Martin Straume

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.

FLOWERING LOCUS C mediates natural variation in the high-temperature response of the Arabidopsis circadian clock.

Temperature compensation contributes to the accuracy of biological timing by preventing circadian rhythms from running more quickly at high than at low temperatures. We previously identified quantitative trait loci (QTL) with temperature-specific effects on the circadian rhythm of leaf movement, including a QTL linked to the transcription factor FLOWERING LOCUS C (FLC). We have now analyzed FLC alleles in near-isogenic lines and induced mutants to eliminate other candidate genes. We showed that FLC lengthened the circadian period specifically at 27°C, contributing to temperature compensation of the circadian clock. Known upstream regulators of FLC expression in flowering time pathways similarly controlled its circadian effect. We sought to identify downstream targets of FLC regulation in the molecular mechanism of the circadian clock using genome-wide analysis to identify FLC-responsive genes and 3503 transcripts controlled by the circadian clock. A Bayesian clustering method based on Fourier coefficients allowed us to discriminate putative regulatory genes. Among rhythmic FLC-responsive genes, transcripts of the transcription factor LUX ARRHYTHMO (LUX) correlated in peak abundance with the circadian period in flc mutants. Mathematical modeling indicated that the modest change in peak LUX RNA abundance was sufficient to cause the period change due to FLC, providing a molecular target for the crosstalk between flowering time pathways and circadian regulation.

Genome-Wide Expression Analysis in DrosophilaReveals Genes Controlling Circadian Behavior

In Drosophila, a number of key processes such as emergence from the pupal case, locomotor activity, feeding, olfaction, and aspects of mating behavior are under circadian regulation. Although we have a basic understanding of how the molecular oscillations take place, a clear link between gene regulation and downstream biological processes is still missing. To identify clock-controlled output genes, we have used an oligonucleotide-based high-density array that interrogates gene expression changes on a whole genome level. We found genes regulating various physiological processes to be under circadian transcriptional regulation, ranging from protein stability and degradation, signal transduction, heme metabolism, detoxification, and immunity. By comparing rhythmically expressed genes in the fly head and body, we found that the clock has adapted its output functions to the needs of each particular tissue, implying that tissue-specific regulation is superimposed on clock control of gene expression. Finally, taking full advantage of the fly as a model system, we have identified and characterized a cycling potassium channel protein as a key step in linking the transcriptional feedback loop to rhythmic locomotor behavior.

Novel Features of Drosophila period Transcription Revealed by Real-Time Luciferase Reporting

The rapid turnover of luciferase and the sensitive, non-invasive nature of its assay make this reporter gene uniquely situated for temporal gene expression studies. To determine the in vivo regulatory pattern of the Drosophila clock gene period (per), we generated transgenic strains carrying a luciferase cDNA fused to the promoter region of the per gene. This has allowed us to monitor circadian rhythms of bioluminescence from pacemaker cells within the head for several days in individual living adults. These high time-resolution experiments permitted neuronal per transcription and opens the door to vastly simplified experiments in general chronobiology and studies of temporally regulated transcription in a wide range of experimental systems.

Circadian rhythms in mouse suprachiasmatic nucleus explants on multimicroelectrode plates

The suprachiasmatic nucleus (SCN) of the mammalian hypothalamus functions as a circadian pacemaker. This study used multimicroelectrode plates to measure extracellular action potential activity simultaneously from multiple sites within the cultured mouse SCN. Neurons within the isolated mouse SCN expressed a circadian rhythm in spontaneous firing rate for weeks in culture.

GABA and Gi/o differentially control circadian rhythms and synchrony in clock neurons

Neurons in the mammalian suprachiasmatic nuclei (SCN) generate daily rhythms in physiology and behavior, but it is unclear how they maintain and synchronize these rhythms in vivo. We hypothesized that parallel signaling pathways in the SCN are required to synchronize rhythms in these neurons for coherent output. We recorded firing and clock-gene expression patterns while blocking candidate signaling pathways for at least 8 days. GABAA and GABAB antagonism increased circadian peak firing rates and rhythm precision of cultured SCN neurons, but Gi/o did not impair synchrony or rhythmicity. In contrast, inhibiting Gi/o with pertussis toxin abolished rhythms in most neurons and desynchronized the population, phenocopying the loss of vasoactive intestinal polypeptide (VIP). Daily VIP receptor agonist treatment restored synchrony and rhythmicity to VIP−/− SCN cultures during continuous GABA receptor antagonism but not during Gi/o blockade. Pertussis toxin did not affect circadian cycling of the liver, suggesting that Gi/o plays a specialized role in maintaining SCN rhythmicity. We conclude that endogenous GABA controls the amplitude of SCN neuronal rhythms by reducing daytime firing, whereas Gi/o signaling suppresses nighttime firing, and it is necessary for synchrony among SCN neurons. We propose that Gi/o, not GABA activity, converges with VIP signaling to maintain and coordinate rhythms among SCN neurons.

