Background: Circadian clocks are internal daily time keeping mechanisms that allow organisms to anticipate daily changes in their environment and to organize their behavior and physiology in a coherent schedule. Although circadian clocks use temperature compensation mechanisms to maintain the same pace over a range of temperatures, they are also capable of synchronizing to daily temperature cycles. This study identifies key properties of this process.

The use of luciferase reporter genes together with luminescence detection has enabled high frequency monitoring of molecular circadian clock function in living tissues. With the help of an intensified CCD camera combined with an inverted epifluorescence microscope, the authors have established a new imaging strategy that makes use of transgenic cell type-specific expression of fluorescent proteins to identify cells of interest for subsequent circadian luminescence recording at single-cell resolution.Keywords circadian clock; Drosophila; imaging; luciferase; luminescence; fluorescence; gene expression Circadian clocks are internal daily time-keeping mechanisms employed by a wide range of organisms to both predict daily environmental rhythms and organize many aspects of their physiology and behavior in a coherent daily schedule. All known circadian clocks rhythmically control biological functions by controlling gene expression and in most known cases the central time-keeping mechanism itself is a gene expression feedback circuit (e.g., Wijnen and Young, 2006). Circadian clock function can, therefore, be studied directly by monitoring circadian gene expression rhythms in tissues and cells from transgenic animals.Traditionally, clock-controlled gene expression has been assayed by sampling from a synchronized population of cells, large heterogenous tissue explants, or whole organisms over the course of multiple days. This approach has at least two important limitations: 1) individual cellular clocks cannot be followed over time, making it difficult to separate defects in synchrony from defects in cell-autonomous clock function, and 2) it is relatively labor intensive, making this approach poorly suited to achieving high temporal resolution of circadian gene expression or high-throughput screening of molecular circadian pheno-types. The development of transgenic reporter constructs that make use of the firefly luciferase gene has provided an alternative method of assaying circadian gene expression that does not suffer from these limitations (Stanewsky, 2007;Welsh et al., 2005;Yamazaki and Takahashi, 2005;Yu and Hardin, 2007). Luciferase enzyme activity is reliably linked to gene expression rhythms and can be monitored noninvasively by assaying luminescence via luminometry or imaging, provided that the enzymatic substrate luciferin is made available to the cells of

Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, we used the genetic tools available in Drosophila to conditionally manipulate the function of the CYCLE component of the positive regulator CLOCK/CYCLE (CLK/CYC) or its negative feedback inhibitor PERIOD (PER). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of CLK/CYC activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of CLK/CYC and both involve the ventral lateral clock neurons (LNvs) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LNvs resulted in abnormal peptidergic small-LNv dorsal projections, and (2) PER expression rhythms in the adult LNvs appeared to be affected by developmental inhibition of CLK/CYC activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex.