Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals.

Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila, exhibit temperature preference rhythm (TPR) behavior to regulate BTR. Here, we demonstrate that the diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. Our results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms.

Drosophila Temperature Preference Rhythms: An Innovative Model to Understand Body Temperature Rhythms.

Human body temperature increases during wakefulness and decreases during sleep. The body temperature rhythm (BTR) is a robust output of the circadian clock and is fundamental for maintaining homeostasis, such as generating metabolic energy and sleep, as well as entraining peripheral clocks in mammals. However, the mechanisms that regulate BTR are largely unknown. Drosophila are ectotherms, and their body temperatures are close to ambient temperature; therefore, flies select a preferred environmental temperature to set their body temperature. We identified a novel circadian output, the temperature preference rhythm (TPR), in which the preferred temperature in flies increases during the day and decreases at night. TPR, thereby, produces a daily BTR. We found that fly TPR shares many features with mammalian BTR. We demonstrated that diuretic hormone 31 receptor (DH31R) mediates Drosophila TPR and that the closest mouse homolog of DH31R, calcitonin receptor (Calcr), is essential for mice BTR. Importantly, both TPR and BTR are regulated in a distinct manner from locomotor activity rhythms, and neither DH31R nor Calcr regulates locomotor activity rhythms. Our findings suggest that DH31R/Calcr is an ancient and specific mediator of BTR. Thus, understanding fly TPR will provide fundamental insights into the molecular and neural mechanisms that control BTR in mammals.

Drosophila DH31 Neuropeptide and PDF Receptor Regulate Night-Onset Temperature Preference.

Body temperature exhibits rhythmic fluctuations over a 24 h period (Refinetti and Menaker, 1992) and decreases during the night, which is associated with sleep initiation (Gilbert et al., 2004; Kräuchi, 2007a,b). However, the underlying mechanism of this temperature decrease is largely unknown. We have previously shown that Drosophila exhibit a daily temperature preference rhythm (TPR), in which their preferred temperatures increase during the daytime and then decrease at the transition from day to night (night-onset) (Kaneko et al., 2012). Because Drosophila are small ectotherms, their body temperature is very close to that of the ambient temperature (Stevenson, 1985), suggesting that their TPR generates their body temperature rhythm. Here, we demonstrate that the neuropeptide diuretic hormone 31 (DH31) and pigment-dispersing factor receptor (PDFR) contribute to regulate the preferred temperature decrease at night-onset. We show that PDFR and tethered-DH31 expression in dorsal neurons 2 (DN2s) restore the preferred temperature decrease at night-onset, suggesting that DH31 acts on PDFR in DN2s. Notably, we previously showed that the molecular clock in DN2s is important for TPR. Although PDF (another ligand of PDFR) is a critical factor for locomotor activity rhythms, Pdf mutants exhibit normal preferred temperature decreases at night-onset. This suggests that DH31-PDFR signaling specifically regulates a preferred temperature decrease at night-onset. Thus, we propose that night-onset TPR and locomotor activity rhythms are differentially controlled not only by clock neurons but also by neuropeptide signaling in the brain. SIGNIFICANCE STATEMENT: Body temperature rhythm (BTR) is fundamental for the maintenance of functions essential for homeostasis, such as generating metabolic energy and sleep. One major unsolved question is how body temperature decreases dramatically during the night. Previously, we demonstrated that a BTR-like mechanism, referred to as temperature preference rhythm (TPR), exists in Drosophila Here, we demonstrate that the diuretic hormone 31 (DH31) neuropeptide and pigment-dispersing factor receptor (PDFR) regulate preferred temperature decreases at night-onset via dorsal neurons 2. This is the first in vivo evidence that DH31 could function as a ligand of PDFR. Although both DH31 and PDF are ligands of PDFR, we show that DH31 regulates night-onset TPR, but PDF does not, suggesting that night-onset TPR and locomotor activity rhythms are controlled by different neuropeptides via different clock cells.

Casein kinase 2 is essential for mitophagy.

Mitophagy is a process that selectively degrades mitochondria. When mitophagy is induced in yeast, the mitochondrial outer membrane protein Atg32 is phosphorylated, interacts with the adaptor protein Atg11 and is recruited into the vacuole with mitochondria. We screened kinase-deleted yeast strains and found that CK2 is essential for Atg32 phosphorylation, Atg32-Atg11 interaction and mitophagy. Inhibition of CK2 specifically blocks mitophagy, but not macroautophagy, pexophagy or the Cvt pathway. In vitro, CK2 phosphorylates Atg32 at serine 114 and serine 119. We conclude that CK2 regulates mitophagy by directly phosphorylating Atg32.

