic SLOB in the absence of a GAL4 driver, whereas the UAS-mtslob1/+ line does not. The blot was probed with an anti-HA antibody that detects only transgenic SLOB. Levels of MAPK were used as loading controls (data not shown). (C) Summary of behavioral data for GMR and Kurs 45 GAL4 flies crossed to various lines. No change in rhythmicity is evident when SLOB is overexpressed only in the Drosophila eye or PI neurons. Page 9 of 14 (page number not for citation purposes) Relative Power FFT Relative Power FFT Relative Power FFT BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 The PI region lies directly beneath the root of the ocellar Discussion We report here that the dSlo binding protein, SLOB, cycles nerve. The PI neurons have large, 15 µm diameter, cell in Drosophila heads. Microarray analyses reported slob bodies, and their axons project along the median bundle transcript cycling with a peak at either ZT 15 [2] or at CT and then bifurcate [27]. One of the branches proceeds 11 [3]. We show by western analysis that SLOB protein ventrally and arborises in the dorsal tritocerebrum region, peaks at ZT 10–14/CT 14, consistent with an earlier peak below the oesophagus. The other branch moves in a for the RNA. Under LD conditions, SLOB continues to posterior direction and enters the cardiac recurrent nerve 01 01 jrk cycle in per , tim and Clk flies in phase with the oscil- in the oesophageal canal. The PI neurons have an exten- lation of wild type flies. It was noted in one of the recent sive network of endoplasmic reticulum and contain secre- microarray studies [2] that there is a cluster of genes, tory granules, suggesting that they are neurosecretory cells named the apterous cluster, that shows rhythmicity in [27]. In insects, peptidergic neurons of the central nervous these three mutants during LD. This cluster has a charac- system regulate the synthesis of developmental hor- teristic peak at ZT 17 and includes such proteins as tran- mones. The PI neurons, in particular, have been impli- scription factors, synaptic regulators and transporters. The cated in hormone production and release in various genes in this group may be regulated not only by the cir- insects [28,29]. Three subsets of PI neurons have been cadian clock, but also by a light-dependent mechanism. identified. We have identified the subgroup PI-3 to be the However in DD, where light is no longer a factor, we find SLOB positive subset of PI neurons. Among the hormones jrk 01 that SLOB does not cycle in Clk and per and exhibits an identified in the PI neurons is insulin, and it has been 01 01 altered phase in tim flies. One might expect tim and proposed that the release of insulin into the hemolymph 01 01 per flies to give similar results, but tim flies may not be is essential for growth control and carbohydrate homeos- true genetic nulls [24]. Consistent with the observation tasis [30]. We have confirmed that the SLOB positive PI jrk that SLOB does not cycle in Clk flies during DD (Figure neurons are also insulin positive (data not shown). 2B) are microarray data from Ueda et al. [5] indicating jrk that slob levels do not change in Clk flies, and other The photoreceptors and the PI neurons express oscillating microarray data from McDonald and Rosbash [3] demon- SLOB protein and intriguingly, the rhythms in the two jrk strating that slob levels are at mid-point in the Clk neuronal types are not in phase with each other. The mutants. Taken together these data demonstrate clearly mechanisms responsible for these phase differences are that clock genes regulate slob mRNA and protein not known. PER and TIM are expressed in the lateral neu- expression. rons in the central brain, in glial cells of the optic lobes, and in the photoreceptor cells [7]. PER and TIM protein Within Drosophila adult heads, slob mRNA is present in the have not been shown to be expressed in the PI neurons, photoreceptors, optic lobe, the neurosecretory cells of the although Kaneko and Hall [31] found that there is expres- PI and the surrounding brain cortex. We also find promi- sion of GAL4 driven by the per promoter in PI neurons. nent immunostaining for SLOB protein in the photore- The photoreceptors in contrast have all the traditional ceptor cells and the PI neurons. In situ hybridization clock genes that might contribute to SLOB cycling [32- experiments with larval brain revealed slob RNA in an area 34]. The presence of these genes in the eye, but not in the of the brain close to PDF-filled projections of the lateral PI, may contribute to the phase differences. In addition, it neurons [2]. This is consistent with our findings of slob is reasonable to expect that there will be molecular differ- transcript in the PI neurons and surrounding cortex. ences in circadian regulation in different cell types. Fur- thermore, the subcellular localization of SLOB is different The Drosophila eye expresses many of the major circadian between the two areas (Figure 4A,4C). SLOB appears to be genes, and is thought to contain an autonomous primarily cytoplasmic in the PI neurons, and nuclear in oscillator that presumably regulates an eye-specific func- the photoreceptors. tion [17]. In addition, the eye contributes to photic entrainment of the pacemaker LN cells [25]. Interestingly, How is SLOB regulated in the PI neurons? Interestingly, the SLOB binding partner, Slo is also expressed in the vis- the PI neurons have been associated with behavioral ual system including the eye, lamina, medulla and lobula rhythmicity and it has been hypothesized that this [26]. Ceriani et al. [1] have demonstrated that the RNA involves hormone release [35]. In fact, the PI neurons of levels of both slob and slo cycle in phase in both LD and Teleogryllus commodus (crickets) have long been hypothe- DD. The dSlo protein was also shown by western blot to sized to serve as a region of coupling between the cycle and peak at ZT 20 [1]. This correlates with the cycling circadian pacemakers and behavioral rhythms [36]. A of SLOB in the photoreceptors, where SLOB peaks at ZT pathway for Drosophila proposed by Kaneko and Hall [31] 14–21 (Figure 5A). suggests that oscillatory signals of the "master pacemaker" in the small LNvs first modulate the oscillatory mecha- nism or neuronal activity operating within neurons in the Page 10 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 dorsal region. The DNs send their oscillatory signals to the mutants such as Pdf flies [13]. Likewise, panneuronal PI, which may lead to rhythmic neurosecretory peptide overexpression of PDF results in a delayed disruption of release. It has been observed in the larval CNS that projec- rhythms, and overexpression of PDF in the PI neurons tions of the DNs terminate near the midline in the PI results in a shortened period and an advance of the morn- region [31]. These DNs express both PER and TIM and ing peak [39]. disconnected (disco) flies, which lack LNs, hence may send robust oscillatory signals to downstream also become arrhythmic only after several days in DD targets such as PI neurons. Interestingly, two neurons of [40]. the DN group express PER and TIM cycling antiphase to the other DNs and LNvs [31]. Regardless of the precise Using GAL4 drivers that direct overexpression of SLOB role of the DNs, it is clear that this dorsal region of the specifically to the eye or PI neurons, we found no obvious brain is important for rest:activity rhythms. For example, circadian locomotion phenotype. It is possible that these PDF release and MAPK activity cycle specifically in this drivers are not strong enough, compared to the panneuro- c155 region, and both participate in behavioral rhythmicity nal driver elav . We note that the overexpression of PER [13,37]. and TIM with the tim-GAL4 driver results in a more severe phenotype than with per-GAL4, even though the expres- Clock mutants alter either SLOB protein oscillation or lev- sion pattern of the two drivers is similar [24], possibly els in both the eye and PI neurons. One striking observa- because the per promoter is weaker [41]. Alternatively, the tion is the ectopic or elevated expression of SLOB in the specific drivers we used may not target all the lamina of tim flies. This suggests that TIM negatively reg- behaviorally-relevant SLOB positive neurons in the eye ulates SLOB. Western analyses show that upregulation of and PI region. Any SLOB positive neurons that are not SLOB does not occur in per flies. Ectopic expression of overexpressing SLOB are therefore wild type, and this may PER occurs in double-time (dbt) flies that are mutant for a prevent rhythmicity breakdown. Similar explanations casein kinase 1ε involved in PER turnover [38]. The inter- have been proposed for the lack of a phenotype when PER pretation in that case is that PER is synthesized in many and TIM are overexpressed by the pdf-GAL4 driver cell types where its expression is normally undetectable although both genes cause arrhythmia when their overex- due to destabilization by the kinase. A similar mechanism pression is driven by the more widely expressed tim and flies. may account for elevated expression of SLOB in tim per-GAL4 drivers [24,42]. The observation that tim alters SLOB expression while per does not could suggest a pathway for slob regulation The widespread distribution of SLOB in the eye and brain that is independent of PER. We also found that SLOB fails suggests that other cells, in addition to or instead of the jrk to cycle in the PI neurons of Clk flies. photoreceptor and PI neurons, may account for SLOB's apparent role in behavioral rhythms. The panneuronal Perhaps most intriguing is the decrease of SLOB in the PI overexpression of SLOB in other SLOB-expressing cells in neurons of Pdf flies. The oscillation of PDF is restricted the brain cortex may explain the rhythmic breakdown. to the dorsal projections emanating from the lateral neu- Alternatively, ectopic expression of SLOB in neurons rons [13]. Dorsal terminals of the LNs express abundant involved in locomotor rhythms might also account for the PDF early in the morning, which is indicative of a block in altered rhythmicity. its release. Thus, PDF release is low during the day while SLOB is at its trough, consistent with PDF being a positive Conclusions regulator of SLOB. In Figure 4H we see that PDF express- In this study, we have demonstrated that SLOB protein ing terminals do not appear to contact the PI neurons. As cycles in a circadian fashion in Drosophila heads. SLOB discussed above, we hypothesize that PDF termini affect oscillates in two discrete areas of the fly head, the photore- the DN, or alternatively, other neurons of the dorsal ceptor cells and the PI neurons. Our results reveal differ- region, which in turn communicate with the PI neurons. ential effects of clock mutations on SLOB expression and jrk Clk flies lack PDF in the small ventral LNs, but still cycling in these two regions. There is a significant decrease express it in the large LNs [13]. This may account for the in SLOB levels in the PI neurons of Pdf flies, thus impli- jrk 01 difference in the phenotype of Clk and Pdf mutants cating PDF as an upstream regulator of SLOB. Along with and would suggest a role for the large LNs in SLOB the observation that flies overexpressing SLOB exhibit a regulation. breakdown of rest:activity patterns, these data suggest that SLOB is a clock controlled protein. The molecular oscillations of the circadian clock proteins result ultimately in behavioral rhythmicity [24]. Our data We showed previously that SLOB, along with dSlo and demonstrate that the panneuronal expression of SLOB Leonardo, participates in a dynamic regulatory complex causes a delayed breakdown of rhythms. Breakdown after in presynaptic nerve terminals. Leonardo binding is several days is characteristic of some circadian output dynamically regulated by phosphorylation of SLOB by the Page 11 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 calcium/calmodulin-dependent protein kinase II (CAM- chemiluminescence detection system (Amersham) was KII) [6]. Intriguingly, CAMKII has recently been impli- used to visualize the proteins. Film exposures of western cated in circadian rhythmicity in vertebrates [43], and it is blots were scanned using Bio Rad Molecular Analyst. The tempting to speculate that this regulatory complex partic- level of SLOB at each time point was calculated as the ipates in the fly circadian output pathway. Not only do SLOB signal minus the background in each lane. The blots dslo mutants have weak rhythms [1,44], but leonardo were stripped and reprobed with anti-MAP Kinase mutants have defects in behavior, synaptic transmission (Sigma). The ratio of SLOB to MAP Kinase was normal- and plasticity [45,46]. Our data are consistent with the ized and averaged between several westerns. hypothesis that SLOB participates in circadian rhythmic- ity by regulating synaptic function and membrane Immunohistochemistry excitability. Brain whole mount: Flies were entrained in LD for three days and then transferred to DD. Fly heads were collected at given time points, fixed in 4% paraformaldehyde Methods Fly stocks and germ line transformation (PFA), and the brains were dissected and kept in cold PBS, D. melanogaster strains Canton S (wild type), y w, ry, tim , and subsequently blocked with 6% normal donkey serum 01 jrk 01 per , Clk , and Pdf and transgenic fly strains were raised in phosphate-buffered saline (PBS)/0.3% Triton X-100 for at 25°C on standard Drosophila medium. slob cDNA was one hour. Samples were then incubated with primary cloned into a pUAST vector and P-element-mediated antibody at a dilution of 1:400 overnight at 4°C. After transformation was performed as described previously washing in PBS/0.3% Triton X-100 three times for 30 min C155 [47]. The transformed lines were crossed to either elav each at room temperature, samples were incubated with (provided by Leslie Griffith), GMR (provided by Konrad the appropriate secondary antibody (Fluorescein (FITC)- Zinsmaier) or Mai 281, Mai 301, Kurs 45, and Kurs 58 conjugated AffiniPure Donkey Anti-Rabbit IgG and Texas GAL4 (provided by Gunter Korge). Red dye-conjugated AffiniPure Donkey Anti-Rabbit IgG from Jackson ImmunoResearch) at a dilution of 1:500 in In situ hybridizations 3% normal donkey serum in PBS/0.3% Triton X-100 for 1 The slob RNA antisense and sense probes were synthesized hour at room temperature, and washed in PBS three times using the DIG RNA Labeling Mix (Boehringer Manheim). for 30 minutes each. Brains were mounted onto slides The sequence used for the RNA probes was made from with mounting medium (Vector H-1200). Wholemounts basepairs 1142–1441 of the slob transcript. In situ hybrid- were visualized using fluorescence microscopy on a Leica ization on adult 12 µm head sections were done according DMIRE2. to the protocols found at http://www.rockefeller.edu/lab heads/vosshall/protocols.php with slight modifications. Section: Flies were collected at given time points, All hybridizations and washes were done at 55°C. Sec- mounted with mounting medium, and sectioned at 12 tions are developed in the dark for 3 days. µm using a cryostat (Leica 3450). Sections were fixed with 4% PFA, washed in PBS/0.3% Triton X-100, and blocked SLOB antibody purification with 6% normal donkey serum in PBS/0.3% Triton X-100 A GST-SLOB fusion protein was used to immunize rabbits for 1 hour, and subsequently incubated with primary anti- as described previously [48]. Polyclonal antibodies body in 6% normal donkey serum in PBS/0.3% Triton X- specific to SLOB were generated by purifying the serum 100 overnight at 4°C. The sections were then washed and using a combination of CNBr conjugated GST and CNBr incubated with secondary antibodies as described above. conjugated GST-SLOB columns. The staining intensity of brain whole mounts and sections Western blotting and quantitation was assessed by blind scoring. A subjective intensity scale Flies were entrained and collected in LD and DD condi- from zero to four was used, with zero being undetectable tions at four hour intervals. Fly heads were lysed in 1% and four being maximal. Statistical analysis of average CHAPS, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 120 staining intensity scores was done using ANOVA and the mM NaCl, 50 mM KCl, 2 mM DTT and protease inhibitors Tukey HSD test. (1 mM PMSF, 1 µg/mL each aprotonin, leupeptin, and pepstatin A (SIGMA)). Protein concentration was deter- Behavioral analysis mined using the BioRad DC Protein Assay. 