The molting cycle is a hallmark of insects and nematodes, but its endocrine control via size, stage, and nutritional inputs, as well as the enzymatic mediators for release of the exoskeleton, are not yet understood. Here, we identify endocrine and enzymatic regulators of molting in C. elegans through a combination of genome-wide RNAi and forward genetic screens. The genes discovered by RNAi encode annotated secreted peptides, transcription factors, membrane signaling molecules, and extracellular matrix enzymes essential for ecdysis. A family of novel, nematode-specific genes regulating molting is revealed via gain-of-function mutations. Fusions between several molting genes and green fluorescent protein show a pulse of expression before each molt in epithelial cells that synthesize the cuticle, indicating that the corresponding proteins are made in the correct time and place to regulate ecdysis. Molting genes are also expressed in neurons and the intestine. We find further that inactivation of particular genes, including the exoribonuclease Y48B6A.3, the secreted peptides W08F4.6 and
dsl-3, and the nuclear hormone receptor
nhr-23, abrogates expression of molting gene GFP reporters in epithelial cells, showing a regulatory network. Together, these results identify many of the signaling molecules and enzymes that regulate the temporal and spatial dynamics of ecdysis, and reveal the regulatory cascades that might couple expression of the corresponding genes to endocrine cues. With the cycling molting gene GFP reporters in hand, we now seek to identify genes that function far upstream in molting pathways, possibly in endocrine or neuroendocrine cells, through genetic and genomic screens. Here, we describe a screen for mutants that undergo supernumerary molts after reproductive maturity as one approach. Particular mutants initiate ecdysis after the terminal differentiation of epithelial cells, a feature distinct from mutants in known heterochronic genes, such as
lin-29. We are cloning the corresponding genes via a combination of SNP mapping and RNAi-based strategies.Diseases caused by parasitic nematodes affect 138 million people in tropical regions of Africa, Asia, and South America. Many genes that we identify as essential for molting are conserved in parasitic nematodes and thus represent attractive targets for pesticide and pharmaceutical development.