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Open Access Highly Accessed Research article

CLOCK expression identifies developing circadian oscillator neurons in the brains of Drosophila embryos

Jerry H Houl12, Fanny Ng123, Pete Taylor124 and Paul E Hardin1*

Author Affiliations

1 Center for Research on Biological Clocks, Department of Biology, Texas A&M University, 3258 TAMU, College Station, TX 77843, USA

2 Department of Biology and Biochemistry, University of Houston, 4800 Calhoun, Houston, TX 77204, USA

3 Department of Neuroscience, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111, USA

4 Department of Pediatrics, MD Anderson Cancer Center, 1515 Holcombe, Houston, TX 77030, USA

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BMC Neuroscience 2008, 9:119  doi:10.1186/1471-2202-9-119

Published: 18 December 2008

Abstract

Background

The Drosophila circadian oscillator is composed of transcriptional feedback loops in which CLOCK-CYCLE (CLK-CYC) heterodimers activate their feedback regulators period (per) and timeless (tim) via E-box mediated transcription. These feedback loop oscillators are present in distinct clusters of dorsal and lateral neurons in the adult brain, but how this pattern of expression is established during development is not known. Since CLK is required to initiate feedback loop function, defining the pattern of CLK expression in embryos and larvae will shed light on oscillator neuron development.

Results

A novel CLK antiserum is used to show that CLK expression in the larval CNS and adult brain is limited to circadian oscillator cells. CLK is initially expressed in presumptive small ventral lateral neurons (s-LNvs), dorsal neurons 2 s (DN2s), and dorsal neuron 1 s (DN1s) at embryonic stage (ES) 16, and this CLK expression pattern persists through larval development. PER then accumulates in all CLK-expressing cells except presumptive DN2s during late ES 16 and ES 17, consistent with the delayed accumulation of PER in adult oscillator neurons and antiphase cycling of PER in larval DN2s. PER is also expressed in non-CLK-expressing cells in the embryonic CNS starting at ES 12. Although PER expression in CLK-negative cells continues in ClkJrk embryos, PER expression in cells that co-express PER and CLK is eliminated.

Conclusion

These data demonstrate that brain oscillator neurons begin development during embryogenesis, that PER expression in non-oscillator cells is CLK-independent, and that oscillator phase is an intrinsic characteristic of brain oscillator neurons. These results define the temporal and spatial coordinates of factors that initiate Clk expression, imply that circadian photoreceptors are not activated until the end of embryogenesis, and suggest that PER functions in a different capacity before oscillator cell development is initiated.