How consistent are the transcriptome changes associated with cold acclimation in two species of the Drosophila virilis group?

This work was financially support by a Marie Curie Initial Training Network grant, “Understanding the evolutionary origin of biological diversity” (ITN-2008–213780 SPECIATION), grants from the Academy of Finland to A.H. (project 132619) and M.K. (projects 268214 and 272927), a grant from NERC, UK to M.G.R. (grant NE/J020818/1), and NERC, UK PhD studentship to D.J.P. (NE/I528634/1).For many organisms the ability to cold acclimate with the onset of seasonal cold has major implications for their fitness. In insects, where this ability is widespread, the physiological changes associated with increased cold tolerance have been well studied. Despite this, little work has been done to trace changes in gene expression during cold acclimation that lead to an increase in cold tolerance. We used an RNA-Seq approach to investigate this in two species of the Drosophila virilis group. We found that the majority of genes that are differentially expressed during cold acclimation differ between the two species. Despite this, the biological processes associated with the differentially expressed genes were broadly similar in the two species. These included: metabolism, cell membrane composition, and circadian rhythms, which are largely consistent with previous work on cold acclimation/cold tolerance. In addition, we also found evidence of the involvement of the rhodopsin pathway in cold acclimation, a pathway that has been recently linked to thermotaxis. Interestingly, we found no evidence of differential expression of stress genes implying that long-term cold acclimation and short-term stress response may have a different physiological basis.PostprintPeer reviewe

Drosophila Rhodopsin 7 can partially replace the structural role of Rhodopsin 1, but not its physiological function

Rhodopsin 7 (Rh7), a new invertebrate Rhodopsin gene, was discovered in the genome of Drosophila melanogaster in 2000 and thought to encode for a functional Rhodopsin protein. Indeed, Rh7 exhibits most hallmarks of the known Rhodopsins, except for the G-protein-activating QAKK motif in the third cytoplasmic loop that is absent in Rh7. Here, we show that Rh7 can partially substitute Rh1 in the outer receptor cells (R1-6) for rhabdomere maintenance, but that it cannot activate the phototransduction cascade in these cells. This speaks against a role of Rh7 as photopigment in R1-6, but does not exclude that it works in the inner photoreceptor cells

Dopamine signaling in wake-promoting clock neurons is not required for the normal regulation of sleep in drosophila

Dopamine is a wake-promoting neuromodulator in mammals and fruit flies. In Drosophila melanogaster, the network of clock neurons that drives sleep/activity cycles comprises both wake-promoting and sleep-promoting cell types. The large ventrolateral neurons (l-LNvs) and small ventrolateral neurons (s-LNvs) have been identified as wake-promoting neurons within the clock neuron network. The l-LNvs are innervated by dopaminergic neurons, and earlier work proposed that dopamine signaling raises cAMP levels in the l-LNvs and thus induces excitatory electrical activity (action potential firing), which results in wakefulness and inhibits sleep. Here, we test this hypothesis by combining cAMP imaging and patch-clamp recordings in isolated brains. We find that dopamine application indeed increases cAMP levels and depolarizes the l-LNvs, but, surprisingly, it does not result in increased firing rates. Downregulation of the excitatory D1-like dopamine receptor (Dop1R1) in the l-LNvs and s-LNvs, but not of Dop1R2, abolished the depolarization of l-LNvs in response to dopamine. This indicates that dopamine signals via Dop1R1 to the l-LNvs. Downregulation of Dop1R1 or Dop1R2 in the l-LNvs and s-LNvs does not affect sleep in males. Unexpectedly, we find a moderate decrease of daytime sleep with downregulation of Dop1R1 and of nighttime sleep with downregulation of Dop1R2. Since the l-LNvs do not use Dop1R2 receptors and the s-LNvs also respond to dopamine, we conclude that the s-LNvs are responsible for the observed decrease in nighttime sleep. In summary, dopamine signaling in the wake-promoting LNvs is not required for daytime arousal, but likely promotes nighttime sleep via the s-LNvs.Fil: Fernández, Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Hermann Luibl, Christiane. University Of Würzburg; AlemaniaFil: Peteranderl, Alina. University Of Wuerzburg; AlemaniaFil: Reinhard, Nils. University Of Wuerzburg; AlemaniaFil: Senthilan, Pingkalai R.. University Of Wuerzburg; AlemaniaFil: Hieke, Marie. University Of Wuerzburg; AlemaniaFil: Selcho, Mareike. University Of Wuerzburg; AlemaniaFil: Yoshii, Taishi. Okayama University; JapónFil: Shafer, Orie T.. City University of New York; Estados UnidosFil: Muraro, Nara Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Helfrich Förster, Charlotte. University Of Wuerzburg; Alemani

