Abstract
BackgroundGreen Fluorescent Protein (GFP) is used extensively as a reporter for transgene expression in Drosophila and other organisms. However, GFP has not generally been used as a reporter for circadian patterns of gene expression, and it has not previously been possible to correlate patterns of reporter expression with 3D movement and behavior of transgenic animals.ResultsWe present a video tracking system that allows tissue-specific GFP expression to be quantified and correlated with 3D animal movement in real time. eyeless/Pax6 reporter expression had a 12 hr period that correlated with fly activity levels.hsp70 and hsp22 gene reporters were induced during fly aging in circadian patterns (24 hr and 18 hr periods, respectively), and spiked in the hours preceding and overlapping the death of the animal. The phase of hsp gene reporter expression relative to fly activity levels was different for each fly, and remained the same throughout the life span.ConclusionThese experiments demonstrate that GFP can readily be used to assay longitudinally fly movement and tissue-specific patterns of gene expression. The hsp22-GFP and hsp70-GFP expression patterns were found to reflect accurately the endogenous gene expression patterns, including induction during aging and circadian periodicity. The combination of these new tracking methods with the hsp-GFP reporters revealed additional information, including a spike in hsp22 and hsp70 reporter expression preceding death, and an intriguing fly-to-fly variability in the phase of hsp70 and hsp22 reporter expression patterns. These methods allow specific temporal patterns of gene expression to be correlated with temporal patterns of animal activity, behavior and mortality.
Highlights
Green Fluorescent Protein (GFP) is used extensively as a reporter for transgene expression in Drosophila and other organisms
These experiments demonstrate that GFP can readily be used to assay longitudinally fly movement and tissue-specific patterns of gene expression
Transgenic fly strains were utilized in which enhanced-fluorescence GFP [23] is expressed in retina tissue in adults by an artificial promoter containing three binding sites for the eyeless/PAX6 homeodomain transcription factor [24]. eGFP was expressed in adult flies using reporters containing the promoters of the stress-response genes hsp70 and hsp22, which are induced in tissue-specific patterns during aging [16,17,25,26]
Summary
Green Fluorescent Protein (GFP) is used extensively as a reporter for transgene expression in Drosophila and other organisms. GFP and related fluorescent proteins [1] have been used extensively as reporter molecules for transgene expression in a variety of cells and transgenic animals, including models for the study of behavior such as Drosophila melanogaster, zebrafish and mouse [2,3,4]. We present a real-time image acquisition system that allows quantification of GFP fluorescence intensity as a read-out of gene expression and the simultaneous tracking of 3D animal movement and behavior. These methods allow specific temporal patterns of gene expression to be correlated with temporal patterns of animal activity, behavior, and mortality
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