Masami Shimoda

A Role for the Drosophila Fragile X-Related Gene in Circadian Output

Mutations that abolish expression of an X-linked gene, FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental retardation. To understand the normal function of the FMR1 protein, we have produced fly strains bearing deletions in a Drosophila homolog of FMR1 (dfmr1). Since fragile X patients show a number of abnormal behaviors including sleep problems, we investigated whether a loss-of-function mutation of dfmr1 affect circadian behavior. Here we show that under constant darkness (DD), a lack of dfmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, their behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila.

Insect reactions to light and its applications to pest management

Insects are able to see ultraviolet (UV) radiation. Nocturnal insects are often attracted to light sources that emit large amounts of UV radiation, and devices that exploit this behavior, such as light traps for forecasting pest outbreaks, and electric insect killers, have been developed. Some diurnal species are attracted to yellow; yellow pan traps are used for conducting surveys for pest outbreaks and yellow sticky plates are used for pest control. Lamps that give off yellow illumination have been used effectively to control the activity of nocturnal moths and thus reduce damage to fruit, vegetables, and flowers. Covering cultivation facilities with film that filters out near-UV radiation reduces the invasion of pests such as whiteflies and thrips into the facilities, thus reducing damage. Reflective material placed on cultivated land can control the approach of flying insects such as aphids. Future development and use of new light sources such as light-emitting diodes is anticipated for promoting integrated pest management.

Nuclear DISC1 regulates CRE-mediated gene transcription and sleep homeostasis in the fruit fly

Disrupted-in-schizophrenia-1 (DISC1) is one of major susceptibility factors for a wide range of mental illnesses, including schizophrenia, bipolar disorder, major depression and autism spectrum conditions. DISC1 is located in several subcellular domains, such as the centrosome and the nucleus, and interacts with various proteins, including NudE-like (NUDEL/NDEL1) and activating transcription factor 4 (ATF4)/CREB2. Nevertheless, a role for DISC1 in vivo remains to be elucidated. Therefore, we have generated a Drosophila model for examining normal functions of DISC1 in living organisms. DISC1 transgenic flies with preferential accumulation of exogenous human DISC1 in the nucleus display disturbance in sleep homeostasis, which has been reportedly associated with CREB signaling/CRE-mediated gene transcription. Thus, in mammalian cells, we characterized nuclear DISC1, and identified a subset of nuclear DISC1 that colocalizes with the promyelocytic leukemia (PML) bodies, a nuclear compartment for gene transcription. Furthermore, we identified three functional cis-elements that regulate the nuclear localization of DISC1. We also report that DISC1 interacts with ATF4/CREB2 and a corepressor N-CoR, modulating CRE-mediated gene transcription.

Expression of Human Gaucher Disease Gene GBA Generates Neurodevelopmental Defects and ER Stress in Drosophila Eye

Gaucher disease (GD) is the most common of the lysosomal storage disorders and is caused by defects in the GBA gene encoding glucocerebrosidase (GlcCerase). The accumulation of its substrate, glucocylceramide (GlcCer) is considered the main cause of GD. We found here that the expression of human mutated GlcCerase gene (hGBA) that is associated with neuronopathy in GD patients causes neurodevelopmental defects in Drosophila eyes. The data indicate that endoplasmic reticulum (ER) stress was elevated in Drosophila eye carrying mutated hGBAs by using of the ER stress markers dXBP1 and dBiP. We also found that Ambroxol, a potential pharmacological chaperone for mutated hGBAs, can alleviate the neuronopathic phenotype through reducing ER stress. We demonstrate a novel mechanism of neurodevelopmental defects mediated by ER stress through expression of mutants of human GBA gene in the eye of Drosophila.