100 µg of pro- Flies aged from 1–5 days were entrained for three days in tein was loaded on 4–15% polyacrylamide gradient gels 12 hr light/dark cycles at 25°C and then kept in constant and transferred to nitrocellulose membranes. After block- darkness for 14 days. Activity was monitored by using the ing with 5% nonfat milk in TBST (0.1% Tween 20 in Tris- Trikinetics system. Individual flies were analyzed for buffered saline), the blots were probed with the appropri- rhythmicity based on their by chi-square periodogram ate primary and secondary antibodies. Enhanced Page 12 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 15. Colwell CS: Circadian modulation of calcium levels in cells in and Fast Fourier Transform (FFT) values [24]. The analy- the suprachiasmatic nucleus. Eur J Neurosci 2000, 12:571-576. ses were performed using ClockLab software. 16. Colwell CS: NMDA-evoked calcium transients and currents in the suprachiasmatic nucleus: gating by the circadian system. Eur J Neurosci 2001, 13:1420-1428. Authors' contributions 17. Cheng Y, Hardin PE: Drosophila photoreceptors contain an AMJ performed the SLOB antibody purification, western autonomous circadian oscillator that can function without period mRNA cycling. J Neurosci 1998, 18:741-750. blotting and quantitation, immunohistochemistry, in situ 18. Edery I, Zwiebel LJ, Dembinska ME, Rosbash M: Temporal phos- hybridization and behavioral analysis. XZ carried out phorylation of the Drosophila period protein. Proc Natl Acad Sci immunohistochemistry and scoring analysis. DAA and AS U S A 1994, 91:2260-2264. 19. Sehgal A, Rothenfluh-Hilfiker A, Hunter-Ensor M, Chen Y, Myers MP, participated in the immunohistochemistry and western Young MW: Rhythmic expression of timeless: a basis for pro- blotting. 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Pattern of distribution and cycling of SLOB, Slowpoke channel binding protein, in Drosophila

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Copyright © 2004 by Jaramillo et al; licensee BioMed Central Ltd.
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Biomedicine; Neurosciences; Neurobiology; Animal Models
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Abstract

Background: SLOB binds to and modulates the activity of the Drosophila Slowpoke (dSlo) calcium activated potassium channel. Recent microarray analyses demonstrated circadian cycling of slob mRNA. Results: We report the mRNA and protein expression pattern of slob in Drosophila heads. slob transcript is present in the photoreceptors, optic lobe, pars intercerebralis (PI) neurons and surrounding brain cortex. SLOB protein exhibits a similar distribution pattern, and we show that it cycles in Drosophila heads, in photoreceptor cells and in neurosecretory cells of the PI. The cycling of SLOB is altered in various clock gene mutants, and SLOB is expressed in ectopic locations in 01 jrk tim flies. We also demonstrate that SLOB no longer cycles in the PI neurons of Clk flies, and that SLOB expression is reduced in the PI neurons of flies that lack pigment dispersing factor (PDF), a neuropeptide secreted by clock cells. Conclusions: These data are consistent with the idea that SLOB may participate in one or more circadian pathways in Drosophila. SLOB/dSlo complex dramatically shifts the channel volt- Background Five independent groups [1-5] recently conducted age range of activation to more depolarized potentials [6]. genome-wide microarray analyses to identify Drosophila transcripts that display circadian oscillations. Each group The circadian system consists of an input pathway, a cen- uncovered slob as a robustly cycling RNA transcript. SLOB, tral clock, and an output pathway [7]. The clock itself is Slowpoke binding protein, is a key component of the Dro- comprised of the transcription factors CLOCK (CLK) and sophila Slowpoke/SLOB/Leonardo dynamic protein com- CYCLE (CYC), which bind to the promoters of period (per) plex [6]. This complex is thought to affect membrane and timeless (tim), inducing their expression. The PER and excitability, as electrophysiological recordings reveal that TIM proteins heterodimerize and feed back to repress SLOB binding to the channel results in an increase in activity of CLK/CYC. Although the expression patterns of channel activity, whereas the addition of Leonardo to the CLK and CYC are not known, the localization of PER and Page 1 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 TIM has been important with respect to identifying neu- for three days in 12:12 hour light/dark cycles (LD). We rons relevant for circadian behavioral rhythms [8]. This found that SLOB protein cycles (Figure 1A) with a peak at molecular loop must transduce signals to surrounding Zeitgeber Time (ZT) 10–14, and a trough around ZT 2 (ZT cells to generate rhythmic behavior [9]. Altering mem- 0 = lights on; ZT 12 = lights off in a 12 hour:12 hour light/ brane excitability is a key mechanism for transducing neu- dark cycle). ronal information, and thus it is reasonable to suspect that the molecular feedback loop might communicate To ensure that rhythms of SLOB expression are circadian with ion channels. and not driven only by external LD cycling we analyzed SLOB protein cycling in conditions of constant darkness. Ion channels may be under direct transcriptional control Flies were entrained for three days in LD, and then placed of the clock genes or modulated by clock-controlled genes in constant darkness (DD) for two days. SLOB protein still [10,11]. Mounting evidence supports the importance of cycles under these conditions, with a slightly dampened electrical activity for the propagation of circadian oscilla- amplitude, but with the same overall phase (Figure 1B). A tions. For example, electrical silencing of clock neurons similar decrease in amplitude has been described for oscil- through targeted expression of potassium channels stops lations of other clock genes in DD [18,19]. the oscillation of PER and TIM proteins and causes arrhythmicity in flies [12]. The cyclic release of neuropep- Expression of SLOB protein is influenced by clock genes tides from clock cells [13] may be a direct consequence of To determine if the clock genes control the cycling of a rhythmic fluctuation in membrane potential [14]. Diur- SLOB, we assayed its expression in different mutant back- jrk nal modulation of pacemaker potentials and calcium cur- grounds. Clk flies, flies mutant for CLK, were entrained rent, intracellular calcium levels and NMDA-evoked in LD cycles for three days and then collected at six time calcium currents have all been observed within a mamma- points. Some flies were transferred from LD to DD for two lian central clock, the suprachiasmatic nucleus (SCN) additional days and likewise collected at six time points. [11,15,16]. In addition, microarray screens have detected Western blot analysis of Drosophila heads shows that jrk the cycling transcripts of ion channels such as Shaker, trpl SLOB protein continues to cycle in Clk adults in LD, with and slowpoke [1,3], and flies mutant in slowpoke have weak a phase and amplitude similar to wild type (Figure 2A). In locomotor rhythms [1]. These observations suggest that DD, the western blot analysis shows SLOB expression to ion channels and their modulators may participate in cir- have no discernable peak indicating that it does not cycle cadian regulation. (Figure 2B). Claridge-Chang et al. [2] noted that slob RNA jrk is down regulated in the