RNA interference (RNAi) pathways, consisting of Argonaute (AGO) proteins and small RNAs (sRNAs) that provide sequence specificity, play key roles in gene regulation across all domains of life. Although AGOs were initially characterized as post-transcriptional modulators of gene expression, studies of AGOs in many species have revealed that they impact gene expression at nearly every stage in the life cycle of a transcript-from transcription to nuclear export and translation. Due to their central role in RNAi and profound impact on development and differentiation in numerous organisms, uncovering new and conserved molecular mechanisms of AGOs advances our fundamental knowledge of cellular function and has the potential to provide more precise means to manipulate gene expression, relevant for biotechnology and therapeutics. C. elegans has long been a model for RNAi research. Its genome encodes an expanded family of 27 ago genes, 21 of which appear to encode functional proteins. We are systematically characterizing all of the AGOs in C. elegans to uncover novel functions for these proteins and to develop an integrated portrait of the molecular mechanisms of these mostly uncharacterized proteins throughout worm development. Utilizing CRISPR/Cas9 genome-editing, we epitope-tagged all 21 AGOs with GFP-3xFLAG. We utilized confocal microscopy to characterize the differential expression patterns of each AGO throughout development, and identified 12 AGOs that are expressed in the germline. By high-throughput sequencing of 1) small RNAs associated with each AGO, and 2) total small RNA pools from ago mutants versus wild type, we have defined the unique and overlapping complements of small RNAs bound by each AGO, revealing surprising plasticity in AGO/sRNA complexes, and novel AGOs that associate with well-studied classes of sRNAs. Furthermore, phenotypic analyses of ago mutants under normal and stressful conditions have revealed previously unappreciated phenotypes; for instance
alg-5,
wago-1 and
ppw-2 display a Mortal Germline (Mrt) fertility defect at elevated temperatures. Thus, overall, our systematic and pioneering studies provide an unprecedented view of the intricacies of sRNA pathway activity throughout the development of a complex animal.