Analysis of residuals: criteria for determining goodness-of-fit.

Publisher Summary When considering residuals, a qualitative approach is often the most revealing and informative. Though, quantitative methods to test more rigorously particular properties of residuals must be considered sometimes to quantitate the statistical significance of conclusions drawn as a result of data analysis. This chapter discusses some of the available methods for considering residuals with the aid of illustrative examples. The definition of an appropriate mathematical model involves considerations of the way to transform the experimentally observed system behavior into a mathematical description that permits a physical interpretation of the model parameter values. The significance of subtle behavior in residuals may suggest the presence of a significant system property that is overlooked by the current mathematical model. After verification that the residuals resulting from a model parameter estimation process to a set of data are indeed free of any systematic trends relative to any variables of significance (that is, dependent or independent variables), a quantitative estimate of the adequacy of the particular model in describing the data is possible. The concepts and approaches outlined in the chapter provide a survey of methods available for qualitatively and quantitatively considering residuals generated from data analysis procedures. In those cases where very precise interpretation of experimental observations is required, a thorough quantitative consideration of residuals may be necessary to address the statistical validity of particularly detailed mathematical models designed to account for the biochemical or biophysical properties of any experimental system of interest.

DNA microarray time series analysis: automated statistical assessment of circadian rhythms in gene expression patterning.

Publisher Summary This chapter describes the use of DNA microarrays in elucidation of network dynamics of circadian gene regulation. Application of this technology to circadian biology is particularly apropos given the molecular basis of intracellular circadian regulation, a system of transcriptional–translational feedback control. The COSOPT algorithm evolved as an extension of the CORRCOS algorithm and has been applied to search for diurnal/circadian gene expression profiling in mammalian and Drosophila microarray time series. Simulated data sets were prepared to approximate gene chip profiles from the experimental examinations of expression time series. The straightforward, direct approximation by nonlinear least squares optimization of a single-frequency cosine function to the data in question produced admirable performance under conditions in which pointwise uncertainties are not considered, but produced the highest false-positive rate of all the methods when pointwise uncertainties were considered. It is found that COSOPT also reports information about mean expression level and can use this information as an exclusionary criterion.

Monte Carlo method for determining complete confidence probability distributions of estimated model parameters

Publisher Summary The implementation of the Monte Carlo confidence probability determination method requires the initial estimation of the set of most probable parameter values that best characterize some set(s) of experimental observations, according to a suitable mathematical model (that is, one capable of reliably describing the data). The Monte Carlo approach is unique in the sense that it is capable of determining confidence interval probability distributions, in principle, to any desired level of resolution and is conceptually extremely easy to implement. The necessary information for the application of the Monte Carlo method for estimating confidence intervals and probability distribution profiles is two-fold: (1) an accurate estimate of the distribution of experimental uncertainties associated with the data being analyzed and (2) a mathematical model capable of accurately characterizing the experimental observations. The Monte Carlo method is then applied by (1) the analysis of the data for the most probable model parameter values, (2) the generation of “perfect” data as calculated by the model, (3) the superposition of a few hundred sets of simulated noise on the perfect data, (4) the analysis of each of the noise containing, simulated data with subsequent tabulation of each set of most probable parameter values, and (5) an assimilation of the tabulated sets of most probable parameter values by generating histograms. The histograms represent discrete approximations of the model parameter confidence probability distributions as derived from the original data set and the distribution of experimental uncertainty contained therein.

Effects of circadian regulation and rest-activity state on spontaneous seizures in a rat model of limbic epilepsy.

Summary: Purpose: Circadian regulation via the suprachiasmatic nuclei and rest–activity state may influence expression of limbic seizures.