Clk mutant, suggesting that CLK We explored a role for SLOB in the circadian system and acts as a transcriptional activator of slob expression. We found that SLOB protein cycles in Drosophila heads during did not observe a dramatic decrease in protein levels in jrk both light/dark and constant darkness conditions. SLOB Clk flies; the level was between the trough and peak lev- oscillates in at least two discrete areas of the fly head, the els seen in wild type flies. photoreceptor cells and the PI neurons. The photorecep- 01 01 tors have their own peripheral circadian oscillator [17], The cycling of SLOB was also analyzed in per and tim whereas the PI neurons, large neurosecretory neurons, are mutants during LD (Figure 2A) and DD (Figure 2C,2D). suspected to play a role in the output pathway that drives In both mutant lines, SLOB continues to cycle in LD with rest:activity rhythms (Kaneko and Hall, 2000). Our results a phase similar to that of wild type (Figure 2A). SLOB may reveal differential effects of clock mutations on SLOB be regulated by a direct light-dependent mechanism, expression and cycling in these two regions. There is a sig- obviating the need for these clock genes in LD. As in the 01 jrk nificant decrease in SLOB levels in the PI neurons of Pdf case of Clk , there appears to be either dampened or no flies, thus implicating PDF as an upstream regulator of protein cycling in per flies under DD conditions (Figure SLOB. SLOB also no longer cycles in the PI neurons of 2C). However, in tim flies SLOB still cycles, but there is jrk Clk flies, supporting the idea that SLOB is a clock con- a shift in the phase of the oscillation in DD (Figure 2D). trolled protein. Together with the observation that flies Instead of peaking at Circadian Time (CT) 10–14, SLOB overexpressing SLOB exhibit a breakdown of rest:activity now peaks at CT 2. This suggests that the regulation of patterns, these data are consistent with the idea that SLOB SLOB by TIM may be different from that by PER, which is participates in circadian rhythms. not unprecedented [20]. Expression pattern of slob transcript in Drosophila heads Results SLOB protein cycles in Drosophila heads To determine the expression pattern of slob transcript, we To determine whether SLOB protein cycles, we performed performed in situ hybridizations on cryosections of Dro- western blot analysis on Drosophila head lysates from wild sophila yellow-white (y w) fly heads. Digoxygenin-labeled type Canton S (CS) or yellow-white (y w) flies entrained antisense and sense RNA probes of slob were used on Page 2 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 A B 2 6 10 14 18 22 2 6 10 14 18 22 SLOB SLOB MK MK Zeitgeber Time (hrs) Circadian Time (hrs) 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 2 6 10 14 18 22 2 6 10 14 18 22 Zeitgeber Time (hrs) Circadian Time (hrs) SLOB protein Figure 1 cycles in both LD and DD SLOB protein cycles in both LD and DD. (A) Canton S flies were entrained in LD for three days and collected at six time points over a 24 hour period. Fly head lysates were prepared and an anti-SLOB antibody was used to identify the 58 kDa band representing SLOB on the top western blot. The bottom blot was probed for MAPK, the protein level of which does not cycle [37] and was used as a loading control. SLOB oscillation has a trough at ZT 2 and a peak between ZT 10–14. The graph below represents the mean ± SEM for each time point from a minimum of three independent experiments. (B) Canton S flies were entrained in LD for three days and transferred to constant darkness. They were collected on the second day of DD at six time points, and SLOB cycling was determined as in (A). frontal sections and the antisense revealed a widespread SLOB is expressed in the photoreceptors and the brain distribution of slob. Expression of slob was detected in the Previously, we demonstrated that SLOB protein is present brain cortex (Figure 3A), photoreceptors, lamina and at the neuromuscular junction of Drosophila larvae [6]. In medulla (Figure 3B) and the PI region of the brain (Figure order to locate SLOB protein in the adult fly, frontal head 3C). A similar pattern of transcript distribution, particu- sections and wholemounts of the brain were prepared larly in the photoreceptors, optic lobes and brain cortex, from Canton S and y w flies. Immunostaining of sections is seen for timeless, clock and cycle [21]. Figure 3D,3E,3F reveals intense staining in the nuclei of the photoreceptor shows the respective areas of Figures 3A,3B,3C using the cells and the basal cells of the eye (Figure 4A). The brain sense RNA probe for slob. wholemounts reveal very bright and discrete cytoplasmic staining of 6–8 PI neurons, with widespread but less intense staining elsewhere in the optic lobe and brain cor- tex (Figure 4B,4C). Although the overall patterns of Page 3 of 14 (page number not for citation purposes) Normalized SLOB Normalized SLOB BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 per Canton S Canton S 0.2 jrk 01 0.2 jrk Clk tim Clk 0.0 0.0 2 6 10 14 18 22 2 6 10 14 18 22 Zeitgeber Time (hrs) Circadian Time (hrs) C D 1.2 1.2 1.0 1.0 0.8 0.8 0.6 0.6 0.4 0.4 Canton S Canton S 0.2 01 0.2 per tim 0.0 0.0 2 6 10 14 18 22 2 6 10 14 18 22 Circadian Time (hrs) Circadian Time (hrs) SL Figure 2 OB cycling is altered under DD conditions in clock mutants SLOB cycling is altered under DD conditions in clock mutants. (A) Western blot analysis of SLOB cycling in Canton S, per , 01 jrk tim and Clk flies under LD conditions. Oscillations in all flies are in phase with one another. The graphs represent the mean ± SEM for each time point from a minimum of three experiments for each genotype. (B, C) Western blot analysis of Canton jrk 01 jrk 01 S, Clk and per flies in DD conditions. There is no obvious cycling of SLOB in Clk and per flies. (D) Western blot analysis 01 01 of Canton S and tim flies in DD conditions. There is a shift in the phase of SLOB cycling in tim flies. The peak is no longer at CT 14 but at CT 2. The same Canton S data are illustrated in panels (B-D). distribution of mRNA and protein are comparable, there identified three distinct subsets of PI neurons (PI 1–3) are apparent differences in the abundance of protein rela- that innervate the corpora cardiaca (a glandular tissue) tive to that of mRNA in some regions. This may simply be and the aorta. To determine which subset of PI neurons is due to the histological differences between head sections SLOB positive we used four of their enhancer trap lines. (Figure 3) and brain wholemounts (Figure 4B), or to the Mai 301, Kurs 58 and Kurs 45 GAL4 lines express GAL4 different detection methods used. It is also possible that exclusively in subsets PI-1, PI-2 and PI-3, respectively. Mai SLOB protein does not accumulate everywhere the mRNA 281 GAL4 expresses GAL4 in two subsets, PI-2 and PI-3. is found. SLOB protein is also expressed in other head tis- We crossed these GAL4 flies to a UAS-GFP transgenic line, sues such as the antenna and proboscis (data not shown). and found by immunostaining that SLOB is expressed exclusively in subset PI-3 (Figure 4F,4G). SLOB does not Siegmund and Korge [22] performed a large scale analysis colocalize with GFP from the enhancer fly lines that of peptidergic neurons of Drosophila larvae. Their study express GFP only in subsets PI-1 or -2 (Figure 4D,4E). Page 4 of 14 (page number not for citation purposes) Normalized SLOB Normalized SLOB Normalized SLOB Normalized SLOB BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 A B DF E Distribution of Figure 3 slob mRNA Distribution of slob mRNA. Frontal sections of Drosophila heads were probed with digoxigenin labeled RNA probes for slob. (A) Low magnification to survey entire head section. Antisense slob probes hybridized to the photoreceptors, optic lobe, PI neurons and the surrounding brain cortex. (B) High levels of expression detected in the photoreceptors, lamina and medulla. (C) slob message appears in the PI region. The