Temperature-dependent resetting of the molecular circadian oscillator in Drosophila.

Circadian clocks responsible for daily time keeping in a wide range of organisms synchronize to daily temperature cycles via pathways that remain poorly understood. To address this problem from the perspective of the molecular oscillator, we monitored temperature-dependent resetting of four of its core components in the fruitfly Drosophila melanogaster: the transcripts and proteins for the clock genes period (per) and timeless (tim). The molecular circadian cycle in adult heads exhibited parallel responses to temperature-mediated resetting at the levels of per transcript, tim transcript and TIM protein. Early phase adjustment specific to per transcript rhythms was explained by clock-independent temperature-driven transcription of per. The cold-induced expression of Drosophila per contrasts with the previously reported heat-induced regulation of mammalian Period 2. An altered and more readily re-entrainable temperature-synchronized circadian oscillator that featured temperature-driven per transcript rhythms and phase-shifted TIM and PER protein rhythms was found for flies of the 'Tim 4' genotype, which lacked daily tim transcript oscillations but maintained post-transcriptional temperature entrainment of tim expression. The accelerated molecular and behavioural temperature entrainment observed for Tim 4 flies indicates that clock-controlled tim expression constrains the rate of temperature cycle-mediated circadian resetting.

Adult circadian behavior in Drosophila requires developmental expression of cycle, but not period.

Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, we used the genetic tools available in Drosophila to conditionally manipulate the function of the CYCLE component of the positive regulator CLOCK/CYCLE (CLK/CYC) or its negative feedback inhibitor PERIOD (PER). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of CLK/CYC activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of CLK/CYC and both involve the ventral lateral clock neurons (LN(v)s) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LN(v)s resulted in abnormal peptidergic small-LN(v) dorsal projections, and (2) PER expression rhythms in the adult LN(v)s appeared to be affected by developmental inhibition of CLK/CYC activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex.

Molecular and Neural Mechanisms of Temperature Preference Rhythm in Drosophila melanogaster.

Temperature influences animal physiology and behavior. Animals must set an appropriate body temperature to maintain homeostasis and maximize survival. Mammals set their body temperatures using metabolic and behavioral strategies. The daily fluctuation in body temperature is called the body temperature rhythm (BTR). For example, human body temperature increases during wakefulness and decreases during sleep. BTR is controlled by the circadian clock, is closely linked with metabolism and sleep, and entrains peripheral clocks located in the liver and lungs. However, the underlying mechanisms of BTR are largely unclear. In contrast to mammals, small ectotherms, such as Drosophila, control their body temperatures by choosing appropriate environmental temperatures. The preferred temperature of Drosophila increases during the day and decreases at night; this pattern is referred to as the temperature preference rhythm (TPR). As flies are small ectotherms, their body temperature is close to that of the surrounding environment. Thus, Drosophila TPR produces BTR, which exhibits a pattern similar to that of human BTR. In this review, we summarize the regulatory mechanisms of TPR, including recent studies that describe neuronal circuits relaying ambient temperature information to dorsal neurons (DNs). The neuropeptide diuretic hormone 31 (DH31) and its receptor (DH31R) regulate TPR, and a mammalian homolog of DH31R, the calcitonin receptor (CALCR), also plays an important role in mouse BTR regulation. In addition, both fly TPR and mammalian BTR are separately regulated from another clock output, locomotor activity rhythms. These findings suggest that the fundamental mechanisms of BTR regulation may be conserved between mammals and flies. Furthermore, we discuss the relationships between TPR and other physiological functions, such as sleep. The dissection of the regulatory mechanisms of Drosophila TPR could facilitate an understanding of mammalian BTR and the interaction between BTR and sleep regulation.

Dorsal clock networks drive temperature preference rhythms in Drosophila.

Animals display a body temperature rhythm (BTR). Little is known about the mechanisms by which a rhythmic pattern of BTR is regulated and how body temperature is set at different times of the day. As small ectotherms, Drosophila exhibit a daily temperature preference rhythm (TPR), which generates BTR. Here, we demonstrate dorsal clock networks that play essential roles in TPR. Dorsal neurons 2 (DN2s) are the main clock for TPR. We find that DN2s and posterior DN1s (DN1ps) contact and the extent of contacts increases during the day and that the silencing of DN2s or DN1ps leads to a lower temperature preference. The data suggest that temporal control of the microcircuit from DN2s to DN1ps contributes to TPR regulation. We also identify anterior DN1s (DN1as) as another important clock for TPR. Thus, we show that the DN networks predominantly control TPR and determine both a rhythmic pattern and preferred temperatures.

