Nematode molting is a remarkable process where the animals must essentially build a new epidermis underneath the old skin and then rapidly shed the old skin. The study of molting provides a window into the developmental program of many core cellular processes, such as oscillatory gene expression, coordinated intracellular trafficking, steroid hormone signaling, developmental timing, and extracellular remodeling. Like many of these processes, molting is coordinated by gene regulatory networks, and the nuclear hormone receptor NHR-23 is a master regulator. An imaging timecourse revealed oscillatory NHR-23::GFP expression in the epithelium which closely follows the reported mRNA expression. Previous work using RNAi depletion of
nhr-23 suggested that molting defects are rarely observed at the first molt. We have previously generated
nhr-23::AID::3xFLAG animals carrying a TIR transgene to allow auxin-dependent depletion in all somatic tissues. When we plated these animals on auxin plates directly following synchronization, 100% of L1 larvae arrested and we did not observe molting defects, such as animals trapped in the cuticle, or dragging partially shed cuticles. Using this same strain, we observed the same L1 arrest phenotype when animals were shifted onto auxin within the first six hours of release from synchronization. However, when we delayed NHR-23 depletion until 6-12 hours after release from synchronization, animals no longer arrested. Instead, we observed defects, such as entrapment in the cuticle, formation of large vacuoles, abnormal tail morphology, and worms with a flattened morphology we termed "splatter". Thus, our AID system has revealed a more significant role for NHR-23 in the L1 molt than previously reported and connected NHR-23 levels during its oscillation with two distinct classes of phenotypes upon depletion. Our data suggest a novel NHR-23 dependent decision point controlling molting.