arrows point to the dorsal medial PI neurons. (D-F) Digoxigenin labeled sense probes of slob reveal no significant labeling. The three panels correspond to the head sections of (A-C). Figure 4H highlights components of the circadian system DNs are immunostained red indicating the presence of in order to illustrate the location of the PI neurons in rela- PER. Projections from the LN s are green due to PDF tion to other clock gene expressing cells. The lateral neu- immunostaining. The PI neurons are the SLOB positive rons (LNs) as mentioned before consist of three clusters, a green cells. cluster of cells located dorsally (LN s), and two other ven- The phase of SLOB protein cycling is different in the trally located clusters differing in the size of their somata, large LN s and small LN s. Another group of clock gene photoreceptor and PI neurons v v expressing cells are the dorsal neurons (DNs), which con- Immunohistochemistry was used to determine whether sists of three subsets as well, DN . This wholemount was SLOB cycles in the photoreceptor and PI neurons. Flies 1–3 immunostained for SLOB, PER and PDF. The LNs and were entrained for three days in LD and collected at four Page 5 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 time points; others were then transferred to DD for two A B additional days and likewise collected at four time points. Antibody against SLOB was used on whole head sections and wholemounts of the brain. We find that SLOB protein cycles in the nuclei of the photoreceptor neurons with a trough at ZT 2 and a peak at ZT 14–21 (Figure 5A). During constant darkness there is a peak at around CT 14 (Figure 5A). This correlates with western blot data collected from the entire Drosophila head (see Figure 2). Intriguingly, in the PI neurons, SLOB cycles with a peak at ZT 3 and a trough at ZT 15–21 (Figure 5B), out of phase to the oscil- lation in the photoreceptor neurons. SLOB continues to oscillate during constant darkness conditions in both the photoreceptor and PI neurons (Figure 5A,5B). DN /DN DN 01 Ectopic expression of SLOB in tim flies PI We noticed an increase in SLOB protein levels, at all time DE LN points examined by western blot, in tim flies. To locate LN v 01 where the increase in SLOB occurs, we stained tim heads with anti-SLOB antibody, and found considerable SLOB F G H expression in the optic lobe, specifically in the outer edges of the lamina (Figure 6A). Samples shown here are at ZT SLOB is n Figure 4 eurons expressed prominently in photoreceptor cells and PI 14. Expression in these regions is much more limited in SLOB is expressed prominently in photoreceptor cells and PI the wild type controls. No increases in SLOB expression neurons. (A) Head frontal section from y w (wild type) flies were detectable in the PI neurons of tim flies (Figure 6B). immunostained for SLOB protein. SLOB is expressed in the nuclei of the photoreceptor cells. (B) Brain wholemounts Since SLOB protein normally appears to be expressed at from y w flies immunostained for SLOB protein. SLOB is relatively low levels in the optic lobe, we infer that the loss expressed prominently in the PI neurons of the protocere- of TIM leads to elevated or ectopic expression of SLOB in brum (box), as well as elsewhere in the brain. (C) Enlarge- specific cells. We cannot exclude the possibility that the ment of the boxed area in (B) to illustrate the cytoplasmic SLOB signal from tim flies seen on western blots repre- localization of SLOB in the PI neurons. Typically 6–8 PI neu- sents other head tissues as well. This elevated expression rons are SLOB positive. (D-G) GAL4 flies, specific for the of SLOB in tim flies further demonstrates the regulation three subsets of PI neurons, were crossed to UAS-GFP flies of SLOB expression by the clock genes. and immunostained for SLOB. (D) Mai 301 GAL4 is expressed in PI-1 neurons [22], shown by GFP expression. SLOB is regulated by a circadian output molecule, PDF UAS-GFP tends to have a nuclear localization. SLOB positive The neuropeptide, PDF, accumulates at dorsal axon termi- PI neurons are immunostained with Texas Red, and SLOB has a cytoplasmic expression pattern. SLOB does not localize nals of the small LN in a cyclic fashion, indicative of vs within PI-1 neurons. (E) Kurs 58 GAL4 is expressed in PI-2 regulated release from these terminals [13]. After two to neurons. SLOB is not found within the PI-2 neurons. (F) Mai three days in constant darkness, the majority of Pdf null 281 GAL4 is expressed in two of the three subsets of PI neu- flies are behaviorally arrhythmic supporting a role for PDF rons (PI-2 and PI-3). SLOB is localized within some of these in clock output [23]. The location of SLOB in PI neurons PI neurons and because SLOB was not found within PI-2 this makes it a candidate for an output molecule. To deter- suggests that the SLOB positive subset is PI-3. (G) Kurs 45 mine whether SLOB is a component of the output path- GAL4 is expressed only in PI-3 neurons. SLOB positive cells way, we analyzed SLOB expression in the PI neurons of a colocalize with the GFP expressing PI-3 cells. (H) Brain jrk 01 clock mutant, Clk , and the output mutant, Pdf . Flies wholemount from y w flies highlighting components of the were entrained and collected at four time points. Western circadian system – the small ventral lateral neurons (LN ), the dorsal lateral neurons (LN ), the dorsal neurons (DN ) analysis of head lysates during DD indicated that SLOB d 1–3 jrk and the pars intercerebralis neurons (PI). The wholemount cycling is dampened or absent in Clk flies (Figure 3B). was immunostained for SLOB, PER and PDF. The red com- Similarly, SLOB cycling in DD is diminished in the PI neu- ponent is PER staining; the green staining in the PI neurons jrk rons in Clk flies (Figure 7A). This suggests that CLK is at corresponds to SLOB, and the green staining elsewhere is least indirectly required for SLOB oscillation in the PI neu- PDF. Previous staining in Pdf flies reveals no PDF in the PI rons. Wholemounts of brains from Pdf flies reveal a neurons (data not shown). PDF filled projections end close decrease in SLOB expression and dampened cycling in the to, but appear to stop short of, the PI neurons. The projec- PI neurons (Figure 7B), consistent with a role for SLOB tions of the DN (not visible) may synapse on the PI neurons. downstream of the PDF-secreting neurons. Page 6 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 ZT2 ZT9 ZT14 ZT21 4 4 ** 3 3 2 2 1 1 0 0 ZT14 ZT2 ZT9 ZT21 CT2 CT9 CT14 CT21 CT3 CT9 CT15 CT21 4 4 3 3 ** 2 2 1 1 0 0 ZT3 ZT9 ZT15 ZT21 CT3 CT9 CT15 CT21 SLOB cycles in the photorec Figure 5 eptor cell and the PI neurons SLOB cycles in the photoreceptor cell and the PI neurons. y w flies were entrained in LD for three days and collected at four time points over a 24 hour period. Some flies, after three days entrainment, were transferred to DD for two additional days. At least 5 heads were assayed per time point. (A) Head frontal sections were immunostained for SLOB. The bars depict mean ± SEM staining intensity scores (*p < 0.05, ** p < 0.001). The left bar graph is for LD and the right is for DD. SLOB cycling in the photoreceptors closely follows that of the western blots for both LD and DD. (B) Brain wholemounts from y w flies immu- nostained for SLOB. SLOB cycles in the PI neurons with an altered phase compared to the photoreceptors. The left bar graph is for LD and the right is for DD. Statistical analysis was done as in (A). Page 7 of 14 (page number not for citation purposes) Relative Intensity Score Relative Intensity Score Relative Intensity Score Relative Intensity Score BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 Optic lobe ** ry jrk Clk CT9 CT14 CT14 CT9 CT9 CT14 jrk Clk y w ry tim Brain wholemount ** y w Pdf CT9 CT15 CT9 CT15 CT9 CT15 y w tim y w Pdf 01 jrk 01 Figure 6 Elevated or ectopic expression of SLOB in tim flies Figure 7 SLOB expression is altered in Clk and Pdf flies jrk 01 Elevated or ectopic expression of