Identification of novel regulators of developmental hematopoiesis using Endoglin regulatory elements as molecular probes

Abstract

Enhancers are the primary determinants of cell identity, and specific promoter/enhancer combinations of Endoglin (ENG) have been shown to target blood and endothelium in the embryo. Here, we generated a series of embryonic stem cell lines, each targeted with reporter constructs driven by specific promoter/enhancer combinations of ENG, to evaluate their discriminative potential and value as molecular probes of the corresponding transcriptome. The Eng promoter (P) in combination with the 28/17/19-kb enhancers, targeted cells in FLK1 mesoderm that were enriched for blast colony forming potential, whereas the P/28-kb enhancer targeted TIE21/c-KIT1/CD412 endothelial cells that were enriched for hematopoieticpotential. These fractions were isolated using reporter expression and their transcriptomes profiled by RNA-seq. There was high concordance between our signatures and those from embryos with defects at corresponding stages of hematopoiesis. Of the 6 genes that were upregulated in both hemogenic mesoderm and hemogenic endothelial fractions targeted by the reporters,LRP2, amultiligandreceptor,wasthe only gene that hadnot previously been associated with hematopoiesis.WeshowthatLRP2is indeedinvolved indefinitivehematopoiesisandbydoingsovalidatetheuseof reportergene-coupled enhancers as probes to gain insights into transcriptional changes that facilitate cell fate transitions.