Analysis of Runx1 expression at the onset of definitive hematopoiesis in the E8.5 mouse conceptus. (A) VE-cadherin⁺Ter119⁻CD45⁻CD41^−/lo putative hemogenic endothelium and VE-cadherin⁺Ter119⁻CD45⁻CD41⁺ definitive hematopoietic cells were isolated from wild-type E8.5 (7-12 sp) YS and PAS (posterior part of embryo including PAS, vitelline, and allantois) by flow cytometry. (B) Quantitative RT-PCR of total Runx1 expression in the cell populations isolated in panel A. Relative Runx1 expression was obtained by normalization to Gapdh. (C) RT-PCR analysis of the ratio of P1-Runx1 and P2-Runx1 transcripts in VE-cadherin⁺Ter119⁻CD45⁻CD41^−/lo putative hemogenic endothelium and VE-cadherin⁺Ter119⁻CD45⁻CD41⁺ committed definitive hematopoietic progenitor cells. Although both promoters actively transcribed Runx1 in all populations analyzed, there was a skewing toward use of the P2 promoter. Note that no intersample comparisons of Runx1 levels can be made; see panel B for this. Data represent the relative Runx1 levels and P1/P2-Runx1 ratio in cell populations isolated from a pool of 24 wild-type YS or PAS and are consistent with results obtained from VE-cadherin⁺Ter119⁻CD45⁻CD41^−/lo and VE-cadherin⁺Ter119⁻CD45⁻CD41⁺ cells isolated from the combined E8.5 YS and PAS (n = 2; data not shown).