SLOB in tim flies. (A) SLOB expression is altered in Clk and Pdf flies. (A) Brain jrk Head sections from y w and tim flies immunostained for wholemount from wild type rosy (ry) and Clk flies. Two time points, taken on the second day of DD, show that SLOB SLOB. There is greater expression of SLOB in the optic lobe 01 jrk in tim flies. The arrowheads point to the outer edges of the does not cycle in the PI neurons of the Clk flies. The accom- lamina. (B) Brain wholemounts from y w and tim flies panying bar graph illustrates relative intensity scores. Data immunostained for SLOB. There is no change in SLOB were analyzed as described in Figure 5. (B) Brain wholem- 01 01 ount from wild type y w and Pdf flies. Two time points, expression within the PI neurons of the tim flies. taken on the second day of DD, demonstrate the decrease in SLOB expression and dampened cycling in the PI neurons of Pdf flies. PDF appears to positively regulate SLOB protein. The bar graph represents relative intensity scores. Overexpression of SLOB alters locomotor activity To determine whether manipulation of SLOB levels can alter behavioral rhythms during constant darkness conditions, we created two transgenic UAS (upstream acti- protein results in the breakdown of rhythms suggesting vational sequence) lines. One line, UAS-slob, expresses a that Leonardo binding is not required for this wild type version of the SLOB protein whereas the second, phenomenon. There is some breakdown in rhythmicity in UAS-mtslob, expresses a mutant form of SLOB that renders UAS-slob1/+ flies (Figure 8B), but this is most likely due to it unable to bind Leonardo [6]. The UAS lines were leaky expression of the UAS-slob transgene (inset in Figure C155 crossed to elav (panneuronal), GMR (eye) and Kurs 45 8B). However, breakdown in rhythmicity does not occur (PI-3) GAL4 drivers. Flies were entrained for three days in in the UAS-mtslob/+ flies (Figure 8B), and western blot LD cycles and then monitored for rest:activity in DD for analysis indicates no such leaky expression of the UAS- 14 days. Progeny of the crosses between UAS-slob or UAS- mtslob transgene (inset in Figure 8B). Fly lines carrying c155 mtslob and elav GAL4 are robustly rhythmic for the first either the GMR or K45 driver along with UAS-slob show no seven days, followed by a breakdown in their rhythms decrease in rhythmicity (Figure 8C), suggesting that SLOB during the last seven days (Figure 8A). This breakdown is overexpression in the eye or PI-3 may not be the cause of not seen in control lines. Thus, the lines that overexpress the behavioral breakdown. These data indicate that SLOB exhibit a decrease in the strength of rhythmicity ectopic SLOB expression is capable of altering a behavio- determined by their Fast Fourier Transform (FFT) value ral rhythm produced by the circadian system. during the last seven days, as compared to the control lines (Figure 8B). Overexpression of either form of SLOB Page 8 of 14 (page number not for citation purposes) Relative Intensity Score Relative Intensity Score BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 c155 c155 c155 elav , df(1)w elav ; UAS-slob/+ elav ; UAS-mtslob/+ Days 1-7 Days 8-14 mtslob1/+ slob1/+ 0.16 0.14 0.12 ** 0.10 ** ** 0.08 0.06 0.04 0.02 0.00 slob2/+ mtslob1/+ elav, elav; elav; elav; slob1/+ Df(1)w slob1/+ slob2/+ mtslob1/+ Days 1-7 Days 1-7 Days 8-14 Days 8-14 0.16 0.25 0.14 0.12 0.20 0.10 0.15 0.08 0.06 0.10 0.04 0.05 0.02 0.00 0.00 GMR/ GMR, GMR/ Kurs 45, Kurs 45; Kurs 45; slob1/+ mtslob1/+ Df(1)w Df(1)w mtslob1/+ slob2/+ Overexpressi Figure 8 on of SLOB alters behavioral rhythms c155 Overexpression of SLOB alters behavioral rhythms. Elav , GMR and Kurs 45 GAL4 flies were crossed to UAS-slob, UAS-mtslob and Df(1)w flies. Progeny were entrained for three days in LD cycles and then transferred to a locomotor monitoring device c155 for 14 days in DD. (A) Actograms representing control, UAS-slob and UAS-mtslob lines crossed to elav . There is an apparent breakdown of rhythmicity after 7 days in flies overexpressing wild type and mutant SLOBs (middle and right panels). (B) Sum- c155 mary of behavioral data for elav flies crossed to control and UAS lines. The FFT value is a measure of rhythmic strength and is plotted in the bar graph for the first and second week periods per fly line. Using Student's t test, all overexpressing SLOB lines exhibit significant breakdown of rhythms during the second seven days (*p < 0.05 and **p < 0.001, respectively). Controls show no significant breakdown except for one line, UAS-slob1/+. However, as demonstrated by the inserted western blot, this line expresses transgenic SLOB in the absence of a GAL4 driver, whereas the UAS-mtslob1/+ line does not. The blot was probed with an anti-HA antibody that detects only transgenic SLOB. Levels of MAPK were used as loading controls (data not shown). (C) Summary of behavioral data for GMR and Kurs 45 GAL4 flies crossed to various lines. No change in rhythmicity is evident when SLOB is overexpressed only in the Drosophila eye or PI neurons. Page 9 of 14 (page number not for citation purposes) Relative Power FFT Relative Power FFT Relative Power FFT BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 The PI region lies directly beneath the root of the ocellar Discussion We report here that the dSlo binding protein, SLOB, cycles nerve. The PI neurons have large, 15 µm diameter, cell in Drosophila heads. Microarray analyses reported slob bodies, and their axons project along the median bundle transcript cycling with a peak at either ZT 15 [2] or at CT and then bifurcate [27]. One of the branches proceeds 11 [3]. We show by western analysis that SLOB protein ventrally and arborises in the dorsal tritocerebrum region, peaks at ZT 10–14/CT 14, consistent with an earlier peak below the oesophagus. The other branch moves in a for the RNA. Under LD conditions, SLOB continues to posterior direction and enters the cardiac recurrent nerve 01 01 jrk cycle in per , tim and Clk flies in phase with the oscil- in the oesophageal canal. The PI neurons have an exten- lation of wild type flies. It was noted in one of the recent sive network of endoplasmic reticulum and contain secre- microarray studies [2] that there is a cluster of genes, tory granules, suggesting that they are neurosecretory cells named the apterous cluster, that shows rhythmicity in [27]. In insects, peptidergic neurons of the central nervous these three mutants during LD. This cluster has a charac- system regulate the synthesis of developmental hor- teristic peak at ZT 17 and includes such proteins as tran- mones. The PI neurons, in particular, have been impli- scription factors, synaptic regulators and transporters. The cated in hormone production and release in various genes in this group may be regulated not only by the cir- insects [28,29]. Three subsets of PI neurons have been cadian clock, but also by a light-dependent mechanism. identified. We have identified the subgroup PI-3 to be the However in DD, where light is no longer a factor, we find SLOB positive subset of PI neurons. Among the hormones jrk 01 that SLOB does not cycle in Clk and per and exhibits an identified in the PI neurons is insulin, and it has been 01 01 altered phase in tim flies. One might expect tim and proposed that the release of insulin into the hemolymph 01 01 per flies to give similar results, but tim flies may not be is essential for growth control and carbohydrate homeos- true genetic nulls [24]. Consistent with the observation tasis [30]. We have confirmed that the SLOB positive PI jrk that SLOB does not cycle in Clk flies during DD (Figure neurons are also insulin positive (data not shown). 