Endoglin Identifies the First Wave of Hematopoietic Progenitors During Embryogenesis.

Abstract 1600

Endoglin (Eng), an ancillary receptor for several members of the Transforming Growth Factor (TGF)-beta superfamily, plays a critical role in early development. Eng^-/- embryos dye around 10.5 dpc due primarily to vascular and cardiac abnormalities. Yolk sacs (YS) of 9.5 dpc Eng^-/- embryos present abnormal vasculature and anemia. However, the mechanism by which endoglin leads to an anemic phenotype is unclear. Using in vitro differentiation of Eng^-/- ES cells, we have previously demonstrated that endoglin is required for proper hemangioblast and primitive hematopoietic development. To test the hypothesis that endoglin might have a direct effect on blood formation, here we investigated the role of endoglin during hematopoiesis in vivo. We first evaluated YS from 9.5 dpc Eng^-/- embryos and observed significantly reduced numbers of hematopoietic colony-forming cells (CFCs), in particular GEMMs and BFU-Es, when compared to both Eng^+/− and Eng^+/+ embryos. Real-time PCR analysis revealed decreased expression of embryonic globin, Gata-1, and SCL in the mutant YS. We then investigated the function of endoglin in wild-type mice by sorting the endoglin positive and negative population from E7.5 to 9.5 CD1 mouse embryos. When cultured on the stromal cell line OP9 in the presence of hematopoietic cytokines, only cell cultures derived from the Eng⁺ fraction gave rise to hematopoietic progenitors, as evidenced by colony assays and FACS analyses. Eng^neg cells did not produce any hematopoietic colonies. We then separated cells into 4 fractions using Eng and Flk-1, and found that from E7.5-9.5, the hematopoietic progenitor activity resided in the Eng⁺Flk-1⁺ double-positive fraction. FACS and microarray analyses of the 4 fractions in E7.5 embryos demonstrated that the Eng⁺Flk-1⁺ fraction highly expresses both endothelial and hematopoietic markers, including VE-Cadherin, Tie-2, PECAM1, Gata-1, SCL, and Lmo2, indicative of hemogenic endothelial potential. Taken together, our findings demonstrate that endoglin marks the first wave of hematopoietic progenitors during development, pointing to a defined role for endoglin in the specification of YS hemogenic endothelium as early as E7.5, in addition to its well defined role in vascular branching and remodeling.