Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in <em>Drosophila</em>

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Cincinnati Childrens Hospital Medical Center

In the basidiomycete Coprinus cinereus (C. cinereus), which shows a highly synchronous meiotic cell cycle, the meiotic prophase I cells demonstrate flap endonuclease-1 activity. To investigate its role during meiosis, we isolated a C. cinereus cDNA homolog of flap endonuclease-1 (CcFEN-1), 1377bp in length with the open reading frame (ORF) encoding a predicted molecular mass of 51 kDa. At amino-acid residues Glu276-Pro345, a specific inserted sequence composed of 70 amino acids rich in polar forms was found to exist, without sequence identity to other eukaryotic FEN-1 or the polar amino acid rich sequences found in C. cinereus PCNA and C. cinereus DNA ligase IV, although the lengths and percentages of polar amino acids were similar. Northern hybridization analysis indicated CcFEN-1 to be expressed not only in the pre-meiotic S phase but also in meiotic prophase I. The roles of CcFEN-1 during meiosis are discussed.

Expression of flap endonuclease-1 during meiosis in a basidiomycete, Coprinus cinereus.

[Latest frontiers of meiosis research in Coprinus cinereus].

A long-standing model postulates that X-chromosome dosage compensation in Drosophila occurs by twofold up-regulation of the single male X, but previous data cannot exclude an alternative model, in which male autosomes are down-regulated to balance gene expression. To distinguish between the two models, we used RNA interference to deplete Male-Specific Lethal (MSL) complexes from male-like tissue culture cells. We found that expression of many genes from the X chromosome decreased, while expression from the autosomes was largely unchanged. We conclude that the primary role of the MSL complex is to up-regulate the male X chromosome.

Global regulation of X chromosomal genes by the MSL complex in Drosophila melanogaster.

Lim15/Dmc1 is a meiosis specific RecA-like protein. Here we propose its participation in meiotic chromosome pairing-related events along with DNA topoisomerase II. Analysis of protein-protein interactions using in vitro binding assays provided evidence that Coprinus cinereus DNA topoisomerase II (CcTopII) specifically interacts with C.cinereus Lim15/Dmc1 (CcLim15). Co-immunoprecipitation experiments also indicated that the CcLim15 protein interacts with CcTopII in vivo. Furthermore, a significant proportion of CcLim15 and CcTopII could be shown to co-localize on chromosomes from the leptotene to the zygotene stage. Interestingly, CcLim15 can potently activate the relaxation/catenation activity of CcTopII in vitro, and CcTopII suppresses CcLim15-dependent strand transfer activity. On the other hand, while enhancement of CcLim15's DNA-dependent ATPase activity by CcTopII was found in vitro, the same enzyme activity of CcTopII was inhibited by adding CcLim15. The interaction of CcLim15 and CcTopII may facilitate pairing of homologous chromosomes.

DNA topoisomerase II interacts with Lim15/Dmc1 in meiosis.

The basidiomycete Coprinus cinereus has many advantages as a model organism for studying sexual development and meiosis, but it has been difficult to investigate using reverse-genetics methods, such as gene disruption by homologous recombination. Here, gene repression by dsRNA-mediated gene silencing was tried as an alternative method for reverse-genetics studies. It was shown that transformation of the LIM15/DMC1 dsRNA expression construct (LIM15dsRNA) resulted in genomic insertion of LIM15dsRNA and paucity of the LIM15/DMC1 transcript. First, LIM15dsRNA was transformed into the homothallic strain AmutBmut to generate a homozygote in which both nuclei had a copy of LIM15dsRNA. The LIM15/DMC1-repressed strain showed abnormal homologous chromosome synapsis during meiosis. Basidiospore production was reduced to 16 % by the induction of dsRNA. However, approximately 60 % of basidiospores were viable. Next, a heterozygote was generated in which one nucleus had a copy of LIM15dsRNA. The phenotype was similar to that of the homozygote. These results are not only the first demonstration of dsRNA-mediated gene silencing in a member of the homobasidiomycete fungi, to which 90 % of mushroom species belong, but also the first successful use of a reverse-genetics approach in C. cinereus research.

Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing.

