New Results

The Circadian Neuropeptide PDF has Sexually Dimorphic Effects on Activity Rhythms

Aishwarya R. Iyer, Eva Scholz-Carlson, Evardra Bell, Grace Biondi, Shlesha Richhariya, Maria P. Fernandez
doi: https://doi.org/10.1101/2024.01.31.578273

Abstract

The circadian system regulates the timing of multiple molecular, physiological, metabolic, and behavioral phenomena. In Drosophila as in other species, most of the research on how the timekeeping system in the brain controls timing of behavioral outputs has been conducted in males, or sex was not included as a biological variable. The main circadian pacemaker neurons in Drosophila release the neuropeptide Pigment Dispersing Factor (PDF), which functions as a key synchronizing factor in the network with complex effects on other clock neurons. Lack of Pdf or its receptor, PdfR, results in most flies displaying arrhythmicity in activity-rest cycles under constant conditions. However, our results show that female circadian rhythms are less affected by mutations in both Pdf and PdfR. Crispr-Cas9 mutagenesis of Pdf specifically in the ventral lateral neurons (LNvs) also has a greater effect on male rhythms. We tested the influence of the M-cells over the circadian network and show that speeding up the molecular clock specifically in the M-cells leads to sexually dimorphic phenotypes, with a more pronounced effect on male rhythmic behavior. Our results suggest that the female circadian system is more resilient to manipulations of the PDF pathway and that circadian timekeeping is more distributed across the clock neuron network in females.

Introduction

The differences in neuronal circadian timekeeping between sexes remain relatively unexplored, despite the expanding body of research highlighting the influence of sex on the mechanisms underlying neuronal control of behavior [1]. In mammals, steroid hormones display daily, clock-driven changes in abundance, and while these sex hormones are not required to maintain rhythms, they influence the amplitude and response to photic stimuli [1, 2]. Furthermore, structural and functional sex differences have been observed in brain areas that receive direct input from the brain’s circadian timekeeping center [2, 3]. Research in humans has also revealed significant sexual dimorphism: men tend to have lower-amplitude endogenous rhythms than women [4], and they are less resilient to nocturnal sleep disruptions and spend less time asleep [5].

In mammals, the main circadian pacemaker resides in the suprachiasmatic nuclei (SCN), which in mice consist of a network of ∼20,000 neurons (reviewed by [6]). The Drosophila circadian clock neuron network involves ∼150 neurons and is the functional equivalent of the mammalian SCN [7, 8]. Each circadian clock neuron contains an intracellular molecular timekeeping mechanism based on a transcriptional-translational feedback loop: the genes Clock (Clk) and cycle (cyc) promote rhythmic transcription of several key genes, including Period (Per) and Timeless (Tim), which inhibit their own transcription [9]. Multiple kinases that act on components of these clock proteins and can affect the pace of the molecular clock have been identified. One such kinase is Doubletime (DBT), which binds to and phosphorylates PER, hindering its nuclear accumulation [10, 11].

The fly’s clock network consists of lateral neurons (LNs), which include ventroateral (LNv), dorsolateral (LNd), and lateral posterior neurons (LPNs), as well as three groups of dorsal neurons (DN1, DN2, and DN3), some of which can be further subdivided [8, 12–16]. The ventral and dorsal LNs are sufficient to produce the normal endogenous bimodal rhythm of sleep and activity [17, 18]. The four small LNvs (s-LNvs) are usually referred to as morning cells (M-cells) since they control the morning peak of activity under light-dark cycles (LD). These cells are also essential to maintain rhythmicity under free-running conditions [19, 20]. The evening peak is controlled by the LNds and a Pdf-negative LNv, the 5th LNv (E-cells) [17, 21, 22]. Some DNs also contribute to timing and amount of sleep via modulation of the M and E cells [23–27].

Release of the circadian neuropeptide Pigment Dispersing Factor (PDF) by the s-LNvs is essential for endogenous circadian timekeeping. A Pdf null mutation, Pdf01, results in a substantial fraction of arrhythmic flies [19], desynchronization of molecular oscillations [28, 29], and phase changes in electrical activity of some clock clusters, most notably the LNds [30]. Loss of PdfR also leads to loss of behavioral rhythms [31–33]. Interestingly, PDF and PDFR also regulate behaviors that are sex-specific or sexually dimorphic. Rival induced long mating duration requires PDF expression in the s-LNvs, PDFR expression in a subset of LNds, and NPF expression in the LNds [34]. PDF controls rhythms in the sexually dimorphic pheromone profiles produced by the oenocytes [35], and is involved in long term mating suppression in males [36]. Both PDF and PDFR contribute to geotactic behaviors [37] and the phenotypes of Pdf01 mutants are sexually dimorphic, with males showing a more extreme negative geotaxis phenotype [38].

Sexual dimorphism in Drosophila sleep/wake cycles has been studied mostly under LD cycles. Males show lower activity and more sleep during the light phase [39–42]. This higher midday activity is due to the activity of a subset of sleep-promoting DN1s, which is more active in males [39] and receives input from the male-specific P1 neurons that control male courtship [40]. Unlike studies on circadian rhythms, Drosophila sleep research often involves only females. Males also have an earlier and more pronounced morning peak and a larger phase angle between the morning and the evening peak [43]. Under conditions of constant darkness and temperature (DD), males of several wild type strains have a small but significant reduction in free-running period relative to females of the same strain [43]. Moreover, males are more likely to retain a bimodal activity pattern in DD [43]. A recent transcriptomic analysis of fruitless (fru) expressing neurons revealed clusters that are enriched in circadian clock genes [44]. Interestingly, only the male dataset had some fru+ clusters with multiple genes having known circadian functions. A previous study reported that the DN1s express the male-specific FruM protein [45], and that the number of cells in the DN1ps cluster is sexually dimorphic in cell number [46]. In addition, the E3 subset of LNds has been shown to be dimorphic in expression of the neuropeptide NPF [47, 48].

Given the sexually dimorphic roles of neuropeptides, including PDF, in other behaviors [49], we asked if females were similarly affected by manipulations of the Pdf/PdfR pathway. We found that female circadian rhythms are less affected by null mutations in both Pdf and PdfR, and that similar effects are observed via CRISPR-Cas9 Pdf mutagenesis specifically in the LNvs. Moreover, speeding up the molecular clock in the LNvs via expression of DBTs leads to an advance of the morning peak in males but not in females, and the pace of the free-running period of activity is significantly shortened only in males. Taken together, our results show that the female circadian system is more resilient to manipulations in the PDF pathway and suggest that the Pdf+ neurons play a more dominant role over the male than over the female circadian network.

Results

Mutations in PDF and PDFR lead to sexually dimorphic phenotypes

A null mutation in Pdf results in molecular, physiological, and behavioral phenotypes in males [50]. We assayed the activity rhythms of Pdf01females under free-running conditions (DD) and found that a remarkably large proportion of the experimental females were still rhythmic (Fig. 1 A-C). We calculated the difference in rhythmic power of the experimental flies relative to their respective controls and found that the effect is less pronounced in females than in males (Fig. 1 D-E), suggesting that experimental females have more consolidated rhythms. Mutant females that were still rhythmic had a slightly but significantly shorter free-running period than did the controls (Table 1). This phenotype, previously described in Pdf01 males, was not observed in experimental males, consistent with the findings of a recent study [51].

Figure 1:
Figure 1: Mutations in Pdf and PdfR lead to sexually dimorphic phenotypes.

(A) Representative actograms of w1118 (left) and Pdf01 (right) male flies for 6 days of LD followed by 7 days of DD. (B) Representative actograms of w1118 (left) and Pdf01 (right) female flies for 6 days of LD followed by 7 days of DD. (C) Percentage of rhythmic flies are plotted for control (w1118) and Pdf01 males (left, n = 86 (w1118), n = 81 (Pdf01)) and females (right, n = 75 (w1118), n = 101 (Pdf01)). Error bars are SEM values plotted across 3 replicate experiments. (D) Rhythmic power of control (w1118) and Pdf01 male and females calculated using the Chi-square periodogram are plotted. (E) The difference in rhythmic power of experimental males and females from their respective controls are plotted. (F) Average sleep plots of flies over 7 days in DD are plotted for male and female control (w1118) and experimental (Pdf01) flies. (G) Average sleep plots under LD 12:12 of control (w1118) and experimental (Pdf01) are plotted for males (left) and females (right). The plots are averaged over flies and days for a period of 3 days under LD 12:12. (H) Total sleep values under LD are plotted for male (left) and female (right) flies of control (w1118) and experimental (Pdf01) flies. (I) The difference in total LD sleep values of experimental males and females from their respective controls are plotted. (J) Percentage of rhythmic flies are plotted for control (w1118) and han5304 males (left, n = 86 (w1118), n = 115 (han5304)) and females (right, n = 74 (w1118), n = 94 (han5304)). (K) Rhythmic power of control (w1118) and han5304 male and females calculated using the Chi-square periodogram are plotted. (L) The difference in rhythmic power of experimental males and females from their respective controls are plotted. (M) Average sleep plots under LD 12:12 of control (w1118) and experimental (han5304) flies are plotted for males (left) and females (right). The plots are averaged over flies and days for a period of 3 days under LD 12:12. (N) Total sleep values under LD are plotted for male (left) and female (right) flies of control (w1118) and experimental (han5304) flies. (O) The difference in total LD sleep values of experimental males and females from their respective controls are plotted. Free-running period and rhythmic power values are calculated using Chi square periodogram in CLOCKLAB for a period of 7 days. Total sleep values are calculated for 3 days in LD using PHASE. Statistical comparisons were performed between the control and experimental flies for both sexes using the Mann-Whitney U test. The box plots extend from the 25th to 75th percentile, with whiskers extending from the smallest to the largest value, and each point representing data from a single fly. Combined data from at least 3 replicate experiments are plotted.

