Quantitative Profiling Identifies Potential Regulatory Proteins Involved in Development from Dauer Stage to L4 Stage in Caenorhabditis elegans.

When Caenorhabditis elegans encounters unfavorable growth conditions, it enters the dauer stage, an alternative L3 developmental period. A dauer larva resumes larval development to the normal L4 stage by uncharacterized postdauer reprogramming (PDR) when growth conditions become more favorable. During this transition period, certain heterochronic genes involved in controlling the proper sequence of developmental events are known to act, with their mutations suppressing the Muv (multivulva) phenotype in C. elegans. To identify the specific proteins in which the Muv phenotype is highly suppressed, quantitative proteomic analysis with iTRAQ labeling of samples obtained from worms at L1 + 30 h (for continuous development [CD]) and dauer recovery +3 h (for postdauer development [PD]) was carried out to detect changes in protein abundance in the CD and PD states of both N2 and lin-28(n719). Of the 1661 unique proteins identified with a < 1% false discovery rate at the peptide level, we selected 58 proteins exhibiting ≥2-fold up-regulation or ≥2-fold down-regulation in the PD state and analyzed the Gene Ontology terms. RNAi assays against 15 selected up-regulated genes showed that seven genes were predicted to be involved in higher Muv phenotype (p < 0.05) in lin-28(n791), which is not seen in N2. Specifically, two genes, K08H10.1 and W05H9.1, displayed not only the highest rate (%) of Muv phenotype in the RNAi assay but also the dauer-specific mRNA expression, indicating that these genes may be required for PDR, leading to the very early onset of dauer recovery. Thus, our proteomic approach identifies and quantitates the regulatory proteins potentially involved in PDR in C. elegans, which safeguards the overall lifecycle in response to environmental changes.