Using highly sensitive RNAseq to examine the whole transcriptome of enriched aortic hematopoietic stem cells and endothelial cells, the authors find G-protein–coupled receptor, Gpr56, is required to generate the first HSCs during endothelial to hematopoietic cell transition.

Interlinked gene regulatory networks (GRNs) are vital for the spatial and temporal control of gene expression during development. The hematopoietic transcription factors (TFs) Scl, Gata2 and Fli1 form one such densely connected GRN which acts as a master regulator of embryonic hematopoiesis. This triad has been shown to direct the specification of the hemogenic endothelium and emergence of hematopoietic stem cells (HSCs) in response to Notch1 and Bmp4-Smad signaling. Here we employ previously published data to construct a mathematical model of this GRN network and use this model to systematically investigate the network dynamical properties. Our model uses a statistical-thermodynamic framework to describe the combinatorial regulation of gene expression and reconciles, mechanistically, several previously published but unexplained results from different genetic perturbation experiments. In particular, our results demonstrate how the interactions of Runx1, an essential hematopoietic TF, with components of the Bmp4 signaling pathway allow it to affect triad activation and acts as a key regulator of HSC emergence. We also explain why heterozygous deletion of this essential TF, Runx1, speeds up the network dynamics leading to accelerated HSC emergence. Taken together our results demonstrate that the triad, a master-level controller of definitive hematopoiesis, is an irreversible bistable switch whose dynamical properties are modulated by Runx1 and components of the Bmp4 signaling pathway.

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hThe bone morphogenetic protein (BMP)/SMAD signaling pathway is a critical regulator of angiogenic sprouting and is involved in vascular development in the embryo. SMAD1 and SMAD5, the core mediators of BMP signaling, are vital for this activity, yet little is known about their transcriptional regulation in endothelial cells. Here, we have integrated multispecies sequence conservation, tissue-specific chromatin, in vitro reporter assay, and in vivo transgenic data to identify and validate Smad1؉63 and the Smad5 promoter as tissue-specific cis-regulatory elements that are active in the developing endothelium. The activity of these elements in the endothelium was dependent on highly conserved ETS, GATA, and E-box motifs, and chromatin immunoprecipitation showed high levels of enrichment of FLI1, GATA2, and SCL at these sites in endothelial cell lines and E11 dorsal aortas in vivo. Knockdown of FLI1 and GATA2 but not SCL reduced the expression of SMAD1 and SMAD5 in endothelial cells in vitro. In contrast, CD31؉ cKit ؊ endothelial cells harvested from embryonic day 9 (E9) aorta-gonad-mesonephros (AGM) regions of GATA2 null embryos showed reduced Smad1 but not Smad5 transcript levels. This is suggestive of a degree of in vivo selection where, in the case of reduced SMAD1 levels, endothelial cells with more robust SMAD5 expression have a selective advantage.