Selective entrainment of the Drosophila circadian clock to daily gradients in environmental temperature.

BACKGROUND: Circadian clocks are internal daily time keeping mechanisms that allow organisms to anticipate daily changes in their environment and to organize their behavior and physiology in a coherent schedule. Although circadian clocks use temperature compensation mechanisms to maintain the same pace over a range of temperatures, they are also capable of synchronizing to daily temperature cycles. This study identifies key properties of this process. RESULTS: Gradually ramping daily temperature cycles are shown here to synchronize behavioral and molecular daily rhythms in Drosophila with a remarkable efficiency. Entrainment to daily temperature gradients of amplitudes as low as 4 degrees C persisted even in the context of environmental profiles that also included continuous gradual increases or decreases in absolute temperature. To determine which elements of daily temperature gradients acted as the key determinants of circadian activity phase, comparative analyses of daily temperature gradients with different wave forms were performed. The phases of ascending and descending temperature acted together as key determinants of entrained circadian phase. In addition, circadian phase was found to be modulated by the relative temperature of release into free running conditions. Release at or close to the trough temperature of entrainment consistently resulted in phase advances. Re-entrainment to daily temperature gradients after large phase shifts occurred relatively slowly and required several cycles, allowing flies to selectively respond to periodic rather than anecdotal signals. The temperature-entrained phase relationship between clock gene expression rhythms and locomotor activity rhythms strongly resembled that previously observed for light entrainment. Moreover, daily temperature gradient and light/dark entrainment reinforced each other if the phases of ascending and descending temperature were in their natural alignment with the light and dark phases, respectively. CONCLUSION: The present study systematically examined the entrainment of clock-controlled behavior to daily environmental temperature gradients. As a result, a number of key properties of circadian temperature entrainment were identified. Collectively, these properties represent a circadian temperature entrainment mechanism that is optimized in its ability to detect the time-of-day information encoded in natural environmental temperature profiles. The molecular events synchronized to the daily phases of ascending and descending temperature are expected to play an important role in the mechanism of circadian entrainment to daily temperature cycles.

Xenopus Rnd1 and Rnd3 GTP-binding proteins are expressed under the control of segmentation clock and required for somite formation.

The process of segmentation in vertebrates is described by a clock and wavefront model consisting of a Notch signal and an fibroblast growth factor-8 (FGF8) gradient, respectively. To further investigate the segmentation process, we screened gene expression profiles for downstream targets of the segmentation clock. The Rnd1 and Rnd3 GTP-binding proteins comprise a subgroup of the Rho GTPase family that show a specific expression pattern similar to the Notch signal component ESR5, suggesting an association between Rnd1/3 and the segmentation clock. Rnd1/3 expression patterns are disrupted by overexpression of dominant-negative or active forms of Notch signaling genes, and responds to the FGF inhibitor SU5402 by a posterior shift analogous to other segmentation-related genes, suggesting that Rnd1/3 expressions are regulated by the segmentation clock machinery. We also show that antisense morpholino oligonucleotides to Rnd1/3 inhibit somite segmentation and differentiation in Xenopus embryos. These results suggest that Rnd1/3 are required for Xenopus somitogenesis.

Genetic screens for mutations affecting development of Xenopus tropicalis.

We present here the results of forward and reverse genetic screens for chemically-induced mutations in Xenopus tropicalis. In our forward genetic screen, we have uncovered 77 candidate phenotypes in diverse organogenesis and differentiation processes. Using a gynogenetic screen design, which minimizes time and husbandry space expenditures, we find that if a phenotype is detected in the gynogenetic F2 of a given F1 female twice, it is highly likely to be a heritable abnormality (29/29 cases). We have also demonstrated the feasibility of reverse genetic approaches for obtaining carriers of mutations in specific genes, and have directly determined an induced mutation rate by sequencing specific exons from a mutagenized population. The Xenopus system, with its well-understood embryology, fate map, and gain-of-function approaches, can now be coupled with efficient loss-of-function genetic strategies for vertebrate functional genomics and developmental genetics.

Neuropeptides PDF and DH31 hierarchically regulate free-running rhythmicity in Drosophila circadian locomotor activity.

