Circadian Expression of PERIOD and the Pigment-Dispersing Factor in the yellow white mutant Drosophila melanogaster

Citation: Egypt. Acad. J. Biolog. Sci. (A. Entomology) Vol. 10(1)pp: 101110 (2017) Egyptian Academic Journal of Biological Sciences is the official English language journal of the Egyptian Society for Biological Sciences, Department of Entomology, Faculty of Sciences Ain Shams University. Entomology Journal publishes original research papers and reviews from any entomological discipline or from directly allied fields in ecology, behavioral biology, physiology, biochemistry, development, genetics, systematics, morphology, evolution, control of insects, arachnids, and general entomology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Most organisms have circadian clocks that synchronize their behavior and physiology to the daily cycling environmental conditions.The core oscillator of these clocks consists of at least three interlocked molecular feedback loops (Tomioka and Matsumoto, 2010) that occurs within clock neurons (Nitabach and Taghert, 2008).A primary loop of these involves period (per), timeless (tim), Clock (Clk), and cycle (cyc) clock genes (Hardin, 2006).CLK and CYC proteins produced by Clk and cyc genes form a heterodimer and bind to the E-box promoter region of per and tim to activate their transcription during late day to early night.The product proteins PER and TIM accumulate during the night to peak at late night.PER and TIM then form a heterodimer that enters the nucleus and repress their own transcription by inhibiting CLK-CYC heterodimer.Suppression of per and tim transcription leads to a reduction in PER and TIM levels, which in turn releases the suppression on CLK-CYC and eventually leads to increasing the transcription of per and tim, which starts a new cycle (Stanewsky, 2002).The output timing signals of the clock are then relayed to overt downstream rhythms mainly via the pigment-dispersing factor (PDF), which is a peptide neurotransmitter that is considered the primary output factor for the clock.
The neuronal network of the circadian machinery are best described in D. melanogaster (Tomioka and Matsumoto, 2010).These clock neurons are about 150 and are divided into two major groups; the lateral neurons (LNs) located in the anterior lateral brain and the dorsal neurons (DNs) that lie in the dorsal protocerebrum (Taghert and Shafer, 2006).Each of them is further divided into three groups.The LNs are divided into: the large ventro-lateral neurons (l-vLNs), the small ventro-lateral neurons (s-vLNs) and the dorso-lateral neurons (dLNs), while the DNs are divided into DN1s, DN2s and DN3s (Helfrich-Förster, 2003).Although, it was thought for a long time that the circadian mechanism is purely cell-autonomous within these individual clock neurons, however, recently, many reports highlight the role of the neuronal interactions among multiple clock neurons of the fly brain in driving rhythmic behavior (Nitabach and Taghert, 2008;Hermann-Luibl and Helfrich-Förster, 2015).
Studying mutant Drosophila provided immense help in understanding the circadian clock machinery and mechanisms (Konopka and Benzer, 1971;Sehgal et al., 1994;Allada et al., 1998;Hardin, 2011).yellow white is one of these mutants.The yellow (y) gene plays a role in melanin pigmentation (Biessmann, 1985), while the white (w) gene encodes a transmembrane ABC transporter protein that is involved in the transport of vital precursors to certain Drosophila pigments (Ewart and Howells, 1998).The abnormal eye pigmentation in addition to reduced and altered levels of clock-related neurotransmitters have adverse effects on the behavior and circadian plasticity in these flies (Campbell and Nash, 2001;Hassaneen, 2015).
This study investigates the effect of yellow white mutation on the rhythmic expression profiles of PER and PDF proteins in the different clock neuronal subsets of the D. melanogaster brain.Results are expected to reveal the effect of possible differential alterations of clock protein expression profiles on behavioral rhythms.The study also aims to identify the specific role of each neuronal subset in regulating circadian locomotor behavior.

Experimental animals
Adult male D. melanogaster flies at the age of 4-7 days after eclosion were used in the experiments.The wild-type strain Canton S (CS) was used as control against the yellow white (y w) mutant D. melanogaster.Both were obtained from the University of California San Diego Drosophila Species Stock Center (DSSC).All flies were reared on standard cornmeal/agar medium with yeast at 18°C and a cycle of 12 hours light -12 hours dark (LD 12:12).

Antibodies
A commercially available monoclonal anti PDF serum, obtained from the (Developmental Studies Hybridoma Bank at the University of Iowa) was used to stain PDF.The antibody was raised by immunizing balb/c mice with the amidated Drosophila PDF peptide (NSELINSLLSLPKNMNDA-NH2) by PickCell Laboratories B.V. (Amsterdam, The Netherlands) (Cyran et al., 2005).The antibody reliably labels only PDF + neurons.No staining is observed in pdf 01 mutants.For PER protein, a polyclonal rabbit anti-PER antibody was used for staining that was raised against full-length PER expressed in a baculovirus expression system (Liu et al., 1992).Fluorescence-conjugated Alexa Fluor antibodies of 635 nm (goat anti-mouse) and 488 nm (goat anti-rabbit) were used as secondary antibodies (Molecular Probes, Carlsbad, CA).

Immunohistochemistry
Adult male brains were used for immunostaining.Brains of flies, entrained to LD12:12 (500 lux) at 20°C for at least 4 days, were collected every four hours at Zeitgeber times (ZT 3,ZT 7,ZT 11,ZT 15,ZT 19,ZT 23); 20 flies each.Flies were fixed for 2.5 hours in 4% paraformaldehyde (PFA) in phosphate buffer (PB; 0.1 M; pH 7.4) with 0.1% Triton-X-100 on a shaker in complete darkness at room temperature.After fixation, the flies were washed three times in PB for 15 minutes and were dissected in PB. 5% normal goat serum (NGS) in PB with 0.5% Triton X-100 was applied onto the brains as blocking buffer at 4°C overnight.The brains were then incubated in the primary antibody solution of anti-PDF (1:1,000) and anti-PER (1:6,000) in PB with 5% NGS and 0.5% Triton X-100) for 48 hours at 4°C.This was followed by six washes in PB with 0.5% Triton X-100 for 10 minutes each.The brains were then incubated in secondary antibodies with a dilution of 1:200 in PB with 5% NGS and 0.5% Triton X-100 for three hours at room temperature.Brains were washed again six times in PB with 0.5% Triton X-100 for 10 minutes then were embedded in Vecta-shield medium (Vector Laboratories, Burlingame, CA) and mounted on glass slides (Hermann et al., 2013).

Microscopy and image analysis
Immunofluorescent brains were analyzed using a laser scanning confocal microscope (Leica TCS SPE; Leica, Wetzlar, Germany).Confocal stacks of 2 µm thickness were recorded.Two diode laser lines were used sequentially for double (488 and 635 nm) immunolabelling that excites the fluorophores of the secondary antibodies.The concentration of PDF and PER was quantified by measuring the staining intensity of the neurons containing the proteins.The two hemispheres of eight to twelve brains were analyzed for each ZT group.Leica Application Suite Advanced Fluorescence Lite Software (LAS AF Lite, 2.2.1 build 4842) was used to view complete confocal stacks.Cropping stacks and overlays generation was done using Fiji distribution of the ImageJ (http://rsb.info.nih.gov/ij), an open-source software freely available for data analysis in life sciences (Schneider et al., 2012).Images were converted to grayscale and the brightness value from zero to 255 was used as a measure of staining intensity.The staining intensity of each neuron was measured minus the average intensity of a similar-sized area from the background to standardize background-staining differences.Each measurement was repeated three times then averaged for each neuron.Intensity staining for each neuron of the LN subsets was measured, while five representatives were selected for each DN neuronal subset.

Data analysis
Data were analyzed and plotted using Microsoft Excel 2016 (Microsoft Corp., Redmond, WA) and SPSS Statistics for Windows version 22.0 (IBM Corp., Armonk, NY).Student t-test was used for statistical analysis at a significance probability of (p<0.05).

PDF expression profile
PDF is expressed only in s-vLNs and the l-vLNs.Statistical analysis using t-test at (p<0.05) between the PDF staining intensity of y w and CS D. melanogaster at each Zeitgeber time tested revealed that in the s-vLNs; PDF expression level in y w was always significantly lower than wild-type CS (Figure 1 (1-3 and 10-14) and Figure 2 (A)).While, in l-vLNs, it was only significantly lower during daytime only at ZT 3, 7, 11 (Figure 1 (1-3 and 10-14) and Figure 2 (B)).

