UCLA

Biological rhythms are ubiquitous in nature. Examples include daily cycles of rest and activity observed among most living organisms and annual cycles of hibernation and migration observed among animals and birds. However, the timekeeping mechanisms that underlie the vast majority of biological rhythms are not well understood. I chose the molting cycle of C. elegans as a scientific model to study this problem. C. elegans molt 4 times at regular time intervals. Each life stage of the animal consists of the intermolt, a 6 – 8 h period of feeding and locomotion, which is followed by the molt, and a 2 h period of sleep-like quiescence and remodeling of the exoskeleton.

I found that transcriptional–post-transcriptional feedback loops among the conserved nuclear hormone receptor NHR-23 and the let-7 family of miRNAs govern both the pace of the molting cycle and the total number of molts. The negative feedback loop acts within a genetic oscillator – the molting cycle timer – to schedule the timing of reiterative molts and within an hourglass timer to schedule the extinction of the molting cycle in adulthood. Knockdown of nhr-23 slowed down the pace of the cycle, whereas forced expression of nhr-23 or inactivation of let-7s accelerated the pace of the cycle. Interactions between NHR-23 and let-7s drive the cyclical expression of nhr-23, let-7s and scores of downstream targets necessary for the process of molting. I also found that the oscillatory expression of nhr-23 is dampened in regular increments over the course of larval development; the dampening was dependent upon the activity of let-7s. Moreover, forced expression of nhr-23 was sufficient to trigger extra molts in sexually mature animals. I propose that the incremental dampening brings the levels of nhr-23 below a critical threshold, preventing entry into another molt. Both the core components and cis-regulatory elements of this timekeeping mechanism are conserved from nematodes to humans.

Going further, I identified additional genes and molecules that either act upstream of NHR-23 and let-7s or in independent pathways to control the number of molts in C. elegans. An as-yet-unidentified hormone is thought to control molting by acting as a ligand for NHR-23. Consistent with this model, I show that dietary supplementation of cholesterol is necessary for forced expression of NHR-23 to trigger extra molts. Thus, cholesterol or a cholesterol-derivative might act as a native ligand for NHR-23. Using a reverse genetics approach, I also uncovered genes that might act in pathways for synthesis of the ligand. My findings might lead to discovery of a hormone that promotes molting in C. elegans—a long-standing question in the field. Further examination of how the molecular and genetic interactions uncovered in this thesis regulate the molting cycle could lead to a better understanding of developmental clocks, as well as the circadian clock and related sleep, metabolic and psychiatric disorders.