SMAD4 has a critical role in erythropoiesis and HSC formation during embryogenesis

Abstract

Hematopoietic stem cells (HSC) are generated during embryogenesis from specialized vascular beds termed hemogenic endothelium (HE), which includes the aortic endothelium of the aortic-gonado-mesonephros (AGM) region. In the aorta, pre-HSCs are organized in clusters attached to the vascular endothelium. The TGF signaling pathway has a pivotal role in both arterial specification and HSC formation. Previous endothelial SMAD4 loss-of-function studies in mice using a Tie2cre have demonstrated a critical role of this protein in the endothelial to hematopoietic transition (EHT), with notable phenotypes of increased cluster formation and embryonic lethality at E10.5. In order to bypass the early lethality seen in other studies, we used an inducible arterial Cre to delete SMAD4 to evaluate the contribution of the TGF signaling pathway to later EHT events, as well as HSC migration, and colonization of adult hematopoietic organs. We noted embryonic lethality between E13.5-E17.5 in animals with SMAD4 loss of function in arterial endothelial cells. The mutants exhibited decreased HSC numbers, enlarged fetal livers, erythropoiesis defects, and the inability to engraft adult recipients. In addition, we evaluated the deletion of SMAD4 using NG2cre which is expressed within the sub-aortic mesenchyme during EHT. The mutant animals did not exhibit any hematopoietic abnormalities and survived until adult life. Our data suggest that the TGF signaling pathway in arterial endothelial cells is critical for definitive hematopoiesis; however other non-endothelial sources of TGF do not contribute to the endothelial to hematopoietic transition or definitive hematopoiesis.