Microrna Modulation Of The Non-Canonical Wnt Signaling Pathway Is Essential For Cellular Morphogenesis
Date
2016-05
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
The Wnt signaling pathways are highly evolutionarily conserved in regulating cell specification, cell polarity and morphogenesis in development. The non-canonical Wnt pathways (ncWnt) consist of the Wnt/Planar Cell Polarity (Wnt/PCP) and the Wnt/calcium (Wnt/Ca2+) pathways. In all Wnt signaling pathways, Dvl transduces Wnt ligand activation. We hypothesize that perturbation of the Wnt/Ca2+ pathway with drugs will impact the morphogenic movements of primary mesenchyme cells (PMCs), which give rise to the skeleton spicules that facilitate larval swimming and feeding in the sea urchin embryo. To investigate specifically the regulation of ncWnt/Ca2+ pathway on the directed migration of PMCs, we treated sea urchin embryos with inhibitor Cyclosporin A (CsA), activator Phorbol 12-myristate 13-acetate (PMA) and inhibitor KN-93 that disrupt the ncWnt/Ca2+ pathway. All drug treatments led to significantly shorter skeleton spicules compared to untreated embryos. PMA and KN-93 treated embryos also resulted in PMC patterning defects while CsA did not. These results indicate that Wnt/Ca2+ pathway is important for the development of PMCs. To further examine the regulation of Wnt pathways, we tested the post-transcriptional regulation of Dvl by microRNAs (miRNAs). miRNAs are non-coding RNA molecules that mediate post-transcriptional regulation by binding to the 3’untranslated regions (3’UTR) of target transcripts. Using luciferase constructs and site-directed mutagenesis, we identified Dvl to be directly regulated by at least one miRNA. To examine the in vivo impact of miRNA regulation on Dvl, we treated newly fertilized sea urchin eggs with Dvl miRNA Target Protector (Dvl miRNA TP) designed to specifically block miRNA binding to the Dvl mRNA. Our results indicate that the removal of miRNA regulation of Dvl led to defects in skeleton spicule formation. While the directed migration of PMCs were unaffected by Dvl miRNA TP treatment PMCs lack membranous connections that allow them to form a syncytium. In summary, this study reveals the important role of Wnt/Ca2+ in regulating PMC morphogenesis and demonstrated that at least one miRNA directly regulates Dvl. These results contribute to a deeper understanding of the Wnt signaling pathway in regulating PMC development and function. This in turn may help us understand underlying causes of developmental defects resulting from failed control of cell migration.
Description
Keywords
biological sciences, wnt signaling