In Drosophila, time-keeping is based on a ~24h transcription feedback loop, in which CLOCK-CYCLE (CLK-CYC) heterodimers activate transcription of genes encoding the feedback repressors PERIOD (PER) and TIMELESS (TIM). Despite the progress that has been made to define the molecular mechanisms that govern feedback loop function in animals, we do not understand how/when clock cells develop, how the clock network is organized, and how the feedback loop is initiated. To determine when pacemaker neurons arise in Drosophila, I used a Clk-GFP transgene because Clk is the first clock gene to be expressed. I found that CLK is expressed in the late pacemaker neuron clusters (neurons with active rhythms in per expression during metamorphosis) by L3 larvae. This delay in per expression is not due to the lack of cyc expression as a GFP-cyc transgene is expressed exclusively in Clkexpressing pacemaker neurons, indicating novel factors are needed to activate circadian oscillator function during metamorphosis. In addressing how Clk promotes CYC expression to initiate clock function, I found that Clk controls CYC accumulation by stabilizing CYC protein in cultured Drosophila Schneider 2 (S2) cells. Likewise, CYC accumulates specifically in ectopic cells expressing Clk, indicating that CLK also stabilizes CYC in vivo. CLK and CYC are, however, not sufficient for clock function in ectopic cells; cryptochrome (cry) is also required to entrain and/or maintain these clocks. To determine how Clk affects the formation of clock network, I generated a Clk21-Gal4 transgene to mark pacemaker neurons in Clk^o^u^t flies. I found that Clk is essential for the maintenance of small ventral lateral neurons (sLNvs) in adults without affecting the development of other groups of pacemaker neurons, which provides a new role of Clk in the sLNv development and/or maintenance besides its role in initiating circadian clock function. Taken together, my work has demonstrated clock cell development, network formation, as well as the mechanisms by which the functions of clock are initiated in Drosophila. Since these features appear to be conserved in eukaryotic clocks, my research will hopefully provide new insights into the development and function initiation of the clock in other organisms.