Survey of the ciliary motility machinery of Drosophila sperm and ciliated mechanosensory neurons reveals unexpected cell-type specific variations: a model for motile ciliopathies

The motile cilium/flagellum is an ancient eukaryotic organelle. The molecular machinery of ciliary motility comprises a variety of cilium-specific dynein motor complexes along with other complexes that regulate their activity. Assembling the motors requires the function of dedicated “assembly factors” and transport processes. In humans, mutation of any one of at least 40 different genes encoding components of the motility apparatus causes Primary Ciliary Dyskinesia (PCD), a disease of defective ciliary motility. Recently, Drosophila has emerged as a model for motile cilia biology and motile ciliopathies. This is somewhat surprising as most Drosophila cells lack cilia, and motile cilia are confined to just two specialized cell types: the sperm flagellum with a 9+2 axoneme and the ciliated dendrite of auditory/proprioceptive (chordotonal, Ch) neurons with a 9+0 axoneme. To determine the utility of Drosophila as a model for motile cilia, we survey the Drosophila genome for ciliary motility gene homologs, and assess their expression and function. We find that the molecules of cilium motility are well conserved in Drosophila. Most are readily characterized by their restricted cell-type specific expression patterns and phenotypes. There are also striking differences between the two motile ciliated cell types. Notably, sperm and Ch neuron cilia express and require entirely different outer dynein arm variants—the first time this has been clearly established in any organism. These differences might reflect the specialized functions for motility in the two cilium types. Moreover, the Ch neuron cilia lack the critical two-headed inner arm dynein (I1/f) but surprisingly retain key regulatory proteins previously associated with it. This may have implications for other motile 9+0 cilia, including vertebrate embryonic nodal cilia required for left-right axis asymmetry. We discuss the possibility that cell-type specificity in ciliary motility machinery might occur in humans, and therefore underlie some of the phenotypic variation observed in PCD caused by different gene mutations. Our work lays the foundation for the increasing use of Drosophila as an excellent model for new motile ciliary gene discovery and validation, for understanding motile cilium function and assembly, as well as understanding the nature of genetic defects underlying human motile ciliopathies

The MAP Kinase p38 Is Part of Drosophila melanogaster's Circadian Clock

All organisms have to adapt to acute as well as to regularly occurring changes in the environment. To deal with these major challenges organisms evolved two fundamental mechanisms: the p38 mitogen-activated protein kinase (MAPK) pathway, a major stress pathway for signaling stressful events, and circadian clocks to prepare for the daily environmental changes. Both systems respond sensitively to light. Recent studies in vertebrates and fungi indicate that p38 is involved in light-signaling to the circadian clock providing an interesting link between stress-induced and regularly rhythmic adaptations of animals to the environment, but the molecular and cellular mechanisms remained largely unknown. Here, we demonstrate by immunocytochemical means that p38 is expressed in Drosophila melanogaster's clock neurons and that it is activated in a clock-dependent manner. Surprisingly, we found that p38 is most active under darkness and, besides its circadian activation, additionally gets inactivated by light. Moreover, locomotor activity recordings revealed that p38 is essential for a wild-type timing of evening activity and for maintaining ∼ 24 h behavioral rhythms under constant darkness: flies with reduced p38 activity in clock neurons, delayed evening activity and lengthened the period of their free-running rhythms. Furthermore, nuclear translocation of the clock protein Period was significantly delayed on the expression of a dominant-negative form of p38b in Drosophila's most important clock neurons. Western Blots revealed that p38 affects the phosphorylation degree of Period, what is likely the reason for its effects on nuclear entry of Period. In vitro kinase assays confirmed our Western Blot results and point to p38 as a potential "clock kinase" phosphorylating Period. Taken together, our findings indicate that the p38 MAP Kinase is an integral component of the core circadian clock of Drosophila in addition to playing a role in stress-input pathways

