Endoglin Is Required for Hemangioblast Development.

A critical role for endoglin (CD105) in early development has been demonstrated in mice deficient for this gene. Embryos homozygous for the endoglin mutation (eng^−/−) fail to progress beyond 10.5 days postcoitum due primarily to vascular and cardiac abnormalities (Bordeau et al, 1999). Analysis of 9.5 dpc eng^−/− embryos revealed abnormal vasculature and anemia of the yolk sac, suggesting that endoglin may be required for both blood and endothelial lineages. The hemangioblast, the bipotent precursor for hematopoietic and endothelial cells, can be assessed through the blast colony assay (BL-CFC) using a model system based on the in vitro differentiation of embryonic stem (ES) cells into embryoid bodies (EBs). To evaluate a role for endoglin in this early precursor, we differentiated eng^−/−, eng^+/−, and eng^+/+ (wild-type) ES cells into EBs. At day 3 of EB differentiation, cells were disrupted and plated for blast colony formation in methylcellulose media containing vascular endothelial growth factor (VEGF), stem cell factor (SCF), and thrombopoietin (TPO). We found no difference in blast colony formation between heterozygous and wild-type ES cells. However, a significant reduction in the number of BL-CFCs was observed in eng^−/− cells when compared to eng^+/− or eng^+/+ BL-CFCs (p < 0.001). Single eng^−/−, eng^+/−, and eng^+/+ BL-CFCs gave rise to secondary hematopoietic colonies as well as endothelial cells, confirming their nature as hemangioblasts. These results suggest that although endoglin is required for hemangioblast development, its absence does not affect the bipotentiality of formed BL-CFCs. Since anemia was a feature of 9.5 dpc eng^−/− yolk sac embryos, we also examined early erythropoiesis using the ES/EB system. For this purpose, eng^−/−, eng^+/−, and eng^+/+ ES cells were differentiated into EBs for 4 days, at which time cells were disrupted and plated for primitive erythroid colonies (EryP) in methylcellulose media containing IL-3, IL-6, SCF, and Epo. We observed a reduction in the number of EryP colonies in eng^−/− (p < 0.01) and eng^+/− (p < 0.05) EBs when compared to controls (eng^+/+). These results corroborate the anemia observed in vivo in the eng^−/− embryos. We used RT-PCR and flow cytometry analysis to detect endoglin expression during a time course of EB differentiation. Endoglin is expressed in ES cells and disappears with differentiation. Expression re-appears at day 3 of differentiation, concomitantly with specification of the hemangioblast. Expression thereafter increases, correlating with mature endothelial cells at later time points. We did not find major differences in gene expression for Brachyury, Flk-1, Tie-2, embryonic and adult globins in a time course of EB differentiation for eng^−/−, eng^+/−, and eng^+/+ ES cells. These data point out a role for endoglin, an ancillary receptor for several members of the transforming growth factor (TGF)-beta superfamily, in hemangioblast development.