Fluorescence/Luminescence Circadian Imaging of Complex Tissues at Single-Cell Resolution

Abstract

The use of luciferase reporter genes together with luminescence detection has enabled high frequency monitoring of molecular circadian clock function in living tissues. With the help of an intensified CCD camera combined with an inverted epifluorescence microscope, the authors have established a new imaging strategy that makes use of transgenic cell type-specific expression of fluorescent proteins to identify cells of interest for subsequent circadian luminescence recording at single-cell resolution.

Keywords

circadian clock Drosophila imaging luciferase luminescence fluorescence gene expression

References

Hall JC ( 2005) Systems approaches to biological rhythms in Drosophila . Methods Enzymol 393:61-185.

Helfrich-Forster C. , Yoshii T. , Wulbeck C. , Grieshaber E. , Rieger D. , Bachleitner W. , Cusamano P. , and Rouyer F. ( 2007) The lateral and dorsal neurons of Drosophila melanogaster: New insights about their morphology and function. Cold Spring Harb Symp Quant Biol 72:517-525.

Lee T. and Luo L. ( 1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22:451-461.

Meyer P. , Saez L. , and Young MW ( 2006) PER-TIM interactions in living Drosophila cells: An interval timer for the circadian clock. Science 311:226-229.

Park JH , Helfrich-Forster C. , Lee G. , Liu L. , Rosbash M. , and Hall JC ( 2000) Differential regulation of circadian pacemaker output by separate clock genes in Drosophila. Proc Natl Acad Sci U S A 97:3608-3613.

Peng Y. , Stoleru D. , Levine JD , Hall JC , and Rosbash M. ( 2003) Drosophila free-running rhythms require intercellular communication. PLoS Biol 1:E13.

Price JL , Blau J. , Rothenfluh A. , Abodeely M. , Kloss B. , and Young MW ( 1998) double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation. Cell 94:83-95.

Sehadova H. , Glaser FT , Gentile C. , Simoni A. , Giesecke A. , Albert JT , and Stanewsky R. ( 2009) Temperature entrainment of Drosophila’s circadian clock involves the gene nocte and signaling from peripheral sensory tissues to the brain. Neuron 64:251-266.

Stanewsky R (2007) Analysis of rhythmic gene expression in adult Drosophila using the firefly luciferase reporter gene. Methods Mol Biol 362:131-142.

10.

Stanewsky R, Kaneko M, Emery P, Beretta B, Wager-Smith K, Kay SA, Rosbash M, and Hall JC (1998) The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila . Cell 95:681-692.

11.

Stoleru D. , Peng Y. , Nawathean P. , and Rosbash M. ( 2005) A resetting signal between Drosophila pacemakers synchronizes morning and evening activity. Nature 438:238-242.

12.

Welsh DK , Imaizumi T. , and Kay SA ( 2005) Real-time reporting of circadian-regulated gene expression by luciferase imaging in plants and mammalian cells. Methods Enzymol 393:269-288.

13.

Wijnen H. and Young MW ( 2006) Interplay of circadian clocks and metabolic rhythms. Annu Rev Genet 40:409-448.

14.

Yamazaki S. and Takahashi JS ( 2005) Real-time luminescence reporting of circadian gene expression in mammals. Methods Enzymol 393:288-301.

15.

Yoshii T. , Wulbeck C. , Sehadova H. , Veleri S. , Bichler D. , Stanewsky R. , and Helfrich-Forster C. (2009) The neuropeptide pigment-dispersing factor adjusts period and phase of Drosophila’s clock. J Neurosci 29:2597-2610.

16.

Yu W. and Hardin PE ( 2007) Use of firefly luciferase activity assays to monitor circadian molecular rhythms in vivo and in vitro. Methods Mol Biol 362:465-480.

Supplementary Material

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