During embryogenesis, the de novo generation of Hematopoietic stem cells (HSCs) is restricted temporally and spatially. In the mouse, HSCs are generated between embryonic day (E) 10.5 and E12. During this period, HSCs arise from the vessels through which they flow. Specifically, HSCs arise from an arterial specific subpopulation of endothelium aptly called hemogenic endothelium. Hemogenic endothelial cells can be distinguished from their non-hemogenic neighbors based on the expression of the transcription factor Runx1. As a master regulator of hematopoiesis, Runx1 initiates the downregulation of endothelial genes and the upregulation of hematopoietic genes, resulting in the direct transition of hemogenic endothelial cells into hematopoietic cells. The specification of hemogenic endothelium during development is not fully understood. Furthermore, it is unknown whether Runx1 activity is sufficient to confer hemogenic properties to non- hemogenic endothelial populations. If the specification of HSC-producing endothelium can be delineated the field will be one step closer to differentiating pluripotent stem cells or adult endothelium into HSCs. The goal of this proposal is to determine if Runx1 is sufficient to induce the transition of non-hemogenic endothelial cells into functional blood cells and to discern the mechanism by which endothelial resistance to Runx1 activity occurs. My preliminary data suggest that when Runx1 expression is induced in all endothelium at E6.75 there are significantly more phenotypic blood cells in the conceptus at E10.5 and a corresponding decrease in vascular density. This suggests that Runx1 alone is sufficient to induce an endothelial to hematopoietic transition in vivo. However, when Runx1 is induced in endothelium slightly later during development at E8.5 there is no effect on blood cell number or vascular density at E10.5. This suggests that at some point after E6.75 endothelium is no longer permissive to Runx1 activity. In this proposal, I will confirm that ectopic Runx1 expression in non-hemogenic endothelium induces functional blood cells and I will discern the mechanism by which endothelial resistance to Runx1 activity occurs. First, I will perform colony forming and transplantation assays to determine if the ectopic blood cells that form as a result of ectopic Runx1 expression are erythroid, myeloid, lymphoid and/or hematopoietic stem cells. Secondly, I will use an in vitro model to determine if ectopically expressing Runx1 from the vascular endothelial cadherin (Cdh5) regulatory sequences influences the differentiation of mouse embryonic stem cells towards a hematopoietic fate. Lastly, I will discern the epigenetic mechanisms that are responsible for conferring resistance to Runx1 activity in endothelial cells. Together these experiments will demonstrate whether Runx1 alone is sufficient to induce the transition of non-hemogenic endothelium into functional blood cells and define the epigenetic mechanisms by which endothelial cells become stabilized and hence insensitive to Runx1 activity.

Public Health Relevance

A common treatment for patients with hematological malignancies such as leukemia and multiple myeloma is hematopoietic stem cell transplantation. Allogeneic hematopoietic stem cell transplantations are complicated by the risk of the life threatening inflammatory disease, graft-versus-host disease (GVHD). Results from this study will elucidate the specification of hemogenic endothelium. Hemogenic endothelium is a transient embryonic population of endothelial cells that give rise to hematopoietic stem cells. Delineating the specification of hemogenic endothelium will provide critical insights necessary for the in vitro production of HSCs from non-hemogenic patient endothelium. The production of patient-specific HSCs will eliminate the possibility of GVHD and the need for a stem cell donor.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Predoctoral Individual National Research Service Award (F31)
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Special Emphasis Panel (ZRG1)
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Welniak, Lisbeth A
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University of Pennsylvania
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Yzaguirre, Amanda D; Howell, Elizabeth D; Li, Yan et al. (2018) Runx1 is sufficient for blood cell formation from non-hemogenic endothelial cells in vivo only during early embryogenesis. Development 145:
Yzaguirre, Amanda D; Speck, Nancy A (2016) Extravascular endothelial and hematopoietic islands form through multiple pathways in midgestation mouse embryos. Dev Biol 415:111-121
Yzaguirre, Amanda D; Speck, Nancy A (2016) Insights into blood cell formation from hemogenic endothelium in lesser-known anatomic sites. Dev Dyn 245:1011-28
Yzaguirre, Amanda D; Padmanabhan, Arun; de Groh, Eric D et al. (2015) Loss of neurofibromin Ras-GAP activity enhances the formation of cardiac blood islands in murine embryos. Elife 4:e07780
Tamplin, Owen J; Durand, Ellen M; Carr, Logan A et al. (2015) Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche. Cell 160:241-52
Li, Yan; Esain, Virginie; Teng, Li et al. (2014) Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production. Genes Dev 28:2597-612