Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation

10.1084/jem.20140767Journal of Experimental Medicine212193-10

Investigations into the regulation and function of Smad1/5 during embryonic hematopoietic and vascular development

Bone morphogenetic protein (Bmp) signalling has been implicated in the formation of both the hematopoietic and vascular systems, however many of its roles and regulatory mechanisms remain undefined. Here I have focused on two of the downstream effectors of the pathway, Smad1 and Smad5, which transduce the signal from the cell surface to alter gene transcription. Together, Smad1 and Smad5 are required for normal vascular development. In this thesis I have identified two enhancers for Smad1 and one promoter element for Smad5 which show activity in endothelial cells. Ets, Gata and Ebox transcription factor (TF) binding motifs were enriched and active at these sites, and members of these families were found to bind the Smad1 and Smad5 regulatory elements in the endothelium. Two of these factors, Fli1 and Gata2, were shown to be key upstream regulators of Smad1 and Smad5 in the endothelium. Further, Fli1 was found to control endothelial cell response to Bmp, partially through Smad1. A third factor, Tal1/Scl, was shown not to be a Smad1/5 regulator. Next, I investigated the role of Smad1 in hematopoietic development, where previous studies have suggested it plays a non-redundant role. Endothelial-specific deletion of Smad1 did not show any serious hematopoietic defects. Constitutive deletion of Smad1 resulted in early hematopoietic defects, with embryos showing significant reduction in transient wave and primitive progenitors at E8.5. At E7.5 Bmp/Smad signalling was found to be required for hematopoietic cell emergence from the hemogenic mesoderm, however, transcriptional analysis of mesoderm cells lacking Smad1 found little difference compared to wild-type. This work adds to our understanding of the complex roles and regulation of Bmp/Smad signalling during embryonic hematopoietic and vascular development

SMAD1 and SMAD5 expression is coordinately regulated by FLI1 and GATA2 during endothelial development.

The 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