Pdf mutants have increased sleep, and this effect is mediated by PDF function on the LNvs themselves [52]. We found that both sexes show an increase in total sleep in LD, but the effect is more pronounced in females (Fig. 1F-I). While the sleep increase in males was most prominent at midday, females exhibited increased sleep throughout most of the light phase (Fig. 1G). To rule out the presence of remnant PDF expression in Pdf01 females we stained brains of control and experimental males and females with an anti-PDF antibody. We did not observe any traces of PDF in experimental flies of either sex (Fig. S1A).

PDF accumulates rhythmically in the s-LNv dorsal termini in a time-of-day dependent manner both in LD and DD [19, 20], and can be released from the projections and the soma [53]. To examine if there are differences in the phase and amplitude of PDF cycling between the sexes in a wild type background, we dissected Canton-S males and females on the third day under DD at 6 timepoints over a 24-hour cycle. Using a COSINOR-based curve fitting method [54], we found that both males and females have a significant 24-hour rhythms in PDF cycling in their dorsal projections, with no significant sex differences in phase or amplitude (Fig. S1B-C, Table 2).

Next, we asked if the effects of a Pdf receptor mutation (PdfR) on activity and sleep were also sexually dimorphic. Expression of PDFR, a GPCR, can be detected in most of the clock neurons except for 3 LNds, half of DN1ps, and some DN3s [55], coinciding with Cryptochrome expression in clock neurons [55]. However, PdfR mRNA appears to be expressed in all clock neurons [56]. The han5304 mutant is a PdfR hypomorph and exhibits behavioral phenotypes under both LD 12:12 and DD [31–33]. Under DD, both Han males and females had a significant reduction in rhythmicity compared to the controls, but there was a greater proportion of rhythmic females (∼65%) than males (∼16%) (Fig. 1J). The free-running period of experimental flies was significantly shorter for both sexes (Table 3), as reported previously for males. Rhythmic power is significantly lower than controls for both han5304 males and females (Fig. 1K), but the effect was more pronounced in males, suggesting that females have more consolidated rhythms (Fig. 1L). Sleep plots for the first week under DD show that both males and females sleep more than controls (Fig. 1E). Similar to the effect of the Pdf mutation, han5304 flies showed significantly higher levels of LD sleep compared to controls, and the effect was again more pronounced in females (Fig. 1 M-O). Taken together, the results suggest that female circadian rhythms are less affected by the loss of both Pdf and PdfR, while at least under LD, the sleep phenotypes are more pronounced in females.

CRISPR-Cas9 mediated Pdf mutagenesis has more pronounced effects on male behavior

In the Pdf null mutant background, other genetic factors could contribute to the sexual dimorphism observed in behavioral rhythms. We therefore employed a tissue-specific CRISPR-Cas9 mediated knockout of Pdf in both males and females, as described in a recent study that focused on males [57]. To assess the efficiency of the manipulation we stained for PDF in flies that constitutively expressed Pdf gRNA and Cas9 in Pdf+ neurons. This experiment was conducted at 28°C, as this temperature was more effective at mimicking the behavioral phenotypes of the Pdf01 mutant than was 25°C (described below), and to allow direct comparison with an adult-specific manipulations.

PDF was significantly reduced in the s-LNvs in both sexes (Fig. 2 A-B). However, in most brains we noted faint staining in the dorsal projections of at least one s-LNv in at least one hemisphere. The PDF signal within the large LNvs was higher and could be detected in two or three l-LNv cell bodies in both hemispheres in the majority of brains. In addition to behavioral phenotypes, the Pdf01 mutation leads to pronounced misrouting of the s-LNv projections in male flies [58]. We next employed a transgene expressing a red fluorescent protein under a Pdf regulatory sequence [59] and observed some s-LNv projections occasionally defasciculating from the main axonal bundle in one or both hemispheres in Pdf >Pdf-g; Cas9 flies of both sexes (Fig. 2A). The extent of the reduction in PDF intensity of experimental flies relative to the controls was similar between the sexes (Fig. 2C).

Figure 2:
Figure 2: Tissue-specific CRISPR mediated Pdf manipulation leads to a reduction in PDF levels and misrouting of s-LNv dorsal termini.

(A) Representative confocal images of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts) (top) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) (middle) male flies stained with RFP and PDF antibodies. Representative confocal images of experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) (bottom) female flies stained with RFP and PDF antibodies. The experimental flies show significant reduction in PDF levels and misrouting of the s-LNv dorsal termini (arrows) as seen in the RFP channel for both the sexes. (B) Quantification of PDF intensity from s-LNv cell bodies in control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) flies are plotted for males (left) and females (right). Data points represent intensity from a single brain sample. n > 12 brains for all genotypes. (C) The difference in PDF intensity values of experimental males and females from their respective controls are plotted.

We then analyzed activity-rest rhythms in Pdf >Pdf-g; Cas9 flies and found that experimental males were mostly arrhythmic, whereas a greater proportion of experimental females remained rhythmic (Fig. 3A-B). The free-running period of experimental males was not significantly different from one of the controls, possibly because of the wide range of periods exhibited. Females had significantly shorter free-running period compared to parental controls (Fig. 3C). The rhythmic power was significantly lower in experimental flies of both sexes (Fig. 3D) but the effect was less pronounced in females (Fig. 3E), indicating that females are able to better consolidate activity rhythms. At 25°C, both males and females exhibited reduction in percent rhythmicity but the majority of the flies were rhythmic (Fig S3A). Both males and females showed a shortening of their free-running period (Fig. S3B). Similar to what we observed at 28°C, the rhythmic power of the experimental flies of both the sexes was significantly reduced (Fig. S3C) but females were less affected (Fig. S3D).

Figure 3:
Figure 3: CRISPR-Cas9 mediated Pdf mutagenesis has more pronounced effects on male behavior.

(A) Representative actograms of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts), and (UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right) are plotted for 6 days of LD followed by 9 days of DD. (B) Percentage of rhythmic flies are plotted for control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts), and (UAS Cas9; Pdf-g), and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left, n = 85 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts), n = 89 (UAS Cas9; Pdf-g), n = 106 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) and females (right, n = 79 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts), n = 78 (UAS Cas9; Pdf-g), n = 85 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g). Error bars are SEM values plotted across 3 replicate experiments. (C) Free-running period of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right) calculated using the Chi-square periodogram are plotted. (D) Rhythmic power of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right) calculated using the Chi-square periodogram are plotted (E) The difference in rhythmic power of experimental males and females from their respective controls are plotted. Flies were kept at 28°C throughout development and as adults. The activity-rest behavior was recorded at 28°C. Free-running period and rhythmic power values are calculated using Chi square periodogram in CLOCKLAB for a period of 7 days. Statistical comparisons were performed between the control and experimental flies for both sexes using the Kruskal-Wallis ANOVA followed by the Dunn’s multiple comparisons test. The box plots extend from the 25th to 75th percentile, with whiskers extending from the smallest to the largest value, and each point representing data from a single fly. Combined data from at least 3 replicate experiments are plotted.

We next conducted an adult specific Crispr-Cas9 knockdown of Pdf (Fig S4). While a previous study conducted in males did not find a correlation between the misrouting induced by the loss of Pdf during development and the loss of behavioral rhythms in adults [60], it is possible that the sex differences in Pdf01and Pdf >Pdf-g; Cas9 flies were due to developmental effects. The AGES system was previously employed to rule of a developmental contribution to the Pdf mutagenesis phenotypes [57], but a recent study showed that AUXIN exposure short-and long-term changes in physiology and behavior [61]. Therefore, we employed a temperature-sensitive Gal80 variant with ubiquitous expression to conditionally inhibit Gal4-mediated expression of the Pdf guide and Cas9 [62]. This allows for the temporal control of UAS transgenes since Gal80ts is active at lower temperatures but inactive at higher temperatures. We raised flies at 18°C and transferred them to 28°C immediately after eclosion, so that Pdf mutagenesis only occurs in adult flies. Consistent with what was reported for males by Gorostiza et al. [60] using RNA mediated interference (RNAi) of Pdf, we did not observe misrouting in flies of either sex (Fig. S4A). While PDF levels were significantly reduced in both males and females, the manipulation was more effective in males (Fig S4A-B). Therefore, although the circadian behavioral phenotypes were again more pronounced in males than in females (Fig S4C-E), this is likely due to higher remaining PDF expression in experimental females.

Speeding up the M-cell clock leads to a coherent period shortening only in males

Next, we sought to determine if the influence of the Pdf releasing cells themselves was sexually dimorphic. While PDF is released from both the large and small LNvs, only the s-LNvs (M-cells) play key roles in regulating free-running rhythm properties [17]. In males, manipulations that change the pace of the clock specifically in the LNvs result in changes in the phase of the morning peak of activity and in free running period [21, 63]. We expressed the Doubletime ‘short’ (DBTs) allele [64] under the Pdf-Gal4 driver (Fig. 4A) and analyzed the effects on behavior in both sexes. Interestingly, we found that Pdf > DBTs males but not females have an advanced phase of the morning peak of activity (M-peak, Fig. 4B-C). This suggests that under LD cycles, the M-oscillator is more effective at setting the phase of male than female behavior.

Figure 4:
Figure 4: Speeding up the clock in M-cells leads to sexually dimorphic phenotypes.