Sumoylation is a post-translational modification system that covalently attaches the small ubiquitin-related modifier (SUMO) to target proteins. Ubc9 is required as the E2-type enzyme for SUMO-1 conjugation to targets. Here, we show that Ubc9 interacts with the meiosis-specific RecA homolog, Lim15/Dmc1 in the basidiomycete Coprinus cinereus (CcLim15), and mediates sumoylation of CcLim15 during meiosis. In vitro protein-protein interaction assays revealed that CcUbc9 interacts with CcLim15 and binds to the C-terminus (amino acids 105-347) of CcLim15, which includes the ATPase domain. Immunocytochemistry demonstrates that CcUbc9 and CcLim15 colocalize in the nuclei from the leptotene stage to the early pachytene stage during meiotic prophase I. Coimmunoprecipitation experiments indicate that CcUbc9 interacts with CcLim15 in vivo during meiotic prophase I. Furthermore, we show that CcLim15 is a target protein of sumoylation both in vivo and in vitro, and identify the C-terminus (amino acids 105-347) of CcLim15 as the site of sumoylation in vitro. These results suggest that sumoylation is a candidate modulator of meiotic recombination via interaction between Ubc9 and Lim15/Dmc1.

Sumoylation of a meiosis-specific RecA homolog, Lim15/Dmc1, via interaction with the small ubiquitin-related modifier (SUMO)-conjugating enzyme Ubc9.

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity.

In eukaryotes, meiosis leads to genetically variable gametes through recombination between homologous chromosomes of maternal and paternal origin. Chromatin organization following meiotic recombination is critical to ensure the correct segregation of homologous chromosomes into gametes. However, the mechanism of chromatin organization after meiotic recombination is unknown. In this study we report that the meiosis-specific recombinase Lim15/Dmc1 interacts with the homologue of the largest subunit of chromatin assembly factor 1 (CAF-1) in the basidiomycete Coprinopsis cinerea (Coprinus cinereus). Using C. cinerea LIM15/DMC1 (CcLIM15) as the bait in a yeast two-hybrid screen, we have isolated the C. cinerea homologue of Cac1, the largest subunit of CAF-1 in Saccharomyces cerevisiae, and named it C. cinerea Cac1-like (CcCac1L). Two-hybrid assays confirmed that CcCac1L binds CcLim15 in vivo. beta-Galactosidase assays revealed that the N-terminus of CcCac1L preferentially interacts with CcLim15. Co-immunoprecipitation experiments showed that these proteins also interact in the crude extract of meiotic cells. Furthermore, we demonstrate that, during meiosis, CcCac1L interacts with proliferating cell nuclear antigen (PCNA), a component of the DNA synthesis machinery recently reported as an interacting partner of Lim15/Dmc1. Taken together, these results suggest a novel role of the CAF-1-PCNA complex in meiotic events. We propose that the CAF-1-PCNA complex modulates chromatin assembly following meiotic recombination.

Interaction between Lim15/Dmc1 and the homologue of the large subunit of CAF-1: a molecular link between recombination and chromatin assembly during meiosis.

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&#x70ED;&#x4F20;&#x611F;&#x5668;&#x5185;&#x90E8;&#x63A7;&#x5236;&#x6E29;&#x5EA6;&#x5728;&#x679C;&#x8747;&#x4E2D;&#x7684;&#x9996;&#x9009;&#x9879;&#x3002;

Animals from flies to humans are able to distinguish subtle gradations in temperature and show strong temperature preferences. Animals move to environments of optimal temperature and some manipulate the temperature of their surroundings, as humans do using clothing and shelter. Despite the ubiquitous influence of environmental temperature on animal behaviour, the neural circuits and strategies through which animals select a preferred temperature remain largely unknown. Here we identify a small set of warmth-activated anterior cell (AC) neurons located in the Drosophila brain, the function of which is critical for preferred temperature selection. AC neuron activation occurs just above the fly's preferred temperature and depends on dTrpA1, an ion channel that functions as a molecular sensor of warmth. Flies that selectively express dTrpA1 in the AC neurons select normal temperatures, whereas flies in which dTrpA1 function is reduced or eliminated choose warmer temperatures. This internal warmth-sensing pathway promotes avoidance of slightly elevated temperatures and acts together with a distinct pathway for cold avoidance to set the fly's preferred temperature. Thus, flies select a preferred temperature by using a thermal sensing pathway tuned to trigger avoidance of temperatures that deviate even slightly from the preferred temperature. This provides a potentially general strategy for robustly selecting a narrow temperature range optimal for survival.

An internal thermal sensor controlling temperature preference in Drosophila.

Animaux de mouches aux humains sont capables de distinguer les nuances subtiles de temp&eacute;rature et montrent des pr&eacute;f&eacute;rences de forte temp&eacute;rature. Animaux se d&eacute;placent aux environnements de temp&eacute;rature optimale et certains manipulent la temp&eacute;rature de leur environnement, comme le font les humains en utilisant des v&ecirc;tements et des abris. Malgr&eacute; l&#39;influence omnipr&eacute;sente de la temp&eacute;rature ambiante sur le comportement animal, les circuits neuronaux et les strat&eacute;gies par lesquelles animaux s&eacute;lectionner une temp&eacute;rature pr&eacute;f&eacute;r&eacute;e demeurent largement inconnus. Ici, nous identifions un petit ensemble de neurones activ&eacute;s par chaleur ant&eacute;rieur cellulaire (AC) situ&eacute; dans le cerveau de la drosophile, dont la fonction est critique pour la s&eacute;lection de la temp&eacute;rature pr&eacute;f&eacute;r&eacute;e. Activation du neurone AC se produit juste au-dessus de la temp&eacute;rature pr&eacute;f&eacute;r&eacute;e de la mouche et d&eacute;pend de la dTrpA1, un canal ionique qui fonctionne comme une sonde mol&eacute;culaire de la chaleur. Les mouches qui expriment s&eacute;lectivement les dTrpA1 dans les neurones de l&#39;AC s&eacute;lectionner des temp&eacute;ratures normales, tandis que les mouches dans lequel dTrpA1 fonction est r&eacute;duite ou &eacute;limin&eacute;e choisissent des temp&eacute;ratures plus chaudes. Cette voie de d&eacute;tection de chaleur interne favorise l&#39;&eacute;vitement des temp&eacute;ratures l&eacute;g&egrave;rement sur&eacute;lev&eacute;es et agit avec une voie distincte pour &eacute;viter froid r&eacute;gler la temp&eacute;rature pr&eacute;f&eacute;r&eacute;e de la mouche. Ainsi, les mouches s&eacute;lectionnez une temp&eacute;rature pr&eacute;f&eacute;r&eacute;e en utilisant une voie de d&eacute;tection thermique &agrave; l&#39;&eacute;coute &agrave; l&#39;&eacute;vitement de la g&acirc;chette des temp&eacute;ratures qui s&#39;&eacute;cartent m&ecirc;me l&eacute;g&egrave;rement la temp&eacute;rature pr&eacute;f&eacute;r&eacute;e. Celle-ci pr&eacute;voit une strat&eacute;gie g&eacute;n&eacute;rale potentiellement robuste en s&eacute;lectionnant une &eacute;troite plage de temp&eacute;rature optimale pour la survie.

Un capteur thermique interne contr&ocirc;lant la pr&eacute;f&eacute;rence de temp&eacute;rature chez la drosophile.

Tiere von fliegen auf den Menschen sind in der Lage, feine Abstufungen in der Temperatur zu unterscheiden und zeigen starke Temperatur-Einstellungen. Tiere in Umgebungen der optimalen Temperatur verschieben und einige, die Temperatur der Umgebung, wie Menschen mit Kleidung und Obdach zu manipulieren. Trotz der allgegenw&auml;rtigen Einfluss der Umgebungstemperatur auf Tierverhalten bleiben die neuronale Schaltkreise und Strategien, die durch die Tiere eine bevorzugte Temperatur W&auml;hlen Sie weitgehend unbekannt. Hier erkennen wir eine kleine Gruppe von W&auml;rme aktivierte anterior Zelle (AC) Neuronen befindet sich im Gehirn Drosophila, deren Funktion ist entscheidend f&uuml;r die Auswahl der gew&uuml;nschten Temperatur. AC-Neuron-Aktivierung tritt oberhalb der Fliege bevorzugte Temperatur und dTrpA1, ein Ionenkanal, die als molekulare Sensor W&auml;rme wirkt, h&auml;ngt. Fliegen, die selektiv dTrpA1 in den AC-Neuronen exprimieren w&auml;hlen Sie normale Temperaturen, w&auml;hrend fliegen in welche, die dtrpa1 Funktion reduziert oder eliminiert wird w&auml;rmere Temperaturen w&auml;hlen. Diese interne W&auml;rme-Sensorik-Stoffwechselweg f&ouml;rdert Vermeidung von leicht erh&ouml;hten Temperaturen und dient zusammen mit einen deutlichen Weg f&uuml;r kalte Vermeidung der Fliege bevorzugte Temperatur festgelegt. So ausw&auml;hlen fliegen eine bevorzugte Temperatur mit thermal Sensor Weg abgestimmt, um Trigger-Vermeidung von Temperaturen, die die bevorzugte Temperatur sogar leicht abweichen. Dieses sieht eine potenziell allgemeine Strategie robust ausw&auml;hlen einen engen Temperaturbereich optimal f&uuml;r &uuml;berleben.

