Formation of transient polykaryons by fusion of erythrocytes of different developmental programs

We report here two methods of fusing erythroid cells from bullfrogs (Rana catesbeiana), using polyethylene glycol or calcium phosphate, which yield masses of polykaryons in which the cytoplasms and nuclei of tadpole and adult frog erythroid cells are intermixed. The masses of fused cells carry out protein synthesis in culture, including the assembly of normal hemoglobin (Hb) tetramers. In these polykaryons there is reactivation of the expression of specific Hbs that have previously been "turned off" in vivo as the result of either a developmental Hb switch or normal cellular differentiation and RBC maturation. For example, the products of fusion of tadpole erythroblasts with adult frog mature RBCs synthesize adult Hb, whereas neither cell population alone does so. Recent experiments have taken advantage of a Hb-expression polymorphism that we discovered in this species, such that some tadpoles have greatly reduced expression of one of the larval Hbs (Hb Td-4). Fusion of erythroblasts from such tadpoles with RBC from frogs that had expressed Hb Td-4 when they were tadpoles produces polykaryons that synthesize Hb Td-4, indicating there is a trans factor that stimulates Td-4 expression. Heterospecific erythroid cell polykaryons can be constructed in an analogous manner, facilitating the study of trans-acting factors that regulate specific globin gene expression during development.