Neuropeptides play pivotal roles in modulating circadian rhythms. Pigment-dispersing factor (PDF) is critical to the circadian rhythms in Drosophila locomotor activity. Here, we demonstrate that diuretic hormone 31 (DH31) complements PDF function in regulating free-running rhythmicity using male flies. We determined that Dh31 loss-of-function mutants (Dh31#51) showed normal rhythmicity, whereas Dh31#51;Pdf01 double mutants exhibited a severe arrhythmic phenotype compared to Pdf-null mutants (Pdf01). The expression of tethered-PDF or tethered-DH31 in clock cells, posterior dorsal neurons 1 (DN1ps), overcomes the severe arrhythmicity of Dh31#51;Pdf01 double mutants, suggesting that DH31 and PDF may act on DN1ps to regulate free-running rhythmicity in a hierarchical manner. Unexpectedly, the molecular oscillations in Dh31#51;Pdf01 mutants were similar to those in Pdf01 mutants in DN1ps, indicating that DH31 does not contribute to molecular oscillations. Furthermore, a reduction in Dh31 receptor (Dh31r) expression resulted in normal locomotor activity and did not enhance the arrhythmic phenotype caused by the Pdf receptor (Pdfr) mutation, suggesting that PDFR, but not DH31R, in DN1ps mainly regulates free-running rhythmicity. Taken together, we identify a novel role of DH31, in which DH31 and PDF hierarchically regulate free-running rhythmicity through DN1ps.

The influence of light on temperature preference in Drosophila.

Ambient light affects multiple physiological functions and behaviors, such as circadian rhythms, sleep-wake activities, and development, from flies to mammals. Mammals exhibit a higher body temperature when exposed to acute light compared to when they are exposed to the dark, but the underlying mechanisms are largely unknown. The body temperature of small ectotherms, such as Drosophila, relies on the temperature of their surrounding environment, and these animals exhibit a robust temperature preference behavior. Here, we demonstrate that Drosophila prefer a ∼1° higher temperature when exposed to acute light rather than the dark. This acute light response, light-dependent temperature preference (LDTP), was observed regardless of the time of day, suggesting that LDTP is regulated separately from the circadian clock. However, screening of eye and circadian clock mutants suggests that the circadian clock neurons posterior dorsal neurons 1 (DN1(p)s) and Pigment-Dispersing Factor Receptor (PDFR) play a role in LDTP. To further investigate the role of DN1(p)s in LDTP, PDFR in DN1(p)s was knocked down, resulting in an abnormal LDTP. The phenotype of the pdfr mutant was rescued sufficiently by expressing PDFR in DN1(p)s, indicating that PDFR in DN1(p)s is responsible for LDTP. These results suggest that light positively influences temperature preference via the circadian clock neurons, DN1(p)s, which may result from the integration of light and temperature information. Given that both Drosophila and mammals respond to acute light by increasing their body temperature, the effect of acute light on temperature regulation may be conserved evolutionarily between flies and humans.

Design and analysis of temperature preference behavior and its circadian rhythm in Drosophila.

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms(1) (,) (2). We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night (3). Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons(3). Drosophila locomotor activity is a well known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms(4). Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR(3). Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies(5-8). We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

The RRASK motif in Xenopus cyclin B2 is required for the substrate recognition of Cdc25C by the cyclin B-Cdc2 complex.

The FLRRXSK sequence is conserved in the second cyclin box fold of B-type cyclins. We show that this conserved sequence in Xenopus cyclin B2, termed the RRASK motif, is required for the substrate recognition by the cyclin B-Cdc2 complex of Cdc25C. Mutations to charged residues of the RRASK motif of cyclin B2 abolished its ability to activate Cdc2 kinase without affecting its capacity to bind to Cdc2. Cdc2 bound to the cyclin B2 RRASK mutant was not dephosphorylated by Cdc25C, and as a result, the complex was inactive. The cyclin B2 RRASK mutants can form a complex with the constitutively active Cdc2, but a resulting active complex did not phosphorylate a preferred substrate Cdc25C in vitro, although it can phosphorylate the non-specific substrate histone H1. The RRASK mutations prevented the interaction of Cdc25C with the cyclin B2-Cdc2 complex. Consistently, the RRASK mutants neither induced germinal vesicle breakdown in Xenopus oocyte maturation nor activated in vivo Cdc2 kinase during the cell cycle in mitotic extracts. These results suggest that the RRASK motif in Xenopus cyclin B2 plays an important role in defining the substrate specificity of the cyclin B-Cdc2 complex.