Essentially all organisms use circadian clocks to control daily rhythms in physiology, metabolism and behavior. These clocks are of great clinical importance since their dysfunction can lead to sleep disorders and certain forms of depression. Molecular and genetic studies in the fruit fly, Drosophila melanogaster, have contributed significantly to our understanding of the circadian clock. The identification and analysis of clock genes in Drosophila revealed that the circadian timekeeping mechanism is comprised of 2 interlocked feedback loops in gene expression, a period (per)/timeless (tim) loop and a Clock (CIk) loop, that are synchronized to environmental light cycles by the blue light photoreceptor CRYPTOCHROME (CRY). As the transcriptional networks that regulate these feedback loops are revealed, 2 important regulatory themes have emerged: CLK-CYCLE (CYC) heterodimers activate their feedback regulators via E-box mediated transcription, and kinases control the accumulation and subcellular localization of specific transcription factors. Although genes that feedback to control E-box dependent transcription are required for oscillator function, it is not known if this is the case for factors that mediate rhythmic CIk transcription. Circadian feedback loop oscillators operate autonomously in specific neuronal and non-neuronal """"""""clock cells', but how this expression pattern is determined is not known. We recently found that CIk, cyc and cry are expressed exclusively in clock cells, which suggests that the factors responsible for their activation determine which cells will become clock cells. Such factors may also be responsible for activating the expression of clock protein kinases in clock cells. Our working model is that CIk, cyc, cry and possibly clock protein kinases are activated in presumptive clock cells during development, and then initiate feedback loop function by activating the necessary E-box dependent feedback regulators or phosphorylating clock proteins. In this proposal, we will test this model by: (1) Identifying factors that activate clock cell specific transcription of CIk, cyc and cry, (2) determining whether factors that activate CIk, cyc and cry in clock cells also activate the expression of clock protein kinases, (3) determining whether the clock developmental program can activate circadian feedback loop function in non-clock cells, and (4) determining whether PDP1 and VRI are necessary for circadian oscillator function. Since the feedback loop mechanism has been conserved between flies and mammals, what we learn here will be relevant to the situation in mammals.
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