LAUSR

Significance The hierarchically organized master and slave clock neurons are widely believed to generate circadian rhythms via an identical molecular clockwork. However, their different roles in regulating those rhythms raise the question of whether their molecular clockworks differ. Here, leveraging systematic model-driven in vivo experiments for an unbiased search for their heterogeneity, we found that the master clock neurons have higher synthesis and turnover rates of repressor and lower activator levels than the slave clock neurons. Further in silico analysis revealed that this distinguished molecular clockwork of the master clock neurons allows them to generate strong rhythms but also to flexibly adjust rhythms upon environmental perturbation. This explains how the circadian clock can have two contradictory properties, robustness and flexibility. In metazoan organisms, circadian (∼24 h) rhythms are regulated by pacemaker neurons organized in a master–slave hierarchy. Although it is widely accepted that master pacemakers and slave oscillators generate rhythms via an identical negative feedback loop of transcription factor CLOCK (CLK) and repressor PERIOD (PER), their different roles imply heterogeneity in their molecular clockworks. Indeed, in Drosophila, defective binding between CLK and PER disrupts molecular rhythms in the master pacemakers, small ventral lateral neurons (sLNvs), but not in the slave oscillator, posterior dorsal neuron 1s (DN1ps). Here, we develop a systematic and expandable approach that unbiasedly searches the source of the heterogeneity in molecular clockworks from time-series data. In combination with in vivo experiments, we find that sLNvs exhibit higher synthesis and turnover of PER and lower CLK levels than DN1ps. Importantly, light shift analysis reveals that due to such a distinct molecular clockwork, sLNvs can obtain paradoxical characteristics as the master pacemaker, generating strong rhythms that are also flexibly adjustable to environmental changes. Our results identify the different characteristics of molecular clockworks of pacemaker neurons that underlie hierarchical multi-oscillator structure to ensure the rhythmic fitness of the organism.