Systematic modeling-driven experiments identify distinct molecular clockworks underlying hierarchically organized pacemaker neurons

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 (sLN(v)s), but not in the slave oscillator, posterior dorsal neuron 1s (DN1(p)s). 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 sLN(v)s exhibit higher synthesis and turnover of PER and lower CLK levels than DN1(p)s. Importantly, light shift analysis reveals that due to such a distinct molecular clockwork, sLN(v)s 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.