One night of disturbed sleep is all it takes for us to realize how essential sleep is. Our long-term objectives are to understand the molecular signals that cause us to wake up and go to sleep. An internal body clock regulates sleep/wake cycles and many other daily (circadian) rhythms. We use the model system Drosophila, which has the best molecular characterization of the molecular clock that governs circadian rhythms. We have found clock roles for two related transcription factors, vrille (vri) and Pdp1 (Par domain protein 1). vri and Pdp1 expression oscillates in a clock-dependent manner. Cycling expression of vri and Pdp1 is required for circadian rhythmicity since flies that continuously express high levels of vri and Pdp1 become arrhythmic. This is because vri and Pdp1 feed back to the clock and regulate expression of the other clock genes, indicating vri and Pdp1 are themselves clock genes that constitute a previously unidentified loop within the central clock. vri and Pdp1 may also connect the central clock to output pathways regulating behavior. For vri, this may be partly by regulating levels of the Pigment Dispersing Factor neuropeptide, which is required for rhythmicity. Other output signals are also predicted. We will take molecular and genetic approaches to understand the roles of vri and Pdp1 in the central clock, and a genomic approach to ask how they may link the clock to behavior. Our specific aims are: (1) To understand how vri and Pdp1 regulate the central oscillator. Which central clock genes do VRT and PDP1 proteins regulate? Do VRI and PDPI proteins form a functional complex or do they compete for binding to target gene promoters? (2) To use a genomic approach to identify VRI and PDP1-regulated genes. Are any of these genes expressed in pacemaker cells, and do they connect cycling clock gene expression with rhythmic behavior? Which VRI target gene explains how increased vri levels indirectly suppress PDF levels post-transcriptionally. Most of the Drosophila clock genes have related mammalian genes that function similarly. Clock output signals we identify in Drosophila may, ultimately, be useful in human therapy.