Calcium and cAMP directly modulate the speed of the Drosophila circadian clock.

Circadian clocks impose daily periodicities to animal behavior and physiology. At their core, circadian rhythms are produced by intracellular transcriptional/translational feedback loops (TTFL). TTFLs may be altered by extracellular signals whose actions are mediated intracellularly by calcium and cAMP. In mammals these messengers act directly on TTFLs via the calcium/cAMP-dependent transcription factor, CREB. In the fruit fly, Drosophila melanogaster, calcium and cAMP also regulate the periodicity of circadian locomotor activity rhythmicity, but whether this is due to direct actions on the TTFLs themselves or are a consequence of changes induced to the complex interrelationship between different classes of central pacemaker neurons is unclear. Here we investigated this question focusing on the peripheral clock housed in the non-neuronal prothoracic gland (PG), which, together with the central pacemaker in the brain, controls the timing of adult emergence. We show that genetic manipulations that increased and decreased the levels of calcium and cAMP in the PG caused, respectively, a shortening and a lengthening of the periodicity of emergence. Importantly, knockdown of CREB in the PG caused an arrhythmic pattern of eclosion. Interestingly, the same manipulations directed at central pacemaker neurons caused arrhythmicity of eclosion and of adult locomotor activity, suggesting a common mechanism. Our results reveal that the calcium and cAMP pathways can alter the functioning of the clock itself. In the PG, these messengers, acting as outputs of the clock or as second messengers for stimuli external to the PG, could also contribute to the circadian gating of adult emergence

Knockdown in the PG of genes in cAMP pathway affects the periodicity of eclosion.

<p>(A) Knockdown of <i>dunce</i> in the PG shortens the periodicity of eclosion (a), whereas knockdown of <i>rutabaga</i> (b), <i>Epac</i> (c), and <i>Rap1</i> (d) in the PG lengthens the periodicity of eclosion, with respect to controls (B). (B) Free-running periodicity for results shown in (A). Each circle indicates results from separate experiments; average is indicated by horizontal line; different letters indicate statistically different groups (<i>p</i><0.05; one-way ANOVA, Tukey’s <i>post hoc</i> multiple comparison analyses). (C) Average rhythmicity index (RI) values (± SEM) for results shown in (A). Numbers in parenthesis indicate number of separate experiments; different letters indicate statistically different groups (<i>p</i><0.05; one-way ANOVA, Tukey’s <i>post hoc</i> multiple comparison analyses). Flies bearing only UAS-RNAi transgenes express normal rhythmicity of emergence (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007433#pgen.1007433.s003" target="_blank">S3 Fig</a>). In all experiments, GAL4 driver was used in combination with UAS-<i>dcr2</i> to potentiate effectiveness of RNAi knockdown.</p

cAMP levels in the PG vary during the course of the day.

<p>(A) <i>Left</i>: Representative image of the signal from the cAMP sensor, <i>Epac1</i>, in the PG before (t = 0), and 10min and 30 min after incubation with 100μM forskolin (FSK); <i>right</i>: quantitation of the signal; numbers in parenthesis indicate number of glands measured. (B) Dynamic range of the <i>Epac1</i> sensor, determined using increasing concentrations of 8-Br-cAMP; n: number of prothoracic gland cells (taken from at least 4 PGs per condition). (C) cAMP levels in the PG in controls and in animals bearing knockdown of <i>dunce</i> and <i>rutabaga</i> in the PG (using <i>phm</i>-GAL4 driver)(all measured at ZT12). The number of cells analyzed is indicated above each circle. For each experiment 9–17 animals were examined. (D-G) cAMP levels in the PG of wildtype animals at different times of day under LD (D) and DD (E), and in <i>per</i>⁰ mutants under LD (F) and DD (G) conditions. Error bars denote SEM. In (A) ***<i>p</i><0.0001 (paired <i>t</i>-test compared with signal measured prior to adding drug); scale bar, 10μm. In B-G, different letters indicate statistically significant values (ANOVA with Tukey’s <i>post hoc</i> test). The number of cells is indicated in brackets and above each error bars. In B-G: Normalized FRET signal is (YFP/CFP).</p

Knockdown of calcium and cAMP pathway in central clock affects the periodicity of adult emergence.

<p>(A)(a-e) <i>Left</i>: Pattern of emergence in DD following knockdown of <i>cacophony</i> (a), <i>Ca-alpha1D</i> (b), <i>rutabaga</i> (c), and <i>dunce</i> (d) in PDF neurons <i>versus</i> controls (e); <i>Right</i>: corresponding MESA analysis with value of free-running period (h) indicated. (B)(a-e) Left: pattern of emergence under DD of corresponding knockdown in all clock cells. (C) Free-running periodicity (a) and rhythmicity index (RI) values (b) for genotypes shown in (A) and for controls. In (a) each circle indicates results obtained in separate experiments; average is indicated by horizontal line; (b) shows the average (± SEM) RI, with different letters indicating statistically different groups (<i>p</i><0.05; one-way ANOVA, Tukey’s <i>post hoc</i> multiple comparison analyses). (D) Free-running periodicity (a) and rhythmicity index (RI) values (b) for genotypes shown in (B) and for controls, represented as in (C). In all experiments, GAL4 driver was used in combination with UAS-<i>dcr2</i> to potentiate effectiveness of RNAi knockdown.</p

Calcium levels in the PG vary during the course of the day.

