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

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.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.Most organisms exhibit daily rhythms in physiology, metabolism, and behavior that persist in the absence of environmental cues. In animals, these ~24 hr rhythms are controlled by circadian oscillators that reside in the central nervous system (CNS) and/or peripheral tissues. These oscillators are comprised of interlocked transcriptional feedback loops that regulate rhythmic gene expression within and downstream of the circadian timekeeping mechanism.In Drosophila, the per/tim and Clk feedback loops control rhythmic transcription that peaks around dusk and dawn, respectively (reviewed in [1-3]). The per/tim feedback loop is initiated durin