A physiological saline for Lepidopterous insects: Effects of ionic composition on heart beat and neuromuscular transmission

Abstract Three species of Lepidoptera, Bombyx mori, Agrius convolvuli and Antheraea yamamai were used to prepare a suitable physiological saline for maintaining heart beat and junctional potentials of somatic muscle cells. A variety of salines designed for Lepidoptera were reexamined. Heart beat in the three species could not be maintained in salines used previously as physiological or culture solutions for Lepidoptera. Ionic compositions were determined for physiological salines that were suitable for maintaining the normal heart beat and normal neuromuscular transmission in body muscle cells. It was found that salines should contain 12–28 mM NaCl, 32-16 mM KCl ([Na+] + [K+ ] = 44) and at least 9 mM CaCl2. In the salines, stimuli to motor nerves evoke action potentials of more than 40 mV in amplitude in ventral muscle cells. The ionic composition of a new physiological saline for Lepidoptera is as follows: NaCl, 12–28; KCl, 32-16([Na+] + [K+] = 44); CaCl2, 9; NaH2PO4, 1.5; Na2HPO4, 1.5; MgCl2, 18; sucrose, 175 (mM), pH 6.5.

Mating rhythms of Drosophila: rescue of tim01 mutants by D. ananassae timeless

Background It is reported that the circadian rhythms of female mating activity differ among Drosophila species and are controlled by an endogenous circadian clock. Here, we found that the mating rhythm of D. ananassae differed from that of D. melanogaster. Moreover, to evaluate the effect of clock gene products on mating activities, we examined the mating activity of D. melanogaster timeless (tim01) transgenic fly harboring heat-shock promotor driven-D. ananassae timeless (tim) gene (hs-AT tim01). Methods Flies were maintained under light/dark (LD) cycles for several days and then they were transferred to constant dark (DD) conditions at 25°C. Transformant flies were heat-shocked for 30 min (PZT 10.5–11.0 or PZT 22.5–23.0; PZT means Projected Zeitgeber Time) at 37°C every day. Daily expressions of D. ananassae TIMELESS (TIM) protein in transgenic flies were measured by western blotting. To examine whether the timing of D. ananassae TIM protein induction by heat shock can change the patterns of the behavior activities of D. melanogaster tim01 flies, we measured locomotor and mating activity rhythms under DD at 25°C ± 0.5°C except when heat shock was applied. Results Heat shock applied at PZT 10.5–11.0 and at PZT 22.5–23.0 induced high TIM levels during subjective night and day, respectively, in hs-AT tim01 flies. The locomotor rhythm of these flies was changed from diurnal to nocturnal by the timing of D. ananassae TIM induction. However, the mating rhythm of these flies could not be entrained by the timing of D. ananassae TIM induction. Conclusion The pattern of mating activity rhythms of D. ananassae and of D. melanogaster differed. The mating activity rhythms of D. melanogaster tim01 flies harboring hs-AT tim appeared after heat-shock but the pattern and phase differed from those of wild-type D. ananassae and D. melanogaster. Moreover, the mating rhythm of these flies could not be entrained by the timing of D. ananassae TIM induction although the locomotor rhythm of hs-AT tim01 was changed from diurnal to nocturnal according to the timing of D. ananassae TIM induction. These data suggest that species-specific mating activities require output pathways different from those responsible for locomotor rhythms.

Circadian phenotypes of Drosophila fragile x mutants in alternative genetic backgrounds.

Abstract Drosophila FMR1 mutants are models of human fragile X syndrome. They show a loss of locomotor activity rhythm and severe degradation of eclosion timing. We analyzed the circadian behavior of FMR1 mutants (dfmr1B55) in two genetic backgrounds, yellow white (yw) and Canton S (CS). The arrhythmic phenotype of circadian locomotor activity in constant darkness (DD) did not significantly change in either genetic background. Surprisingly, eclosion timing was completely restored by backcrossing dfmr1B55 with yw or CS flies. Morphological analysis of the small ventrally located lateral neurons of FMR1 mutants revealed that the dorsal-projection area was significantly larger in arrhythmic than rhythmic flies. In addition, dfmr1B55 mutants in both genetic backgrounds had a significantly lower evening peak in the light-dark (LD) cycle. These results indicate that lack of FMR1 does not affect eclosion timing, but alters locomotor activity patterns in both LD and DD conditions by affecting the arborization of small ventrally located lateral neurons. Thus, the FMR1 gene may regulate the circadian-related locomotor activity of Drosophila.

