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Dr Erica Hutchins

California Instirute of Technology

Using live imaging of RNA to unravel the mechanisms of the neural crest epithelial—mesenchymal transition

The neural crest (NC) is a multipotent stem cell population that undergoes a developmentally regulated epithelial—mesenchymal transition (EMT). Cranial NC cells, which arise in the head region of the embryo and are the only NC population in vivo with the ability to differentiate into craniofacial skeleton and cartilage, are indispensable for the development of the face. In the cranial NC, transient expression of the Wnt antagonist, Draxin, is critical for NC EMT and cell migration. How its expression is precisely temporally regulated has been unclear. Here, using an in vivo reporter construct, we show that the rapid degradation of Draxin mRNA is mediated post-transcriptionally via its 3’-untranslated region (3’-UTR). Using an adapted MS2-MCP reporter system and time lapse imaging, we further demonstrate that the MS2 construct containing the 3’-UTR of Draxin (MS2-Draxin 3’-UTR) localizes to small cytoplasmic granules resembling processing bodies (P-bodies) in migrating cranial NC cells. These granules not only co-localize with a fluorescently-tagged P-body component (DCP1a), but MS2-Draxin 3’-UTR also localizes to and is dissolved within P-bodies in migrating NC. Furthermore, knockdown of the RNA helicase DDX6, known to dissolve P-bodies, disrupted the granular localization of MS2-Draxin 3’-UTR, which instead appeared broadly cytoplasmic and failed to be degraded. Importantly, disruption of P-bodies via DDX6 knockdown also inhibited endogenous Draxin mRNA degradation and impeded cranial NC migration in vivo. This work provides the first description of P-bodies in vertebrate NC through an adapted RNA live imaging approach and identifies an mRNA that is targeted to and degraded within P-bodies. Together, our data highlight a novel and important role for P-bodies in an intact organismal system— playing an essential role in NC EMT and cell migration via targeted RNA decay. This work was funded by the US National Institutes of Health K99 DE028592 (EJH) and R01 DE024157 (MEB).