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

Abstract

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.