PER circadian expression
PER is expressed in all clock neurons of D. melanogaster.PER expression profile in y w mutants exhibited the general profile known in wild-type CS, by degrading during the light phase (ZT 3 to 11) then accumulating again during the dark phase (ZT 15 to 23) (Figure 1 and 3), however with some differences.PER staining in y w against CS at each ZT time tested were statistically analyzed using t-test at (p<0.05) (Figure 3).During the light phase (daytime; ZT 3-11), PER was found significantly higher in y w than CS always in s-vLNs (Figure 3(A)), while it was higher in early and midday only (ZT 3 and 7) in all other neuronal subsets (Figure 3(B-E)), except in DN3s where they were not significantly different (Figure 3(F)).
On the other hand, during the dark phase (night, ZT 15-23), PER was found significantly higher in y w than CS in mid and late night (ZT 19 and 23) in the l-vLNs, 5 th s-vLNs, DN1s, and DN3s (Figure 3 (B, C, E, and F)), respectively.However, in dLNs (Figure 3 (D)), PER was higher in y w than CS only at late night (ZT 23).On the contrary, in s-vLNs, PER of y w and CS were not significantly different (Figure 3 (A)), while in the 5 th s-vLNs, PER was significantly lower in y w compared to CS at early night (ZT 15) (Figure 3(C)).

DISCUSSION
In the natural light-dark cycle, D. melanogaster exhibits a bimodal crepuscular locomotor rhythmic activity, with a morning peak of activity (M) and an evening peak (E), separated by a resting siesta period (S).The various neuronal subsets of the circadian clock contribute differentially to regulate these activity bouts (Grima et al., 2004).An earlier model of the clock, namely the "double-oscillator" model assumed that the s-vLNs drive (M), while the 5 th s-vLN and some dLNs drive (E) (Grima et al., 2004;Stoleru et al., 2004).However, later studies proposed that the s-vLNs (M cells) contribute to the regulation of (E), and thus suggested that their abbreviation (M) should denote "Main" rather than only "Morning" pacemaker (Rieger et al., 2006;Yoshii et al., 2012).A previous behavioral study of y w mutants revealed that they have a functional circadian clock, but their (M) was significantly delayed by about 2.5 hours, (E) was advanced by 1.7 hours, and (S) was shortened by 1.5 hours compared to wild-type CS (Hassaneen, 2015).The objective of this study was to investigate if these behavioral alterations are reflected on the molecular level.

Reduced PDF expression
In this study, PDF in y w flies was significantly reduced compared to CS, always in s-vLNs and during daytime only in l-vLNs (Figure 2).This result explains the advance of (E) and also the degraded morning anticipation in our previous study of y w flies (Hassaneen, 2015).That is because (Renn et al., 1999;Grima et al., 2004) found that pdf 01 mutant flies with no PDF or flies in which v-LNs is genetically ablated, loose morning anticipation and exhibit a 2-3 hour advance of the evening activity.On the contrary, injecting the related pigment-dispersing hormone produced phase delays in cockroaches (Petri and Stengl, 1997), which is similar to the natural role of PDF in delaying (E) in the wild-type CS.Since the s-vLNs are primary circadian control neurons, the behavioral changes in y w flies and their reflected molecular alterations, are expected to affect them, which was manifested by reduced PDF at all time points.However, in l-vLNs, PDF reductions occurred only in daytime (Figure 3 (B)), probably because the l-vLNs are not essential for circadian control, but rather suggested to perceive light input and communicate that to the dLNs and consequently determining the phase of the evening peak (Potdar and Vasu, 2012), through their massive innervation to the optic medulla (Taghert and Shafer, 2006).Their involvement with photic input may be the reason why PDF decreased in them during daytime but not at night.

