RA signaling is not required for primitive hematopoiesis. (A,C) FACS analysis of the primitive hematoendothelial compartment in E8.0 Raldh2^−/− and WT yolk sac. All population data were calculated as a percentage of the total live cell population plus or minus SEM (n ≥ 3). Statistical analysis of significance was determined by Student t test, with a confidence interval of 95% (P ≤ .05). (B) Benzidine staining reveals normal blood island development in E8.0 Raldh2^−/− and WT embryos. (D) qPCR analysis of hematopoietic gene expression in E8.0 Raldh2^−/− versus WT littermate yolk sacs. All data points were calculated as gene expression relative to endogenous β-actin expression (n = 3). (E) Quantitative measurement of intravascular plasma flow in precirculation E8.0 Raldh2^−/− and WT embryos. FRAP was used to determine the average rate of plasma flow in caudal vessels of 6- to 8-somite Raldh2^−/− (n = 12) and WT (n = 63) yolk sacs, before entry of blood cells into the systemic circulation. Plasma flow was calculated as the mean per embryo plus or minus SEM.