Dysregulation of Wnt-Signaling and a Candidate Set of miRNAs Underlie the Effect of Metformin on Neural Crest Cell Development

Neural crest cells (NCC) are a population of epithelial cells that arise from the dorsal tube and undergo epithelial-mesenchymal transition (EMT) eventually generating tissues from peripheral nervous system, melanocytes, craniofacial cartilage, and bone. The antidiabetic drug metformin reportedly inhibits EMT in physiological conditions like cancer and fibrosis. We hypothesize that perturbation of EMT may also contribute to developmental disabilities associated with neural crest (NC) development. To understand the molecular network underlying metformin action during NC formation, we first differentiated murine embryonic stem (ES) cells into NCC and characterized them by demonstrating spatiotemporal regulation of key markers. Metformin treatment prompted a delay in delamination of NCC by inhibiting key markers like Sox-1, Sox-9, HNK-1, and p-75. We then revealed that metformin impedes Wnt axis, a major signaling pathway active during NC formation via DVL-3 inhibition and impairment in nuclear translocation of beta-catenin. Concomitantly we identified and tested a candidate set of miRNAs that play a crucial role in NC cell fate determination. Further studies involving loss and gain of function confirmed that NCC specifiers like Sox-1 and Sox-9 are direct targets of miR-200 and miR-145, respectively and that they are essentially modulated by metformin. Our in vitro findings were strongly supported by in vivo studies in zebrafish. Given that metformin is a widely used drug, for the first time we demonstrate that it can induce a delayed onset of developmental EMT during NC formation by interfering with canonical Wnt signaling and mysregulation of miR-145 and miR-200.