Properties of the earliest clonogenic hemopoietic precursors to appear in the developing murine yolk sac.

Abstract

We have found that a variety of clonogenic hemopoietic cells can be obtained in a viable state from mouse conceptuses as early as day 7 of gestation when tissues are disaggregated in a crude collagenase solution containing fetal bovine serum. Examination of the time course of colony formation, and the ultimate size and lineages represented in colonies produced in semisolid medium containing methylceflulose, together with analysis of individual erythroid colonies stained with rabbit antisera specific for adult (HbA) and embryonic (HbE) mouse hemoglobins, revealed the presence on days 7 and 8 of gestation (but not later) of erythropoietic progenitors that give rise to mature erythroid colonies containing up to 100 HbE-containing erythroblasts after 4-6 days of growth in culture. These progenitors are highly sensitive to the disaggregation conditions used. Clonogenic progenitors of exclusively HbA-positive erythroblasts can also be detected in the day-7 conceptus. Assays of progenitors from separately disaggregated yolk sacs and embryos from day-8 conceptuses yielded colonies only from yolk sac suspensions, and again these contained either HbE-and HbA-positive erythroblasts or only HbA-positive erythroblasts. These findings demonstrate the very early appearance in the yolk sac of a population of erythroid progenitors with a number of unique properties. Although most of these yield HbE-positive erythroblasts in vitro, some produce erythroblasts containing HbA only. Such a developmental pattern is consistent with the hypothesis that definitive erythropoiesis in the mammalian fetal liver is derived from stem cells that originate in the yolk sac blood islands.In the developing mouse embryo, hemopoiesis begins with the formation of the yolk sac blood islands on the seventh day of gestation (1). Two days later the circulatory system is complete, and "primitive" erythroblasts are the predominant cell type found in the embryonic blood at that time (1-4). Unlike adult erythroid cells, these circulating primitive erythroid cells remain nucleated until they disappear around the 15th day of gestation (1-5). In addition, they are unique in their ability to synthesize embryonic hemoglobin (HbE), although with time they also produce some adult hemoglobin (HbA) (6). Erythrocytes containing exclusively HbA first appear in the fetal liver, which is formed shortly after day 10 of gestation (7,8).The restriction of HbE production to a gestationally early cohort of erythroid cells that show other unique differentiation features has led to the concept of a developmentally separate lineage of primitive hemopoietic cells. In previous studies, we showed that progenitors of exclusively HbApositive erythroid cells were already present in the 9-day-old conceptus, at a time when the fetal liver is not yet formed (9). In addition these studies revealed an unusual sensitivity of gestationally early precursors to conventional methods for obtaining single-cell suspensions, although organ cultures of day-8 conceptuses did suggest the existe...

Yolk-sac hematopoiesis

“…These progenitor colonies, called "day-5 CFU-E," consisted of approximately 100 red cells expressing both embryonic and adult globins. Day-5 CFU-E progenitors arose early on the eighth gestational day exclusively in the yolk sac and disappeared by E9 [40]. It was subsequently confirmed that day 5 CFU-E are primitive erythroid progenitors (EryP-CFC) that generate colonies in semisolid media with a distinct timing of differentiation, morphology, and pattern of gene expression compared to the definitive BFU-E and CFU-E [41].…”

“…Wong et al [40] confirmed that BFU-E arose late on the eighth day of gestation and continued to increase in number at E9 and E10. Unexpectedly, these definitive erythroid progenitors were found exclusively in the yolk sac at E8.5, but by E9.0 were also present in the bloodstream and the embryo proper [40,44]. Recently, an analysis of carefully staged mouse embryos revealed that BFU-E originate in the yolk sac of early somite pair embryos at E8.25, just prior to the onset of circulation [42].…”

“…Early erythroid progenitors in the murine bone marrow are recognized by the generation of large colonies of red cells in methylcellulose (burst-forming unit erythroid; BFU-E) following 7-10 days in culture, while more mature erythroid progenitors generate smaller colonies (colony-forming unit erythroid; CFU-E) within 2-3 days in culture. Wong et al identified a unique intermediate erythroid progenitor type during gastrulation in the mouse embryo [29,40]. These progenitor colonies, called "day-5 CFU-E," consisted of approximately 100 red cells expressing both embryonic and adult globins.…”

“…EryP-CFC are confined to the yolk sac, where they expand in numbers at E8.25 (early somite pair stages). However, by E9.0 (20-25 somite pair stages) they were no longer present in the conceptus ( [40,42] and Fig. 3).…”

Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis

“…These primitive erythroid progenitors generate large nucleated red cells that sustain embryonic growth until E12.5 when definitive enucleate red cells begin to enter the bloodstream from the liver. The first definitive erythroid progenitors [burst-forming units-erythroid (BFU-E)] originate in the yolk sac at early somite-pair stages at E8.25 (9,10). It is not known whether these unilineage primitive and definitive erythroid progenitors are derived from multipotential hematopoietic precursors, because multipotential precursors are detected first at E8.5 (11) and CFU-S are not detected until E9.5 (12,13).…”