18 Circulation Plays an Essential Role in Distributing Hematopoietic Progenitors from the Yolk Sac to the Embryo Proper; Lessons from the Ncx1-Null Mouse.

Abstract

The yolk sac is the lone site of primitive hematopoiesis. However, the role of the yolk sac in generating definitive hematopoietic progenitors has remained controversial since the onset of circulation alters the localization of hematopoietic progenitors. The Ncx1 knockout mouse fails to initiate a heartbeat and thus permits an analysis of the temporal and spatial distribution of definitive hematopoietic progenitor cells in an embryonic environment lacking circulation. Embryos were harvested from timed Ncx1 heterozygote crosses beginning at the onset of circulation at embryonic day 8.5 (E8.5) and ending 36 hours later (E10). The developmental stage of each embryo was determined by counting somite pairs. All embryos were carefully separated from their yolk sac (YS) and the paraaortic splanchnopleura (PSp) was dissected from the embryo proper. The tissues were digested and plated in methylcellulose. Colonies were counted after seven days. At E8.5, definitive hematopoietic progenitors are enriched in the YS more than 28-fold compared to the PSp in both wild type and Ncx1-null embryos. This ratio drops to 3-fold enrichment in the YS following progenitor redistribution by a functional circulation (E10) in wild type embryos, as reported previously (McGrath, et al. Blood 2003). The YS of Ncx1-null embryos, which lack a functional circulation, contain as many definitive progenitors as wild type YS and PSp combined. Few hematopoietic progenitors are found in the Ncx1-null PSp as late as E10, resulting in a 72-fold enrichment of progenitors in the Ncx1-null YS. We conclude that the PSp does not appear to function as a site of active hematopoiesis prior to E10. Furthermore, our findings support a model in which large numbers of definitive hematopoietic progenitors are generated in the YS between E8.5 and E10 and are redistributed to the embryo proper of the mouse embryo once cardiac function initiates a functional circulation.