Fluorescent Reporters for Studying Circadian Rhythms in Drosophila melanogaster

Abstract

Circadian rhythms are daily oscillations in physiology and gene expression that are governed by a molecular feedback loop known as the circadian clock. In Drosophila melanogaster, the core clock consists of transcription factors clock (Clk) and cycle (cyc) which form protein heterodimers that activate transcription of their repressors, period (per) and timeless (tim). Once produced, protein heterodimers of per/tim repress Clk/cyc activity. One cycle of activation and repression takes approximately ("circa") 24-h ("diem") and repeats even in the absence of external stimuli. The circadian clock is active in many cells throughout the body; however, tracking it dynamically represents a challenge. Traditional fluorescent reporters are slowly degraded and consequently cannot be used to assess dynamic temporal changes exhibited by the circadian clock. The use of rapidly degraded fluorescent protein reporters containing destabilized GFP (dGFP) that report transcriptional activity in vivo at a single-cell level with ~1-h temporal resolution can circumvent this problem. Here we describe the use of circadian clock reporter strains of Drosophila melanogaster, Clock^PER and Clock^TIM, to track clock transcriptional activity using the intestine as a tissue of interest. These methods may be extended to other tissues in the body.

Keywords: Circadian clock; Circadian rhythms; Drosophila melanogaster; Intestine; Regeneration; Transcriptional reporters.