(A) Depiction of the adult Drosophila brain hemisphere indicating the clock cell subsets (colored) having a faster running molecular clock. (B) Average activity plots of control (Pdf-Gal4) and (UAS-DBTs) and experimental (Pdf > DBTs) flies are plotted for males (left) and females (right). The plots are averaged over flies and days for a period of 3 days under LD 12:12. (C) The phase of the morning peak of activity under LD 12:12 is plotted for control (Pdf-Gal4), (UAS-DBTs) and experimental (Pdf >DBTs) males (left, n = 116 (Pdf-Gal4), n = 120 (UAS-DBTs), n = 88 (Pdf > DBTs)) and females (right, n = 85 (Pdf-Gal4), n = 109 (UAS-DBTs), n = 73 (Pdf > DBTs)). (D) Percentage of rhythmic flies are plotted for controls (Pdf-Gal4), (UAS-DBTs), and experimental (Pdf > DBTs) males (left, n = 90 (Pdf-Gal4), n = 108 (UAS-DBTs), n = 93 (Pdf > DBTs) and females (right, n = 66 (Pdf-Gal4), n = 83 (UAS-DBTs), n = 70 (Pdf > DBTs). Error bars are SEM values plotted across 3 replicate experiments. (E) Representative actograms of control (UAS-DBTs) and experimental (Pdf > DBTs) males (left) and females (right) plotted for 6 days of LD followed by 10 days of DD. (F) Free-running period of control (Pdf-Gal4), (UAS-DBTs) and experimental (Pdf > DBTs) males (left) and females (right) calculated using the Chi-square periodogram are plotted. (G) Rhythmic power of control (Pdf-Gal4), (UAS-DBTs) and experimental (Pdf > DBTs) males (left) and females (right) calculated using the Chi-square periodogram are plotted. (H) The difference in rhythmic power of experimental males and females from their respective controls are plotted. Free-running period and rhythmic power values are calculated using Chi-square periodogram in Clocklab for a period of 7 days. Activity plots and phase of the M-peak were calculated using PHASE. Statistical comparisons were performed between the control and experimental flies for both sexes using the Kruskal-Wallis ANOVA followed by the Dunn’s multiple comparisons test. The box plots extend from the 25th to 75th percentile, with whiskers extending from the smallest to the largest value, and each point representing data from a single fly. Combined data from at least 3 replicate experiments are plotted.

Under free-running conditions, both Pdf > DBTs males and females flies showed a significantly lower percentage of rhythmic flies compared to their controls (Fig. 4D-E), but there were fewer rhythmic females (∼40%) than males (∼65%) (Fig. 4D). A possible reason for this could be that the rest of the clock network, where the pace of the molecular clock remained unaltered, is able to resist the influence of the M-cells to a greater extent in females. The free-running period (FRP) of Pdf > DBTs males was ∼18.5 hours, with very few exceptions (Fig. 4F). However, Pdf > DBTs females showed a bimodal distribution, with some individuals showing a FRP consistent with the pace of the M-cells (∼18-18.5 hrs.), whereas a large proportion of females had FRP of ∼24 hours, consistent with the pace of the molecular clock in the rest of the clock network, which was not affected by the genetic manipulation (Fig. 4F). Among rhythmic flies, both Pdf > DBTs males and females had lower rhythmic power than the controls (Fig. 4G), with no difference between the sexes (Fig. 4H). These results support the notion that M-cells are more dominant over the male than the female circadian network. Previous studies have shown that M-cells’ influence over the rest of the clock neurons is mediated via PDF [65], so it is possible that the differential role of M cells in males and females is precisely due to the sexually dimorphic effects of PDF.

Speeding up the E-cell clock leads to a more advanced evening peak in females

We next asked if changing the pace of the clock in the evening cells (E-cells) via DBTS expression also had sexually dimorphic effects on behavior. These cells can be subdivided into at least 3 different clusters, based on their anatomy [8], physiology [63], transcriptomics profiles [14] and connectivity patterns [16]. The PDFR-expressing E1 and E2 clusters have been shown to regulate evening activity under LD [65, 66] and to be able to maintain free-running activity rhythms in the absence of a functional clock in the M cells [67, 68], while the behavioral role of the E3 cluster remains unknown. We used the MB122-B split-gal4 driver to target the E1 and E2 subsets (Fig. 5A). We found that expressing DBTS in the E1+E2 subsets of evening cells significantly advanced the phase of E-peak in experimental flies of both sexes (Fig. 5B-C). Remarkably, this effect was more pronounced in females (Fig. 5D).

Figure 5:
Figure 5: Speeding up the clock in a E-cell subset leads to a more advanced evening peak in females.

(A) Depiction of the adult Drosophila brain hemisphere indicating the clock cell subsets (colored) having a faster running molecular clock. (B) Average activity plots of control (MB122B-Gal4) and (UAS-DBTs) and experimental (MB122B > DBTs) flies are plotted for males (left) and females (right). The plots are averaged over flies and days for a period of 3 days under LD 12:12. (C) Phase of the evening peak of activity under LD 12:12 for controls (MB122B-Gal4) and (UAS-DBTs) and experimental (MB122B > DBTs) males (left, n = 92 (MB122B-Gal4), n = 93 (UAS-DBTs), n = 93 (MB122B > DBTs)) and females (right, n = 85 (MB122B-Gal4), n = 87 (UAS-DBTs), n = 89 ( MB122B > DBTs)). (D) The difference in the phase of E-peak of experimental males and females from their respective controls are plotted. (E) Free-running period of control (MB122B-Gal4 and UAS-DBTs) and experimental (MB122B > DBTs) males (left) and females (right) calculated using the Chi-square periodogram are plotted. (F) Rhythmic power of control (MB122B-Gal4 and UAS-DBTs) and experimental (MB122B > DBTs) males (left) and females (right) calculated using the Chi-square periodogram are plotted. Free-running period and rhythmic power values are calculated using Chi-square periodogram in CLOCKLAB for a period of 7 days. Activity plots and phase of the M-peak were calculated using PHASE. Statistical comparisons were performed between the control and experimental flies for both sexes using the Kruskal-Wallis ANOVA followed by the Dunn’s multiple comparisons test. The box plots extend from the 25th to 75th percentile, with whiskers extending from the smallest to the largest value, and each point representing data from a single fly. Combined data from at least 3 replicate experiments are plotted.

Speeding up of clocks in the E1 + E2 LNds did not significantly alter the free-running period or rhythmic power of experimental flies of either sex (Fig. 5E-F). M-cells have been shown to be the most dominant oscillators in DD and to regulate rhythm properties like persistence and free-running period to a large extent [17, 21, 22, 63], although manipulations of other clock cells can affect rhythm properties to some extent. Thus, speeding up the clock in the PDFR+ E1 and E2 clusters leads to similar behavioral phenotypes in males and females under free-running conditions.

Discussion

The critical role of sex as a biological variable has gained increasing recognition in biomedical research [69, 70]. Male bias is particularly prevalent in neuroscience, with single-sex studies using male animals outnumber those using females by a ratio of 5.5 to 1 [71]. This disparity extends to chronobiology, leading to a lack of knowledge regarding how temporal organization in the nervous system is influenced by sex. However, work from several laboratories has revealed sexual dimorphism in the SCN, as well as in its input and output pathways [1, 72]. Sex differences in the SCN morphology have been described in both animal models and humans, as well as sex differences in SCN electrical activity and steroid hormone receptors. In addition, sex differences in the number of SCN neurons that express the key neuropeptide vasoactive intestinal polypeptide (VIP) and in Vip mRNA expression have also been reported (reviewed in [2]).The roles of the mammalian VIP and the Drosophila PDF to circadian physiology are highly similar, although neither these peptides nor their receptors are sequence orthologs [73].

Several studies over the years have shown the importance of PDF in generating coherent rhythms of ∼2hour periodicity. Here, we report that females lacking Pdf or its receptor PdfR are more likely to be able to maintain consolidated activity-rest behavior than males. This could be because of sex differences in PDF signaling mechanisms, PDFR expression, or the roles of other clock neurons within the network. We did not detect sex differences in the overall levels or temporal pattern of PDF accumulation, suggesting that these differences might not contribute to the sexually dimorphic effects of the loss of Pdf expression. Another possibility is that in the female circadian system, other neuropeptides or neurotransmitters can maintain network synchronization even in the absence of PDF. In males, other neuropeptides have been shown to act in concert with PDF to maintain consolidated rhythms in the network, although none of them have as profound an effect as PDF in regulating activity-rest rhythms in DD [51, 74]. Single mutants of DH31 and CCHamide1 do not affect activity rhythms by themselves but the double mutants of these neuropeptides along with Pdf01 (DH3101Pdf01 and Pdf01/CCHaSK8) are almost completely arrhythmic, indicating that these neuropeptides act hierarchically in the network, with PDF being on top of that hierarchy [51, 74]. The importance of PDF relative to other peptides released by clock neurons may also be sexually dimorphic.

To overcome possible pleiotropic effects of Pdf01 and PdfR mutations, we employed a constitutive knockout of Pdf in the ventral lateral neurons using the CRISPR-Cas9 method. Although the Crispr manipulation was only partially effective at eliminating PDF expression, it was able to replicate the phenotypes of the Pdf01 mutation. We observed faint staining in the dorsal projections of at least one s-LNv in at least one hemisphere in most brains, and PDF staining in a single s-LNv projection reaching the dorsal brain has been shown to be sufficient for behavioral rhythms [75]. Experimental flies in which Pdf was mutagenized starting at the onset of the promoter expression early in development showed extensive misrouting of their dorsal termini, similar to what has been reported for Pdf01 males [60]. Instances of s-LNv misrouting have also been observed in other core clock mutants such as per01 and tim01 [76], and cyc01 [77]. We did not find evidence of sex differences in the severity of the misrouting phenotype caused by the loss of Pdf, but the percentage of brains with misrouting was higher in experimental females (data not shown), and no correlation between misrouting and behavioral phenotypes was found by others for pdf01 males [60]. Importantly, the Pdf > Pdfg;Cas9 manipulation recapitulates the sexually dimorphic circadian phenotypes of Pdf01mutants: a larger fraction of females are rhythmic and females exhibit a higher rhythm power.