<p>(A) <i>Left</i>: Representative images of GCaMP fluorescence in the PG before (t = 0), and 10 min and 30 min after incubation with 50μM calcimycin (external [calcium]: 1mM) and <i>Right</i>: quantitation of the fluorescence in the PG; numbers in parenthesis indicate number of PG glands measured; *<i>p</i><0.05 (paired <i>t</i>-test compared with signal measured before adding drug). (B) Dose response of GCaMP sensor measured using increasing extracellular calcium concentrations; n: number of PG cells (taken from at least 4 PGs per condition). (C) Levels of fluorescence in controls and in animals bearing knockdown of <i>calcium channels</i> (<i>cacophony</i> [<i>cac</i>] and <i>Ca-alpha1D</i>), <i>IP3</i> receptor (<i>IP3R</i>) and <i>SERCA</i> in the PG (using <i>phm</i>-GAL4 driver)(all measured at ZT12). The number of cells analyzed is indicated above colored circle. For each experiment 4–8 animals were examined. Error bars denote SEM. (D-G) Levels of fluorescence in the PG of wildtype animals at different times of day under LD (D) and DD (E) conditions, and in the PG of <i>per</i>⁰ mutants under LD (F) and DD (G) conditions. Different letters indicate statistically significant differences (ANOVA with Tukey’s <i>post hoc</i> test). The number of cells is indicated in brackets and above each error bars. In A: PG: prothoracic gland, CA: <i>corpus allatum</i>, RG: ring gland. Scale bar 10μm; in A-G: Normalized fluorescence is quantitated in arbitrary units.</p

Locomotor activity rhythmicity phenotypes following genetic manipulations of calcium and cAMP pathways in clock cells.

<p>Locomotor activity rhythmicity phenotypes following genetic manipulations of calcium and cAMP pathways in clock cells.</p

Knockdown of genes in calcium pathway in the PG affects the periodicity of eclosion.

<p>(A) Knockdown of calcium channels <i>cacophony</i> (a), <i>Ca-alpha1D</i> (b), and of IP3 receptor (<i>IP3R</i>) (c) in the PG lengthen the periodicity of eclosion with respect to controls (d). Records show time course of emergence of a single population in DD (left) and corresponding MESA analyses (right); principal periodicity is indicated. (B) Knockdown of <i>SERCA</i> in the PG shortens the periodicity of eclosion (a) compared to control (b). (C) Knockdown of <i>CaMKII</i> (a) and <i>CASK</i> (b) in the PG lengthens the periodicity of eclosion. (D) Free-running periodicity for results shown in A-C. Each point indicates results from separate experiments; average is indicated by horizontal line; different letters indicate statistically different groups (<i>p</i><0.05; one-way ANOVA, Tukey’s <i>post hoc</i> multiple comparison analyses). (E) Average rhythmicity index (RI) values (± SEM) for results shown in A-C. Numbers in parenthesis indicate number of separate experiments; different letters indicate statistically different groups (<i>p</i><0.05; one-way ANOVA, Tukey’s <i>post hoc</i> multiple comparison analyses). In A-C, GAL4 driver was used in combination with UAS-<i>dcr2</i> to potentiate effectiveness of RNAi knockdown. Flies bearing only UAS-RNAi transgenes express normal rhythmicity of emergence (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007433#pgen.1007433.s001" target="_blank">S1 Fig</a>).</p

Orcokinin neuropeptides regulate reproduction in the fruit fly, Drosophila melanogaster

In animals, neuropeptidergic signaling is essential for the regulation of survival and reproduction. In insects, Orcokinins are poorly studied, despite their high level of conservation among different orders. In particular, there are currently no reports on the role of Orcokinins in the experimental insect model, the fruit fly, Drosophila melanogaster. In the present work, we made use of the genetic tools available in this species to investigate the role of Orcokinins in the regulation of different innate behaviors including ecdysis, sleep, locomotor activity, oviposition, and courtship. We found that RNAi-mediated knockdown of the orcokinin gene caused a disinhibition of male courtship behavior, including the occurrence of male to male courtship, which is rarely seen in wildtype flies. In addition, orcokinin gene silencing caused a reduction in egg production. Orcokinin is emerging as an important neuropeptide family in the regulation of the physiology of insects from different orders. In the case of the fruit fly, our results suggest an important role in reproductive success.Fil: Silva, Valeria. Universidad de Valparaíso; ChileFil: Palacios Muñoz, Angelina. Universidad de Valparaíso; ChileFil: Volonté, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Endocrinología Experimental y Aplicada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Endocrinología Experimental y Aplicada; Argentina. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; ArgentinaFil: Frenkel, Lia. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ewer, John. Universidad de Valparaíso; ChileFil: Ons, Sheila. Universidad Nacional de La Plata. Centro Regional de Estudios Genómicos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Endocrinología Experimental y Aplicada. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Endocrinología Experimental y Aplicada; Argentin

Central and peripheral clocks are coupled by a neuropeptide pathway in Drosophila

Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals

Keratitis-Ichthyosis-Deafness Syndrome-Associated Cx26 Mutants Produce Nonfunctional Gap Junctions but Hyperactive Hemichannels When Co-Expressed With Wild Type Cx43

Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like the Keratitis-Ichthyosis-Deafness syndrome (KID). Because in the human skin connexin 26 (Cx26) is co-expressed with other connexins, like Cx43 and Cx30, and as the KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild-type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channel (GJC) functions remain unknown. In this study, we demonstrate that syndromic mutations, at the N terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) shows exacerbated hemichannel activity but nonfunctional GJCs; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca(2+) overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin