Ldb1 deletion leads to defects in primitive hematopoiesis and hemangioblast development. (A) At 9.5 dpc, blood and a vascular network in the Ldb1^−/− embryo yolk-sac is completely absent. (B) Ldb1^+/+ and Ldb1^−/− ES cells were differentiated into EBs. After 3 and 4 days, Ldb1^+/+ and Ldb1^−/− EBs look similar. After 8 days, Ldb1^−/− EBs lack erythroid clusters (gray arrows). (C) CFC assay with cell suspensions from Ldb1^+/+ and Ldb1^−/− day 6 EBs. Primitive erythroid and macrophage colonies were able to grow from Ldb1^+/+ but not Ldb1^−/− cultures. (D) CFC assay with cell suspensions from Ldb1^+/+ and Ldb1^−/− day 6 EBs as well as Ldb1^−/− EBs at day 6 expressing exogenous Ldb1. The colonies that formed were counted as primitive erythroid or macrophage colonies according to morphology and color. The figure shows that although Ldb1 absence impairs the formation of primitive erythroid and macrophage colonies, this phenotype can be rescued with Ldb1 expression. (E) FACS analysis of day 6 and day 8 Ldb1^+/+ and Ldb1^−/− EBs. CD41⁺ primitive erythroid progenitors are not present in the Ldb1^−/− EBs that contain less than half the number of CD31⁺ endothelial cells compared with the Ldb1^+/+ EBs. Cell suspensions were stained with either CD41-PE or CD31-FITC. CD41-PE⁺ cells were detected through the Fl2 channel and CD31-FITC⁺ cells were detected through the Fl1 channel. (F) FACS analysis of day 4 EBs. Ldb1^−/− EBs contain approximately 50% fewer Flk1⁺ BL-CFCs than Ldb1^+/+ EBs. (G) Ldb1^+/+ Flk1⁺ BL-CFCs give fully-grown blast colonies but Ldb1^−/− Flk1⁺ BL-CFCs do not.