In males, changing the speed of the M cell clock leads to phase changes in the morning peak under LD [65]. To determine if M-cell manipulations also have sexually dimorphic effects on behavior, we sped up the molecular clock by expressing the Doubletime short allele, DBTs. Surprisingly, our results showed that speeding up the clock of M-cells advances the phase of the morning peak in males, while the female morning peak phase is not affected. These results support previous studies conducted in males on the role of M-cells in regulating the morning peak of activity [17, 22, 65], and suggest that the M cells are unable to regulate the phase of the morning activity in the same way in females. In DD, males had largely coherent short period rhythms and the majority (65%) were rhythmic. In contrast, only 40% of the females were rhythmic, and their FRP showed a bimodal distribution, with most flies exhibiting a ∼24-hour period. This further supports the notion that the M-cells are less dominant over the circadian clock network in females. One possible explanation is that other clock neurons are able to “resist” their influence, and the conflict between the fast-paced M cell clock and the ∼24 clock other clock neurons is what leads to higher arrhythmicity in the females.

The Cryptochrome and PdfR-expressing clusters of evening cells – the sNPF expressing E1 cluster and the ITP-expressing E2 cluster [78] – have roles in setting the phase of the E-peak under LD and sustaining behavioral rhythms in the absence of a functional molecular clock in the M-cells [67, 68]. To test if these cells have a differential influence over the network in males and females, we expressed DBTS under a driver that is expressed specifically in the E1 and E2 subsets of LNds. Our results showed that speeding up the clocks in the E1+E2 clusters resulted in a phase advance in evening peak of activity in both the sexes, but the effect was more pronounced in the females. Neither MB122 > DBTS males nor females had phenotypes under free running conditions. A possible reason for the behavioral differences seen between the sexes could be redundancy in females, such that the network is not as dependent on PDF and therefore on M-cells for timekeeping. This would mean that the female network could have more distributed mode of timekeeping throughout the circadian clock network.

Across species, sex differences in the circadian timing system are largely related to the regulation of reproduction relevant behaviors. In mammals, the SCN determines the timing of the release of reproductive hormones and influences timing of mating (reviewed in [72]) and aggression [79]. In Drosophila, the circadian clock controls the timing of sex-specific and sexually dimorphic behaviors, such as male courtship [80] and aggression [81] and female sexual receptivity [82] and egg-laying [83]. This regulation of rhythmic behaviors requires connectivity between clock neurons and downstream sex specific circuits. For example, the DN1p cluster, which has been shown to be more active in males [39], is functionally connected to the male-specific fru-expressing P1 neurons that regulate male courtship [40]. In females, Allostatin C-producing DN1ps have been shown to connect to downstream targets to control rhythms in oogenesis [84], and the Janelia female hemibrain connectome revealed that the LNds form connections doublesex-expressing PC1 cluster [16]. Our data suggest that the relative hierarchy of circadian oscillators is sexually dimorphic, with a less dominant M oscillator in females. This pattern of circadian timekeeping may serve an adaptive purpose, ensuring precise timing of essential female-specific behaviors crucial for reproductive fitness, such as sexual receptivity and egg laying.

Materials and Methods

Fly lines and rearing

All genotypes were reared on standard cornmeal medium under LD (12 hr Light: 12 hr Dark) cycles and 25°C, unless specified otherwise (see figure legends for details). The fly lines used in this study were Canton-S, w1118, Pdf01, PdfR01, w; Pdf-RFP, Pdf-Gal4; tub-Gal80ts, w;UAS-Cas9; UAS-Pdfg, w; Pdf-Gal4, w;;UAS-DBTs, and w;MB122B-Gal4;.

Activity-rest behavior recording and analysis

Individual male and virgin female flies (3–5 days old) were housed in glass locomotor tubes with sucrose agar food on one end and yarn on the other end. Locomotor activity was recorded using the Drosophila Activity Monitors (DAM, Trikinetics, Waltham, United States of America). Experiments were conducted in Tritech or Percival incubators under controlled light and temperature conditions. Flies were entrained to 12:12 LD cycles for at least 5 days, and then transferred to constant darkness (DD) for at least 7 days, at a constant temperature of 25°C, unless otherwise specified (see figure legends for details). Raw data obtained from the DAM system were scanned and binned into activity counts of 15 min interval using DAM File scan. Data was analyzed using the CLOCKLAB software (Actimetrics, Wilmette, IL).

Values of period and rhythmic power were calculated for a period of 8 days using the Chi-square periodogram with a cut-off of p =0.01. The Rhythmic Power for each designated rhythmic fly was determined by subtracting the Chi-squared significance value from the Power of the Periodogram. Flies that did not exhibit a periodicity peak above the significance threshold were categorized as “arrhythmic,” and their period and rhythmic power were not included in the analysis. Values of the Morning and Evening peaks was calculated using the PHASE software [85]. Total LD sleep values for all genotypes were calculated for a period of 3 days (LD days 2-4). Representative actograms were generated using ClockLab and activity plots were generated using PHASE. The period, rhythmic power, total sleep, phase values of all the flies for a particular experimental genotype were compared against the background or parental controls either using the Mann-Whitney test or the Kruskal-Wallis ANOVA followed by the Dunn’s multiple comparisons test. The details on the statistical comparisons and the no. of flies used in a given experiment are indicated in their respective figure legends. The number of rhythmic flies of the experimental genotype were compared against their respective background or parental controls using the Fisher’s exact test. All statistical analysis was done using GraphPad Prism 9.0.

Immunohistochemistry

Brains from adult male or female flies (6-8 days old) were dissected in ice-cold Schneider’s Insect media (S2) and fixed immediately after dissection in 2%Paraformaldehyde (PFA) in S2 media for 30 minutes at room temperature. The fixed brains were washed (3 washes of 10 mins each) with 0.3% PBS-Triton X 100 (PBS-TX) and then treated with the blocking solution (5% Normal Goat Serum made in 0.3% PBS-TX) for 1-hr at room temperature. The brains were then incubated with primary antibodies at 4°C for 24-48 hrs. The primary antibodies used were anti-PDF (mouse, 1:3000, C7, DSHB), anti-RFP (rabbit, 1:2000, Rockland). After incubation, the brains were given 6 washes with 0.3% PBS-TX and incubated with Alexa-fluor conjugated secondary antibodies overnight or for 24-48 hrs. at 4°C. The following secondary antibodies were used, goat anti-mouse 488 (1:3000, Invitrogen), goat anti-rabbit 568 (1:3000, Invitrogen). After incubation, the brain samples were washed 6 times with 0.3% PBS-TX, cleaned and mounted on a clean, glass slide using Vectashield mounting media.

Image acquisition and analysis

The slides were imaged using a confocal microscope (Olympus FV3000) using the 20X objective. Image analysis was performed using the Fiji software [86]. In the samples, small and large ventral lateral neurons were classified based on their anatomical locations and expression of PDF. PDF intensity in these cells were measured by selecting the slice of the Z-stack that showed the maximum intensity, drawing a Region of Interest (ROI) around the cells and measuring their intensities. 3–4 separate background values were also measured around each cell and the final intensity was taken as the difference between the cell intensity and the background. For quantification of PDF in the dorsal projections, a rectangular box was drawn as ROI starting from the point where the PDF projection turns into the dorsal brain and intensity was measured. 3–4 background values were also measured around the projection. The intensity values obtained from both the hemispheres for each cell type for each brain was averaged and used for statistical analysis. PDF intensity from the s-LNv were compared between the experimental and control genotypes using the Mann-Whitney test. To estimate different aspects of rhythmicity in PDF oscillations in the dorsal termini of s-LNv in males and females, we used a COSINOR based curve-fitting method [54] COSINOR analysis was implemented using the CATCosinor function from the CATkit package written for R [87].

Supplementary figures

Figure S1:
Figure S1: PDF rhythmic accumulation is similar in male and female brains. (Related to Figure 1.)

(A) Representative confocal images of control (w1118) (top) and experimental (Pdf01) male (middle) and female (bottom) flies stained with PDF antibodies. (B) Scatterplots of PDF staining intensities in s-LNv dorsal projections of both male and female flies plotted at different time-points over a 24-hr cycle on DD day3. Each dot represents the mean PDF intensity value averaged over both the hemispheres of one brain. The cyan and pink lines are the best fit Cosine curves from the parameters that were extracted using COSINOR analysis. See Table 2 for more details. (C) Amplitude values of PDF oscillation obtained from COSINOR curve fits are plotted for male and female flies. Error bars are 95% CI values calculated from the standard error obtained from COSINOR analysis. Overlapping error bars indicate that amplitude values of males and females are not significantly different from controls. COSINOR analysis was implemented using the CATCosinor function from the CATkit package written for R [87]. n > 7 brain samples.

Figure S2:
Figure S2: CRISPR-Cas9 mediated Pdf mutagenesis has similar effects on male and female sleep. (Related to Figure 3.)

