The identification and analysis of clock genes in the fruit fly, Drosophila melanogaster, revealed that the circadian clock is based on autoregulatory feedback loops in gene expression. Similar feedback loops serve to keep circadian time in essentially all eukaryotes, and the genes that mediate feedback loop function are well conserved from insects to mammals. A key function of these feedback loops is that they drive circadian rhythms in transcription that are required to keep circadian time and drive overt rhythms in physiology, metabolism and behavior. Consequently, these clocks are of great clinical importance because their dysfunction can lead to sleep problems, metabolic disorders, and even cancer. Despite the progress that has been made over the last 21 years to define the molecular mechanisms that govern feedback loop function in animals, we do not understand how the feedback loop determines core clock properties such as circadian period and phase or activates specific sets of output genes in different tissues. To answer these important questions we propose to develop cell lines from Drosophila clock cells that can be used to quickly and efficiently screen for clock genes which were missed using traditional behavior-based screening approaches and that enable biochemical analysis of circadian clocks from cell types that have been heretofore inaccessible. Existing Drosophila cell lines were derived spontaneously from embryo or imaginal disc cultures through a process that often took years, but a new method for generating Drosophila cell lines using the rasV12 oncogene increases the rate and efficiency of cell line production, and can be targeted to specific tissues. We will use the rasV12 technique to create and characterize circadian cell lines from brain pacemaker neurons and olfactory sensory neurons (OSNs) from Drosophila.
In Specific Aim 1 we will drive rasV12 expression in dividing brain pacemaker neurons in embryos and dividing OSNs in eye-antennal imaginal discs to generate circadian cell lines that are marked by GFP expression and contain a rhythmically expressed luciferase reporter gene.
In Specific Aim 2 we will determine whether these cell lines display the cardinal clock properties of self-sustained ~24h periodicity, phase shifting by environmental cues, and periods that are essentially invariant at different temperatures, and will identify the brain pacemaker neuron and OSN subclasses that gave rise to these cell lines. Success in generating circadian cell lines from Drosophila would immediately enable fast and efficient RNAi screens for the remaining clock components, the identification of clock cell-specific output genes in different clock tissues, and chemical library screening for drugs that alter core clock properties, as well as pave the way for producing circadian cell lines that incorporate additional genetic tools or originate from other circadian cell types. Thus, creating circadian cell lines from Drosophila will advance our understanding of the circadian timekeeping mechanism and tissue-specific clock outputs in flies as well as mammals.
The identification and analysis of clock genes in fruit flies revealed that transcriptional feedback loops keep circadian time and control rhythmic behavior, physiology and metabolism in essentially all organisms including humans. These clocks are of great clinical importance since their dysfunction can lead to sleep problems, metabolic disorders, and even cancer. This project will provide tools necessary to characterize the circadian timekeeping mechanism in sufficient detail to begin developing treatments for clock-dependent disorders.