The effect ofHexdeletion on embryonic hematopoiesis. (A) RT-PCR and morphology of BL-CFC hemangioblast colonies. In vitro formation of definitive hemangioblasts from day-3 EBs expresses low levels of Brachyury, but high levels of Runx1 and Flk-1, and has characteristic BL-CFC colony morphology.^1,4  Hex is not expressed in these BL-CFCs by RT-PCR. BL-CFC colony morphology is shown at × 40 magnification. (B) Primitive and definitive hematopoietic progenitor assays from in vitro differentiation of Hex^-/-, Hex^+/-, and Hex^+/+ EBs. There was no effect of the deletion of the homeoprotein Hex on in vitro formation in day-3 EB hemangioblasts (i) and day-6 EB EryPs (ii). However, there was a dose-dependent requirement for the presence of Hex for the proper development of in vitro EB definitive BFU-E (iii), CFU-GEMM (iv), CFU-GM (v), and CFU-Meg (vi) hematopoietic progenitors. All assays were performed 3 times in triplicate. (C) Measurement of in vivo day-8.5 yolk sac hematopoietic progenitor formation. Both primitive and definitive hematopoietic progenitors are present at this stage and are measured together in this assay. The lack of Hex also reduces the ability of yolk sacs to develop in vivo in hematopoietic progenitors (n = 41 embryos). Error bars represent SEM.