An RNA interference approach for functional studies in the sea urchin and its use in analysis of Nodal signaling gradients

Abstract

AbstractDicer substrate interfering RNAs (DsiRNAs) destroy targeted transcripts using the RNA-Induced Silencing Complex (RISC) through a process called RNA interference (RNAi). This process is ubiquitous among eukaryotes. Here we report the utility of DsiRNA in embryos of the sea urchinLytechinus variagatus (Lv).Specific knockdowns phenocopy known morpholino and inhibitor knockdowns, and DsiRNA offers a useful alternative to morpholinos. Methods for designing and obtaining specific DsiRNAs that lead to destruction of targeted mRNA are described. DsiRNAs directed againstpks1, an enzyme necessary for pigment production, show how successful DsiRNA perturbations are monitored by RNAin situanalysis and by qPCR to determine relative destruction of targeted mRNA. DsiRNA-based knockdowns phenocopy morpholino- and drug-based inhibition ofnodalandlefty. Other knockdowns demonstrate that the RISC operates early in development as well as on genes that are first transcribed hours after gastrulation is completed. Thus, DsiRNAs effectively mediate destruction of targeted mRNA in the sea urchin embryo. The approach offers significant advantages over other widely used methods in the urchin in terms of cost, and ease of procurement, and offers sizeable experimental advantages in terms of ease of handling, injection, and knockdown validation.HighlightsDsiRNA provides an RNAi approach for perturbation of sea urchin embryos. A dilution series of DsiRNA oligos reveals properties of the Nodal gradient in establishing the Dorsal-Ventral axis.