Effects of TWIN-OF-EYELESS on Clock Gene Expression and Central-Pacemaker Neuron Development in Drosophila

Circadian oscillators are autonomous molecular rhythms that reside in cells to align whole-organism physiology and behavior to the 24-h day. In flies, as in mammals, the oscillator operates in cells that coexpress CLOCK (CLK) and CYCLE (CYC). Recent work in Drosophila has shown that CLK is unique in its ability to generate heterologous oscillators, indicating that Clk gene expression defines the circadian cell fate. Here, using standard in vitro and in vivo techniques, we show that TWIN-OF-EYELESS (TOY; dPax6) regulates Clk expression in small ventrolateral neurons (s-LN_vs) that coordinate sleep-wake cycles. Crucially, toy binds multiple sites at the Clk locus, is expressed independent of CLK-CYC in LN_vs, regulates CLK protein levels under optimal photoperiodic conditions, and sets clock-speed during endogenous free-run. Furthermore, TOY is necessary for the onset of Clk expression in LN_vs during embryogenesis. We propose that TOY contributes to a transcription complex that functions upstream of the oscillator to promote Clk expression in s-LN_vs.