ETV2 (Ets Variant Transcription Factor 2)- Rhoj Cascade Regulates Endothelial Progenitor Cell Migration during Embryogenesis

Objective: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. The regulatory mechanisms that govern their migration are not completely defined. Here, we describe a novel role for ETV2 (Ets variant transcription factor 2) in cell migration and provide evidence for an ETV2-Rhoj network as a mechanism responsible for this process. Approach and Results: Analysis of RNAseq datasets showed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 chromatin immunoprecipitation-seq and assay for transposase accessible chromatin-seq datasets, which showed enrichment of chromatin immunoprecipitation-seq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Migratory assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells, embryoid bodies, and mouse embryonic fibroblasts. Knockout of Etv2 led to migratory defects of Etv2-EYFP⁺angioblasts to their predefined regions of developing embryos relative to wild-type controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds the promoter region of Rhoj serving as an upstream regulator of cell migration. Single-cell RNAseq analysis of Etv2-EYFP⁺sorted cells revealed coexpression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both embryoid bodies and mouse embryonic fibroblasts, whereas, its expression was abolished in the Etv2 knockout embryoid bodies. Finally, shRNA-mediated knockdown of Rhoj resulted in migration defects, which were partially rescued by overexpression of ETV2. Conclusions: These results define an ETV2-Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration.