Etv2-mediated hemangiogenic fate commitment of mesoderm

A comprehensive understanding of a lineage map and molecular mechanisms underlying lineage specification is fundamental to development. To this end, ETS transcription factor Etv2 functions as the master regulator of hematopoietic and endothelial cell formation. As such, Etv2 regulated hemangiogenesis provides an excellent model for assessing cell lineage specification. Herein, we generated several reporter embryonic stem (ES) cell lines to map developmental route pertaining to hemangiogenesis. We performed transcriptome analysis and high throughput CRISPR screening to further delineate molecular mechanisms regulating hemangiogenic lineage specification. We show a distinct lineage map of hemangiogenesis, in which hemangiogenic fate is specified not simply by the onset of Etv2 expression, but in a threshold-dependent manner. Importantly, VEGF-FLK1 signaling is necessary for efficiently achieving ETV2-threshold. Moreover, we find forkhead transcription factor Foxh1 to be required for FLK1 mesoderm formation. These studies provide a roadmap in hematopoietic and vascular cell generation and applications in regenerative medicine. We utilized T(Brachyury), Etv2 and Scl expression together with PDGFR-alpha and FLK1 mesodermal markers to track mesoderm differentiation. (1) We performed transcriptome analysis of different stages in the generation of Etv2-high cells. 7 samples were included. (2) We performed Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated screening for genes implicated in hemangiogenic cell lineage specification, in which 4 samples were included.