Abstract Body: Aims: Mechanisms that govern the emergence of multiple cell lineages from common mesodermal progenitors remain incompletely understood due to their limited number and accessibility. The pioneer transcription factor ETV2 sits at the top of the hematoendothelial (HE) lineage development molecular networks and directs both differentiation and reprogramming of endothelial lineage. In this study, we uncovered the transcriptional and epigenetic changes orchestrated by ETV2 to promote the HE lineage while suppressing other mesodermal lineages.

Methods and Results: We isolated cells poised for skeletal muscle, cardiac and HE progenitor lineages by fluorescence activated cell sorting using the mouse embryonic stem cell-embryoid body (ES-EB) differentiation system over multiple time points with and without ETV2 overexpression. Analyzing these progenitor cells primed for distinct and different fate choices by singe-cell RNA-sequencing and transposase-accessible chromatin sequencing (ATAC-seq), we defined the enhancers, transcriptional and epigenetic regulatory networks employed by ETV2 for its pioneer activity during HE lineage development. Overexpression of ETV2 directed the pluripotent cells towards a highly efficient generation of HE lineage at the expense of cardiac and skeletal muscle cell lineages. Dosage of ETV2 expression determined the sub-lineages of the HE progenitors. Tlr3 and Tlr4 were discovered as direct downstream targets of ETV2 contributing to the HE lineage development and their expression was dependent on ETV2 using overexpressing and null EBs. Master regulators of the cardiac lineage including Mesp1 were identified to be directly repressed by ETV2 in a context dependent fashion to suppress cardiac development in ETV2 overexpression cells. Repression of Mesp1 expression by ETV2 was further validated using Etv2 knock out (KO) EBs as well as stage-matched wild type (WT) and Etv2 null mouse embryos. Direct binding and down-regulation of Mesp1 by ETV2 overexpression was verified by ChIP-seq, electrophoretic mobility shift assay (EMSA) and qPCR.

Conclusion: Collectively, our results define the functional role(s) played by ETV2 expression levels in the differential promotion and suppression of alternate fate choices of the mesodermal progenitor cells.