Casein kinase Iε Does Not Rescue double-time Function in Drosophila Despite Evolutionarily Conserved Roles in the Circadian Clock

double-time (dbt) is a casein kinase gene involved in cell survival, proliferation, and circadian rhythms in the fruit fly, Drosophila melanogaster. Genetic and biochemical studies have shown that dbt and its mammalian ortholog casein kinase Iε (hckIε) regulate the circadian phosphorylation of period (per), thus controlling per subcellular localization and stability. Mutations in these kinases can shorten the circadian period in both mammals and Drosophila. Since similar activities in circadian clock have been described for these kinases, we investigated whether the expression of mammalian casein kinase I can replace the activity of dbt in flies. Global expression of the full-length dbt rescued lethality of the null mutant dbt revVIII and rescued flies showed normal locomotor activity rhythms. Global expression of dbt also restored the locomotor activity rhythm of the arrhythmic genotype, dbtar /dbtrevVIII . In contrast, global expression of hckIε or hckIα did not rescue lethality or locomotor activity of dbt mutants. Furthermore dbt overexpression in wild-type clock cells had only a small effect on period length, whereas hckIε expression in clock cells greatly lengthened period to ~30.5 hours and increased the number of arrhythmic flies. These results indicate that hckIε cannot replace the activity of dbt in flies despite the high degree of similarity in primary sequence and kinase function. Moreover, expression of hckIε in flies appears to interfere with dbt activity. Thus, caution should be used in interpreting assays that measure activity of mammalian casein kinase mutants in Drosophila, or that employ vertebrate CKI in studies of dPER phosphorylations.

Spermatogenesis in the Testes of Diapause and Non-Diapause Pupae of the Sweet Potato Hornworm, Agrius convolvuli (L.) (Lepidoptera: Sphingidae)

Abstract Dichotomous spermatogenesis was examined in relation to diapause in the sweet potato hornworm, Agrius convolvuli. In non-diapause individuals, eupyrene metaphase began during the fifth larval instar and eupyrene spermatids appeared in wandering larvae. Bundles of mature sperm were found after pupation. Apyrene spermatocytes also appeared during the fifth larval instar, but meiotic divisions occurred irregularly and their nuclei were discarded from the cells during spermiogenesis. Morphometric analyses of flagellar axonemes showed a variable sperm number in apyrene bundles. The variation ranging from 125 to 256 sperm per bundle indicated abnormal divisions or the elimination of apyrene spermatocytes. In diapause-induced hornworms, spermatogenesis progressed similarly during the larval stages. The cessation of spermatogenesis during diapause is characterized by 1) secondary spermatocytes and sperm bundles degenerating gradually as the diapause period lengthens, and 2) spermatogonia or primary spermatocytes appearing throughout diapause. A TUNEL (TdT-mediated dUTP-biotin nick end-labeling) assay revealed that DNA fragmentation occurred in the nuclei of secondary spermatocytes and early spermatids. Aggregates of heterochromatin along the nuclear membrane indicated the onset of apoptosis, and condensed chromatin was confirmed by electron microscopy to be the apoptotic body. These results show that the degenerative changes in spermatogenic cells during pupal diapause were controlled by apoptosis.

Highly conserved Drosophila ananassae timeless gene functions as a clock component in Drosophila melanogaster

The behavior and physiology of Drosophila are subject to rhythms that are controlled by the circadian clock genes, period, timeless, clock and cycle, all of which are thought to participate in central pacemaker control. The molecular mechanism of rhythm in Drosophila has been studied in detail. However, rhythm and clock genes have mostly been analyzed in Drosophila melanogaster. To confirm whether the tim gene exists and works as a clock component in other Drosophila species, we cloned a tim homolog from Drosophila ananassae that shared 85.9% similarity with Drosophila melanogaster tim at the amino acid level. In addition, the PER interaction domains and NLS were highly conserved. Introduction of the D. ananassae tim homolog rescued the rhythm of the locomotor activity of about 44% of a population of D. melanogaster tim(01) flies. At the molecular level, hs-tim introduced not only TIM but PER oscillation in transgenic flies. These results indicate that the tim gene in D. ananassae functions as a component of the circadian clock in D. melanogaster.