(A) Average sleep plots under LD 12:12 of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right) are plotted. The plots are averaged over flies and days for a period of 3 days under LD 12:12 at 28°C. (B) Total sleep values under LD are plotted for control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right). (C) Average sleep plots of flies over 7 days in DD are plotted for male and female control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) flies. (D) Total sleep values under DD are plotted for control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right). (E) The difference in the total DD sleep values of experimental males and females from their respective controls are plotted.

Figure S3:
Figure S3: CRISPR-Cas9 mediated Pdf mutagenesis has more pronounced effects on male circadian behavior at 25C. (Related to Figure 3.)

(A) Percentage of rhythmic flies are plotted for control (Pdf-Gal4), and (UAS Cas9; Pdf-g), and experimental (Pdf > Cas9; Pdf-g) males (left, n = 36 (Pdf-Gal4), n = 49 (UAS Cas9; Pdf-g), n = 52 (Pdf > Cas9; Pdf-g) and females (right, n = 33 (Pdf-Gal4), n = 46 (UAS Cas9; Pdf-g), n = 35 (Pdf > Cas9; Pdf-g). Error bars are SEM values plotted across 2 replicate experiments. (B) Free-running period of control (Pdf-Gal4 and UAS Cas9; Pdf-g) and experimental (Pdf > Cas9; Pdf-g) males (left) and females (right) calculated using the Chi-square periodogram are plotted. (C) Rhythmic power of control (Pdf-Gal4 and UAS Cas9; Pdf-g) and experimental (Pdf > Cas9; Pdf-g) males (left) and females (right) calculated using the Chi-square periodogram are plotted. (D) The difference in the rhythmic power values of experimental males and females from their respective controls are plotted.

Figure S4:
Figure S4: Adult specific CRISPR-Cas9 mediated Pdf mutagenesis has more pronounced effects on male circadian behavior. (Related to Figure 3.)

(A) Representative confocal images of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts) (top) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) male (middle) and female (bottom) flies stained with RFP and PDF antibodies. The experimental flies show significant reduction in PDF levels but no misrouting of the s-LNv dorsal termini. (B) Quantification of PDF intensity from s-LNv cell bodies in control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) flies are plotted for males (left) and females (right). Data points represent intensity from a single brain sample. n > 10 brains for all genotypes. (C) The difference in PDF intensity values of experimental males and females from their respective controls are plotted. (D) Percentage of rhythmic flies are plotted for control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts), and (UAS Cas9; Pdf-g), and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left, n = 103 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts), n = 109 (UAS Cas9; Pdf-g), n = 115 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) and females (right, n = 96 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts), n = 90 (UAS Cas9; Pdf-g), n = 119 (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g). Error bars are SEM values plotted across 3 replicate experiments. (E) Free-running period of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right) calculated using the Chi-square periodogram are plotted. (F) Rhythmic power of control (Pdf-RFP, Pdf-Gal4; tub-Gal80ts and UAS Cas9; Pdf-g) and experimental (Pdf-RFP, Pdf-Gal4; tub-Gal80ts > Cas9; Pdf-g) males (left) and females (right) calculated using the Chi-square periodogram are plotted. Flies were raised at 18°C until eclosion and were then transferred to 28°C as adults. Adult flies were kept at 28°C for a period of 7 days before recording activity-rest behavior at 28°C. Free-running period and rhythmic power values are calculated using Chi square periodogram in CLOCKLAB for a period of 7 days. Statistical comparisons were performed between the control and experimental flies for both sexes using the Kruskal-Wallis ANOVA followed by the Dunn’s multiple comparisons test. The box plots extend from the 25th to 75th percentile, with whiskers extending from the smallest to the largest value, and each point representing data from a single fly. Combined data from at least 3 replicate experiments are plotted.

Acknowledgments

We are grateful to Abhilash Lakshman, Jeff Price, Orie Shafer, and Paul Taghert for helpful discussions members of the Fernandez Lab for helpful comments on the manuscript. We also thank Justin Blau, Aljoscha Nern, Michael Rosbash, Gerry Rubin, and Paul Taghert for sharing fly lines. The mouse anti-PDF antibody was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This work was supported by NIH grant (R01NS118012), an NSF Grant (IOS-2239994) to M.P.F. and Barnard Internal Funds (SRI) to E.S-C., G.B. and E.B.

References

  1. 1.↵
    Anderson ST, FitzGerald GA. Sexual dimorphism in body clocks. Science. 2020;369(6508):1164-5. Epub 2020/09/05. doi: 10.1126/science.abd4964. PubMed PMID: 32883849.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    Bailey M, Silver R. Sex differences in circadian Vming systems: implicaVons for disease. Front Neuroendocrinol. 2014;35(1):111–39. Epub 2013/11/30. doi: 10.1016/j.yfrne.2013.11.003. PubMed PMID: 24287074; PubMed Central PMCID: PMCPMC4041593.
    OpenUrlCrossRefPubMed
  3. 3.↵
    Vida B, Hrabovszky E, KalamaVanos T, Coen CW, Liposits Z, Kalló I. Oestrogen Receptor α and β ImmunoreacVve Cells in the SuprachiasmaVc Nucleus of Mice: DistribuVon, Sex Differences and RegulaVon by Gonadal Hormones. Journal of Neuroendocrinology. 2008;20(11):1270–7. doi: pmcid:10.1111/j.1365-2826.2008.01787.x.
    OpenUrlCrossRefPubMedWeb of Science
  4. 4.↵
    Lyall LM, Wyse CA, Graham N, Ferguson A, Lyall DM, Cullen B, et al. AssociaVon of disrupted circadian rhythmicity with mood disorders, subjecVve wellbeing, and cogniVve funcVon: a cross-secVonal study of 91 105 parVcipants from the UK Biobank. Lancet Psychiatry. 2018;5(6):507-14. Epub 2018/05/20. doi: 10.1016/S2215-0366(18)30139-1. PubMed PMID: 29776774.
    OpenUrlCrossRefPubMed
  5. 5.↵
    Bixler EO, Papaliaga MN, Vgontzas AN, Lin HM, Pejovic S, Karataraki M, et al. Women sleep objecVvely beger than men and the sleep of young women is more resilient to external stressors: effects of age and menopause. J Sleep Res. 2009;18(2):221–8. Epub 2009/03/24. doi: 10.1111/j.1365-2869.2008.00713.x. PubMed PMID: 19302341; PubMed Central PMCID: PMCPMC3594776.
    OpenUrlCrossRefPubMedWeb of Science
  6. 6.↵
    HasVngs MH, Maywood ES, Brancaccio M. The Mammalian Circadian Timing System and the SuprachiasmaVc Nucleus as Its Pacemaker. Biology (Basel). 2019;8(1). Epub 20190311. doi: 10.3390/biology8010013. PubMed PMID: 30862123; PubMed Central PMCID: PMCPMC6466121.
    OpenUrlCrossRefPubMed
  7. 7.↵
    Helfrich-Forster C. The neuroarchitecture of the circadian clock in the brain of Drosophila melanogaster. Microsc Res Tech. 2003;62(2):94–102. Epub 2003/09/11. doi: 10.1002/jemt.10357. PubMed PMID: 12966496.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    Schubert FK, Hagedorn N, Yoshii T, Helfrich-Forster C, Rieger D. Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their funcVonal role in the circadian system. J Comp Neurol. 2018;526(7):1209–31. Epub 2018/02/10. doi: 10.1002/cne.24406. PubMed PMID: 29424420; PubMed Central PMCID: PMCPMC5873451.
    OpenUrlCrossRefPubMed
  9. 9.↵
    Yu W, Hardin PE. Circadian oscillators of Drosophila and mammals. J Cell Sci. 2006;119(Pt 23):4793–5. Epub 2006/11/30. doi: 10.1242/jcs.03174. PubMed PMID: 17130292.
    OpenUrlFREE Full Text
  10. 10.↵
    Kloss B, Price JL, Saez L, Blau J, Rothenfluh A, Wesley CS, et al. The Drosophila clock gene double-Vme encodes a protein closely related to human casein kinase Iepsilon. Cell. 1998;94(1):97–107. Epub 1998/07/23. doi: 10.1016/s0092-8674(00)81225-8. PubMed PMID: 9674431.
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    Price JL, Blau J, Rothenfluh A, Abodeely M, Kloss B, Young MW. double-Vme is a novel Drosophila clock gene that regulates PERIOD protein accumulaVon. Cell. 1998;94(1):83–95. Epub 1998/07/23. doi: 10.1016/s0092-8674(00)81224-6. PubMed PMID: 9674430.
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    Helfrich-Forster C. OrganizaVon of endogenous clocks in insects. Biochem Soc Trans. 2005;33(Pt 5):957–61. Epub 2005/10/26. doi: 10.1042/BST20050957. PubMed PMID: 16246020.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.
    Dubowy C, Sehgal A. Circadian Rhythms and Sleep in Drosophila melanogaster. GeneVcs. 2017;205(4):1373–97. Epub 2017/04/01. doi: 10.1534/geneVcs.115.185157. PubMed PMID: 28360128; PubMed Central PMCID: PMCPMC5378101.
    OpenUrlCrossRefPubMed
  14. 14.↵
    Ma D, Przybylski D, Abruzzi KC, SchlichVng M, Li Q, Long X, et al. A transcriptomic taxonomy of Drosophila circadian neurons around the clock. eLife. 2021;10:e63056. doi: 10.7554/eLife.63056.
    OpenUrlCrossRef
  15. 15.
    Shafer OT, Helfrich-Forster C, Renn SC, Taghert PH. ReevaluaVon of Drosophila melanogaster’s neuronal circadian pacemakers reveals new neuronal classes. J Comp Neurol. 2006;498(2):180–93. Epub 2006/07/21. doi: 10.1002/cne.21021. PubMed PMID: 16856134; PubMed Central PMCID: PMCPMC2596765.
    OpenUrlCrossRefPubMedWeb of Science
  16. 16.↵
    Shafer OT, GuVerrez GJ, Li K, Mildenhall A, Spira D, Marty J, et al. Connectomic analysis of the Drosophila lateral neuron clock cells reveals the synapVc basis of funcVonal pacemaker classes. Elife. 2022;11. Epub 2022/06/30. doi: 10.7554/eLife.79139. PubMed PMID: 35766361; PubMed Central PMCID: PMCPMC9365390.
    OpenUrlCrossRefPubMed
  17. 17.↵
    Grima B, Chelot E, Xia R, Rouyer F. Morning and evening peaks of acVvity rely on different clock neurons of the Drosophila brain. Nature. 2004;431(7010):869-73. Epub 2004/10/16. doi: 10.1038/nature02935. PubMed PMID: 15483616.
    OpenUrlCrossRefPubMedWeb of Science
  18. 18.↵
    Rieger D, Shafer OT, Tomioka K, Helfrich-Forster C. FuncVonal analysis of circadian pacemaker neurons in Drosophila melanogaster. J Neurosci. 2006;26(9):2531–43. Epub 2006/03/03. doi: 10.1523/JNEUROSCI.1234-05.2006. PubMed PMID: 16510731.
    OpenUrlAbstract/FREE Full Text
  19. 19.↵
    Renn SC, Park JH, Rosbash M, Hall JC, Taghert PH. A pdf neuropepVde gene mutaVon and ablaVon of PDF neurons each cause severe abnormaliVes of behavioral circadian rhythms in Drosophila. Cell. 1999;99(7):791–802. Epub 2000/01/05. PubMed PMID: 10619432.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Park JH, Helfrich-Forster C, Lee G, Liu L, Rosbash M, Hall JC. DifferenVal regulaVon of circadian pacemaker output by separate clock genes in Drosophila. Proc Natl Acad Sci U S A. 2000;97(7):3608–13. Epub 2000/03/22. doi: 10.1073/pnas.070036197. PubMed PMID: 10725392; PubMed Central PMCID: PMCPMC16287.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    Stoleru D, Peng Y, Nawathean P, Rosbash M. A reseong signal between Drosophila pacemakers synchronizes morning and evening acVvity. Nature. 2005;438(7065):238-42. Epub 2005/11/11. doi: 10.1038/nature04192. PubMed PMID: 16281038.
    OpenUrlCrossRefPubMedWeb of Science
  22. 22.↵
    Stoleru D, Peng Y, Agosto J, Rosbash M. Coupled oscillators control morning and evening locomotor behaviour of Drosophila. Nature. 2004;431(7010):862-8. Epub 2004/10/16. doi: 10.1038/nature02926. PubMed PMID: 15483615.
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    Fernandez MP, Peobone HL, Bogart JT, Roell CJ, Davey CE, Pranevicius A, et al. Sites of Circadian Clock Neuron PlasVcity Mediate Sensory IntegraVon and Entrainment. Curr Biol. 2020;30(12):2225–37 e5. Epub 2020/05/11. doi: 10.1016/j.cub.2020.04.025. PubMed PMID: 32386535; PubMed Central PMCID: PMCPMC7314648.
    OpenUrlCrossRefPubMed
  24. 24.
    Guo F, Chen X, Rosbash M. Temporal calcium profiling of specific circadian neurons in freely moving flies. Proc Natl Acad Sci U S A. 2017;114(41):E8780-E7. Epub 2017/10/05. doi: 10.1073/pnas.1706608114. PubMed PMID: 28973886; PubMed Central PMCID: PMCPMC5642695.
    OpenUrlAbstract/FREE Full Text
  25. 25.
    Lamaze A, Stanewsky R. DN1p or the “Fluffy” Cerberus of Clock Outputs. Front Physiol. 2019;10:1540. Epub 2020/01/24. doi: 10.3389/fphys.2019.01540. PubMed PMID: 31969832; PubMed Central PMCID: PMCPMC6960142.
    OpenUrlCrossRefPubMed
  26. 26.
    Collins B, Kane Elizabeth A, Reeves David C, Akabas Myles H, Blau J. Balance of AcVvity between LNvs and Glutamatergic Dorsal Clock Neurons Promotes Robust Circadian Rhythms in Drosophila. Neuron. 2012;74(4):706–18. doi: pmcid:10.1016/j.neuron.2012.02.034.
    OpenUrlCrossRefPubMedWeb of Science
  27. 27.↵
    Women, MinoriVes, and Persons with DisabiliVes in Science and Engineering: 2021. Special Report NSF 21-321. Alexandria, VA: NaVonal Science FoundaVon: NaVonal Center for Science and Engineering StaVsVcs, 2021.
  28. 28.↵
    Yoshii T, Wulbeck C, Sehadova H, Veleri S, Bichler D, Stanewsky R, et al. The neuropepVde pigment-dispersing factor adjusts period and phase of Drosophila’s clock. J Neurosci. 2009;29(8):2597–610. Epub 2009/02/27. doi: 10.1523/JNEUROSCI.5439-08.2009. PubMed PMID: 19244536.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    Peng Y, Stoleru D, Levine JD, Hall JC, Rosbash M. Drosophila free-running rhythms require intercellular communicaVon. PLoS Biol. 2003;1(1):E13. Epub 2003/09/17. doi: 10.1371/journal.pbio.0000013. PubMed PMID: 12975658; PubMed Central PMCID: PMCPMC193604.
    OpenUrlCrossRefPubMed
  30. 30.↵
    Liang X, Holy TE, Taghert PH. A Series of Suppressive Signals within the Drosophila Circadian Neural Circuit Generates SequenVal Daily Outputs. Neuron. 2017;94(6):1173–89 e4. Epub 2017/05/30. doi: 10.1016/j.neuron.2017.05.007. PubMed PMID: 28552314; PubMed Central PMCID: PMCPMC5502710.
    OpenUrlCrossRefPubMed
  31. 31.↵
    Mertens I, Vandingenen A, Johnson EC, Shafer OT, Li W, Trigg JS, et al. PDF Receptor Signaling in Drosophila Contributes to Both Circadian and GeotacVc Behaviors. Neuron. 2005;48(2):213–9. doi: pmcid:10.1016/j.neuron.2005.09.009.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.
    Hyun S, Lee Y, Hong ST, Bang S, Paik D, Kang J, et al. Drosophila GPCR Han is a receptor for the circadian clock neuropepVde PDF. Neuron. 2005;48(2):267–78. Epub 2005/10/26. doi: 10.1016/j.neuron.2005.08.025. PubMed PMID: 16242407.
    OpenUrlCrossRefPubMedWeb of Science
  33. 33.↵
    Lear BC, Merrill CE, Lin JM, Schroeder A, Zhang L, Allada R. A G protein-coupled receptor, groom-of-PDF, is required for PDF neuron acVon in circadian behavior. Neuron. 2005;48(2):221–7. Epub 2005/10/26. doi: 10.1016/j.neuron.2005.09.008. PubMed PMID: 16242403.
    OpenUrlCrossRefPubMedWeb of Science
  34. 34.↵
    Kim WJ, Jan LY, Jan YN. A PDF/NPF neuropepVde signaling circuitry of male Drosophila melanogaster controls rival-induced prolonged maVng. Neuron. 2013;80(5):1190–205. doi: 10.1016/j.neuron.2013.09.034. PubMed PMID: 24314729; PubMed Central PMCID: PMCPMC3867312.
    OpenUrlCrossRefPubMedWeb of Science
  35. 35.↵
    Krupp JJ, Billeter JC, Wong A, Choi C, Nitabach MN, Levine JD. Pigment-dispersing factor modulates pheromone producVon in clock cells that influence maVng in drosophila. Neuron. 2013;79(1):54–68. Epub 2013/07/16. doi: 10.1016/j.neuron.2013.05.019. PubMed PMID: 23849197; PubMed Central PMCID: PMCPMC3955580.
    OpenUrlCrossRefPubMed
  36. 36.↵
    Inami S, Sato S, Kondo S, Tanimoto H, Kitamoto T, Sakai T. Environmental Light Is Required for Maintenance of Long-Term Memory in Drosophila. J Neurosci. 2020;40(7):1427–39. Epub 2020/01/15. doi: 10.1523/JNEUROSCI.1282-19.2019. PubMed PMID: 31932417; PubMed Central PMCID: PMCPMC7044726.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    Mertens I, Vandingenen A, Johnson EC, Shafer OT, Li W, Trigg JS, et al. PDF receptor signaling in Drosophila contributes to both circadian and geotacVc behaviors. Neuron. 2005;48(2):213–9. Epub 2005/10/26. doi: 10.1016/j.neuron.2005.09.009. PubMed PMID: 16242402.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    Toma DP, White KP, Hirsch J, Greenspan RJ. IdenVficaVon of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait. Nat Genet. 2002;31(4):349–53. Epub 2002/06/04. doi: 10.1038/ng893. PubMed PMID: 12042820.
    OpenUrlCrossRefPubMedWeb of Science
  39. 39.↵
    Guo F, Yu J, Jung HJ, Abruzzi KC, Luo W, Griffith LC, et al. Circadian neuron feedback controls the Drosophila sleep–acVvity profile. Nature. 2016;536:292. doi: 10.1038/nature19097 https://www.nature.com/arVcles/nature19097#supplementary-informaVon.
    OpenUrlCrossRefPubMed
  40. 40.↵
    Chen D, Sitaraman D, Chen N, Jin X, Han C, Chen J, et al. GeneVc and neuronal mechanisms governing the sex-specific interacVon between sleep and sexual behaviors in Drosophila. Nat Commun. 2017;8(1):154. Epub 2017/07/30. doi: 10.1038/s41467-017-00087-5. PubMed PMID: 28754889; PubMed Central PMCID: PMCPMC5533705.
    OpenUrlCrossRefPubMed
  41. 41.
    Kunst M, Hughes ME, Raccuglia D, Felix M, Li M, Barneg G, et al. Calcitonin gene-related pepVde neurons mediate sleep-specific circadian output in Drosophila. Curr Biol. 2014;24(22):2652–64. Epub 2014/12/03. doi: 10.1016/j.cub.2014.09.077. PubMed PMID: 25455031; PubMed Central PMCID: PMCPMC4255360.
    OpenUrlCrossRefPubMed
  42. 42.↵
    Asahina K. Sex differences in Drosophila behavior: QualitaVve and QuanVtaVve Dimorphism. Curr Opin Physiol. 2018;6:35–45. Epub 2018/11/06. doi: 10.1016/j.cophys.2018.04.004. PubMed PMID: 30386833; PubMed Central PMCID: PMCPMC6205217.
    OpenUrlCrossRefPubMed
  43. 43.↵
    Helfrich-Forster C. DifferenVal control of morning and evening components in the acVvity rhythm of Drosophila melanogaster--sex-specific differences suggest a different quality of acVvity. J Biol Rhythms. 2000;15(2):135–54. Epub 2000/04/13. doi: 10.1177/074873040001500208. PubMed PMID: 10762032.
    OpenUrlCrossRefPubMedWeb of Science
  44. 44.↵
    Palmateer CM, ArVkis C, Brovero SG, Friedman B, Gresham A, Arbeitman MN. Single-cell transcriptome profiles of Drosophila fruitless-expressing neurons from both sexes. eLife. 2023;12:e78511. doi: 10.7554/eLife.78511.
    OpenUrlCrossRef
  45. 45.↵
    Fujii S, Amrein H. Ventral lateral and DN1 clock neurons mediate disVnct properVes of male sex drive rhythm in Drosophila. Proc Natl Acad Sci U S A. 2010;107(23):10590–5. Epub 2010/05/26. doi: 10.1073/pnas.0912457107. PubMed PMID: 20498055; PubMed Central PMCID: PMCPMC2890825.
    OpenUrlAbstract/FREE Full Text
  46. 46.↵
    Hanafusa S, Kawaguchi T, Umezaki Y, Tomioka K, Yoshii T. Sexual interacVons influence the molecular oscillaVons in DN1 pacemaker neurons in Drosophila melanogaster. PLoS One. 2013;8(12):e84495. Epub 2013/12/25. doi: 10.1371/journal.pone.0084495. PubMed PMID: 24367668; PubMed Central PMCID: PMCPMC3867508.
    OpenUrlCrossRefPubMed
  47. 47.↵
    Lee G, Bahn JH, Park JH. Sex-and clock-controlled expression of the neuropepVde F gene in Drosophila. Proc Natl Acad Sci U S A. 2006;103(33):12580–5. Epub 20060807. doi: 10.1073/pnas.0601171103. PubMed PMID: 16894172; PubMed Central PMCID: PMCPMC1567921.
    OpenUrlAbstract/FREE Full Text
  48. 48.↵
    Hermann C, Yoshii T, Dusik V, Helfrich-Forster C. NeuropepVde F immunoreacVve clock neurons modify evening locomotor acVvity and free-running period in Drosophila melanogaster. J Comp Neurol. 2012;520(5):970–87. Epub 2011/08/10. doi: 10.1002/cne.22742. PubMed PMID: 21826659.
    OpenUrlCrossRefPubMed
  49. 49.↵
    Nässel DR, Zandawala M. Recent advances in neuropepVde signaling in Drosophila, from genes to physiology and behavior. Prog Neurobiol. 2019;179:101607. Epub 2019/03/25. doi: 10.1016/j.pneurobio.2019.02.003. PubMed PMID: 30905728.
    OpenUrlCrossRefPubMed
  50. 50.↵
    Shafer OT, Yao Z. Pigment-Dispersing Factor Signaling and Circadian Rhythms in Insect Locomotor AcVvity. Curr Opin Insect Sci. 2014;1:73–80. Epub 2014/11/12. doi: 10.1016/j.cois.2014.05.002. PubMed PMID: 25386391; PubMed Central PMCID: PMCPMC4224320.
    OpenUrlCrossRefPubMed
  51. 51.↵
    Kuwano R, Katsura M, Iwata M, Yokosako T, Yoshii T. Pigment-dispersing factor and CCHamide1 in the Drosophila circadian clock network. Chronobiol Int. 2023;40(3):284–99. Epub 2023/02/15. doi: 10.1080/07420528.2023.2166416. PubMed PMID: 36786215.
    OpenUrlCrossRefPubMed
  52. 52.↵
    Parisky KM, Agosto J, Pulver SR, Shang Y, Kuklin E, Hodge JJ, et al. PDF cells are a GABA-responsive wake-promoVng component of the Drosophila sleep circuit. Neuron. 2008;60(4):672-82. Epub 2008/11/29. doi: 10.1016/j.neuron.2008.10.042. PubMed PMID: 19038223; PubMed Central PMCID: PMCPMC2734413.
    OpenUrlCrossRefPubMedWeb of Science
  53. 53.↵
    Klose MK, Bruchez MP, Deitcher DL, Levitan ES. Temporally and spaVally parVVoned neuropepVde release from individual clock neurons. Proc Natl Acad Sci U S A. 2021;118(17). Epub 2021/04/21. doi: 10.1073/pnas.2101818118. PubMed PMID: 33875606; PubMed Central PMCID: PMCPMC8092580.
    OpenUrlAbstract/FREE Full Text
  54. 54.↵
    Cornelissen G. Cosinor-based rhythmometry. TheoreVcal Biology and Medical Modelling. 2014;11(1):16. doi: 10.1186/1742-4682-11-16.
    OpenUrlCrossRefPubMed
  55. 55.↵
    Im SH, Li W, Taghert PH. PDFR and CRY signaling converge in a subset of clock neurons to modulate the amplitude and phase of circadian behavior in Drosophila. PLoS One. 2011;6(4):e18974. Epub 2011/05/12. doi: 10.1371/journal.pone.0018974. PubMed PMID: 21559487; PubMed Central PMCID: PMCPMC3084726.
    OpenUrlCrossRefPubMed
  56. 56.↵
    Reinhard N, Fukuda A, Manoli G, Derksen E, Saito A, Möller G, et al. SynapVc and pepVdergic connectomes of the <em>Drosophila</em> circadian clock. bioRxiv. 2023:2023.09.11.557222. doi: 10.1101/2023.09.11.557222.
    OpenUrlAbstract/FREE Full Text
  57. 57.↵
    Richhariya S, Shin D, Le JQ, Rosbash M. DissecVng neuron-specific funcVons of circadian genes using modified cell-specific CRISPR approaches. Proc Natl Acad Sci U S A. 2023;120(29):e2303779120. Epub 2023/07/10. doi: 10.1073/pnas.2303779120. PubMed PMID: 37428902; PubMed Central PMCID: PMCPMC10629539.
    OpenUrlCrossRefPubMed
  58. 58.↵
    GorosVza EA, Depetris-Chauvin A, Frenkel L, Pírez N, Ceriani María F. Circadian Pacemaker Neurons Change SynapVc Contacts across the Day. Current Biology. 2014;24(18):2161–7. doi: pmcid:10.1016/j.cub.2014.07.063.
