Comparative in situ characterization of erythroid cells found throughout the developing tongue tissue, circulation, and liver of fetal mice

Background

Erythroid cells contribute to embryonic organ development and adult tissue repair supplying oxygen to tissues. During mouse development, the primitive erythroid cells produced in the extraembryonic blood islands of the yolk sac begin to circulate as immature and nucleated erythroblasts with the onset of cardiac contractions around embryonic day 9.5 (E9.5). On the other hand, the definitive erythroid progenitors derived from the yolk sac and arterial vessels colonize the fetal liver, where they mature into small, enucleated definitive erythrocytes that are released into circulation at E11.5. In many cases, however, erythroblasts are also generated in situ during the development of tissue vasculature. We hypothesized that the properties of erythroid cells generated by peripheral tissue hematopoiesis may differ from those of contemporaneously circulating erythroid cells because hematopoiesis is spatiotemporally distinct from typical primitive and definitive hematopoiesis.

Methods

Comparative in situ protein expression analyses of erythroid cells in developing tongue, circulation, and liver of fetal mice were performed at E12.5 and E14.5, using immunofluorescence staining of several marker proteins for erythroid and endothelial cells.

Results

At E12.5, irregularly elongated immature vascular endothelial cells, many of which were in contact with nucleated erythroblasts, were distributed in the developing tongue. In the three fetal locations examined (tongue, circulation, and liver), most erythroid cells at E12.5 expressed CD31, which is involved in cell–cell interactions; however, the expression was downregulated by E14.5. Interestingly, at E12.5, several erythroblasts strongly expressing CD31 were found in the tongue, but less abundant in the circulation and fetal liver. Nearly all erythroblasts in E12.5 fetuses expressed primitive erythroid cell-specific βH1-globin; however, those strongly expressing the embryonic globin were scattered, particularly in the tongue, fewer in the circulation and fetal liver. On the contrary, erythroid cells expressing adult β1-globin were scarcely found in the tongue, which is in stark contrast to those in the circulation and fetal liver, where nearly all erythroid cells weakly expressed β1-globin at the embryonic stage. At E14.5, the number of βH1-globin-expressing cells drastically decreased; however, it was still significantly higher in the tongue than in the circulation and fetal liver. In all three locations, β1-globin accumulation and the enucleation of erythroid cells progressed uniformly and rapidly from E12.5 to E14.5.

Conclusion

The results indicate that primitive erythroblasts in the tongue highly express CD31, mature more slowly, and are replaced by definitive erythroid cells more slowly than those in the circulation and fetal liver. Primitive erythroblasts produced together with immature vascular endothelial cells during vasculogenesis in the developing tongue may perform functions other than oxygen supply, such as regulating vascular remodeling.