2B) are microarray data from Ueda et al. [5] indicating jrk that slob levels do not change in Clk flies, and other The photoreceptors and the PI neurons express oscillating microarray data from McDonald and Rosbash [3] demon- SLOB protein and intriguingly, the rhythms in the two jrk strating that slob levels are at mid-point in the Clk neuronal types are not in phase with each other. The mutants. Taken together these data demonstrate clearly mechanisms responsible for these phase differences are that clock genes regulate slob mRNA and protein not known. PER and TIM are expressed in the lateral neu- expression. rons in the central brain, in glial cells of the optic lobes, and in the photoreceptor cells [7]. PER and TIM protein Within Drosophila adult heads, slob mRNA is present in the have not been shown to be expressed in the PI neurons, photoreceptors, optic lobe, the neurosecretory cells of the although Kaneko and Hall [31] found that there is expres- PI and the surrounding brain cortex. We also find promi- sion of GAL4 driven by the per promoter in PI neurons. nent immunostaining for SLOB protein in the photore- The photoreceptors in contrast have all the traditional ceptor cells and the PI neurons. In situ hybridization clock genes that might contribute to SLOB cycling [32- experiments with larval brain revealed slob RNA in an area 34]. The presence of these genes in the eye, but not in the of the brain close to PDF-filled projections of the lateral PI, may contribute to the phase differences. In addition, it neurons [2]. This is consistent with our findings of slob is reasonable to expect that there will be molecular differ- transcript in the PI neurons and surrounding cortex. ences in circadian regulation in different cell types. Fur- thermore, the subcellular localization of SLOB is different The Drosophila eye expresses many of the major circadian between the two areas (Figure 4A,4C). SLOB appears to be genes, and is thought to contain an autonomous primarily cytoplasmic in the PI neurons, and nuclear in oscillator that presumably regulates an eye-specific func- the photoreceptors. tion [17]. In addition, the eye contributes to photic entrainment of the pacemaker LN cells [25]. Interestingly, How is SLOB regulated in the PI neurons? Interestingly, the SLOB binding partner, Slo is also expressed in the vis- the PI neurons have been associated with behavioral ual system including the eye, lamina, medulla and lobula rhythmicity and it has been hypothesized that this [26]. Ceriani et al. [1] have demonstrated that the RNA involves hormone release [35]. In fact, the PI neurons of levels of both slob and slo cycle in phase in both LD and Teleogryllus commodus (crickets) have long been hypothe- DD. The dSlo protein was also shown by western blot to sized to serve as a region of coupling between the cycle and peak at ZT 20 [1]. This correlates with the cycling circadian pacemakers and behavioral rhythms [36]. A of SLOB in the photoreceptors, where SLOB peaks at ZT pathway for Drosophila proposed by Kaneko and Hall [31] 14–21 (Figure 5A). suggests that oscillatory signals of the "master pacemaker" in the small LNvs first modulate the oscillatory mecha- nism or neuronal activity operating within neurons in the Page 10 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 dorsal region. The DNs send their oscillatory signals to the mutants such as Pdf flies [13]. Likewise, panneuronal PI, which may lead to rhythmic neurosecretory peptide overexpression of PDF results in a delayed disruption of release. It has been observed in the larval CNS that projec- rhythms, and overexpression of PDF in the PI neurons tions of the DNs terminate near the midline in the PI results in a shortened period and an advance of the morn- region [31]. These DNs express both PER and TIM and ing peak [39]. disconnected (disco) flies, which lack LNs, hence may send robust oscillatory signals to downstream also become arrhythmic only after several days in DD targets such as PI neurons. Interestingly, two neurons of [40]. the DN group express PER and TIM cycling antiphase to the other DNs and LNvs [31]. Regardless of the precise Using GAL4 drivers that direct overexpression of SLOB role of the DNs, it is clear that this dorsal region of the specifically to the eye or PI neurons, we found no obvious brain is important for rest:activity rhythms. For example, circadian locomotion phenotype. It is possible that these PDF release and MAPK activity cycle specifically in this drivers are not strong enough, compared to the panneuro- c155 region, and both participate in behavioral rhythmicity nal driver elav . We note that the overexpression of PER [13,37]. and TIM with the tim-GAL4 driver results in a more severe phenotype than with per-GAL4, even though the expres- Clock mutants alter either SLOB protein oscillation or lev- sion pattern of the two drivers is similar [24], possibly els in both the eye and PI neurons. One striking observa- because the per promoter is weaker [41]. Alternatively, the tion is the ectopic or elevated expression of SLOB in the specific drivers we used may not target all the lamina of tim flies. This suggests that TIM negatively reg- behaviorally-relevant SLOB positive neurons in the eye ulates SLOB. Western analyses show that upregulation of and PI region. Any SLOB positive neurons that are not SLOB does not occur in per flies. Ectopic expression of overexpressing SLOB are therefore wild type, and this may PER occurs in double-time (dbt) flies that are mutant for a prevent rhythmicity breakdown. Similar explanations casein kinase 1ε involved in PER turnover [38]. The inter- have been proposed for the lack of a phenotype when PER pretation in that case is that PER is synthesized in many and TIM are overexpressed by the pdf-GAL4 driver cell types where its expression is normally undetectable although both genes cause arrhythmia when their overex- due to destabilization by the kinase. A similar mechanism pression is driven by the more widely expressed tim and flies. may account for elevated expression of SLOB in tim per-GAL4 drivers [24,42]. The observation that tim alters SLOB expression while per does not could suggest a pathway for slob regulation The widespread distribution of SLOB in the eye and brain that is independent of PER. We also found that SLOB fails suggests that other cells, in addition to or instead of the jrk to cycle in the PI neurons of Clk flies. photoreceptor and PI neurons, may account for SLOB's apparent role in behavioral rhythms. The panneuronal Perhaps most intriguing is the decrease of SLOB in the PI overexpression of SLOB in other SLOB-expressing cells in neurons of Pdf flies. The oscillation of PDF is restricted the brain cortex may explain the rhythmic breakdown. to the dorsal projections emanating from the lateral neu- Alternatively, ectopic expression of SLOB in neurons rons [13]. Dorsal terminals of the LNs express abundant involved in locomotor rhythms might also account for the PDF early in the morning, which is indicative of a block in altered rhythmicity. its release. Thus, PDF release is low during the day while SLOB is at its trough, consistent with PDF being a positive Conclusions regulator of SLOB. In Figure 4H we see that PDF express- In this study, we have demonstrated that SLOB protein ing terminals do not appear to contact the PI neurons. As cycles in a circadian fashion in Drosophila heads. SLOB discussed above, we hypothesize that PDF termini affect oscillates in two discrete areas of the fly head, the photore- the DN, or alternatively, other neurons of the dorsal ceptor cells and the PI neurons. Our results reveal differ- region, which in turn communicate with the PI neurons. ential effects of clock mutations on SLOB expression and jrk Clk flies lack PDF in the small ventral LNs, but still cycling in these two regions. There is a significant decrease express it in the large LNs [13]. This may account for the in SLOB levels in the PI neurons of Pdf flies, thus impli- jrk 01 difference in the phenotype of Clk and Pdf mutants cating PDF as an upstream regulator of SLOB. Along with and would suggest a role for the large LNs in SLOB the observation that flies overexpressing SLOB exhibit a regulation. breakdown of rest:activity patterns, these data suggest that SLOB is