    OpenUrlCrossRefPubMed
  59. 59.↵
    Ruben M, Drapeau MD, Mizrak D, Blau J. A mechanism for circadian control of pacemaker neuron excitability. J Biol Rhythms. 2012;27(5):353–64. doi: 10.1177/0748730412455918. PubMed PMID: 23010658; PubMed Central PMCID: PMCPMC4019749.
    OpenUrlCrossRefPubMed
  60. 60.↵
    GorosVza EA, Ceriani MF. Retrograde Bone MorphogeneVc Protein Signaling Shapes a Key Circadian Pacemaker Circuit. The Journal of Neuroscience. 2013;33(2):687–96. doi: 10.1523/jneurosci.3448-12.2013.
    OpenUrlAbstract/FREE Full Text
  61. 61.↵
    Fleck SA, Biswas P, DeWig ED, Knuteson RL, Eisman RC, Nemkov T, et al. Auxin Exposure Disrupts Feeding Behavior and Fagy Acid Metabolism in Adult Drosophila. bioRxiv. 2023. Epub 2023/08/30. doi: 10.1101/2023.08.15.553385. PubMed PMID: 37645868; PubMed Central PMCID: PMCPMC10462055.
    OpenUrlAbstract/FREE Full Text
  62. 62.↵
    McGuire SE, Mao Z, Davis RL. SpaVotemporal gene expression targeVng with the TARGET and gene-switch systems in Drosophila. Sci STKE. 2004;2004(220):pl6. Epub 2004/02/19. doi: 10.1126/stke.2202004pl6. PubMed PMID: 14970377.
    OpenUrlAbstract/FREE Full Text
  63. 63.↵
    Yao Z, Shafer OT. The Drosophila circadian clock is a variably coupled network of mulVple pepVdergic units. Science. 2014;343(6178):1516-20. Epub 2014/03/29. doi: 10.1126/science.1251285. PubMed PMID: 24675961; PubMed Central PMCID: PMCPMC4259399.
    OpenUrlAbstract/FREE Full Text
  64. 64.↵
    Muskus MJ, Preuss F, Fan J-Y, Bjes ES, Price JL. Drosophila DBT Lacking Protein Kinase AcVvity Produces Long-Period and Arrhythmic Circadian Behavioral and Molecular Rhythms. Molecular and Cellular Biology. 2007;27(23):8049–64. doi: 10.1128/mcb.00680-07.
    OpenUrlAbstract/FREE Full Text
  65. 65.↵
    Yao Z, Benneg AJ, Clem JL, Shafer OT. The Drosophila Clock Neuron Network Features Diverse Coupling Modes and Requires Network-wide Coherence for Robust Circadian Rhythms. Cell Rep. 2016;17(11):2873–81. Epub 2016/12/16. doi: 10.1016/j.celrep.2016.11.053. PubMed PMID: 27974202; PubMed Central PMCID: PMCPMC5161247.
    OpenUrlCrossRefPubMed
  66. 66.↵
    Brown MP, Verma S, Palmer I, Zuniga AG, Rosensweig C, Keles MF, et al. A subclass of evening cells promotes the switch from arousal to sleep at dusk. bioRxiv. 2023. Epub 2023/09/11. doi: 10.1101/2023.08.28.555147. PubMed PMID: 37693540; PubMed Central PMCID: PMCPMC10491161.
    OpenUrlAbstract/FREE Full Text
  67. 67.↵
    Delventhal R, O’Connor RM, Pantalia MM, Ulgherait M, Kim HX, Basturk MK, et al. DissecVon of central clock funcVon in Drosophila through cell-specific CRISPR-mediated clock gene disrupVon. Elife. 2019;8. Epub 2019/10/16. doi: 10.7554/eLife.48308. PubMed PMID: 31613218; PubMed Central PMCID: PMCPMC6794090.
    OpenUrlCrossRefPubMed
  68. 68.↵
    SchlichVng M, Diaz MM, Xin J, Rosbash M. Neuron-specific knockouts indicate the importance of network communicaVon to Drosophila rhythmicity. Elife. 2019;8. Epub 2019/10/16. doi: 10.7554/eLife.48301. PubMed PMID: 31613223; PubMed Central PMCID: PMCPMC6794074.
    OpenUrlCrossRefPubMed
  69. 69.↵
    Shansky RM, Murphy AZ. Considering sex as a biological variable will require a global shi{ in science culture. Nature Neuroscience. 2021;24(4):457–64. doi: 10.1038/s41593-021-00806-8.
    OpenUrlCrossRefPubMed
  70. 70.↵
    Kokras N, Dalla C. Sex differences in animal models of psychiatric disorders. BriVsh Journal of Pharmacology. 2014;171(20):4595–619. doi: pmcid:10.1111/bph.12710.
    OpenUrl
  71. 71.↵
    Beery AK, Zucker I. Sex bias in neuroscience and biomedical research. Neurosci Biobehav Rev. 2011;35(3):565–72. Epub 2010/07/14. doi: 10.1016/j.neubiorev.2010.07.002. PubMed PMID: 20620164; PubMed Central PMCID: PMCPMC3008499.
    OpenUrlCrossRefPubMed
  72. 72.↵
    Joye DAM, Evans JA. Sex differences in daily Vmekeeping and circadian clock circuits. Semin Cell Dev Biol. 2022;126:45–55. Epub 20210514. doi: 10.1016/j.semcdb.2021.04.026. PubMed PMID: 33994299; PubMed Central PMCID: PMCPMC8589873.
    OpenUrlCrossRefPubMed
  73. 73.↵
    Taghert PH, Nitabach MN. PepVde neuromodulaVon in invertebrate model systems. Neuron. 2012;76(1):82–97. Epub 2012/10/09. doi: 10.1016/j.neuron.2012.08.035. PubMed PMID: 23040808; PubMed Central PMCID: PMCPMC3466441.
    OpenUrlCrossRefPubMed
  74. 74.↵
    Goda T, Umezaki Y, Alwagari F, Seo HW, Hamada FN. NeuropepVdes PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor acVvity. ScienVfic Reports. 2019;9(1):838. doi: 10.1038/s41598-018-37107-3.
    OpenUrlCrossRef
  75. 75.↵
    Helfrich-Forster C. Robust circadian rhythmicity of Drosophila melanogaster requires the presence of lateral neurons: a brain-behavioral study of disconnected mutants. J Comp Physiol A. 1998;182(4):435–53. Epub 1998/04/08. PubMed PMID: 9530835.
    OpenUrlCrossRefPubMed
  76. 76.↵
    Fernandez MP, Berni J, Ceriani MF. Circadian remodeling of neuronal circuits involved in rhythmic behavior. PLoS Biol. 2008;6(3):e69. Epub 2008/03/28. doi: 10.1371/journal.pbio.0060069. PubMed PMID: 18366255; PubMed Central PMCID: PMCPMC2270325.
    OpenUrlCrossRefPubMed
  77. 77.↵
    Biondi G, McCormick G, Fernandez MP. The ȼem>Drosophila</em> Circadian Clock Gene <em>Cycle</em> Controls Development of Clock Neurons. bioRxiv. 2023:2023.10.29.564626. doi: 10.1101/2023.10.29.564626.
    OpenUrlAbstract/FREE Full Text
  78. 78.↵
    Johard HAD, Yoishii T, Dircksen H, Cusumano P, Rouyer F, Helfrich-Förster C, et al. PepVdergic clock neurons in Drosophila: Ion transport pepVde and short neuropepVde F in subsets of dorsal and ventral lateral neurons. The Journal of ComparaVve Neurology. 2009;516(1):59–73. doi: 10.1002/cne.22099.
    OpenUrlCrossRefPubMedWeb of Science
  79. 79.↵
    Todd WD, Fenselau H, Wang JL, Zhang R, Machado NL, Venner A, et al. A hypothalamic circuit for the circadian control of aggression. Nat Neurosci. 2018;21(5):717–24. Epub 20180409. doi: 10.1038/s41593-018-0126-0. PubMed PMID: 29632359; PubMed Central PMCID: PMCPMC5920747.
    OpenUrlCrossRefPubMed
  80. 80.↵
    Fujii S, Krishnan P, Hardin P, Amrein H. Nocturnal male sex drive in Drosophila. Curr Biol. 2007;17(3):244–51. Epub 2007/02/06. doi: 10.1016/j.cub.2006.11.049. PubMed PMID: 17276917; PubMed Central PMCID: PMCPMC2239012.
    OpenUrlCrossRefPubMedWeb of Science
  81. 81.↵
    Chowdhury B, Wang M, Gnerer JP, Dierick HA. The Divider Assay is a high-throughput pipeline for aggression analysis in Drosophila. Commun Biol. 2021;4(1):85. Epub 2021/01/21. doi: 10.1038/s42003-020-01617-6. PubMed PMID: 33469118; PubMed Central PMCID: PMCPMC7815768.
    OpenUrlCrossRefPubMed
  82. 82.↵
    Sakai T, Ishida N. Circadian rhythms of female maVng acVvity governed by clock genes in Drosophila. Proc Natl Acad Sci U S A. 2001;98(16):9221–5. Epub 2001/07/27. doi: 10.1073/pnas.151443298. PubMed PMID: 11470898; PubMed Central PMCID: PMCPMC55401.
    OpenUrlAbstract/FREE Full Text
  83. 83.↵
    T M, Hari Dass S, Sharma VK. Egg-laying rhythm in Drosophila melanogaster. J Genet. 2008;87(5):495–504. Epub 2009/01/17. doi: 10.1007/s12041-008-0072-9. PubMed PMID: 19147938.
    OpenUrlCrossRefPubMedWeb of Science
  84. 84.↵
    Zhang C, Daubnerova I, Jang YH, Kondo S, Žitňan D, Kim YJ. The neuropepVde allatostaVn C from clock-associated DN1p neurons generates the circadian rhythm for oogenesis. Proc Natl Acad Sci U S A. 2021;118(4). doi: 10.1073/pnas.2016878118. PubMed PMID: 33479181; PubMed Central PMCID: PMCPMC7848730.
    OpenUrlAbstract/FREE Full Text
  85. 85.↵
    Persons JL, Abhilash L, Lopatkin AJ, Roelofs A, Bell EV, Fernandez MP, et al. PHASE: An Open-Source Program for the Analysis of DrosophilaPhase, AcVvity, and Sleep Under Entrainment. J Biol Rhythms. 2022;37(4):455–67. Epub 2022/06/22. doi: 10.1177/07487304221093114. PubMed PMID: 35727044; PubMed Central PMCID: PMCPMC10362883.
    OpenUrlCrossRefPubMed
  86. 86.↵
    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source pla∼orm for biological-image analysis. Nature Methods. 2012;9:676. doi: 10.1038/nmeth.2019 https://www.nature.com/arVcles/nmeth.2019#supplementary-informaVon.
    OpenUrlCrossRefPubMedWeb of Science
  87. 87.↵
    Lee Gierke C, Cornelissen G. Chronomics analysis toolkit (CATkit). Biol Rhythm Res. 2016;47(2):163–81. doi: 10.1080/09291016.2015.1094965.
    OpenUrlCrossRef
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Posted February 01, 2024.
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The Circadian Neuropeptide PDF has Sexually Dimorphic Effects on Activity Rhythms
Aishwarya R. Iyer, Eva Scholz-Carlson, Evardra Bell, Grace Biondi, Shlesha Richhariya, Maria P. Fernandez
bioRxiv 2024.01.31.578273; doi: https://doi.org/10.1101/2024.01.31.578273
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