Harold B. Dowse

Quantitative Analysis of Drosophila period Gene Transcription in Living Animals

To determine the in vivo regulatory pattern of the clock gene period (per), the authors recently developed transgenic Drosophila carrying a luciferase cDNA fused to the promoter region of per. They have now carried out noninvasive, high time-resolution experiments allowing high-throughput monitoring of circadian bioluminescence rhythms in individual living adults for several days. This immediately solved several problems (resulting directly from individual asyn chrony within a population) that have accompanied previous biochemical ex periments in which groups of animals were sacrificed at each time point. Furthermore, the authors have developed numerical analysis methods for auto matically determining rhythmicity associated with bioluminescence records from single flies. This has revealed some features of per gene transcription that were previously unappreciated and provides a general strategy for the analysis of rhythmic time series in the study of molecular rhythms.

Signal analysis of behavioral and molecular cycles.

BackgroundCircadian clocks are biological oscillators that regulate molecular, physiological, and behavioral rhythms in a wide variety of organisms. While behavioral rhythms are typically monitored over many cycles, a similar approach to molecular rhythms was not possible until recently; the advent of real-time analysis using transgenic reporters now permits the observations of molecular rhythms over many cycles as well. This development suggests that new details about the relationship between molecular and behavioral rhythms may be revealed. Even so, behavioral and molecular rhythmicity have been analyzed using different methods, making such comparisons difficult to achieve. To address this shortcoming, among others, we developed a set of integrated analytical tools to unify the analysis of biological rhythms across modalities.ResultsWe demonstrate an adaptation of digital signal analysis that allows similar treatment of both behavioral and molecular data from our studies of Drosophila. For both types of data, we apply digital filters to extract and clarify details of interest; we employ methods of autocorrelation and spectral analysis to assess rhythmicity and estimate the period; we evaluate phase shifts using crosscorrelation; and we use circular statistics to extract information about phase.ConclusionUsing data generated by our investigation of rhythms in Drosophila we demonstrate how a unique aggregation of analytical tools may be used to analyze and compare behavioral and molecular rhythms. These methods are shown to be versatile and will also be adaptable to further experiments, owing in part to the non-proprietary nature of the code we have developed.

A new role for cryptochrome in a Drosophila circadian oscillator

Cryptochromes are flavin/pterin-containing proteins that are involved in circadian clock function in Drosophila and mice. In mice, the cryptochromes Cry1 and Cry2 are integral components of the circadian oscillator within the brain and contribute to circadian photoreception in the retina. In Drosophila, cryptochrome (CRY) acts as a photoreceptor that mediates light input to circadian oscillators in both brain and peripheral tissue. A Drosophila cry mutant, cryb, leaves circadian oscillator function intact in central circadian pacemaker neurons but renders peripheral circadian oscillators largely arrhythmic. Although this arrhythmicity could be caused by a loss of light entrainment, it is also consistent with a role for CRY in the oscillator. A peripheral oscillator drives circadian olfactory responses in Drosophila antennae. Here we show that CRY contributes to oscillator function and physiological output rhythms in the antenna during and after entrainment to light–dark cycles and after photic input is eliminated by entraining flies to temperature cycles. These results demonstrate a photoreceptor-independent role for CRY in the periphery and imply fundamental differences between central and peripheral oscillator mechanisms in Drosophila.

The search for hidden periodicities in biological time series revisited

In the study of rhythmicity in biological systems, two critical questions must be addressed: whether the process under investigation is significantly cyclic, and if so, what is the best estimate of the period of the oscillation. The Whittaker-Robinson “periodogram” has been used extensively to answer both of these questions, Fourier Analysis to a lesser extent. Advances in digital signal processing have produced techniques superior to both, and we have applied one of these (Maximum Entropy Spectral Analysis or MESA) to biological data. We have additionally developed a novel method for analyzing signal-to-noise ratios in biological rhythm data using the autoregressive model underlying MESA. We review here the current methodology for the analysis of biological time series and describe our application of these techniques. The superior performance of this combination of techniques is demonstrated using previously published data. In addition, employing an empirical approach, we have demonstrated that cyclic but aperiodic (i.e. chaotic) systems may be distinguished from noisy periodic or stochastic ones using a combination of these analyses. The implications of this for work on ultradian and circadian rhythms are discussed.

Circadian and ultradian rhythms in period mutants of Drosophila melanogaster.

Using digital techniques for signal analysis—the correlogram and a high-resolution analysis of time series, maximum-entropy spectral analysis (MESA)—we have detected both circadian and ultradian rhythms in the locomotor activity of free-runningper0 males and in females lacking theper locus (per−; heterozygous for two deficiencies, each of which deletes the gene). Over half theper0 individuals and half theper− individuals tested were rhythmically active, with dominant periods ranging from 4 to 22 h; most of the significantly rhythmicper0 andper− flies clearly exhibited multiple periodicities. This novel pattern of rhythmic behavior is thoroughly distinct from the wild-type pattern. One hypothesis suggested by our observations is that theper+ gene products mediate the coupling of multiple ultradian oscillators to produce wild-type circadian rhythms.

Further Evidence that the Circadian Clock in Drosophila is a Population of Coupled Ultradian Oscillators

We hypothesize that ultradian oscillators are coupled to yield a composite circadian clock in Drosophila. In such a system, period would be a function of the tightness of coupling of these oscillators, increasing as coupling loosens. Ultradian oscillations would become appar ent under weak coupling or in the absence of coupling. A new technique for calculating signal- to-noise ratio (SNR) for biological rhythms to characterize their precision has yielded support for this hypothesis. SNR of rhythms of the allelic series of mutations at the period ( per) locus of Drosophila melanogaster were compared. per0 was the noisiest, grading through perL, per+ , and pers, the least noisy. SNR decreases significantly with increasing period in pers, per+, and perL; pero typically has multiple ultradian oscillations and the lowest SNR. At least 70% of perL individuals also exhibit ultradian periodicities.

Modulation of Drosophila heartbeat by neurotransmitters.

Abstract The heart of Drosophila melanogaster is a simple muscular tube with a posterior pulsatile portion and a thoracic-cranial vessel. The pacemaker, located caudally, is myogenic. Its rate of firing is modulated by neurotransmitters. Serotonin, octopamine, norepineph-rine, dopamine, and acetylcholine accelerate the heart, in that order of potency. Dihydroxyphenylalanine, γ-aminobutyric acid, glutamate, and glycine have no effect. Generally, the regularity of the heartbeat is not adversely affected by treatment with any of these neurotransmitters. We show here that amnesiac, a neurological mutation, and Dihydroxyphenylalanine decarboxylasetemperature sensitive, a mutation that interferes with synthesis of dopamine, norepinephrine, and serotonin, result in slower heart rate and reduced regularity across a normal range of temperatures for these flies. Dopamine-N-acetyltransferase, which is on the catabolic route to dopamine, serotonin, and octopamine, has no effect. hypoactiveC reduces the rate of the heart, but its mechanism of action is unknown.

Advanced analysis of a cryptochrome mutation's effects on the robustness and phase of molecular cycles in isolated peripheral tissues of Drosophila

BackgroundPreviously, we reported effects of the cryb mutation on circadian rhythms in period and timeless gene expression within isolated peripheral Drosophila tissues. We relied on luciferase activity driven by the respective regulatory genomic elements to provide real-time reporting of cycling gene expression. Subsequently, we developed a tool kit for the analysis of behavioral and molecular cycles. Here, we use these tools to analyze our earlier results as well as additional data obtained using the same experimental designs.ResultsIsolated antennal pairs, heads, bodies, wings and forelegs were evaluated under light-dark cycles. In these conditions, the cryb mutation significantly decreases the number of rhythmic specimens in each case except the wing. Moreover, among those specimens with detectable rhythmicity, mutant rhythms are significantly weaker than cry+ controls. In addition, cryb alters the phase of period gene expression in these tissues. Furthermore, peak phase of luciferase-reported period and timeless expression within cry+ samples is indistinguishable in some tissues, yet significantly different in others. We also analyze rhythms produced by antennal pairs in constant conditions.ConclusionsThese analyses further show that circadian clock mechanisms in Drosophila may vary in a tissue-specific manner, including how the cry gene regulates circadian gene expression.

A Congenital Heart Defect in Drosophila Caused by an Action-Potential Mutation

The mutation no action potential (nap) induces arrhythmia in the heartbeat of Drosophila melanogaster larvae at temperatures above 20 degrees C; heartbeat becomes normally rhythmic again after a shift back to 20 degrees C. For this phenotype, napa is almost completely recessive to the wild type, napa also reduces the temperature-sensitivity of heart rate over a wide range of temperature, for this phenotype, napa is dominant over the wild type, napa causes reversible paralysis in adults by epistatic effects on the expression of paralyrica, a gene encoding a voltage-dependent sodium channel. However, the paramutation, which induces paralysis in adults at 29 degrees C, has no effect on larval heartbeat at temperatures between 20 degrees and 37.5 degrees C. The period gene, contra earlier reports, has no effect on heartbeat.

The role of courtship song in sexual selection and species recognition by female Drosophila melanogaster

Male fruit flies, Drosophila melanogaster, produce a complex courtship display, which females may use to identify conspecifics and to aid in choosing a high-quality mate. Courtship song, consisting of sine and pulse elements, is an important acoustic component of this display. Whereas characteristics that differ between species have been identified, the signals used for intraspecific mate choice remain unknown, as does the function of sine song. To investigate further the role of pulse and sine song, we varied characteristics of artificial and edited songs and played them to groups of flies to determine whether sine or pulse song parameters contribute to female mate choice. Playing artificial songs did not substantially change the amount of male courtship behaviour. Sine song in any proportion had no effect on female mating propensity, nor was mating affected by sine-song frequency. The amount of pulse song offered positively increased mating, up to a threshold, without regard to the structure of the song, pulse carrier frequency, or interpulse interval. These results indicate that females use pulse song, not sine song, for both species identification and intraspecific mate choice. Males may continue to produce sine song as a relic, as a by-product of a physiological process or because it is inextricably linked to a mate-choice signal in a different modality. The role of song memory is discussed briefly.