Primitive erythroid cells enucleate in association with macrophages. (A) Late-stage primitive erythroblasts derived from fetal blood do not autonomously enucleate when cultured in vitro. In control cultures, immature definitive erythroblasts derived from the fetal liver (E14.5 liver, solid blue line) continue to divide as they mature. This maturation includes autonomous enucleation as evidenced by the increasing accumulation of erythrocytes (dashed blue line). The nucleated cells in E14.5 blood are late-stage primitive erythroblasts that have ceased dividing. Unlike definitive cells, late-stage primitive erythroblasts did not complete their maturation by enucleating in vitro as evidenced by the persistence of nucleated cells (solid red line) and the lack of increasing numbers of enucleated erythrocytes (dashed red line). (B) E14.5 primitive erythroblasts enucleate in vitro when cocultured with macrophage cells. The enucleation of primitive blood cells attached to macrophages was significantly higher than that of the same blood cells cultured in parallel without macrophages. The mean plus or minus SEM of 7 independent experiments is plotted. (C) Nuclear engulfment by macrophages is evident after reconstitution of erythroid islands with E14.5 primitive erythroid cells. E14.5 blood was prelabeled with cytotracker dyes to determine the source of nuclei. The left panels are Hoffman contrast images overlayed with HO staining of nuclei. The right panels are fluorescent images revealing the prestained cytotracker green (top) or cytotracker red (bottom) cells. After 48 hours of reconstitution, most of the attached cells from E14.5 blood (top panels) are primitive orthochromatic erythroblasts with occasional enucleated primitive erythrocytes (*). Additional small nuclei, independent of orthochromatic erythroblasts, are also evident (arrowheads). Stripping of erythroblast islands (lower panels) reveals the persistence of labeled nuclei engulfed by macrophage cells.