a clock controlled protein. The molecular oscillations of the circadian clock proteins result ultimately in behavioral rhythmicity [24]. Our data We showed previously that SLOB, along with dSlo and demonstrate that the panneuronal expression of SLOB Leonardo, participates in a dynamic regulatory complex causes a delayed breakdown of rhythms. Breakdown after in presynaptic nerve terminals. Leonardo binding is several days is characteristic of some circadian output dynamically regulated by phosphorylation of SLOB by the Page 11 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 calcium/calmodulin-dependent protein kinase II (CAM- chemiluminescence detection system (Amersham) was KII) [6]. Intriguingly, CAMKII has recently been impli- used to visualize the proteins. Film exposures of western cated in circadian rhythmicity in vertebrates [43], and it is blots were scanned using Bio Rad Molecular Analyst. The tempting to speculate that this regulatory complex partic- level of SLOB at each time point was calculated as the ipates in the fly circadian output pathway. Not only do SLOB signal minus the background in each lane. The blots dslo mutants have weak rhythms [1,44], but leonardo were stripped and reprobed with anti-MAP Kinase mutants have defects in behavior, synaptic transmission (Sigma). The ratio of SLOB to MAP Kinase was normal- and plasticity [45,46]. Our data are consistent with the ized and averaged between several westerns. hypothesis that SLOB participates in circadian rhythmic- ity by regulating synaptic function and membrane Immunohistochemistry excitability. Brain whole mount: Flies were entrained in LD for three days and then transferred to DD. Fly heads were collected at given time points, fixed in 4% paraformaldehyde Methods Fly stocks and germ line transformation (PFA), and the brains were dissected and kept in cold PBS, D. melanogaster strains Canton S (wild type), y w, ry, tim , and subsequently blocked with 6% normal donkey serum 01 jrk 01 per , Clk , and Pdf and transgenic fly strains were raised in phosphate-buffered saline (PBS)/0.3% Triton X-100 for at 25°C on standard Drosophila medium. slob cDNA was one hour. Samples were then incubated with primary cloned into a pUAST vector and P-element-mediated antibody at a dilution of 1:400 overnight at 4°C. After transformation was performed as described previously washing in PBS/0.3% Triton X-100 three times for 30 min C155 [47]. The transformed lines were crossed to either elav each at room temperature, samples were incubated with (provided by Leslie Griffith), GMR (provided by Konrad the appropriate secondary antibody (Fluorescein (FITC)- Zinsmaier) or Mai 281, Mai 301, Kurs 45, and Kurs 58 conjugated AffiniPure Donkey Anti-Rabbit IgG and Texas GAL4 (provided by Gunter Korge). Red dye-conjugated AffiniPure Donkey Anti-Rabbit IgG from Jackson ImmunoResearch) at a dilution of 1:500 in In situ hybridizations 3% normal donkey serum in PBS/0.3% Triton X-100 for 1 The slob RNA antisense and sense probes were synthesized hour at room temperature, and washed in PBS three times using the DIG RNA Labeling Mix (Boehringer Manheim). for 30 minutes each. Brains were mounted onto slides The sequence used for the RNA probes was made from with mounting medium (Vector H-1200). Wholemounts basepairs 1142–1441 of the slob transcript. In situ hybrid- were visualized using fluorescence microscopy on a Leica ization on adult 12 µm head sections were done according DMIRE2. to the protocols found at http://www.rockefeller.edu/lab heads/vosshall/protocols.php with slight modifications. Section: Flies were collected at given time points, All hybridizations and washes were done at 55°C. Sec- mounted with mounting medium, and sectioned at 12 tions are developed in the dark for 3 days. µm using a cryostat (Leica 3450). Sections were fixed with 4% PFA, washed in PBS/0.3% Triton X-100, and blocked SLOB antibody purification with 6% normal donkey serum in PBS/0.3% Triton X-100 A GST-SLOB fusion protein was used to immunize rabbits for 1 hour, and subsequently incubated with primary anti- as described previously [48]. Polyclonal antibodies body in 6% normal donkey serum in PBS/0.3% Triton X- specific to SLOB were generated by purifying the serum 100 overnight at 4°C. The sections were then washed and using a combination of CNBr conjugated GST and CNBr incubated with secondary antibodies as described above. conjugated GST-SLOB columns. The staining intensity of brain whole mounts and sections Western blotting and quantitation was assessed by blind scoring. A subjective intensity scale Flies were entrained and collected in LD and DD condi- from zero to four was used, with zero being undetectable tions at four hour intervals. Fly heads were lysed in 1% and four being maximal. Statistical analysis of average CHAPS, 20 mM Tris-HCl (pH 7.5), 10 mM EDTA, 120 staining intensity scores was done using ANOVA and the mM NaCl, 50 mM KCl, 2 mM DTT and protease inhibitors Tukey HSD test. (1 mM PMSF, 1 µg/mL each aprotonin, leupeptin, and pepstatin A (SIGMA)). Protein concentration was deter- Behavioral analysis mined using the BioRad DC Protein Assay. 100 µg of pro- Flies aged from 1–5 days were entrained for three days in tein was loaded on 4–15% polyacrylamide gradient gels 12 hr light/dark cycles at 25°C and then kept in constant and transferred to nitrocellulose membranes. After block- darkness for 14 days. Activity was monitored by using the ing with 5% nonfat milk in TBST (0.1% Tween 20 in Tris- Trikinetics system. Individual flies were analyzed for buffered saline), the blots were probed with the appropri- rhythmicity based on their by chi-square periodogram ate primary and secondary antibodies. Enhanced Page 12 of 14 (page number not for citation purposes) BMC Neuroscience 2004, 5 http://www.biomedcentral.com/1471-2202/5/3 15. Colwell CS: Circadian modulation of calcium levels in cells in and Fast Fourier Transform (FFT) values [24]. The analy- the suprachiasmatic nucleus. Eur J Neurosci 2000, 12:571-576. ses were performed using ClockLab software. 16. Colwell CS: NMDA-evoked calcium transients and currents in the suprachiasmatic nucleus: gating by the circadian system. Eur J Neurosci 2001, 13:1420-1428. Authors' contributions 17. Cheng Y, Hardin PE: Drosophila photoreceptors contain an AMJ performed the SLOB antibody purification, western autonomous circadian oscillator that can function without period mRNA cycling. J Neurosci 1998, 18:741-750. blotting and quantitation, immunohistochemistry, in situ 18. Edery I, Zwiebel LJ, Dembinska ME, Rosbash M: Temporal phos- hybridization and behavioral analysis. XZ carried out phorylation of the Drosophila period protein. Proc Natl Acad Sci immunohistochemistry and scoring analysis. DAA and AS U S A 1994, 91:2260-2264. 19. Sehgal A, Rothenfluh-Hilfiker A, Hunter-Ensor M, Chen Y, Myers MP, participated in the immunohistochemistry and western Young MW: Rhythmic expression of timeless: a basis for pro- blotting. AMJ and IL drafted the manuscript. AMJ, XZ, YZ, moting circadian cycles in period gene autoregulation. Science IL and AS conceived of the study, and participated in its 1995, 270:808-810. 20. Stempfl T, Vogel M, Szabo G, Wulbeck C, Liu J, Hall JC, Stanewsky R: design and coordination. All authors read and approved Identification of circadian-clock-regulated enhancers and the final manuscript. genes of Drosophila melanogaster by transposon mobiliza- tion and luciferase reporting of cyclical gene expression. Genetics 2002, 160:571-593. 21. So WV, Sarov-Blat L, Kotarski CK, McDonald MJ, Allada R, Rosbash Acknowledgements M: takeout, a novel Drosophila gene under circadian clock This work was supported by grants from the National Institutes of Health transcriptional regulation. Mol Cell Biol 2000, 20:6935-6944. to I.B.L. and A.S., an NRSA to A.M.J. and the US Army Medical Research 22. Siegmund T, Korge G: Innervation of the ring gland of Dro- Command to A.S. 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