Ein interner Temperatursensor Steuerung Temperatur Pr&auml;ferenz in Drosophila.

Animali dalle mosche agli esseri umani sono in grado di distinguere le sottili gradazioni di temperatura e mostrare preferenze forte temperatura. Animali muovono agli ambienti della temperatura ottima e alcuni manipolare la temperatura dell&#39;ambiente circostante, come fanno gli esseri umani usando abbigliamento e riparo. Nonostante l&#39;onnipresente influenza della temperatura ambientale sul comportamento degli animali, i circuiti neurali e le strategie attraverso le quali gli animali selezionare una temperatura preferita rimangano in gran parte sconosciuti. Qui ci identifichiamo un piccolo insieme di neuroni attivati dal calore cella anteriore (AC) si trova nel cervello della drosofila, la cui funzione &egrave; fondamentale per la selezione della temperatura preferita. Attivazione dei neuroni AC si verifica appena sopra temperatura preferito di volo e dipende dalla dTrpA1, un canale ionico che funziona come un sensore molecolare del calore. Mosche che esprimono selettivamente dTrpA1 nei neuroni AC selezionare temperature normali, mentre vola in cui dTrpA1 funzione &egrave; ridotto o eliminato sceglie temperature pi&ugrave; calde. Questo percorso di rilevazione del calore interno favorisce evasione delle temperature leggermente sopraelevate e agisce insieme ad un percorso distinto per evitare freddo impostare la temperatura preferita di volare. Cos&igrave;, mosche selezionare una temperatura preferita utilizzando un percorso di telerilevamento termico sintonizzato ad evitare di innesco delle temperature che si discostano anche leggermente dalla temperatura preferita. Questo fornisce una strategia potenzialmente generale per robustamente selezionando una gamma di temperature stretta ottima per la sopravvivenza.

Un sensore termico interno controllando la preferenza di temperatura in drosofila.

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Animales de moscas a los seres humanos son capaces de distinguir sutiles gradaciones de temperatura y mostrar preferencias de temperatura fuerte. Animales que se mueven a ambientes de temperatura &oacute;ptima y algunos manipulan la temperatura de su entorno, tal como lo hacen los seres humanos utilizando ropa y abrigo. A pesar de la influencia omnipresente de la temperatura ambiental en comportamiento animal, los circuitos neuronales y estrategias a trav&eacute;s del cual animales seleccionar una temperatura preferida siguen siendo en gran parte desconocidos. Aqu&iacute; identificamos un peque&ntilde;o conjunto de neuronas de activado por calor de la c&eacute;lula anterior (AC), localizada en el cerebro de la Drosophila, cuya funci&oacute;n es fundamental para la selecci&oacute;n de la temperatura preferida. Activaci&oacute;n de la neurona de AC se produce justo por encima de la temperatura preferida de la mosca y depende de dTrpA1, un canal de iones que funciona como un sensor molecular del calor. Moscas que expresan selectivamente dTrpA1 en las neuronas AC seleccione temperaturas normales, mientras que las moscas que dTrpA1 funci&oacute;n es reducida o eliminada elegir las temperaturas m&aacute;s c&aacute;lidas. Esta v&iacute;a de detecci&oacute;n de calor interna promueve evitar temperaturas ligeramente elevadas y act&uacute;a junto a un camino distinto para evitar fr&iacute;o fijar la temperatura preferida de la mosca. As&iacute;, moscas seleccionar una temperatura preferida mediante una v&iacute;a de detecci&oacute;n t&eacute;rmica con gatillo evitaci&oacute;n de temperaturas que se desv&iacute;an ligeramente de la temperatura preferida. Esto proporciona una estrategia potencialmente general para seleccionar s&oacute;lidamente un estrecho temperatura &oacute;ptima para la supervivencia.