Circadian PER expression
Results showed that all neuronal subsets in y w flies exhibited elevated PER levels compared to CS at the transitions from dark to light (ZT 3 and 23) (Figure 3), except the s-vLNs that didn't change significantly from wild-type at ZT 23, probably because they are more involved in precise determination of (M).To interpret that, we have to recall two things.First, in D. melanogaster, PER increase is correlated with rest phase, because PER accumulates during the night phase when the flies are resting.Second, the circadian clock was found to be highly light-sensitive to dim light transitions at dawn and dusk and this is very important for morning arousal, also, exposure to bright light during early night (dusk) delays the phase of the clock, while during late night (dawn) advances it (Bachleitner et al., 2007).Furthermore, recent studies have revealed that even human circadian clocks are more sensitive at dawn time, since simulations of low light intensities at dawn phase advanced both melatonin and activity rhythms (Danilenko et al., 2000;Danilenko et al., 2000).Since y w mutants have impaired vision due to disturbed eye pigmentation (Krstic et al., 2013), they seem unable to receive enough light for the morning arousal, consequently they sleep longer and the PER degradation is delayed and remains elevated during early morning compared to CS. Collectively these changes result in delay of (M), advance of (E) and shortening of (S).
A similar shift of activity was observed in nocturnal Eulemur fulvus albifrons (White-fronted lemurs) to be more day-active when they were subjected to reduced nocturnal illumination (Erkert and Cramer, 2006).Also, nocturnal Mus musculus mice shifted their activity towards diurnality after mutations, genetic manipulations and brain lesions that interfered with photoreception to the circadian clock (Mrosovsky and Hattar, 2005).On the other hand, natural prolonged light exposure in the summer advances the morning activity of flies (speed up) to the beginning of the day and delays the evening activity (slow down) to the end of the day to avoid the midday heat (Majercak et al., 1999;Rieger et al., 2006) which is important for survival, even in humans (Foster and Roenneberg, 2008).
The 5 th s-vLN and the dLNs are the (E) pacemaker cells (Grima et al., 2004;Stoleru et al., 2004).They determine the phase and amplitude of (E) in LD cycles (Hermann et al., 2012).In y w mutants, start of PER accumulation in the 5 th s-vLN was much delayed and is significantly lower than CS at ZT 15, but then quickly rises at ZT 19 (Figure 3 (C)), leading to an advanced start of (E) (Hassaneen, 2015).This can also be attributed to the reduced photic reception of y w flies that might erroneously translated to early night start.In y w dLNs, PER cycles in phase with the 5 th s-vLN, however their non-significant PER decrease at ZT 15 and increase at ZT 19 compared to CS, might indicate a higher level for the 5 th s-vLN compared to dLNs in the hierarchy of the circadian control.
The DNs receive circadian phase information from the s-vLNs and there is a seasonally dependent phase difference between them (Menegazzi et al., 2013).They both have fiber projections to the same area in the dorsal protocerebrum, that is suggested to be a locus for coupling between different subsets of clock neurons (Kouji and Meinertzhagen, 2009).Especially that G-protein coupled PDF receptors has been localized in neurons in the DN1s and DN3s (Lin et al., 2004).However, DNs function is not yet fully understood.In this study, PER expression in DNs is advanced in y w mutants compared to CS at the transitions from dark to light at (ZT 3 and 23) in DN1s and at ZT 23 in DN3s (Figure 3(E and F)), respectively.This might be due to reduced PDF expression and results in advance of (E).These results are similar to the (E) phase advance in the pdf 01 mutants (Renn et al., 1999).

CONCLUSION
The behavioral changes in y w mutant D. melanogaster flies are reflected on the molecular oscillations of their circadian clock machinery.These changes reduce the plasticity and robustness of their circadian clock and expose these flies to higher levels of environmental risk, especially, desiccation by the high temperature of midday.

Fig. 1 :
Fig.1: Double immuno-labeling of PER (Green) and PDF (Cyan) showing their circadian expression in the clock cell clusters of representative D. melanogaster's brains.Separated staining of PER (1) and PDF (2) in y w flies at ZT 23, when cells are best visible, are shown as an example of single staining, while all other images are shown as composite double-staining of CS (4-9) and y w (3 and 10-14), every four hours.Cells are labelled in detail in (1-4) and are applicable to all images, with more description in the text.The scale bar of 10 µm shown in (14) applies to all images.

Fig. 3 :
Fig. 3: Circadian profiles of PER expression in clock neuronal subsets in the y w and CS D. melanogaster brain every four hours.Data represents the average staining intensity of neurons in two hemispheres of 8-12 brains ± SEM. * indicates a significant difference between y w and CS at the given Zeitgeber time (ZT) using t-test at (p<0.05).