Circulation is required to distribute hematopoietic progenitors and differentiated cells to the embryo proper. (A) Primitive progenitor colony number is not altered by the lack of flow in Ncx1^−/− embryos. WT embryos produced an average of 1617 (± 255; mean ± SEM) colonies while Ncx1^−/− embryos produced 1697 (± 227) colonies. (B) ζ-globin in situ hybridization specifically labels the primitive erythroblast lineage. At 6 sp, just after the first heart beats, no difference in blood distribution is detectable in Ncx1^−/− or WT embryos. Note the cluster of cells in the embryo proper at the site of connection of the vitelline vasculature (*). At 14 sp, a significant amount of ζ-globin staining is detected in the embryo proper of the WT while the Ncx1^−/− embryo remains devoid of erythroblasts. (C) Embryos (23 sp) were stained with benzidine, which stains all hemoglobin-containing cells dark blue/black. At this stage of development, all hemoglobin-containing erythroblasts are derived from primitive progenitors from the yolk sac (YS) and the vast majority of staining occurs in the YS. Note Ncx1^−/− EP has no stained erythroblasts compared with age-matched WT littermates. (D) (Representative definitive progenitor numbers) There is an expansion of definitive HPCs in both the YS and embryo proper from E8.5 of development through E9.5 in the WT embryos (Table S1). Definitive HPCs in the YS expand from 82 to 578 (colonies/YS) and in the PSp from 5 to 115 (colonies/PSp) during this time period. In the absence of a circulation in the Ncx1^−/− embryo, definitive HPC numbers in the YS range from 79 to 559 and are not significantly different from WT littermates. The Ncx1^−/− PSp ranges from 0 to 3 definitive HPCs, which is a significant decrease (P < .05 by paired Student t test) from WT at all timepoints examined. Images in panels B,C acquired as for Figures 1A and B. Original magnification, ×60.