Un sensor t&eacute;rmico interno controlando la preferencia de la temperatura en Drosophila.

Mer3 is an evolutionarily conserved DNA helicase that has crucial roles in meiotic recombination and crossover formation. We have identified the MER3 homolog in Coprinus cinereus (Ccmer3) and show that it is expressed in zygotene and pachytene meiocytes. Immunostaining analysis indicated that CcMer3 was localized on chromosomes at zygotene and pachytene and CcMer3 foci were more frequent on paired than unpaired chromosomes. We generated a C. cinereus mer3 mutant (#1) and found that it showed abnormal meiosis progression and underwent apoptosis after prophase I. Basidiospore production in #1 was reduced to 0.8% of the wild-type level; the spores showed slower germination at 25 degrees C but were similar to the wild type at 37 degrees C. Electron microscopic analysis of chromosome spreads revealed that axial elements were formed in the mutant but that synapsis was defective, resulting in a reduction in spore production. Our results demonstrate that CcMer3 is required for synaptonemal complex formation after axial elements align and is thus essential for homologous synapsis.

Coprinus cinereus Mer3 is required for synaptonemal complex formation during meiosis.

A daily body temperature rhythm (BTR) is critical for the maintenance of homeostasis in mammals. Whereas mammals use internal energy to regulate body temperature, ectotherms typically regulate body temperature behaviorally [1]. Some ectotherms maintain homeostasis via a daily temperature preference rhythm (TPR) [2], but the underlying mechanisms are largely unknown. Here, we show that Drosophila exhibit a daily circadian clock-dependent TPR that resembles mammalian BTR. Pacemaker neurons critical for locomotor activity are not necessary for TPR; instead, the dorsal neuron 2 s (DN2s), whose function was previously unknown, is sufficient. This indicates that TPR, like BTR, is controlled independently from locomotor activity. Therefore, the mechanisms controlling temperature fluctuations in fly TPR and mammalian BTR may share parallel features. Taken together, our results reveal the existence of a novel DN2-based circadian neural circuit that specifically regulates TPR; thus, understanding the mechanisms of TPR will shed new light on the function and neural control of circadian rhythms.

Circadian rhythm of temperature preference and its neural control in Drosophila.

Temperature sensation has a strong impact on animal behavior and is necessary for animals to avoid exposure to harmful temperatures. It is now well known that thermoTRP (transient receptor potential) channels in thermosensory neurons detect a variable range of temperature stimuli. However, little is known about how a range of temperature information is relayed and integrated in the neural circuits. Here, we show novel temperature integration between two warm inputs via Drosophila TRPA channels, TRPA1 and Pyrexia (Pyx). The internal AC (anterior cell) thermosensory neurons, which express TRPA1, detect warm temperatures and mediate temperature preference behavior. We found that the AC neurons were activated twice when subjected to increasing temperatures. The first response was at ∼25°C via TRPA1 channel, which is expressed in the AC neurons. The second response was at ∼27°C via the second antennal segments, indicating that the second antennal segments are involved in the detection of warm temperatures. Further analysis reveals that pyx-Gal4-expressing neurons have synapses on the AC neurons and that mutation of pyx eliminates the second response of the AC neurons. These data suggest that AC neurons integrate both their own TRPA1-dependent temperature responses and a Pyx-dependent temperature response from the second antennal segments. Our data reveal the first identification of temperature integration, which combines warm temperature information from peripheral to central neurons and provides the possibility that temperature integration is involved in the plasticity of behavioral outputs.

Fumika N. Hamada

The Visual Systems Group,
Division of Pediatric Ophthalmology,
The Visual Systems Group, Division of Pediatric Ophthalmology

Cincinnati Childrens Hospital Medical Center

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

Fumika N. Hamada

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}The Visual Systems Group,Division of Pediatric Ophthalmology,The Visual Systems Group, Division of Pediatric Ophthalmology

Cincinnati Childrens Hospital Medical Center

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The Visual Systems Group,
Division of Pediatric Ophthalmology,
The Visual Systems Group, Division of Pediatric Ophthalmology