Identifizierung und Charakterizierung von Taubheitsgene in Drosophila melanogaster

Das auditorische Organ der Fruchtfliege Drosophila, das im zweiten Antennensegment befindliche Johnstonsche Organ (JO) ist ein nützliches System um Hörprozesse zu untersuchen. JO Neurone und die Haarsinneszellen der Säugetiere werden durch homologe Gene der atonal-Familie genetisch spezifiziert und besitzen funktional äquivalente Transduktionsmechanismen. In dieser Studie wurde eine neuartige Reverse Genetik Methode angewandt um Gene zu identifizieren, die im Johnstonschem Organ exprimiert werden. Hierfür wurden die Transkriptome im zweiten Antennensegment von atonal Mutanten mit die der Kontrollfliegen verglichen. Es wurden 274 Kandidatengene identifiziert, die in JO exprimiert werden. Zusätzlich zu vier bereits bekannten Hörgene wurden etliche bisher unbekannte hörrelavante Gene entdeckt. Hierzu gehören drei TRP Kanäle, sieben axonemale Dyneinmotoren und Gene, die in der Chemo- und Phototransduktion beteiligt sind, wie z.B. ionotropische Rezeptoren (IR) und Rhodopsine. Die den Transkriptomen zugrunde liegenden Mikroarray Daten wurden anhand von quantitativer real-time PCR (qPCR) und Cluster Analysen validiert. Genexpression wurde mit in situ Hybridisierung, Antikörperfärbung, und Gal4-Konstrukten überprüft. Es wurden 30 neue Gene identifiziert, deren Störung die JO Funktion beeinträchtigte. Hierbei wurde die Anzahl der bisher bekannten Fliegenhörgene verdoppelt. Es wurde gezeigt, dass Proteine, die in der Photo-und Chemotransduktion beteiligt sind - wie ionotropische Rezeptoren und Rhodopsine - im Johnstonschen Organ exprimiert und an der Funktion des Organs beteiligt sind. Diese Ergebnisse erweitern die bestehenden genetischen Parallelen der Entwicklung um weitere genetische Gemeinsamkeiten in der Signalverarbeitung

Rhodopsin 7-The unusual Rhodopsin in Drosophila

Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1–Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a “vertebrate-melanopsin-type”–cluster, and Rh3, Rh4 and Rh5 form an “insect-type”-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins

Rhodopsin 7-The unusual Rhodopsin in Drosophila

Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1–Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a “vertebrate-melanopsin-type”–cluster, and Rh3, Rh4 and Rh5 form an “insect-type”-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins

Rhodopsin 7-The unusual Rhodopsin in Drosophila

ABSTRACT Rhodopsins are the major photopigments in the fruit fly Drosophila melanogaster. Drosophila express six well-characterized Rhodopsins (Rh1-Rh6) with distinct absorption maxima and expression pattern. In 2000, when the Drosophila genome was published, a novel Rhodopsin gene was discovered: Rhodopsin 7 (Rh7). Rh7 is highly conserved among the Drosophila genus and is also found in other arthropods. Phylogenetic trees based on protein sequences suggest that the seven Drosophila Rhodopsins cluster in three different groups. While Rh1, Rh2 and Rh6 form a "vertebrate-melanopsin-type"-cluster, and Rh3, Rh4 and Rh5 form an "insecttype"-Rhodopsin cluster, Rh7 seem to form its own cluster. Although Rh7 has nearly all important features of a functional Rhodopsin, it differs from other Rhodopsins in its genomic and structural properties, suggesting it might have an overall different role than other known Rhodopsins