Extracellular RNAs are found in the circulation of plants and animals. While RNAs that move between cells can regulate physiology and development in plants, identities and roles of such RNAs in animals are unknown. Evidence for the movement of RNA between cells has been obtained in C. elegans through transgenic expression of double-stranded RNAs (dsRNAs) within tissues. Specifically, when a transgene is used to express dsRNA in one tissue, silencing of a matching gene can be detected in other tissues. This silencing requires the dsRNA-selective importer SID-1, suggesting movement of dsRNA between cells. However, multiple models can be envisioned to explain the observed silencing. For example, dsRNA within a cell could be packaged during early embryogenesis into intracellular vesicles that are then inherited through cell divisions, such that dsRNA can later enter into the cytosol and cause silencing. In this model, dsRNA is produced in one cell and causes silencing in another without becoming extracellular. Alternatively, an embryonic cell could deliver dsRNA into an adjacent embryonic cell. In this model, dsRNA enters the extracellular space before the formation of the pseudocoelom. Finally, cells in mature tissues could release dsRNA into circulation and distant tissues could then import dsRNA. In this model, dsRNA transits through the pseudocoelom. Because each model requires a different developmental setting, determining when the SID-1-dependent movement of dsRNA is supported in C. elegans could constrain possibilities. To restrict production, export, and import of dsRNA to specific periods during development, we are implementing optogenetic methods that can enable precise control of these processes. To control export, we will damage neurons expressing dsRNA at different time points during development using the photosensitizer protein miniSOG. Expressing miniSOG in neurons and exposing the worm to blue light will generate reactive oxygen species, causing neuronal damage. Evaluating silencing after the resultant damage will indicate when in development neurons producing dsRNA must be functional to observe gene silencing in a non-neuronal tissue. Preliminary data suggest that the extent of silencing differs depending on the targeted gene and the developmental time point when worms are exposed to light. Finally, to control production of dsRNA and its import, we will use a light-activated gene expression system to express dsRNA in neurons and the dsRNA-selective importer SID-1 in non-neuronal tissues of
sid-1(-) worms, respectively. Progress towards implementing these optogenetic systems will be presented at the meeting.