Constitutively active form of EpoR induces premature expression of the adult β major globin gene and production of EryD.

(A) E8.25 chimeric embryos injected with EpoR^R129C/− ES cells show high level of adult β major globin expression in the YS blood islands (BI) by whole-mount in situ hybridization (top left). In contrast, β major expression is absent in embryos injected with WT or EpoR^−/− blue ES cells or stage-matched noninjected controls (top right). Original magnifications, top panels, ×10. Histologic sections from EpoR^R129C/− chimeric embryos show positive staining of β major expression in the blood islands (lower panels). No signal can be detected in the embryonic tissues (EP) or when probes derived from sense strand of β major globin cDNA fragment were used on matching samples (data not shown). Original magnification, bottom left, × 20; bottom right, × 100. (B) In vitro organ culture experiments (i-viii). E8 YS isolated from chimeric (chi) embryos injected with EpoR^R129C/− ES cells were cultured with or without Epo (iii-iv). Epo-independent erythropoiesis (arrows) could be observed in cultures even without Epo (iii). When cultured YS in (iii) were dissociated and stained with Wright-Giemsa, the presence of enucleated erythrocytes (arrow), corresponding to mature definitive RBCs, was clearly evident. As controls, no mature erythrocytes were formed in the WT YS cultures without exogenous Epo (i), and no erythropoiesis could be detected when EpoR^−/− YS were parallel cultured (v-vi). E8.5 YS from chimeric embryos without culturing also contained mature EryDs (viii). Original magnifications, panels i-vi, × 10; panels vii-viii, × 20; 2 insets, × 100.