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

Abstract

We report the first RNAi-based gene knockdowns in a sea urchin. Dicer-Substrate interfering RNAs (DsiRNAs) successfully disrupted expression of multiple transcripts in embryos of the sea urchinLytechinus variagatus,phenocopying known morpholino and inhibitor knockdowns. We describe our approach for the design of DsiRNAs most likely to target mRNAs of interest. An examination of DsiRNAs directed againstpks1, an enzyme necessary for pigment production, reveal destruction of thepks1mRNA without destroying the now-albino pigment cells. Application of DsiRNA on Nodal-Lefty signaling in this species confirms well-known functional studies. DsiRNA-based knockdowns ofnodalandleftyphenocopy morpholino- and drug-based inhibition of expression of these genes. A dilution series of DsiRNAs directed againstnodalandleftyreveals new diffusion properties of these signals in specifying dorsal vs ventral mesoderm.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.